Showing papers in "Plant Cell Tissue and Organ Culture in 2020"
TL;DR: The current status of understanding the genetic and epigenetic changes that occur during tissue culture are described and variations appeared in regenerated plants as well as variation inherited by generative progeny of regenerants are described.
Abstract: The in vitro tissue cultures are, beyond all difficulties, an essential tool in basic research as well as in commercial applications. Numerous works devoted to plant tissue cultures proved how important this part of the plant science is. Despite half a century of research on the issue of obtaining plants in in vitro cultures, many aspects remain unknown. The path associated with the reprogramming of explants in the fully functioning regenerants includes a series of processes that may result in the appearance of morphological, physiological, biochemical or, finally, genetic and epigenetic changes. All these changes occurring at the tissue culture stage and appearing in regenerants as tissue culture-induced variation and then inherited by generative progeny as somaclonal variation may be the result of oxidative stress, which works at the step of explant preparation, and in tissue culture as a result of nutrient components and environmental factors. In this review, we describe the current status of understanding the genetic and epigenetic changes that occur during tissue culture. Variation appeared in regenerated plants as well as variation inherited by generative progeny of regenerants can may many, positive or negative impact, of gained plant materials. This review focused on factors that triggered this phenomenon with underlying oxidative stress.
69 citations
TL;DR: Increase the efficiency of chromosome doubling and fast-reliable screening of desired phenotypes are prerequisites for production of custom-designed medicinal and ornamental plants through artificial polyploidy induction.
Abstract: Green plants provide food, fuel, materials, and medicine for humans. Amongst these, medicinal plants contain unique molecules with therapeutic applications. However, our understanding of these organisms is in its infancy. Landscaping plants also have a great impact on the human soul and emotional perception. These two groups of plants are part of natural wealth and have great economic value. One of the remarkable breeding strategies to improve the valuable properties of plants is artificial polyploidy induction. Medicinal and ornamental plants with duplicated whole sets of chromosomes, although not always, have more distinctive characteristics such as modified phytochemical profile, higher content of desired pharma molecules, plant form, flower color, size and style, fragrance, vase life, and prolonged flowering period. Therefore, artificial chromosome doubling (ACD) of ornamental and medicinal plants could have significant economic consequences. In medicinal plants with an ornamental flower/inflorescence, and/or in ornamental plants bearing essential oils, ACD is a dual beneficial breeding strategy. Working with both in vitro and in vivo chromosome doubling pathways, researchers are able to produce custom-designed plants with higher marketability. Some critical factors must be considered to establish a successful ACD protocol. The plant genotype and explant type are important parameters in this procedure. Type, dosage and duration of application (exposure time) of antimitotic agent (AMA) should also be considered as the main factors. Antimitotic agents can be classified according to their efficacy and toxicity. Although colchicine is the most applied and well-known AMA, there are other alternative mitotic spindle inhibitors with higher specificity for tubulin binding sites in plant materials and possessing less toxicity for animal tubulin. Testing the interaction effects of plant and AMA parameters is necessary for establishing an ACD breeding program and promoting the economic values of medicinal and ornamental plants. This paper reviews significant developments in polyploidization using different antimitotic agents over the last decade in the field of ornamental and medicinal plants. Artificial polyploidy induction is one of the applicable breeding methods in ornamental and medicinal plants. Increasing the efficiency of chromosome doubling and fast-reliable screening of desired phenotypes are prerequisites for production of custom-designed medicinal and ornamental plants through artificial polyploidy induction.
38 citations
TL;DR: An efficient and reproducible in vitro regeneration protocol was established for chickpea and the in vitro regenerated plants from all four explants were found to be the true to type with their mother plant.
Abstract: High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l−1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l−1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l−1 BAP along with 0.05 mg l−1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l−1), GA3 (1.0 mg l−1) and BAP (1.0 mg l−1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l−1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation. An efficient and reproducible in vitro regeneration protocol was established for chickpea. Application of different concentrations and combinations of PGRs was found to enhance
multiple shoot induction, shoot elongation, rooting and acclimatization of in vitro regenerated
plants in field conditions, and further evaluated genetic fidelity using molecular markers.
37 citations
TL;DR: The MARS statistical approach was used to predict macro nutrient related growth responses of three strawberry species and provides an alternative statistical technique for tissue culture data analyses.
Abstract: Strawberry micropropagation is generally based on Murashige and Skoog mineral salts, and many cultivars grow well on this medium. However, the diverse species found in germplasm collections often do not thrive, which indicates a need to optimize the mineral nutrients. In this study, Multivariate Adaptive Regression Splines (MARS), was employed to predict shoot quality, multiplication, and leaf color responses of three strawberry species in response to the major tissue culture nutrients by generating functional associations. MARS is a non-parametric approach that can be used to deal with continuous and categorical data without requiring the strict distributional assumptions of the basic linear models. The MARS algorithm is capable of capturing non-linear patterns between the input and target variables. NH4NO3, CaCl2·2H2O, MgSO4·7H2O, KNO3 and KH2PO4 were tested in a range of 0.5 × to 3 × MS medium, within a computer-generated optimal design that consisted of 32 treatment combinations. The plant responses were affected by all of the major salts tested and the genotype factor. Multivariate Adaptive Regression Splines captured the significant factors and their interactions to predict optimal major salts suitable for all three strawberry species: 3300 mg L−1 NH4NO3, 862.4 mg L−1 CaCl2, 1110 mg L−1 MgSO4, 3439 mg L−1 KNO3, and 329.8 mg L−1 KH2PO4. This study identified the major nutrient needs of the three strawberry species and provides an alternative statistical technique for tissue culture data analyses. The MARS statistical approach was used to predict macro nutrient related growth responses of three strawberry species. The objective of the study was to make a gentle introduction to the MARS algorithm and show its potential application to tissue culture research.
36 citations
TL;DR: The aim of this work was to check the effect of various culture systems on the production of phenolic compounds in E. alpinum shoots cultured in vitro and the best system for shoot proliferation and phenolic acids and flavonoids accumulation was solid culture on MS medium with BAP, IAA, and GA3.
