Showing papers in "Plant Cell Tissue and Organ Culture in 2016"

Chemical elicitors versus secondary metabolite production in vitro using plant cell, tissue and organ cultures: recent trends and a sky eye view appraisal

Abstract: Plant secondary biosynthetic pathways are exceedingly inducible by elicitors and facilitate enhanced metabolite production using plant cell tissue and organ cultures. Elicitors can regulate large number of control points and trigger the expression of key genes with increased cellular activities at biochemical and molecular level involving signal compounds. A large number of chemical elicitors viz: jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), acetyl salicylic acid (ASA), ethylene (ET) and ethrel (Ethe), heavy metals (HM), many types of chemical compounds (natural or synthetic) and their combinations are used for elicitation studies. Cell suspensions and hairy roots are commonly used culture systems followed by adventitious roots and multiple shoots for elicitation experiments. Amongst the elicitors and concentrations used 100 µM MeJA was found optimum for secondary metabolite enhancement in majority of experiments compared to SA and JA. Elicitor treatments promoted yield enhancements starting from 1.0 to maximum of 2230-fold across plant species studied. Elicitors singly with media additives, combination of elicitors and elicitors other than signal compounds along with hormones were found beneficial for enhanced secondary metabolite production. Further, a combination of target gene over expression and elicitor treatment also supported higher secondary product yield. The present communication presents information exclusively about the use of chemical elicitors for secondary metabolites production in vitro covering approximately more than a decade of research at one place in one review. Further, this extensive appraisal will be useful for the understanding and manipulation of secondary metabolites for enhanced production in vitro.

Cis-regulatory elements used to control gene expression in plants

Abstract: Plant biotechnology is a dynamically developing science, which comprises many fields of knowledge. Novel plant genetic engineering findings highly influence the improvement of industrial production. These findings mostly concern cis-regulatory elements, which are sequences controlling gene expression at all developmental stages. They comprise of promoters, enhancers, insulators and silencers, which are used to construct synthetic expression cassettes. Examples of most important cis-regulatory elements are reviewed in the present paper. Variability among core promoters content and distal promoter regions impedes evaluation of interactions between them during the artificial promoters construction. Synthetic promoters and artificial expression cassettes trigger a significant increase in gene expression level, better properties and quality of a product. Accumulating knowledge about gene promoters, cis sequences and their cooperating factors allows uniform expression systems and highly predictable results.

A grape bHLH transcription factor gene, VvbHLH1, increases the accumulation of flavonoids and enhances salt and drought tolerance in transgenic Arabidopsis thaliana

Abstract: In plants, transcriptional regulation is the most important tool for modulating flavonoid biosynthesis. The basic helix-loop-helix transcription factors are only one example how then flavonoid pathway is regulated. There are other transcription factors as well. In this study, the codon-optimized VvbHLH1 gene from grape was chemically synthesized. Overexpression of VvbHLH1 significantly increased the accumulation of flavonoids and enhanced salt and drought tolerance in transgenic Arabidopsis thaliana plants. Real-time quantitative PCR analysis showed that overexpression of VvbHLH1 resulted in the up-regulation of genes involved in flavonoid biosynthesis, abscisic acid (ABA) signaling pathway, proline biosynthesis, stress responses and ROS scavenging under salt and drought stresses. Further analyses under salt and drought stresses showed significant increases of ABA and proline content, superoxide dismutase and peroxidase activities, as well as significant reduction of hydrogen peroxide (H2O2) and malonaldehyde content. The results demonstrate the explicit role of VvbHLH1 in conferring salt and drought tolerance by increasing the accumulation of flavonoids and ABA signalling in transgenic A. thaliana. The VvbHLH1 gene has the potential to be used to increase the content of valuable flavonoids and improve the tolerance to abiotic stresses in A. thaliana and other plants.

