Showing papers in "Plant Cell Tissue and Organ Culture in 2019"

Abnormalities in somatic embryogenesis caused by 2,4-D: an overview

Abstract: Somatic embryogenesis is a morphogenetic event where somatic cells have the ability to produce embryos without gamete fusion. It is used as a technique for plant mass propagation. It is a process that has six well defined steps such as induction, expression, development, maturation, germination and plant conversion. These steps are characterized by distinct physiological, morphological and molecular events. Although somatic embryogenesis has been established in several plant species, there remains many problems to be solved. The main problem in somatic embryogenesis is the large number of abnormal embryos produced which cannot germinate nor convert into normal plants. Abnormalities in somatic embryos (SE) can be generated by genetic or epigenetic changes in the DNA. These changes in the DNA can be influenced by external factors such as the use of plant growth regulators and mutagenic substances or stress factors applied to the plant tissue such as high and low temperatures, drought, salinity, and heavy metals. Abnormalities generated by genetic changes in the DNA are hardly reversible; however, abnormalities generated by epigenetic changes may be reversible and the abnormal embryos are able to produce normal plants in most cases. This review focuses on the identification of the main factors that can cause abnormal SE development in different plant species, suggest how SE abnormalities are related to somaclonal variations and identify which genes may be involved with embryo abnormalities. Zygotic embryo abnormalities in Arabidopsis thaliana mutants are listed with the aim to understand the main genetic mechanisms involved in embryo aberrations. The abnormalities in somatic embryos are related to the use of 2,4-D in most of the published protocols, this sintetic auxin disrupts the endogenous auxin balance and the auxin polar transportation interfering with the embryo apical-basal polarity.

The effects of chitosan and salicylic acid on elicitation of secondary metabolites and antioxidant activity of safflower under in vitro salinity stress

Abstract: This study investigated the effects of different concentrations of two elicitors, namely chitosan (CHT) (25 and 50 mg L−1) and salicylic acid (SA) (50 and 100 mg L−1) on the production of secondary metabolites (SMs) and antioxidant activity of safflower callus under salinity stress. The content of total phenolics, total flavonoids, total flavonols, anthocyanin and antioxidant activity with 1,1-diphenyl-2 picrylhydrazyl assay (DPPH), significantly increased under salinity stress including 1.5% (w/v) sodium chloride. Under salinity stress, the highest content of total phenolics and total flavonoids were observed under elicitation by 50 (mg L−1) of SA and 25 (mg L−1) of CHT, but the highest callus growth rate (0.048 mm day−1), highest content of total flavonols (4.2 mg RE g−1 FW) and the highest DPPH activity (61.48% of inhibition) were observed under elicitation by 50 (mg L−1) of SA under salinity stress. This indicated partial superiority elicitation of SA over CHT for callus growth and SMs production of safflower under salinity stress. This new elicitation opens new avenues for the selection and exploitation of the best elicitors and their dosages for the enhancement of commercially important SMs in safflower as an important medicinal plant at cellular level. Effects of chitosan and salicylic acid on elicitation of SMs of safflower under in vitro salinity stress.

Elicitors derived from endophytic fungi Chaetomium globosum and Paraconiothyrium brasiliense enhance paclitaxel production in Corylus avellana cell suspension culture

Abstract: Paclitaxel is an impressive chemotherapeutic agent that shows biological activity against a wide range of cancers. Cell suspension culture of Corylus avellana has been reported as an alternative source for production of the valuable secondary metabolite, paclitaxel. The fungal elicitors have been considered as the most impressive strategy for enhancement of secondary metabolites in plant cell culture. In this study, two endophytic fungal strains YEF20 and HEF114 were isolated from Taxus baccata and C. avellana, respectively. The isolates YEF20 and HEF114 were identified as Chaetomium globosum and Paraconiothyrium brasiliense, respectively by sequencing of ITS1-5.8S-ITS2 rDNA region. This is the first report of P. brasiliense on C. avellana tree. Also, this study presents the positive effect of fungal elicitors on paclitaxel production in C. avellana for the first time. The effect of fungal elicitors on paclitaxel production was dependent on fungal species, and also type, concentration and adding time of elicitors to cell culture. The highest total yield of paclitaxel in cell culture treated with cell extract (CE) of C. globosum (291.5 µg L−1) was obtained by using 10% (v/v) of this fungal elicitor on 17th day of cell culture cycle (late log-phase), which was about 4.1 times obtained in the control culture. The cell culture treated with C. globosum CE had an average growth rate of 0.491 g L−1 day−1, i.e. 12.3% lower than that in the control. Among the fungal elicitors, 10% (v/v) CE of C. globosum on 17th day of culture cycle showed the best results with respect to extracellular paclitaxel portion (44.0%). Paclitaxel secretion to culture medium is essential for large-scale steady production of paclitaxel.