Abstract: In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. Shoots of Eryngium alpinum L. were multiplied on Murashige and Skoog (MS) medium in various systems, namely on the solid media and in two liquid cultures—stationary and agitated, as well as via regeneration from callus. The biomass increments were closely correlated with the number of shoots arising from one explant, which was connected with the supplementation of the culture media with the studied plant growth regulators. The methanolic extracts from shoots grown in the tested systems were subjected to phenolic acids and flavonoids qualitative and quantitative analysis. Biomass from in vitro shoot cultures accumulated from 19.59 to 32.95 times more phenolic acids [the total content ranged from 272.52 to 458.38 mg/100 g dry weight (DW)] and from 3.02 to 4.43 times more flavonoids (the total content ranged from 100.03 to 146.98 mg/100 g DW), depending on the culture system, than the extracts from basal leaves from the intact plant (13.91 and 33.16 mg/100 g DW, respectively). The phenolics present in shoot cultures include seven phenolic acids—3,4-dihydroxyphenylacetic, caftaric, caffeic, neochlorogenic, chlorogenic, isochlorogenic, and rosmarinic acids, and three flavonoids—isoquercetin, quercitrin and robinin. The best system for shoot proliferation resulting in the highest biomass growth and phenolic acids and flavonoids accumulation was solid culture on MS medium with BAP, IAA, and GA3 (each 1.0 mg/l). The aim of this work was to check the effect of various culture systems (stationary and agitated, on solidified and in liquid media) on the production of phenolic compounds in E. alpinum shoots cultured in vitro. This is the first report on phenolic acids and flavonoids estimation in Eryngium alpinum in vitro biomass from different culture systems.
30 citations
TL;DR: Governments should consider the regulatory framework of genome editing technologies and establish appropriate regulations, if necessary, without representing an obstacle to the commercialization of products derived from them.
Abstract: Precision biotechnologies have appeared on the horizon resulting in a plethora of possibilities to modify the genome of different organisms with relatively easy application, low cost, and high precision. These technologies make it possible to work with a very simple biological system and have great potential for medicine, and agriculture. Latin American is embracing the technology and researchers are already developing tropical products from its use. The following article explains the operation of these technologies, and some considerations about its regulation among counties in Latin America and the Caribbean region. Survey results demonstrated that seven countries (Argentina, Brazil, Colombia, Chile, Guatemala, Honduras, and Paraguay) have a clearly defined and operational legal framework for new breeding technologies. Nevertheless, the majority of countries in the region have no experience regarding these technologies and lack legal clarity. Therefore, these countries require regulatory clarity to legally differentiate those products of gene editing that are comparable to conventional breeding and those that can be legally defined as a genetically modified organism. New precision biotechnologies could introduce advantageous traits for the improvement of crops, which could be available for the consumers in Latin America and the Caribbean region very soon. Nevertheless, governments should consider the regulatory framework of genome editing technologies and establish appropriate regulations, if necessary, without representing an obstacle to the commercialization of products derived from them.
29 citations
TL;DR: Cobalt and silver nanoparticles (CoNPs, AgNPs) were used to overcome leaf abscission, enhance growth and increase the survival rate of plantlets at the nursery stage and was the most suitable factor for shoots mass propagation.
Abstract: Leaf abscission of rose (Rosa hybrida L. ‘Baby Love’) cultured in vitro due to ethylene gas accumulation and enzymatic hydrolytic activity, is one of the abnormal phenomena affecting the shoots quality. In this study, silver nanoparticles (AgNPs) and cobalt nanoparticles (CoNPs) were used to overcome leaves abscission as well as, the effect of shoot mass propagation, rooting and acclimatization at the nursery stage. The results showed that shoots cultured on MS (Murashige and Skoog, in Physiol Plantarum 15(3):473–497, 10.1111/j.1399-3054.1962.tb08052.x, 1962) medium supplemented with 2 mg/L AgNPs gave the highest shoot multiplication coefficient, shoot height, fresh weight, dry weight and chlorophyll index (5.33 shoots; 3.06 cm; 451.00 mg; 58.33 mg; 32.28; respectively) than CoNPs (replace CoCl2 in the MS medium) and basal MS medium. Meanwhile in the rooting stage, MS medium added of 4.65 µg/L CoNPs was the most optimal with dry mass ratio (10.28%), number of roots (5.67 roots), root length (2.17 cm) and SPAD (41.07 nmol/cm2) as well as reduced the ethylene gas content (0.11 ppm) and enzymes activity such as pectinase (0.07 UI/mL) and cellulase (0.25 UI/mL) in comparision to the other treatments after 4 weeks culture. The plantlets derived from in vitro culture on medium added 4.65 µg/L CoNPs gave the highest survival rate (96.67%) as well as growth and development at the nursery stage. Cobalt and silver nanoparticles (CoNPs, AgNPs) were used to overcome leaf abscission, enhance growth and increase the survival rate of plantlets at the nursery stage. CoNPs helps reduce the ethylene gas content, enzymatic hydrolytic activity such as pectinase and cellulase. AgNPs was the most suitable factor for shoots mass propagation.
27 citations
TL;DR: MeJA is an effective elicitor for stimulating valuable phenolic acids production in M. spicata hairy root cultures and implied that MeJA has a significant impact on RA, CA, CGA, and CIA accumulation, which might be the consequence of gene activation from the phenylpropanoid pathway.
Abstract: Mentha spicata L., as a rich source of phenolic acids, is known for its therapeutic importance. Elicitors play a crucial role in biosynthetic pathways to improve plant secondary metabolites production. The purpose of this study was to investigate the effect of methyl jasmonate (MeJA) as a potent elicitor, on phenolic acids production and the relative changes in expression of their biosynthesis-related key genes (PAL, TAT, C4H, HPPR, and 4CL) in M. spicata hairy root cultures, to elucidate the interrelation between phenolic acids accumulation and the regulation mechanism of gene expression. The results showed that the relative expression levels of phenylpropanoid pathway genes, i.e., PAL, C4H, 4CL, and HPPR in the tyrosine-derived pathway increased 4.04-fold, 3.62-fold, 1.75-fold, and 1.45-fold in comparison to untreated controls, respectively. High-performance liquid chromatography analysis indicated that MeJA dramatically increased rosmarinic acid content (55.44 µg g−1 dry wt, about 11.84-fold) after 6 h exposure to elicitor. Moreover, caffeic acid, chlorogenic acid, and cinnamic acid contents were also enhanced significantly (p < 0.05) in response to MeJA treatment. On the other hand, the application of MeJA had a negative effect on both TAT expression level and lithospermic acid B accumulation. Our results implied that MeJA has a significant impact on RA, CA, CGA, and CIA accumulation, which might be the consequence of gene activation from the phenylpropanoid pathway (PAL, C4H, and 4CL) and HPPR, a key regulatory enzyme of the tyrosine-derived pathway in the elicitor-treated hairy root cultures of M. spicata. MeJA is an effective elicitor for stimulating valuable phenolic acids production in M. spicata hairy root cultures.