Elicitor induced stevioside production, in vitro shoot growth, and biomass accumulation in micropropagated Stevia rebaudiana

Abstract: For the purpose of enhancing the secondary metabolite content in micropropagated Stevia rebaudiana plants without inhibiting plant growth, node explants were cultured on woody plant medium (WPM) containing alginate (ALG), casein hydrolysate (CH), pectin (PEC), yeast extract (YE), methyl jasmonate (MeJA), salicylic acid (SA), or chitosan (CHI). The highest shoot number; shoot length, node number, and leaf number; leaf length; and stem diameter were observed on WPM containing 1.0 g/L YE; 100 µM CHI; 0.5 g/L CH; and 1.0 g/L ALG, respectively. The root regeneration frequency reached 100 % on WPM supplemented with 0.5, 1.0, or 2.0 g/L PEC, 1.0 g/L YE, or 50 µM CHI and the control. The highest root number was obtained on WPM containing 0.5 g/L PEC, while the longest root length was observed on WPM containing 1.0 g/L YE. The highest biomass accumulation was observed with treatment of 100 µM CHI. According to the high-performance liquid chromatography results, except for the treatments with 200 µM CHI, 100 or 200 µM MeJA, and 200 µM SA, the remaining elicitor treatments increased stevioside production compared to the control. The production of stevioside increased from 1.56 mg/g dry weight (DW) to 14.69 and 14.54 mg/g DW in the in vitro plantlets exposed to 0.5 g/L ALG and 2.0 g/L YE, respectively. Rebaudioside A was observed on only 0.5 g/L ALG-treated plants as 0.55 mg/g DW. The stevioside content of field-grown plants was identified as 15.06 mg/g DW. The present findings provide important information regarding the effect of elicitors on plant growth and secondary metabolite production of in vitro micropropagated S. rebaudiana.

Carbon nanotubes impact on date palm in vitro cultures

Abstract: Among the recent line of technological innovations, nanotechnology takes a promising position in agriculture and food production. Nanotechnology permits definite advances in agricultural research, such as reproductive science and technology. This investigation is interested in studying the in vitro effect of carbon nanotubes (CNTs) on callus, embryogenesis, embryo germination and elongation as well as rooting stage of date palm. Carbon nanotubes concentrations were investigated as 0.0, 0.05 and 0.1 mg/l. Results showed that CNTs affect all stages of micropropagation of date palm. Callus fresh weight showed the optimum value at 0.05 mg/l. In embryogenesis stage, CNTs decreased the number of embryos compared with the control while, increased number of germinated embryos and root number. Carbon nanotubes gave a significant enhancement for shoot length and leaf number in elongation stage. Similarly, it enhanced root number, root length, plantlet length and hairy roots. Chemical analysis as chlorophyll a, b, carotenoids, flavonoids, antioxidant enzymes and nutrients concentration and uptake were determined.

Enhanced camptothecin production induced by elicitors in the cell suspension cultures of Ophiorrhiza mungos Linn.

Abstract: Production of camptothecin (CPT), an anticancer compound was enhanced in the cell suspension cultures of Ophiorrhiza mungos Linn. through elicitor treatment. Cell suspension culture was established using the friable callus tissues induced from the field grown leaf explants cultured in MS solid media supplemented with 3 % sucrose, 3 mg L−1 1-Naphthaleneacetic acid (NAA), 1 mg L−1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 mg L−1 kinetin (KIN). The callus tissues were used for establishing cell suspension culture in half-strength MS (1/2X MS) liquid media supplemented with the same hormone concentration. NAA was found to be essential for the prolific growth of O. mungos cells in suspension culture. Influence of different elicitors such as yeast extract (YE) and silver nitrate (AgNO3) on cell growth, CPT accumulation and cell viability was studied and found that YE and AgNO3 caused a significant increase in biomass and CPT yield according to their concentration, incubation time and feeding time. A maximum of 13.3-fold increment in CPT production and threefold increase in cell growth were recorded in cell cultures elicited with 50 mg L−1 YE on the 10th day of incubation. Cell growth and CPT level were found to decrease in the cultures treated with high concentration of elicitors. CPT was estimated using high performance liquid chromatography (HPLC). The results obtained in the present investigation suggest the use of elicitation as a promising alternative method to increase CPT production and cell growth in the cell suspension cultures of O. mungos.