The effects of novel synthetic cytokinin derivatives and endogenous cytokinins on the in vitro growth responses of hemp (Cannabis sativa L.) explants

Abstract: The biotechnological utilization (genetic transformation, gene editing) of industrial hemp (Cannabis sativa L.) has been hampered by a lack of robust regeneration/in vitro multiplication protocols. In order to break this barrier we propose an approach combining standard application of exogenous growth regulators (auxins, cytokinins) with the knowledge of (1) endogenous cytokinin concentrations in primary explants based on latest analytical techniques and (2) the exogenous application of novel synthetic cytokinins or the regulators of their activity/function. We have tested eight explant types isolated from aseptically germinated seedlings in order to induce shoot regeneration/multiple shoot formation. The main problems appeared were callus formation and strong apical dominance. The latter phenomenon was suppressed using the auxin antagonist α-(2-oxo-2-phenylethyl)-1H-indole-3-acetic acid (PEO-IAA), and multiple shoot cultures were established from isolated apical meristems using media containing the novel synthetic cytokinin derivative 6-benzylamino-9-(tetrahydroxypyranyl)purin (BAP9THP). Endogenous cytokinin concentrations were measured in primary explants and explants cultured under diverse conditions to clarify the interactions between endogenous and exogenously applied cytokinins and their synthetic derivatives. These measurements were subsequently used to optimize the applied concentrations and timing of application of specific cytokinin derivatives to achieve desirable in vitro responses and regeneration including inhibition of callus formation, single shoot formation, and induction of multiple shoots. The well-developed shoots were rooted on media containing auxin (α-naphthaleneacetic acid). The protocol was developed using the variety USO-31 and has been successfully applied to four other industrial hemp varieties: Tygra, Monoica, Bialobrzeskie, Fibrol.

Evaluation of secondary metabolites and antioxidant activity in Dracocephalum polychaetum Bornm. cell suspension culture under magnetite nanoparticles and static magnetic field elicitation

Abstract: Suspension-cultured Dracocephlum polychaetum Bornm. is a wild medicinal herb native to Iran, treated with a static magnetic field (SMF) and Fe2O3 magnetic nanoparticles (MNP). The effect of SMF (30 mT), Fe2O3 MNP (100 ppm) and the combination of these treatments on phenolic metabolism and the medicinal compounds were examined by high performance liquid chromatography and UV–Vis spectrophotometer. The activity of polyphenol oxidase and phenylalanine ammonialyase, as well as the content of total phenolics, flavonoid, anthocyanins, lignin and malondialdehyde in all treated cells, showed a significant difference with control. The intracellular content of rosmarinic acid, naringin, apigenin, thymol, carvacrol, quercetin and rutin, in all treated cells increased considerably. Their quantities also increased significantly in the treated cell culture media. These results suggest this increase is due to the increased production and secretion of the medicinal compounds from cells to the culture media upon application of SMF and Fe2O3 MNP.

Ocimum basilicum suspension culture as resource for bioactive triterpenoids: yield enrichment by elicitation and bioreactor cultivation

Abstract: The competence of Ocimum basilicum suspension culture for the production of pharmacologically distinguished triterpenoids had been explored to meet their escalating demand with inequitable natural resources. The production of three pentacyclic triterpenes—betulinic acid (BA), ursolic acid (UA) and oleanolic acid (OA), could be achieved for the first time in the O. basilicum suspension culture, together with the prevalently occurring rosmarinic acid (RA). The production of RA was most abundant (15.73 ± 0.28 mg/g DW) followed by that of BA (14.63 ± 0.21 mg/g DW), UA (4.71 ± 0.23 mg/g DW) and OA (0.91 ± 0.02 mg/g DW) in reducing order on their respective optimum cultivation period. Compared to the in-vivo control leaves, the suspension cell revealed almost 3.25-, 2.89-, and 1.79-folds higher OA, UA and RA contents, respectively. The BA synthesizing power of suspension cells exemplified notable trend as its presence in the control leaves remained obscured. By and large, cultivation in a 10 l bioreactor and elicitation with methyl jasmonate in bioreactor and shake flask improved the cumulative productivities of the targeted metabolites with reciprocated divergence in the amounts of the two isomers, i.e., UA and OA. The overall findings of the present study bear special merit in realising the global demand of these multiple terpenoids in a single culture system of O. basilicum, which remained unrevealed so far. First report towards the in vitro production of multiple pharmacologically distinguished, high demand triterpenoids—betulinic acid (BA), ursolic acid (UA) and oleanolic acid (OA), along with rosmarinic acid (RA) in suspension culture of Ocimum basilicum. Notable yield enhancement through bioreactor cultivation and strategic elicitation with methyl jasmonate in shake flask and bioreactor added additional merit to this study in realizing the global demand of these multiple terpenoids in a single culture system of O. basilicum.

Genome editing of potato using CRISPR technologies: current development and future prospective

Abstract: Potato (Solanum tuberosum L.) has tremendous significance due to its nutritional quality. The mounting pressure of increasing population further reinforces its importance as potato is believed to be a vital crop to meet food needs for population growth. Although conventional approaches of breeding, irradiation/mutagens and introgression of quality and yield related traits have improved potato yield, biotic and abiotic stresses continue to impose crop damages. Modern tools such as CRISPR/Cas have assisted plant scientists in accelerating breeding processes by providing new, simple, versatile and robust technologies. These tools make it possible to eliminate traits that are involved in negative regulation of quality and yield parameters. Besides that, genes of interest can also be introduced in close proximity to specific loci that may remain linked throughout the generations. This review focuses on the endeavors, applications and prospects of CRISPR/Cas-based approaches in potato with the potential to increase sustainable crop productivity. This manuscript focuses the endeavors, applications and prospects of CRISPR/Cas-based approaches in potato with the potential to increase sustainable crop productivity.