27 citations
TL;DR: It is suggested that light quality strongly influences medicinally valuable phenylpropanoid metabolites biosynthesis along with antioxidant potential in in vitro cultures of purple basil.
Abstract: Ocimum basilicum L. var purpurascens (purple basil) contains medicinally valuable metabolites. Light greatly influences the physiological processes, including biomass accumulation and secondary metabolites production in medicinal plants. Herein, we investigated the influence of different spectral lights on the biosynthesis of phenylpropanoid metabolites in purple basil callus cultures. Growth kinetics was ostudied for a total of 49 days, with 7 days of sampling time. Among the various treatments, blue light resulted in maximum biomass accumulation, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant DPPH, FRAP and ABTS activities, as compared to controls. Moreover, blue light also encouraged higher superoxide dismutase activity while the red light was found effective for enhanced peroxidase activity. HPLC analysis revealed enhanced rosmarinic acid (87.62 mg/g DW) and anthocyanins (cyanidin: 0.15 mg/g DW and peonidin: 0.13 mg/g DW) contents under dark grown callus cultures which were almost 1.55, 1.25 and 1.18–fold greater than controls, respectively. Conversely, red light caused maximum production of cichoric acid (14.65 mg/g DW). Moreover, a positive correlation occurred among the accumulation of phenolic and flavonoids and antioxidant activities. These results suggest that light quality strongly influences medicinally valuable phenylpropanoid metabolites biosynthesis along with antioxidant potential in in vitro cultures of purple basil. Light-enhanced precious metabolites in callus of Basil.
26 citations
TL;DR: It is shown that most Azospirillum strains cannot utilize sucrose as the sole carbon source and that their use to inoculate in vitro-grown plants does not lead to bacterial growth in the culture medium.
Abstract: The response of potato microplants to Azospirillum inoculation is highly variable both in vitro and ex vitro. Plant inoculation with plant-growth-promoting rhizobacteria (PGPR) is widely used to increase the effectiveness of clonal micropropagation. Azospirillum rhizobacteria are model subjects to investigate associative plant–microbe interactions. Here we show that most Azospirillum strains cannot utilize sucrose as the sole carbon source and that their use to inoculate in vitro-grown plants does not lead to bacterial growth in the culture medium. Of the eleven surveyed strains, seven gave a significant increase in at least one growth variable of in vitro-grown potato (Solanum tuberosum L. cv. Nevsky) microplants. Inoculation with six strains led to better survival of the microplants in soil. Only with three strains (A. brasilense Sp245, SR80, and A. halopraeferens Au4T) did inoculation in vitro significantly promote plant growth ex vitro. The inoculation results were correlated with the biochemical activity of the strains. Indole-3-acetic acid production ranged from 3.74 μg ml−1 with A. brasilense S27 to 87.3 μg ml−1 with A. brasilense Sp245. Active indole-3-acetic acid producers, but not nitrogen fixers, were better plant-growth-promoters. Inoculation in vitro with A. brasilense strains Sp245 and SR80 can be recommended for increasing the effectiveness of clonal micropropagation of potato.
25 citations
TL;DR: This article reviews the advances in tree peony micropropagation during the past three decades, providing a detailed analysis of the conditions required for the four stages of micro Propagation (initiation, multiplication, in vitro rooting, and acclimatization).
Abstract: Tree peony is a well-known ornamental plant that is also valued for its medical uses and edible oil production. A long breeding period and low propagation rate are the main hurdles hindering the development of the tree peony industry, for which micropropagation can offer a solution. This article reviews the advances in tree peony micropropagation during the past three decades, providing a detailed analysis of the conditions required for the four stages of micropropagation (initiation, multiplication, in vitro rooting, and acclimatization). Additionally, potential of tree peony micropropagation for commercial application was evaluated. This review provides valuable information for the successful micropropagation of tree peony, permitting more targeted and in-depth research into tree peony micropropagation. The review summarized in detail the results achieved in tree peony micropropagation over the last three decades. Furthermore, the potential of the existing micropropagation protocol was evaluated and the problems and the future perspectives were summarized.
TL;DR: In this article, different combinations of plant growth regulators NAA, 2,4-d and KIN were evaluated on Nicotiana tabacum callus induction from root, internode, petiole and leaf explants.
Abstract: Nano-technology has changed the properties of metal elements’ delivery into and effect on living systems. The current study, first, evaluated different combinations of plant growth regulators NAA, 2,4-d and KIN on Nicotiana tabacum callus induction from root, internode, petiole and leaf explants. Two mg L−1 NAA with 0.1 mg L−1 KIN on Murashige and Skoog (MS) medium induced calli in all explant sources. Then, the effect of different concentrations of nP-ZnO (0.015, 0.03, 0.06, 0.12, 0.24 mM) and µP-ZnO (0.03, 0.06, 0.12, 0.24 mM) compared to ZnSO4 (0.03 mM) on cell toxicity was investigated. SEM microscopy, XRD and EDX analyses were used to determine particles characteristics. Higher zinc content was accumulated in calli under nP-ZnO concentrations compared with µP-ZnO, which was positively correlated with calli fresh and dry weights. The nP-ZnO induced oxidative stress more significantly than µP-ZnO. Protein content did not increase under µP-ZnO, while it was raised at all concentrations of nP-ZnO that had positive correlation with MDA and ROS. SOD enzyme activity increased at all levels of µP-ZnO but remained unchanged under all levels of nP-ZnO compared to control. Ferric reducing antioxidant power (FRAP) was more sensitive than α, α-diphenyl-β-picrylhydrazyl (DPPH) for determining plant antioxidant capacity under nP-ZnO stress. The result showed that nP-ZnO induced a combined growth promoting and stress-induction effect in tobacco callus cells in a dose-dependent manner. The toxicity level of nP-ZnO was ameliorated compared to similar µP-ZnO concentrations. The chemical bio-reactivity of nP-ZnO-based Zn2+ could cause its fast-responsive and modified influence in tobacco cells. ZnO nanoparticles increased Zn2+ bioavailability in tobacco callus but ameliorated metal toxicity. Zinc nanoparticles induced dose-dependent and combined oxidative and growth promoting effects. FRAP was recommended for nP-ZnO oxidative studies.