The microRNA167 controls somatic embryogenesis in Arabidopsis through regulating its target genes ARF6 and ARF8

Abstract: MicroRNAs (miRNAs) are single-stranded small, endogenous RNAs that can downregulate gene expression in plants and animals. Plant miRNAs are already known to function in many biological processes during plant development by targeting mRNA for degradation or repression. Somatic embryogenesis is used as a model to investigate miRNAs associated with embryogenesis and also as an important pathway for plant propagation. To determine the roles of miRNAs in Arabidopsis somatic embryogenesis, we analyzed miRNA microarrays representing known miRNAs in embryonic callus before and after somatic embryo (SE) induction. After identifying several differentially expressed miRNAs from the microarray data, we focused on MIR167c encoding miR167 because of its potential involvement in SE induction. Overexpression of miR167 inhibited SE formation, showing that miR167 negatively regulates SE induction. The negative effects of miR167 on SE formation are characterized by the alteration of auxin response and local auxin transport in embryonic callus. Overexpression of miR167 resulted in low levels of auxin response factor 6 (ARF6) and ARF8 transcripts. Genetic analysis revealed that arf6 and arf8 single mutants and the arf6-2 arf8-3/+ sesquimutant displayed SE defects. Our results thus suggest that miR167 regulates somatic embryogenesis through auxin signaling components ARF6 and ARF8. This study provides important information concerning miRNA-mediated somatic embryogenesis.

Production and gene expression of morphinan alkaloids in hairy root culture of Papaver orientale L. using abiotic elicitors

Abstract: Nowadays, hairy root culture elicitation is an excellent platform with many advantages which make it an attractive system for using in plant biochemistry and molecular biology. Here, an efficient system for A. rhizogenes transformation in oriental poppy, Papaver orientale, was developed. Meanwhile, the expression level of various genes (COR, SalAT, SalR, T6ODM, CODM and Salsyn) in the morphinan pathway for response to methyl jasmonate (MJ) and salicylic acid (SA) at different time points (6, 12, 24 and 48 h) was examined. Afterwards, in vitro production of thebaine, morphine and codeine was investigated. The wild-type A. rhizogenes strain ATCC15834 was able to produce transformed roots with the highest frequency on the hypocotyl explant. Quantitative reverse-transcription PCR analysis showed that the expression of SalR, Salsyn, SalAT, T6ODM and CODM were significantly up-regulated with MJ treatment at 48 h. Moreover, the expression levels of Salsyn, T6ODM and CODM was up-regulated considerably in SA at 48 h treatment, while, the expression of SalR was down-regulated at 24 h. Furthermore, high-performance liquid chromatography analysis showed that MJ elicitation enhanced thebaine and morphine at 48 h by 2.63-fold (3.08 mg g−1) and 6.18-fold (5.38 mg g−1), respectively, and also codeine was increased at 24 h by 3.67-fold (2.57 mg g−1). In addition, the accumulation of morphine and thebaine was increased by 4.22-fold (2.87 mg g−1) and 2-fold (1.66 mg g−1) in comparison with control treatment after SA treatment for 48 h. Our findings suggest that oriental poppy hairy roots can be used as a good source for producing commercial alkaloids.

The importance of applied light quality on the production of lignans and phenolic acids in Schisandra chinensis (Turcz.) Baill. cultures in vitro

Abstract: Shoot-differentiating callus cultures of Schisandra chinensis were cultivated under different spectral properties of monochromatic light (far-red, red, blue, UV-A), and in darkness and under white light (control conditions). The applied lighting conditions influenced biomass growth and the accumulation of secondary metabolites—dibenzocyclooctadiene lignans (fourteen compounds) and phenolic acids (seven compounds). Total amounts of the estimated metabolites increased 1.71 and 1.98 times, respectively, depending on light quality. Blue light was found to be the most effective lighting to the production of both groups of compounds. Their total amounts reached the maximum values of 376.41 mg/100 g DW and 46.57 mg/100 g DW, and were correspondingly 1.31 and 1.37 times greater than under white light. The amounts of individual compounds from the tested groups increased from 1.51 to 3.38 times (lignans), and from 1.74 to 2.72 times (phenolic acids), depending on the lighting conditions. The main bioactive compounds obtained in high amounts were: schisandrin (67.70 mg/100 g DW), deoxyschisandrin (55.19 mg/100 g DW), gomisin A (36.97 mg/100 g DW), and also chlorogenic acid (15.33 mg/100 g DW) and protocatechuic acid (13.11 mg/100 g DW). This is the first report providing evidence of the importance of light quality on the production of dibenzocyclooctadiene lignans and phenolic acids in the biomass of S. chinensis cultivated in vitro.