Striking effects of melatonin on secondary metabolites produced by callus culture of rosemary ( Rosmarinus officinalis L.)

Abstract: In recent years, phytomelatonin has become even more important because of its numerous useful properties in plant metabolism. In this study, different concentrations of melatonin (MEL) (0, 100 and 200 µM) were used to investigate the changes in the accumulation of phenolic compounds, and the aromatic content of rosemary (Rosmarinus officinalis L.) leaf explants by callus culture. MS medium supplemented with 1.0 mg/l BAP and 2.0 mg/l NAA was used to induce callus production for determining the changes in secondary metabolites. Phenolic compounds such as rosmarinic acid, caffeic acid, p-coumaric acid and hesperidin and forty-five aromatic compounds were identified from rosemary leaf calli and measured by HPLC and GC/MS, respectively. 200 μΜ MEL increased the amount of rosmarinic acid (680 µg/g) compared to the control group (275 µg/g). However, 100 μΜ MEL increased the caffeic acid content (34 µg/g) two times higher than in the control group (17 µg/g). These findings were very remarkable for rosemary phenolic compound accumulation and it can be said that MEL could be a useful elicitor for rosemary. Totally, 200 μΜ MEL was more effective than 100 μΜ MEL in changing the aromatic content of rosemary calli. Also, some important aromatic compounds such as linalool, styrene and methional were only detected in MEL treated calli and not seen in the control group. The use and function of melatonin in callus culture systems as an elicitor to detect the change in secondary metabolites of rosemary leaf explants.

Adventitious shoot regeneration from leaf, petiole and root explants in triploid ( Populus alba × P. glandulosa )× P. tomentosa

Abstract: Leaf, petiole and root of triploid (Populus alba × P glandulosa) × P tomentosa were cultured on a medium containing benzyladenine (BA) and naphthaleneacetic acid (NAA) either alone or in combination with thidiazuron (TDZ) for the study of adventitious shoot regeneration The study demonstrated that TDZ played a primary and distinctive role in shoot regeneration: the frequency of shoot organogenesis of explants cultured with TDZ (7116%) far surpassed those cultured without TDZ (3042%) Among explant types, high variation was observed in the regeneration capacity: petiole and leaf had better regeneration capacity than that of root To understand this difference, an analysis of endogenous hormones (zeatin (ZT), abscisic acid (ABA) and indole-3-acetic acid (IAA)) was conducted on leaf, petiole and root explants The results showed that the regeneration ability of explants varied dependently on their IAA and ABA contents The organogenic capacity varied significantly with the position of the explant along the shoot axis The highest regeneration potential (9833%) and shoot number (up to 94 shoots/explant) were obtained in leaf explants harvested from the intermediate node (node 3) Regenerated shoots transferred to the medium containing 05 mg l−1 indole-3-butyric acid (IBA) achieved the highest frequency of rooting (100%), with means of 533 roots per shoot and 632 cm length per root The shoots with an abundant root system were later transferred to the greenhouse for further growth and development The effect of plant growth regulators (PGRs), explant type, endogenous hormone contents and maturity of explant was evaluated on adventitious shoot regeneration of triploid (Populus alba × P glandulosa) × P tomentosa in this study

Antimicrobial peptides as effective tools for enhanced disease resistance in plants

Abstract: Plants being exposed to a variety of pathogenic infections, acquire the natural mechanism of combating the pathogens leading to an increased level of defense. Several pathogenesis-related (PR) proteins are expressed by plants; approximately 17 families have been discovered till now. Most of them confer resistance to fungal pathogens and some of the PR proteins are bactericidal. These PR proteins consist of a short sequence of amino acids thus called antimicrobial peptides (AMPs). These AMPs permeabilize the pathogenic membrane via pore formation leading to cell death. The specific characteristic of these peptides is conserved domain and the disulfide bond pattern. Several types of AMPs such as defensins, thionins, cyclotides, lipid transfer proteins (LTPs) and several others, with a diverse mode of action, have been characterized from different plant species. These AMPs have been playing an important role in the host plant’s defense against the pathogens. In this review we summarized types of AMPs, their structural conformation and mode of action, their expression and co-expression in transgenic plants for conferring elevated resistance against the phytopathogens.

Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

Abstract: Cell suspension culture of Linum usitatissimum is a great source of the novel and multipurpose medicinal compounds lignans and neolignans. Conventional culturing practices usually result in low yield of plant secondary metabolites; therefore, we conceived a successful mechanism to elicit production of lignans and neolignans in cell suspension cultures, simply, by addition of chemogenic Ag-NPs into the culture medium. A three stage feeding strategy (day 10, 10 and 15, and 10 and 20, respectively, after inoculation) spanning the log growth phase (day 10–20), was implemented to elicit cell suspension cultures of Linum usitatissimum. Though enhancing effects of Ag-NPs were observed at each stage, feeding Ag-NPs at day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Furthermore, a linear trend in accumulation of lignans and neolignans was observed throughout log phase as compared to control, wherein growth non-associated trend in biosynthesis of these metabolites was observed. Optimum production of both lignans and neolignans occurred on day 20 of culture; a ten fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, five fold increase in dehydrodiconiferyl alcohol glucoside and 1.75 fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively.