TL;DR: The application of jasmonic acid can be suggested as an effective way to alleviate deleterious impacts dehydration stress on strawberry under in vitro conditions.
Abstract: To investigate the influence of jasmonic acid (JA) on morpho-physiological and biochemical characteristics of strawberry cv. Queen Elisa, under in vitro PEG-induced water stress, a factorial experiment based on completely randomized design (CRD) was conducted. PEG-induced water stress was imposed using polyethylene glycol (PEG6000) at three levels (0, 5 and 7% w/v), under three levels of JA application (0, 0.01 and 0.05 mM). The results indicated that PEG-induced water stress negatively impacted all studied morphological traits except for shoot fresh and dry weight. Furthermore, PEG-induced water stress had a decreasing effect on physiological traits including relative water content (RWC), photosynthesis pigments, membrane-stability index (MSI), and total proteins while it increased the activity of peroxidase (POD) and superoxide dismutase (SOD) enzyme and the content of malondialdehyde (MDA), hydrogen peroxide (H2O2), and proline. JA application showed an interaction effect with PEG-induced water stress on all studied traits except for SOD, POD, and total carbohydrate. Besides, based on the heat-map and principal component analysis results, three distinct groups were observed for physio-biochemical traits. Accordingly, the most effective treatment was the application of 0.05 mM JA under non-stressed condition. Altogether, the strawberry plants treated with higher JA concentrations could effectively cope with PEG-induced water stress adverse effects. Meanwhile, JA application improved strawberry growth attributes under both stress and non-stress conditions. In conclusion, the application of JA can be suggested as an effective way to alleviate deleterious impacts dehydration stress on strawberry under in vitro conditions. In vitro culture was used to study the effects of JA in strawberry under stress. PCA and heat map showed that relationships among studied parameters changed.
TL;DR: Silver nanoparticles added to the culture medium affect the regeneration and metabolism of plants and may be a source of significant genetic and phenotype variation in the in vitro-propagated chrysanthemum breeding.
Abstract: Despite the tremendous progress in breeding, novel and user-friendly techniques of plant improvement are desirable. The study aimed to analyze the usefulness of silver nanoparticles (AgNPs) in the breeding of chrysanthemum: one of the top ornamental plant species. In vitro regeneration of adventitious shoots from internodes of chrysanthemum ‘Lilac Wonder’ was induced on the modified Murashige and Skoog (MS) medium supplemented with 0.6 mg L−1 6-benzylaminopurine (BAP), 2 mg L−1 indole-3-acetic acid (IAA) and AgNPs at 0, 5, 10 and 20 ppm concentration. The efficiency of callogenesis and caulogenesis were analyzed after 10 weeks of culture. The concentration of chlorophylls, carotenoids, and phenolic compounds in shoots and calli were estimated. Plants obtained from 20 ppm AgNPs treatment were additionally analyzed on the genetic level using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. In vitro rooted shoots were acclimatized in the glasshouse and subjected to biochemical and phenotype stability evaluation. AgNPs at the highest concentration (20 ppm) suppressed both callogenesis and caulogenesis in vitro. The concentration of metabolites in callus was stable, regardless of AgNPs treatment, except for carotenoids which production was enhanced by 20 ppm AgNPs. In contrast, the content of chlorophyll a and b in shoots varied depending on AgNPs treatment. Polymorphic loci were detected in 12 and 9 AgNPs-treated-plants by RAPD and ISSR markers, respectively (one of which was common to both marker systems). Rooting and acclimatization were fully successful in all experimental combinations. Phenotype alternations were detected in six plants; one from 10 ppm AgNPs treatment and five from 20 ppm treatment. They included variation in pigment content (anthocyanins and carotenoids) and/or inflorescence shape. Interestingly, only two plants revealed both genetic and phenotype polymorphisms. No genetic or phenotype variation was detected in the control plants. In conclusion, AgNPs can be used in chrysanthemum breeding. Silver nanoparticles added to the culture medium affect the regeneration and metabolism of plants. Moreover, they may be a source of significant genetic and phenotype variation in the in vitro-propagated chrysanthemum.
TL;DR: It is demonstrated that long-term culture with 2,4- d decreases embryogenic competence in sugarcane embryogenic callus, which is induced via changes in the synthesis/mobilization of reserves and protein abundance.
Abstract: Somatic embryogenesis is a biotechnological tool with high application potential in the in vitro propagation and regeneration of crop plants, such as sugarcane. However, decreasing embryogenic competence is observed in embryogenic callus with successive subcultures, especially when the callus are maintained in the presence of 2,4-dichlorophenoxyacetic acid (2,4-d). Thus, the aim of this work was to investigate the morphological and molecular changes associated with the decreasing embryogenic competence in sugarcane embryogenic callus under long-term culture with or without 2,4-d. Sugarcane embryogenic callus were subcultured with 10 μM 2,4-d every 21 days for eight subcultures, and during each subculture, the callus were matured. In addition, the embryogenic callus from the first subculture after induction (S1) and after six subcultures with (S6) or without 2,4-d [S6(-)] were matured, and their histomorphological and histochemical features, endogenous polyamine levels and proteomic profiles were examined. The callus cultured with 2,4-d for a long time period showed reduced embryogenic competence after six subcultures, while callus cultivated in culture medium without 2,4-d maintained high embryogenic competence. In contrast to the callus in S6, those in the first subculture (S1) and S6(-) presented prominent nuclei, a high nucleus/cytoplasm ratio. Long-term culture with 2,4-d also affected polyamine (PA) metabolism, leading to high concentrations of putrescine (Put) and spermidine (Spd) in particular, and the synthesis and regulation of proteins, such as late embryogenesis abundant protein, chitinase, oleosin, and heat shock proteins. Therefore, we demonstrate that long-term culture with 2,4-d decreases embryogenic competence in sugarcane embryogenic callus. Loss in embryogenic competence in sugarcane callus by the long exposure to 2,4-d, is induced via changes in the synthesis/mobilization of reserves and protein abundance.