Time to flowering of temperate pulses in vivo and generation turnover in vivo–in vitro of narrow-leaf lupin accelerated by low red to far-red ratio and high intensity in the far-red region

Abstract: Understanding the role light quality plays on floral initiation is key to a range of pre-breeding tools, such as accelerated single-seed-descent. We have elucidated the effect of light quality on early flowering onset in cool-season grain legumes and developed predictive models for time to flowering under the optimised light conditions. Early and late flowering genotypes of pea, chickpea, faba bean, lentil and lupin were grown in controlled environments under different light spectra (blue and far red-enriched LED lights and metal halide). All species and genotypes showed a positive response to a decreasing red to far-red ratio (R:FR). In general, ratios above 3.5 resulted in the longest time to flowering. In environments with R:FR below 3.5, light with the highest intensity in the FR region was the most inductive. We demonstrate the importance of considering both relative (R:FR) and absolute (FR photons) light values for flower induction in grain legumes. Greater response to light spectra was observed in the later flowering genotypes, enabling a drastic compression of time to flowering between phenologically diverse genotypes. A novel protocol for robust in vitro germination of immature seeds was developed for lupin, a species known for its recalcitrance to in vitro manipulation. We show how combining this protocol with growth under conditions optimized for early flowering drastically speeds generation turnover. The improved understanding of the effect of light on flowering regulation and the development of robust in vitro culture protocols will assist the development and exploitation of biotechnological tools for legume breeding.

Synergistic effects of light and temperature on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana)

Abstract: We established a red callus from the leaves of a red-fleshed apple individual, which was a hybrid offspring of the cross between Malus sieversii f. niedzwetzkyana and Malus domestica cv. ‘Fuji’. We analyzed callus growth and anthocyanin biosynthesis/metabolism under different combinations of temperature and light conditions. Incubation in darkness resulted in decreased anthocyanin accumulation, while it promoted callus growth. Exposure to light and low temperature (16 °C) induced the expression of MYB10 and bHLH3/33, which are responsible for coordinating the regulation of anthocyanin biosynthesis, as well as the expression of other structural genes. Treatments with light and high temperature (32 °C) induced MYB16 expression, which repressed anthocyanin biosynthesis. Additionally, low temperature (16 °C) inhibited the expression of MYB111. We analyzed the expression patterns of MYB and bHLH transcription factor genes by quantitative real-time polymerase chain reaction. Our data suggest that light-induced regulation of anthocyanin biosynthesis is primarily caused by altered MYB10 transcript levels, while temperature-induced regulation is the result of changes to the expression of bHLH3/33, MYB16, MYB17, MYB111, and other repressors. In conclusion, we investigated the reciprocal effects of light and temperature on anthocyanin biosynthesis in red-fleshed apple calli. Our findings may provide a theoretical basis for breeding red-fleshed apple varieties with high anthocyanin contents.

Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas

Abstract: Nardostachys jatamansi (D. Don) DC., a small, perennial, rhizomatous herb of immense medicinal importance since ancient times, is restricted to specialized habitats of alpine Himalayas ranging from 3000 to 5200 m asl. The species has been recently listed as critically endangered under IUCN Red list of threatened species due to over exploitation of its rhizomes for medicinal uses, habitat degradation, trade and other biotic and anthropogenic interferences. An efficient protocol using both indirect and direct shoot organogenesis has been optimized for N. jatamansi. Best callusing was achieved from the cut ends of leaf and petiole explants within 15 days of culture in MS medium supplemented with 1.5 mg/l α-naphthalene acetic acid and 1.0 mg/l meta-Topolin. Culturing the explants at low temperature (13 ± 1 °C) resulted in better callus growth, shoot regeneration, hyperhydricity control and improvement in photosynthetic pigment content in regenerated shoots. Also, direct organogenesis from shoot tip and petiole explants was achieved in MS medium containing 1.0 mg/l meta-Topolin. Optimum rooting was achieved in the same medium supplemented with 1.0 mg/l indole acetic acid wherein averages of 4.52 roots/shoot were induced. Genetic stability of in vitro-derived plantlets was assessed and compared to mother plant using molecular markers and flow cytometry. Intron Splice Junction (ISJ) and Start Codon Targeted polymorphism (SCoT) marker based profiling revealed uniform banding profile in case of direct shoot organogenesis (DSO)-derived plants while callus mediated organogenesis (CMO)-derived plants showed slight variations as compared to mother plant. The genome size of N. jatamansi was found to be 2C = 1.40 ± 0.01 pg and therefore 684.6 Mbp (1C). Although organogenic calli showed mixoploidy but no major phenotypic and genetic rearrangements were detected by flow cytometry in callus-derived plants. Significantly higher antioxidant activity was observed in callus-derived plants as compared to mother and DSO-derived plants. Plant parts, regeneration pathways and various solvent systems greatly affected the yields of total phenolics, flavonoids, alkaloids, tannins contents present in the in vitro raised plantlets.