Synergistic effect of coronatine and sorbitol on artemisinin production in cell suspension culture of Artemisia annua L. cv. Anamed

Abstract: Artemisinin is an efficient anti-malarial drug and it possesses biological activity against a wide range of cancers. The combined application of two different elicitors can be an efficient way to increase the production of secondary metabolite in plant cell cultures. The results of coronatine (Cor) pretreatment and three concentrations of sorbitol were assessed on the growth, biochemical traits, expression of artemisinin biosynthetic genes, and artemisinin production in Artemisia annua cell suspension culture (CSC). After pretreating CSC with 0.05 µM Cor [on the 14th day (three days before the stationary phase) for 48 h], liquid medium in the culture flasks was decanted and replaced with fresh medium (containing 30 g/L sucrose) plus or minus sorbitol at selected concentrations (0, 20, 30, and 40 g/L) on day 16th (one day before the stationary phase). The sorbitol treatment enhanced the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and resulted in oxidative stress. Cor-pretreatment increased the activity of antioxidant enzymes and consequently it reduced H2O2 content and oxidative stress which resulted in decreased MDA content and better growth. The application of Cor plus sorbitol resulted in a dramatic enhancement in the expression of artemisinin biosynthetic genes and artemisinin production at all concentrations. The expression levels of artemisinin biosynthetic genes (about 7.66, 8.67, 8.67, and 8.33-fold in ADS, CYP71AV1, ALDH1, and DBR2 genes, respectively at 4 h after sorbitol treatment) and artemisinin production (9.33 mg/L, 8-fold) peaked at 30 g/L sorbitol plus Cor and decreased at 40 g/L sorbitol, probably because of higher oxidative stress. The simultaneous application of Cor and sorbitol resulted in a dramatic enhancement in the expression of artemisinin biosynthetic genes and artemisinin production owing to a synergistic or potentiating result.

A high production of flavonoids and anthraquinones via adventitious root culture of Oplopanax elatus and evaluating antioxidant activity

Abstract: Adventitious root (AR) culturing is an effective approach for obtaining bioactive compounds from the endangered plant species of Oplopanax elatus Nakai. In the present study, flavonoids and anthraquinones, both important bioactive compounds were identified via HPLC-ESI–MS/MS, and flavonoid monomers of quercetin and kaempferide and anthraquinone monomers of aloe-emodin, rhein, and emodin were detected in O. elatus AR cultures. To enhance synthesis of both compounds, methyl jasmonate (MeJA) was used as the elicitor to treat ARs. At 200 µM, MeJA significantly increased the contents of quercetin, aloe-emodin, rhein, and emodin, whereas 225 µM was the optimal concentration for kaempferide accumulation. The antioxidant property in ARs was evaluated by determining activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and alkyl radical scavenging using the an electron spin resonance spectrometer. The IC50 of DPPH (6.2 µg/mL) and alkyl (20.2 µg/mL) radical scavenging activities with MeJA treatment were lower than that of the control, thereby indicating that ARs treated with MeJA possess stronger antioxidant activity. Hence, AR cultures can be deployed as raw materials or additives in the production of antioxidant-related O. elatus products using the MeJA as elicitor.

Morphological, anatomical, physiological, and cytological studies in diploid and tetraploid plants of Plantago psyllium

Abstract: Plantago psylliumis is under development as the source of mucilage, but has not been entirely domesticated. Since mucilage content is low and variable in the medicinal plant, it is imperative to apply breeding approaches to accelerate the domestication process and improve the mucilage content in the seeds. One approach to achieve these purposes is polyploidy induction, which can increase the size of plants and their phyto-medicines. To conduct this approach and evaluate the effect of in vitro-induced polyploidy on different traits of P. psylliumis, a gradient of colchicine concentrations, 0.1, 0.3 and 0.5% (w/v), was used to treat the terminal buds for 6, 12 and 24 h. The terminal buds were separately treated with different trifluralin concentrations, 7.5, 15 and 22.5% (w/v), for 12, 24 and 48 h. The optimal treatments of 0.5% colchicine for 24 h, and of 22.5% (w/v) trifluralin for 72 h resulted in an induction efficiency of 23% and 19%, respectively. Chromosome counting revealed successful chromosome duplication in tetraploids (2n = 4x = 24), in contrast with intact diploids (2n = 2x = 12). Duplication of DNA size in tetraploids was also proved by flow-cytometry analysis. The tetraploids size was larger than their intact diploids for spike, seed and pollen grain, leaf thickness, and plant height. The stomata of tetraploids were larger with lower density than diploids. The chloroplast number in guard cells, along with chlorophyll and carotenoid content all were higher in tetraploids. In final, our study suggests that tetraploidization could be useful to improving the seed mucilage content for commercial use. We firstly fulfilled the in vitro induced tetraploidy in P. psyllium by 0.5% colchicine for 24 h and 22.5% (w/v) trifluralin for 72 h, and improved the mucilage content in the seeds.