TL;DR: Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.
Abstract: The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 µl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species. Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.
TL;DR: This study sought to optimize the isolation of protoplasts from chrysanthemums by manipulating the mannitol and cellulase levels, the incubation period, and the purification method, followed by the conversion of the protoplASTs into calli and shoots, and achieved regenerating chrysantshemum protoplast-derived calli into whole plants.
Abstract: In this study, we sought to optimize the isolation of protoplasts from chrysanthemums by manipulating the mannitol and cellulase levels, the incubation period, and the purification method, followed by the conversion of the protoplasts into calli and shoots. A high protoplast yield was achieved using 0.5 M mannitol, 1.5% cellulase, and a 4 h incubation period. Cell wall regeneration was observed after 3 days, with the first cell division occurring approximately 4–5 days after culturing. The addition of sucrose to the culture media was more beneficial than glucose; in sucrose media the protoplasts grew more rapidly and successfully reached the colony and microcalli stage. The addition of activated charcoal to the culture improved colony and microcalli formation. Greater proliferation of microcalli was also achieved using solid Murashige & Skoog (MS) media supplemented with 1 mg l−1 6-Benzylaminopurine (BA) and 2 mg l−1 Naphthaleneacetic acid (NAA). The calli produced shoots THE on media supplemented with 2 mg l− 1 BA and 0.5 mg l−1 NAA. These findings could facilitate further chrysanthemum protoplast-based research. The regeneration of chrysanthemum protoplasts into whole plants is difficult due to the recalcitrant nature of these plants and because of their genotype-dependent response. This study is appropriate for protoplast isolation and callus formation of Chrysanthemum cv. White ND, and it also achieved regenerating chrysanthemum protoplasts-derived calli into whole plants.
TL;DR: Information could be useful in the commercial production of GAL as a valuable anti-AD compound through in vitro mass bulblet proliferation of N. tazetta through photoperiod and GAL production ability of the regenerated bulblets.
Abstract: Narcissus tazetta L., a bulbous plant belongs to the Amaryllidaceae family, contains alkaloid galantamine (GAL) with acetylcholinesterase inhibitory activity which has been recently considered to treat Alzheimer’s disease (AD). In the current work, the effect of photoperiod (16/8 h light/dark and 24 h dark) and various concentrations of NAA, BAP, and GA3 (0, 0.5, 1 and 2 mg l‒1) on the in vitro mass bulblet regeneration of N. tazetta was studied. The GAL production ability of the regenerated bulblets was assessed by HPLC-UV-MS. Light treatments significantly affected the number of bulblet and leaf, the ratio of bulblet/leaf, and leaf length. The maximum number of bulblet (31.0 ± 1.58) and leaf (13.3 ± 1.33) was recorded from the cultures fortified with NAA and BAP (2 mg l‒1) kept in 16/8 h light/dark, while the maximum leaf length (2.1 ± 0.92 cm) was measured on the MS medium containing 0.5 mg l‒1 NAA and 2 mg l‒1 BAP incubated in the same photoperiod. The average ratio of bulblet proliferation per explant was significantly different between studied photoperiod (1.1 ± 0.86) and 24 h dark (0.62 ± 0.31). The regenerated bulblets contained 40 and 20 µg g‒1 DW GAL underexposed photoperiod and 24 h dark, respectively. This information could be useful in the commercial production of GAL as a valuable anti-AD compound through in vitro mass bulblet proliferation of N. tazetta. The regenerated mass bulblets of Narcissus tazetta (Amaryllidaceae) on MS medium containing 2 mg l‒1 NAA and BAP kept in 16/8 h light/dark are recommended to produce galanatamine and lycorine.
TL;DR: This is the first report on effective in vitro propagation and bulblets formation in C. malabaricum and is the foremost report on in vitro regeneration and genetic fidelity analysis for restoration of this critically endangered aquatic plant using twin scale technique.
Abstract: This study intended to develop a significant in vitro regeneration protocol for sustainable propagation, conservation and re-establishment of critically endangered aquatic plant species Crinum malabaricum Lekhak & Yadav (Malabar river lily). This plant is the natural source of galanthamine, the drug to treat Alzheimer’s disease. We present a scientific understanding, emphasizing the use of twin scales (separated from the large parent bulb) in direct regeneration of new shoots and proliferation of bulblets assisted by nutrients supply. The meristematic region of the bulb plate, present between the scales was activated using cytokinins to produce shoots (maximum 12 shoots per twin scale) on full strength Murashige and Skoog’s (MS) medium augmented singularly with 2.0 mg L−1 6-benzylaminopurine (BAP). Upon subculturing of shoots on diverse concentrations of plant growth regulators (BAP and IAA/NAA), BAP alone at 2.0 mg L−1 was served optimum for the better proliferation of shoots (53 shoots). The regenerated shoots were rooted in vitro on half strength MS medium fortified with various types of auxins. Highest number of roots (11.6 within 4 weeks) and bulblets (after 3 months) resulted with 1.0 mg L−1 Indole-3-butyric acid (IBA) under in vitro conditions. The rooted plants were hardened in the greenhouse and finally transferred to the natural stream with 83% survival rate. The SCoT (start codon targeted) and ISSR (inter simple sequence repeats) marker analysis of in vitro raised and mother plants confirmed the genetic stability of tissue cultured plants and the reliability of present protocol for C. malabaricum. It is the foremost report on in vitro regeneration and genetic fidelity analysis for restoration of this critically endangered aquatic plant using twin scale technique. The study could help in ex situ conservation, reintroduction and restoration of C. malabaricum population in its natural habitat. Crinum malabaricum is a critically endangered aquatic plant species and natural source of galanthamine. It is endemic to India and about 1000 plants only available in a private estate in the Western Ghats of India. In vitro regeneration protocol has been developed utilizing twin scales for conservation of this plant. It is the first report on effective in vitro propagation and bulblets formation in C. malabaricum.
TL;DR: The complex relationship between the regulatory molecules and the profile of nitrogenous compounds enhanced the phenolics accumulation in hairy roots of Linum album is proposed.
Abstract: Due to medicinal value, phenolics are of great interest. Many investigations have already been conducted on Linum album Kotschy ex Boiss to increase these valuable compounds especially lignans. In the present work, the hairy roots of the L. album were treated with 200 mg L−1 chitosan for 12, 24, 48, and 72 h and the lignan contents were determined by high performance liquid chromatography (HPLC). To understand the governing upstream routes, the contents of hydrogen peroxide (H2O2), nitric oxide (NO), salicylic acid (SA), the activity of antioxidant enzymes, the amino acids, polyamines, and phenylpropanoids were determined by HPLC and spectroscopy techniques. The levels of regulatory molecules (H2O2, NO, and SA) enhanced at first hours of treatment and the antioxidant enzymatic activities increased in correlation with them. This observation might be attributed to adjustment of the oxidative status and direction of the elicited cells toward specific defensive responses. The content of amino acids, polyamines, and phenylpropanoids underwent a time-dependent reprogramming. The amino acid metabolism shifted toward the elevated levels of glutamate, aspartate, arginine, methionine, phenylalanine, and tyrosine after 24 h as well as correlated with the modulation of polyamines and phenylpropanoids. After 72 h, there was a channeling of the cell metabolism towards the accumulation of secondary metabolites, mainly podophyllotoxin, 6-methoxy podophyllotoxin, catechin, and vitexin. These results propose the complex relationship between the regulatory molecules and the profile of nitrogenous compounds enhanced the phenolics accumulation. Chitosan treatment of Linum album hairy roots activates signaling network accompanied by modulation of nitrogenous compounds and alteration of defensive pathways towards production of phenylpropanoids.
TL;DR: The polyploid induction system of Zingiber Officinale Roscoe cv.
Abstract: ‘Fengtou’ ginger is a high quality ginger (Zingiber Officinale Roscoe) cultivar only planted in Laifeng County, Enshi, Huhei, China, and is a National Product of Geographical Indication originated. ‘Fengtou’ ginger yield is low compared with most ginger. Therefore, it is imperative to apply breeding methods to improve yield. One strategy to accelerate breeding is polyploid induction, which could improve yield and maintain quality. In this study, different concentrations of colchicine (50, 100, 150, 200 mg/L) and different durations of exposure (3, 5, 7 days) were applied to stem segments of adventitious buds in ginger. Among obtained polyploidy plantlets were identified by flow cytometric analysis. The optimal induction condition of tetraploids was that the concentrations of colchicine was 150 mg/L, induced for 7 days, the chromosome doubling rate reached 18%. The tetraploid plants achieved were larger than their diploid plants for leaf length, leaf width, leaf thickness, stem diameter and guard cell. The soluble sugar, soluble protein, proline and other substances were also significant difference, especially carotenoids concentration was 1.375 times in contrast with diploid plants, which showed that tetraploids ginger had potentiality to increase yield and better adaptability. We explored the polyploid induction system of Zingiber Officinale Roscoe cv. ‘Fengtou’ ginger. Studies of morphological characteristics and secondary metabolites have shown that tetraploids ginger have potentiality to increase yield and better adaptability.
TL;DR: The current investigation confirms the effectiveness of FeNPs in growth media and offers a suitable prospect for commercially desirable production of S. rebaudiana biomass with higher sweet glycosides profiles in vitro.
Abstract: Application of nanomaterials is becoming the most effective strategy of elicitation to produce a desirable level of plant biomass with complex medicinal compounds. This study was, designed to check the influence of commercial iron nanoparticles (FeNPs) on physical growth characteristics, antioxidant status and production of steviol glycosides of in vitro grown Stevia rebaudiana. Results indicated that lower concentrations of FeNPs (45 µg/L) had a positive influence on morphological growth parameters. At a higher dose (90, and 135 µg/L) FeNPs in culture media were found detrimental to growth characteristics and development. Furthermore, the stress caused by FeNPs at 135 µg/L in cultures produced higher levels of total phenolic content (3.2 ± 0.042 mg/g dry weight: DW), total flavonoid content (1.6 ± 0.022 mg/g DW and antioxidant activity (73 ± 4.6%). In addition, plants grown in the presence of FeNPs at 90 µg/L resulted in higher enzymatic antioxidant activities (SOD = 3.2 ± 0.042 U/mg; POD = 2.1 ± 0.026 U/mg; CAT = 2.6 ± 0.034 U/mg and APx = 3.3 ± 0.043 U/mg), respectively. Furthermore, exposure to a low dose of FeNPs (45 µg/L) exhibited the maximum amount of stevioside (stevioside: 4.2 ± 0.058 mg/g (DW) and rebaudioside A: 4.9 ± 0.068 mg/g DW) as compared to high doses. The current investigation confirms the effectiveness of FeNPs in growth media and offers a suitable prospect for commercially desirable production of S. rebaudiana biomass with higher sweet glycosides profiles in vitro. Lower concentrations of Iron nano particles (FeNPs) had a positive influence on morphological growth parameters, production of antioxidant secondary metabolites and natural calorie free steviol glycosides in Stevia rebaudiana.
TL;DR: The results showed that explant genotypes, ZEs stages, sucrose and plant growth regulators (PGRs) have greatly influences on in vitro morphogenesis.
Abstract: Tree peony (Paeonia sect. Moutan) is a world famous ornamental and economically important species, but it is recalcitrant to in vitro regeneration. Here, we present a protocol for induction of direct somatic embryogenesis (SE) and direct shoot organogenesis (SO) from zygotic embryos (ZEs) of Paeonia rockii and P. ostii. The results showed that explant genotypes, ZEs stages, sucrose and plant growth regulators (PGRs) have greatly influences on in vitro morphogenesis. The highest frequency of SE (48%) and mean number of somatic embryos (5) was obtained from mature ZE of P. rockii ‘Jing Hong’ when cultured on modified Murashige and Skoog (mMS) medium supplemented with 2.22 µM BA and 0.23 M sucrose.The proliferation can be achieved by recurrent production of embryogenic callus and somatic embryos on mMS medium supplemented with 0.89 µM BA. The germinated rate of somatic embryos was 45% on WPM medium containing 2.22 µM BA and 1.44 µM GA3 after dormancy release at 4 °C under dark for 20 days. In addition, direct SO was obtained firstly in tree peony by using a variety of cytokinins. Morpho-histological analysis confirmed the initiation, development and reserve accumulation of somatic embryos and shoots. The study will be benificial to the propagation and breeding of tree peony. Tree peony (Paeonia sect. Moutan) is a world famous ornamental and economically important species which is recalcitrant to in vitro regeneration. Direct somatic embryogenesis protocol of P. rockii ‘Jing Hong’ was developed and morpho-histological analysis confirmed the initiation, development and reserve accumulation during in vitro morphogenesis. The study will provide valuable reference for propagation and breeding of tree peony.