Improved effects of polyethylene glycol on the growth, antioxidative enzymes activity and taxanes production in a Taxus baccata L. callus culture

Abstract: Application of abiotic stress-inducing elicitors is one of the effective strategies in order to increase the production of secondary metabolites in plant tissues. Here, the effect of in vitro polyethylene glycol (PEG)-induced drought stress on morphological and biochemical changes as well as the production of paclitaxel and 10-deacetyl baccatin III in a Taxus baccata callus culture was investigated. The results showed that increased PEG concentration in the culture medium enhanced the levels of H2O2 and the membrane lipid peroxidation in the calli. The activity of catalase and guaiacol peroxidase as antioxidant responses were increased significantly in all PEG concentrations. At 6% of PEG concentration, the levels of fresh weight (FW) and dry weight (DW) in the calli were declined significantly by dropping in the amount of water content in the calli and rising the level of oxidative stress, and the morphological changes occurred following the increased browning index of the calli. However, the PEG at low to moderate concentrations reduced the accumulation and oxidation of phenolic compounds in the calli through the absorption of phenolic compounds and the reduction in the activity of the polyphenol oxidase. As a result, improved growth and viability of the calli resulted in the enhanced levels of FW and DW at the concentrations of 1% up to 3% (w/v) PEG. The highest contents of 10-deacetyl baccatin III and taxol were obtained at concentrations of 2% and 3% PEG, respectively. The concentration of 3% PEG increased the production of taxol by 2.5 times more than the control. In conclusion, PEG can be proposed as an effective elicitor in the production of taxanes in the Taxus cell and tissue cultures.

Effects of ZnO, CuO and γ-Fe3O4 nanoparticles on mature embryo culture of wheat (Triticum aestivum L.)

Abstract: In this study, the effects of ZnO, CuO, and γ-Fe3O4 nanoparticles on two different wheat (Triticum aestivum L.) genotypes, namely, Kirik and ES-26, were investigated as micro elements in mature embryo culture. In this direction, nanoparticle constructions were tested with both the normal amount (1 ×) and the two (2 ×) and three (3 ×) quantities available in the Murashige and Skoog media. The study was supported by negative and positive controls. Obtained findings suggest that wheat embryos left to develop in applications containing nanoparticles do not actively utilize nanoparticle structures. As a result, the development of nanoparticle-containing applications was less than control. It has been determined that media containing 3 × CuO nanoparticles and media containing 3× ZnO nanoparticles are more successful than controls in terms of callus formation rate among all applications. The most successful group in terms of plant-building ability has been the control group. Plant regeneration did not increase with nanoparticle application compared to control. However, it is thought that this situation is mainly related to the utilization process of the plant by the nanoparticles. However, since the properties of nano-sized elements are variable, it is considered that the obtained data may be related to toxicity. This study is a first in the test of NP structures obtained by green synthesis in mature embryo culture. It is thought that this study contributes to the literature in determining the effects nanoparticle’s have on tissue culture development stages of embryos.

The influential role of polyamines on the in vitro regeneration of pea ( Pisum sativum L.) and genetic fidelity assessment by SCoT and RAPD markers

Abstract: A proficient and reliable in vitro plant regeneration protocol was established for pea by utilizing cotyledonary node as an explants, which excised from 3-days old imbibed seeds. The present research work explains the positive role of polyamines (PAs) accompanied by plant growth regulators (PGRs) on the multiple shoot induction and rooting in pea plant regeneration. The highest multiple shoots (65.1 shoots/explant) was attained from the cotyledonary node explant on Murashige and Skoog (MS) medium accompanied with 20 mg l−1 spermidine (SPD) along with 1.5 mg l−1 6-benzyladenine (BA). Moreover, the highest elongation of multiplied shoots (10 cm length/shoot) was noted on MS medium enriched with 1 mg l−1 of gibberellic acid (GA3). The elongated shoots produced the highest number of roots (33.66 roots/shoot) on MS medium augmented with 30 mg l−1 putrescine (PUT) along with 0.6 mg l−1 1-naphthaleneacetic acid (NAA). Rooted plants were hardened and acclimatized in the greenhouse with an existence rate of 98%. The standardized procedure with the use of PAs has enhanced the multiple shoot induction to threefold higher than the plants raised from PGRs treatments. The regenerated pea plants revealed the fivefold enhanced photosynthetic pigment, twofold enhanced antioxidant profile and the substantial growth in chloroplast count have been achieved in optimized SPD and BA supplemented MS medium compared to control plant. Start Codon Targeted polymorphism and Random Amplified Polymorphic DNA molecular markers recognized the genetic purity of the in vitro regenerated pea plants for their true type of nature. An enhanced and consistence in vitro regeneration protocol was established for pea, by the application of polyamines along with PGRs, which significantly improved the multiple shooting, shoot elongation, rooting, total chlorophyll and antioxidant profile of regenerated plants, further not hampering their genetic fidelity.

In vitro culture of the endangered plant Eryngium viviparum as dual strategy for its ex situ conservation and source of bioactive compounds

Abstract: Different Eryngium species have been used with ornamental, agricultural and medicinal purposes, as a consequence of their chemical constituents. In the southwest Europe the endemic Eryngium viviparum, presents a high risk of extinction and ex situ strategies are high recommended for efficient conservation and re-introduction program. The objective of this study was to satisfy a dual objective: (i) to develop an ex situ conservation strategy through micropropagation and (ii) taking advantage of the extraordinary potential of plant tissue culture, produce a considerable amount of plant material to carry out a preliminary phytochemical study, based on the accumulation of phenolic compounds and their associated antioxidant activity. First a factorial design was conducted in order to study the effect of two cytokinins (6- benzylaminopurine, BAP, and kinetin, KIN), at three levels (0, 1 and 2 mg L−1), on shoot multiplication. Later another factorial design was applied, by using three levels of MS medium salt strength (full, half and quarter- strength) and four sucrose levels (0, 1, 2, and 3%) for improving shoot elongation and rooting. In parallel, a preliminary quantification of total phenolic and flavonoid contents from E. viviparum aerial parts was determined. The simple micropropagation protocol designed allowed obtaining a high rates of shoot multiplication (5.1–5.8 new shoots), rooting (100%) with healthy long roots (3.1–3.5 cm) and plantlet acclimatization (96%). Moderate antioxidant activity was recorded in hydromethanolic extracts from E. viviparum aerial parts. High correlation between total phenolic content and BAP levels in the culture media was found. In conclusion, the micropropagation procedure described here for the endangered E. viviparum can be used as new and very efficient ex situ conservation strategy, and as potential source of antioxidants, conferring an added-value to this plant. In this work, we addresses the development of an efficient in vitro culture procedure of Eryngium viviparum as ex situ conservation methodology, which leads to a plant reintroduction programs, and as new source for secondary metabolites (mainly phenolic compounds), without ecological impact in their limited populations (either using seeds or wild plants as source materials).

The WUSCHEL-related homeobox transcription factor MtWOX9-1 stimulates somatic embryogenesis in Medicago truncatula

Abstract: Somatic embryogenesis is widely used in plant biotechnology, but regulatory networks underlying this process are not fully understood. This process is regulated by many protein and hormonal factors, among which WUSCHEL-related homeobox proteins are believed to play key roles. In this study, we aimed to identify the role of Medicago truncatula WOX9-1 (MtWOX9-1), an Arabidopsis thaliana WOX9 (STIMPY) homolog, in somatic embryogenesis. We demonstrated that the MtWOX9-1 promoter is active in somatic embryos, and overexpression of MtWOX9-1 leads to the increase in embryogenic capacity, as well as to the changes in expression levels of two MADS-box genes (AGL15 and AGL8 homologs), associated with embryogenesis. The data obtained suggest that stimulation of MtWOX9-1 expression may have positive impact on plant biotechnology in improving the transformation and regeneration capacity of recalcitrant plants. MtWOX9-1 participates in somatic embryogenesis and can stimulate this process in tissue culture changing the expression level of different embryogenesis-associated genes.

Enhanced production of cichoric acid in cell suspension culture of Echinacea purpurea by silver nanoparticle elicitation

Abstract: This study was conducted to optimize callus induction, cell suspension culture, and enhance cichoric acid production in Echinacea purpurea. For this purpose, leaf and root explants were grown in ½ MS medium supplemented with different concentrations of plant growth regulators. The highest percentage of callus induction (100%) was obtained with different concentrations of Kin in combination with 2,4-D and or NAA, in both explants. Then interaction slicing method was applied to analyze the effects of different concentrations of Kin in combination with 2,4-D and or NAA on total phenol contents. The maximum amount of total phenol was observed in 0.5 mg/L kin and 1 mg/L 2,4-D, in both leaf and root-derived callus. Data from HPLC–UV analysis showed that cichoric, chlorogenic, and caffeic acids are present in the callus. While cichoric acid content was 2.11 and 4.73 mg/g dry weight (DW) in leaf explant and its derived callus, it was 3.47 and 5.67 mg/g DW in root explant and its derived callus, respectively. Cell suspension culture was established and treated by AgNO3 and AgNPs (0, 2 and 4 mg/L). Overall, AgNPs’ elicitation was more effective in cichoric acid production compared to AgNO3. The highest amount of cichoric acid (9.54 mg/g DW) was found in root cell suspension culture after 48 h of exposure to 2 mg/L AgNPs. But leaf cell suspension culture reached the maximum cichoric acid level (8 mg/g DW) 72 h after treatment with 2 mg/L AgNPs. The current protocol can be used for the production of cichoric acid from callus and cell suspension cultures. Optimization of callus induction, cell suspension culture and cichoric acid production by AgNPs elicitation in Echinacea purpurea.

Methyl jasmonate enhances apple’ cold tolerance through the JAZ–MYC2 pathway

Abstract: Improving the cold resistance of plants is important, because their growth and development are negatively affected by cold stress. In this study, exogenous applications of methyl jasmonate could enhance the cold resistance of ‘Orin’ apple (Malus × domestica) calli by increasing the expression levels of the cold-signal response genes MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3. In addition, yeast two-hybrid and pull-down assays demonstrated that MdCIbHLH1 interacts with MdJAZ1/4 and MdMYC2 in vitro and in vivo. Protein degradation experiments demonstrated that the stability of the MdJAZ1/4 proteins were affected by the application of exogenous methyl jasmonate, which was followed by their degradation by the 26S proteasome. MdJAZ1/4 act as repressors, binding MdMYC2 in the jasmonate-signaling pathway. The overexpression of MdMYC2 in ‘Orin’ calli increased the expression levels of MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3, resulting in an increased freeze tolerance. Furthermore, the overexpression of MdJAZ1 or MdJAZ4 in transgenic red-fleshed apple calli weakened the promotive effect of MdMYC2 on cold tolerance. Yeast one-hybrid and chromatin immunoprecipitation-PCR analyses showed that MdMYC2 could bind the G-box element found in the MdCBF1 promoter. Thus, jasmonate may function as a critical upstream signal in the ICE–CBF/DREB1 pathway to positively regulate apple freeze tolerance.