TL;DR: Cloned and functionally identified a new OpWRKY1 TF from O. pumila that was preferentially expressed in stems and responded to exogenous elicitors such as gibberellin, acetylsalicylic Acid, salicylic acid, abscisic acid and 1-aminocyclopropane-carboxylic acid and elucidated how OpWR KY1 directly down-regulated the transcription of OpCPR and inhibited CPT biosynthesis in O
Abstract: Camptothecin (CPT) has unique anti-tumor activities, and its analogs such as irinotecan and topotecan are clinically used as anti-cancer drugs. Recently, CPT biosynthesis pathway is gradually being resolved. However, few studies have been conducted on transcription factors (TFs), which regulate CPT biosynthesis in Ophiorrhiza pumila. In this study, we cloned and functionally identified a new OpWRKY1 TF from O. pumila that was preferentially expressed in stems and responded to exogenous elicitors such as gibberellin, acetylsalicylic acid, salicylic acid, abscisic acid and 1-aminocyclopropane-carboxylic acid. Subcellular localization assay showed that OpWRKY1 was specifically expressed in the nucleus. Transgenic results revealed that over-expression of OpWRKY1 significantly repressed CPT accumulation in the hairy roots by inhibiting the expression of several CPT biosynthesis genes, especially OpCPR. Furthermore, Dual-Luciferase assay and yeast-one-hybrid assay showed that OpCPR was a target of OpWRKY1. The above results elucidated how OpWRKY1 directly down-regulated the transcription of OpCPR and inhibited CPT biosynthesis in O. pumila. Our findings indicate that OpWRKY1 acts as a negative regulator of CPT biosynthesis in O. pumila and provide new insights on metabolic engineering of CPT metabolism in the future. Overexpression of OpWRKY1 inhibits CPT accumulation in transgenic O. pumila hairy roots by reducing
the expression of CPT biosynthetic gene OpCPR.
TL;DR: The results of this study demonstrate that indirect regeneration of F. carica L. by the use of TCL system is a reliable and promising approach for future mass propagation programs as well as possible in vitro breeding objectives.
Abstract: The fig (Ficus carica L.), is known as a precious fruit tree for its nutrition and medicinal values, economic importance and for sustainable production in the semi-arid and arid areas. Expanding the cultivation of fig in new vulnerable areas and the breeding programs in fig need a reliable high-efficient system for in vitro morphogenesis to meet future demands. This study was carried to develop an efficient protocol for indirect regeneration of F. carica L. cultivars ‘Sabz’ and ‘Torsh’ using thin cell layer (TCL) technique. The genetic fidelity of the regenerated plants was also evaluated using flow cytometry technique and ISSR markers. Stem segments of 10 mm in diameter were taken from mature plants, then explants were transversally cut into layers of 0.5–0.8 mm thickness. Callus induction was successful using Murashige and Tucker (MT) medium supplemented with 9.08 μM TDZ plus 9.8 μM IBA (IM3 medium) which resulted in 50 ± 6.11% calli in ‘Sabz’ cultivar. Morphogenic calli were cut into small pieces and cultured on Murashige and Skoog (MS) medium for shoot development. Maximum shoot regeneration (45%) was observed in 17.68 μM BAP in combination with 4.54 μM TDZ and 1.07 μM NAA (RM2 medium), with an average of 6.9 shoots per explant. Flow cytometry and ISSR molecular marker analyses confirmed the stability of ploidy level and genetic identity of indirectly regenerated plants in both cultivars. The results of this study demonstrate that indirect regeneration of F. carica L. by the use of TCL system is a reliable and promising approach for future mass propagation programs as well as possible in vitro breeding objectives. A rapid and high-efficient in vitro method for mass propagation via callus culture in two F. carica cultivars was established by using TCL technique for the first time. Flow cytometry and ISSR molecular markers confirmed the clonal identity of regenerants in both cultivars.
TL;DR: It is suggested that well-characterized chitosan could be used as an alternative to JAS and BAP in orchid cultures and induce similar effects with jasmonic acid and 6-benzylaminopurine inOrchid cultures.
Abstract: This study aimed to compare the in vitro effects of chitosan oligomers and polymer with commonly-used plant growth regulators (PGRs) on seed germination, protocorm formation, and organ development in Serapias vomeracea. The effects of N-acetylated (10%) chitosan oligomer mixture (CHI-OM) with a degree of polymerization (DP) between 2 and 15 (5, 10, 15, and 20 mg L−1) and chitosan polymer (CHI-P) with a DP of 70 were compared with commonly-used cytokinins [6-benzylaminopurine (BAP) and kinetin (KIN)], auxins [indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA)], and jasmonic acid (JAS) at 0.25, 0.5, 1.0, and 2.0 mg L−1. The medium supplemented with CHI-P at 5 mg L−1 gave the highest seed germination rate, whereas JAS and CHI-OM triggered protocorm formation better than the all treatments tested. The JAS and IAA treatments resulted in intense browning of the roots. The CHI-P treatments at high concentrations and JAS treatments at moderate concentrations increased mean shoot length while the medium containing KIN at 0.5 mg L−1 induced root elongation significantly. The root elongation-inhibitory effect of chitosan was observed at the higher concentrations of CHI-OM, but the media containing 10 mg L−1 CHI-OM and 15 mg L−1 CHI-P triggered adventitious rooting. However, the highest tuberization success was found after 10 mg L−1 CHI-OM and 0.5 mg L−1 JAS treatments. Tuber development was achieved in the media supplemented with BAP and CHI-OM. This study suggested that well-characterized chitosan could be used as an alternative to JAS and BAP in orchid cultures. Well-characterized chitosans at the right concentration induce similar effects with jasmonic acid and 6-benzylaminopurine in orchid cultures.
TL;DR: Ectopic expression of MdD14 in Arabidopsis is resulted in a phenotype that inhibited shoot branching and hypocotyl elongation, as well as increased tolerance to salt, drought and low temperature stresses.
Abstract: Strigolactones (SLs) are phytohomones that regulate shoot branching and hypocotyl elongation. Here, the strigolactone receptor gene MdD14 in apple was cloned. MdD14 was localized to the nucleus and cytoplasm. GUS staining showed that MdD14 was expressed in various tissues. A promoter analysis revealed that MdD14 contained multiple response elements and its expression levels were induced by various abiotic stress treatment. The post-translational of MdD14 was regulated by the SL analog GR24. Ectopic expression of MdD14 in Arabidopsis is resulted in a phenotype that inhibited shoot branching and hypocotyl elongation, as well as increased tolerance to salt, drought and low temperature stresses. These findings reveal the functions of MdD14 and lay a foundation for studying SLs in apple. The apple MdD14 plays a key role in the SL signal transduction pathway and is involved in various abiotic stress resistance.
TL;DR: The present study showed that argan callus culture could be envisaged for sustainable and continuous production of bioactive compounds, and that each extract analyzed had unique and distinct characteristics.
Abstract: Argan (Argania spinosa (L.) Skeels) is an endangered and endemic agroforestry species of Morocco highly appreciated for its nutraceutical properties. Herein, the antioxidant activity, total phenolic content and fatty acids were evaluated in different extracts obtained from callus culture, seeds and leaves of four argan genotypes: G25, G36, G41 and G84. Callus induction, proliferation and morphology varied depending on genotype and explant type. The highest callus induction rate (97.5%) was observed in the cotyledon explants of genotype G84. The radical scavenging activity values ranged from 76.5 to 98.1%. The essential oils extracted from calli induced from seedling-derived leaves exhibited a slightly higher radical scavenging activity (91.7%) than those extracted from field-grown leaves (90–91.1%). The total phenolic content ranged from 0.72 mg/g dry weight gallic acid equivalent in the methanolic extracts of G84 callus obtained from seedling-derived leaves to 198.26 mg/g dry weight gallic acid equivalent in the essential oils of G84 seeds. The fatty acid composition varied significantly among the different samples. The essential oils extracted from seeds and callus obtained from cotyledon explants have high contents in oleic and linoleic acids (26–37.9% and 25–36.8%, respectively), while the major fatty acid found in the essential oils of leaves and callus obtained from seedling-derived leaves was eicosenoic acid (18.8–45.4%). The present study showed that argan callus culture could be envisaged for sustainable and continuous production of bioactive compounds, and that each extract analyzed had unique and distinct characteristics. The essential oils and other extracts obtained from argan callus induced in vitro were characterized and were compared with those obtained from seeds and field-grown leaves
TL;DR: Results provided an effective method for the regulation of flavonoid biosynthesis in M. chamomilla cell suspension culture, and the use of SMF as a tool for the induction of apigenin production.
Abstract: This study represents an optimized protocol for callus establishment and cell suspension culture of Matricaria chamomilla, and the impact of the static magnetic field (SMF) on flavonoid metabolism and antioxidant activity were examined for the first time. The effect of growth regulators was investigated to enhance biomass growth and apigenin production. Murashige and Skoog medium supplemented with 2,4-D (1.5 mg l−1) and Kinetin (0.5 mg l−1) showed the highest callus induction rate (100%), fresh weight, apigenin (0.82%) and apigenin-7-glucoside (1.57%) contents. Cell suspension culture was established, and the optimum subculture time was found to 13–15 days. SMF induced cell leaching and oxidative stress in all treated cells by an increase in H2O2 content and more stimulation of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) enzymes activities. Total phenolic, flavonoid and DPPH activity increased in cells treated to SMF, and the maximum content of apigenin (1.3%) and apigenin-7-glucoside (2.1%) were identified in cell treated to 4 mT. These results provided an effective method for the regulation of flavonoid biosynthesis in M. chamomilla cell suspension culture, and the use of SMF as a tool for the induction of apigenin production. Cell suspension cultures of Matricaria chamomilla contain valuable medicinal flavonoids. Static magnetic field promoted apigenin production and antioxidative enzyme activities in M. chamomilla cell suspension.
TL;DR: Results obtained here indicate that RNAi-mediated transcriptional regulation of targeted eIF4E gene family using plant tissue-specific promoters in marker-free intragenic potato plants is a promising strategy for improving the viral resistance in clonally propagated crops without affecting plant phenotype and productivity.
Abstract: Among various defence strategies used to protect potato plants from a viral infection, a deficiency of the eukaryotic translation initiation factors (eIF) provided by the host-mediated translational suppression has been characterized as a promising approach in modern crop breeding. The strategy used in the present study relies on knocking down the genes of the eIF4E-encoding family in a susceptible potato cultivar by the RNAi silencing mechanism. Taking into account the high concern regarding the potential effects of biotech crops on product safety and public acceptance, no bacterial/viral sequences and selectable marker genes were used. A hairpin construct carrying the fragment of potato eIF4E1 gene was created under control of potato Lhca3 (photosystem I 24 kDa light-harvesting protein) gene promoter and terminator. As a result of marker-free Agrobacterium-mediated transformation, two independent events were identified; one was confirmed to be clean from vector-backbone DNA sequences. Due to high homology between the genes encoding eIF4E1 and eIF4E2 potato factors, the silencing of both genes was achieved. During the two seasonable experiments, the intragenic line with higher production of small RNAs due to the expression of hpRNAi cassette showed a strong level of resistance after the mechanical inoculation with agriculturally significant PVYNTN strain and produced tubers phenotypically similar to non-infected control. Overall, the results obtained here indicate that RNAi-mediated transcriptional regulation of targeted eIF4E gene family using plant tissue-specific promoters in marker-free intragenic potato plants is a promising strategy for improving the viral resistance in clonally propagated crops without affecting plant phenotype and productivity. This study is a proof-of-concept for successful generation of viral resistance in potato by the RNAi-mediated transcriptional regulation of targeted eIF4E gene family using intragenic approach.