Application of the salt stress to the protoplast cultures of the carrot ( Daucus carota L.) and evaluation of the response of regenerants to soil salinity

Abstract: This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm−1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot. Salt stress applied to carrot protoplasts generates variability manifested in differences in cellular response and variation in ploidy. The adaptation of carrot regenerants to soil salinity was associated with accumulation of anthocyanins and increased hairiness.

Isolation and functional identification of an apple MdCER1 gene

Abstract: The outermost layer of the plant epidermis is covered by cuticular wax, which resists UV radiation, insects, and pathogens, and protects the plant from various environmental stressors. We cloned MdCER1 to further study the molecular regulatory pathway of cuticular wax in apple (Malus × domestica Borkh.). The results showed that MdCER1 is a homolog of Arabidopsis CER1, which is essential for wax synthesis. A subcellular localization study revealed that MdCER1 was localized on the endoplasmic reticulum. We examined the expression pattern of MdCER1 in different tissues and found that it was constitutively expressed in roots, stems, leaves, flowers, and fruits, with the highest expression in leaves. Ectopic expression of MdCER1 promoted the accumulation of cuticular wax by changing the permeability of the epidermis and the response to abscisic acid, as well as by improving drought resistance in Arabidopsis.

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Abstract: Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

Positive effect of AgNPs and AuNPs in in vitro cultures of Lavandula angustifolia Mill.

Abstract: The aim of this study was determined how an addition of gold nanoparticle (AuNPs) and silver NPs (AgNPs) into culture media affects plant development and formation of oil glands in narrow-leaved lavender (Lavandula angustifolia) cv. ‘Munstead’. Plant shoots were propagated on media supplemented with 1, 2, 5, 10, 20 and 50 mg dm−3 AuNPs or AgNPs (diameter of 24.2 ± 2.4 nm and 27.5 ± 4.8 nm). Both of NPs positively influenced the growth and development of lavender propagated in vitro. The culture media with NPs stimulated formation of shoots and increased plant weight. Roots of plants propagated on the media supplemented with NPs were usually longer than those in the control. Only high concentrations of NPs (20 and 50 mg dm−3) in the culture media were toxic to plants, as demonstrated by restricted shoot length and gradual decrease in the value of other morphological features. Increases in AgNPs concentration caused the number of secretory trichomes to decrease. The diameter of the trichomes on both sides of the leaf blade was larger when the plants were propagated on the media supplemented with 1 and 2 mg dm−3 NPs. The diameter of trichomes formed on the adaxial surface of the leaf blade was greatest in the media enriched with 2 mg dm−3 AgNPs and 5 mg dm−3 AuNPs, and smallest in the media enriched with 5 mg dm−3 AgNPs. The diameter of trichomes formed on the abaxial surface was largest in plants exposed to 1, 2, 5 and 10 mg dm−3 AuNPs, 1 mg dm−3 AgNPs, and smallest in plants exposed to 5 mg dm−3 AgNPs. Silver and gold nanoparticles had a significantly improved the growth and development of narrow-leaved lavender propagated in vitro. The number and size of secretory trichomes formed on the leaves of narrow-leaved lavender grown in in vitro cultures depends on the concentration of silver or gold nanoparticles in the media.

Identification and expression profiling of rosmarinic acid biosynthetic genes from Satureja khuzistanica under carbon nanotubes and methyl jasmonate elicitation

Abstract: Satureja khuzistanica is a medicinal herb endemic to Iran which can serve as a source of rosmarinic acid (RA). In the present study, the effect of different concentrations of methyl jasmonate (MeJA) and multi-walled carbon nanotubes (MWCNTs) on the rosmarinic acid accumulation and the expression of genes involved in its biosynthetic pathway was evaluated in nodal segment cultures of S. khuzistanica. The concentration of RA varied in plant extracts derived from the field, plants obtained under in vitro solid media, calli, suspension cultures and nodal segment cultures in liquid medium, with the highest amount recorded in nodal segment cultures. Phenylalanine ammonia-lyase (PAL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR) and RA synthase (RAS) cDNA clones as key biosynthetic genes of RA production were identified and expression patterns were assayed in response to MeJA and MWCNTs, exogenously applied at a range of 0, 50, 100, 250 mg L−1. The expression levels of HPPR, PAL and TAT were up-regulated at 100 mg L−1 MWCNTs, whereas down-regulated levels were observed at 250 mg L−1. RAS was up-regulated in all MWCNTs treatments. HPPR, PAL and TAT expression increased sharply at 250 mg L−1 MeJA. The highest levels of RAS transcripts were observed at 100 mg L−1 MeJA. The highest HPPR, PAL and TAT expression levels were observed at 250 mg L−1 MeJA. In accordance, HPLC analysis showed a high amount of RA under MeJA and MWCNT elicitors. Our results provide helpful information on the expression profiles of biosynthetic genes in connection with RA accumulation.

Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis.

Abstract: The aim of this protocol was to develop an alternative in vitro propagation system for Cannabis sativa L. by mimicking nursery-based vegetative propagation. Photoautotrophic micropropagation (PAM) was achieved on rockwool blocks as substrate combined with commercially available fertilizer suitable for cannabis cultivation. Stock plants were initiated after sterilisation in forced-ventilated glass jars which then provided a continuous supply of shoot tip and nodal cuttings. A 97.5% rooting rate of in vitro shoot tip cuttings and successful acclimatisation were achieved within 3 weeks in glass vessels with passive ventilation.

Bioreactor type affects the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis (Turcz.) Baill.

Abstract: Microshoots of the East Asian medicinal plant species Schisandra chinensis (Chinese magnolia vine) were grown in bioreactors characterized by different construction and cultivation mode. The tested systems included two continuous immersion systems—a cone-type bioreactor (CNB) and a cylindric tube bioreactor (CTB), a nutrient sprinkle bioreactor (NSB), and two temporary immersion systems (TIS)—RITA® and Plantform. Microshoots were grown for 30 and 60 days in the MS medium enriched with 1 mg l−1 NAA and 3 mg l−1 BA. The accumulation of two groups of phenolic compounds: phenolic acids and flavonoids in the bioreactor-grown S. chinensis biomass, was evaluated for the first time. In the microshoot extracts, seven phenolic acids: chlorogenic, gallic, p–hydroxybenzoic, protocatechuic, syringic, salicylic and vanillic, and three flavonoids: kaempferol, quercitrin and rutoside, were identified. The highest total amount of phenolic acids (46.68 mg 100 g−1 DW) was recorded in the biomass maintained in the CNB for 30 days. The highest total content of flavonoids (29.02 mg 100 g−1 DW) was found in the microshoots maintained in the NSB for 30 days. The predominant metabolites in all the tested systems were: gallic acid (up to 10.01 mg 100 g−1 DW), protocatechuic acid (maximal concentration 16.30 mg 100 g−1 DW), and quercitrin (highest content 21.00 mg 100 g−1 DW). The influence of bioreactor type on the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis was proven and optimized.

Does somaclonal variation play advantageous role in conservation practice of endangered species?: comprehensive genetic studies of in vitro propagated plantlets of Viola stagnina Kit. (Violaceae)

Abstract: In vitro regeneration of Viola stagnina Kit., endangered in most part of its European distribution range, was successfully obtained based on the newly developed protocol. Adventitious shoots via direct and indirect organogenesis were induced on leaf blade and petiole fragments on solidified Murashige and Skoog (MS) medium supplemented with 0.5 or 1 mg l−1 thidiazuron, respectively. Shoots were rooted on half-strength MS medium with 2% sucrose and 0.5 mg l−1 indole-3-acetic acid (IAA), and plantlets were successfully acclimatized. Sixty-five of the regenerated plants (72% of isolated shoots cultured on rooting medium) survived at the experimental plot conditions. Among recovered via organogenesis plants, individuals ‘true-to-type’ derived from initial plant G1 and also plants genetically distant from initial plants were detected by ISSR markers. In all groups of clones genetic indices (number of genotypes, polymorphic markers, gene diversity, total gene diversity, mean gene diversity) were lower than in natural populations. Regenerated plantlets had the same genome size estimated by flow cytometry as initial material and plants from natural populations. They developed chasmogamous flowers with highly viable pollen grains (over 90%), cleistogamous flowers, and set seeds from both flower types in the first and second seasons cultivated at experimental plots. This is the first report of a successfully developed micropropagation protocol of V. stagnina, and the first detailed genetic analysis of recovered plants with the use of ISSR markers and genome size measurements allowing to discuss the advantageous role of somaclonal variation in ex situ plant conservation with the use of in vitro micropropagation.

Millets genetic engineering: the progress made and prospects for the future.

Abstract: Millets comprise a highly variable small-seeded group of Poaceae members that can grow in extreme environmental conditions of drought, high temperature and low soil fertility hence, recognized as climate-resilient. Among millets, the phylogenetic closeness of Setaria with other agronomically important grasses like maize, sugarcane, and sorghum helped in its adoption as a translational model plant. Established efficient gene transfer methodology is a prerequisite for embracing plant species as models. However, genetic engineering of some of the economically important millets has been started in the 1990s, but inadequate progress made this group lag behind other members of Poaceae as rice, maize and wheat. Genetic transformation in millets has generally been achieved by a physical method of microprojectile bombardment, recently Agrobacterium-mediated gene transfer technique has also established in some of the millets but with very few reports. The central hindrance in millet transformation is its recalcitrant nature to regeneration through tissue culture techniques. Optimization of highly efficient regeneration procedure for each millet species is thus, necessary to establish advanced transformation system for them. The possibility of alternative transformation approaches is also discussed. The establishment of robust gene transfer methods whether it’s conventional in-vitro tissue culture dependent or in-planta are important for functional validation studies and would enable development of crop improvement strategies. This review presents the progress made on millet genetic transformation, discussing the major challenges that need to be overcome and future opportunities of transgenic techniques in various millets.