A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins

Abstract: Plant protoplasts constitute a versatile system for transient gene expression and have been widely used with several plant species for the functional characterization of genes and studies of diverse signaling pathways. However, such a system has not been developed for grapevine (Vitis vinifera L.) due to the challenges of large-scale isolation of viable grapevine protoplasts. Here, we report a simplified method for obtaining high yields and excellent viability of isolated protoplasts from young grapevine leaves. In addition, both the conditions for isolation and transfection of grapevine mesophyll protoplasts were modified, and the system was shown to be suitable for protein expression and studies of protein subcellular localization and protein–protein interactions. In addition, we heterologously and transiently expressed the Arabidopsis thaliana disease resistance protein RPW8.2, which has previously been reported to confer broad-spectrum resistance to several biotrophic pathogens in different plant families, as a fluorescent fusion protein in grapevine protoplasts. We observed that expression of the RPW8.2 fusion protein was induced in response to application of exogenous salicylic acid and following infection by the grapevine downy mildew pathogen, Plasmopara viticola. These results illustrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and investigation of the activity of disease resistance proteins in grapevine.

In vitro induction and characterization of tetraploid Patchouli ( Pogostemon cablin Benth.) plant

Abstract: Patchouli (Pogostemon cablin Benth.) is one kind of plant from Indonesia which is producing oil. In vitro induction of tetraploid in Patchouli plant and cytological and morphological characterization were conducted. Induced polyploidy was done by culturing leaf explant on MS medium added with a varying concentration of colchicine. It was observed that colchicine on MS medium mostly affect to growth of explant and shoot regeneration. Tetraploid plants were determined by observation of chromosome number. The tetraploid plants have chromosome number (2n = 4x = 128), twice higher than those of diploid plants (2n = 2x = 64). The tetraploid plants exhibited much larger stomata size but less than diploid plants. The tetraploid plants also showed more glandular hairs than diploid plants. Significant different in morphological characters between the diploid and tetraploid plants were noted. Leaf size and stem diameter of the tetraploids plant are larger than the diploid plants. In this study, the tetraploids Patchouli have a promising potential as a superior variety.

Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants

Abstract: This study reports Agrobacterium rhizogenes-mediated transformation of three cultivars of Ocimum basilicum for hairy root establishment, screening and selection for the production of rosmarinic acid and antioxidants. Hairy root development was found to be explant-specific and virulence-dependent. Distinct inter-cultivar morphological variability was found between the seven axenically developed hairy root lines and morphological traits were found to be correlated with the presence of aux2 genes, their expression and endogenous IAA content. Further inter-cultivar variability in the content of total phenolics, rosmarinic acid and caffeic acid was also found. Production of rosmarinic acid was found to be age-dependent and cultivar-specific. Chemiluminescence analysis showed the hairy roots to be rich in antioxidants and that rosmarinic acid was the major antioxidant molecule. The concentration of rosmarinic acid was found to be positively correlated with the total antioxidant potential of the hairy root extracts. On the basis of origin, morphology and metabolite content, three elite hairy root lines were selected that had significantly higher rosmarinic acid production, biomass and antioxidant potential than non-transformed roots. These new lines are rich reserves of both antioxidants and rosmarinic acid.

High-frequency somatic embryogenesis and artificial seeds for mass production of true-to-type plants in Ledebouria revoluta: an important cardioprotective plant

Abstract: Scaly bulb of Ledebouria revoluta is a natural source of several cardiac glycosides and is traditionally used in Indian and South African ethno-medicinal system from ancient age. High-frequency indirect somatic embryogenesis protocol via callus culture had been developed from bulb scale explant. Optimum embryogenic calli was induced on Murashige and Skoog (MS) medium supplemented with 3.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.75 mg l−1 β-naphthoxyacetic acid. A maximum of 93.3 % of the cultures responded after 6 weeks of culture on MS medium containing 3.0 mg l−1 of thidiazuron, 0.75 mg l−1 α-naphthalene acetic acid and 1.75 mM spermidine forming 46.7 ± 0.58 somatic embryos per 500 mg callus. Individual somatic embryo was encapsulated in calcium alginate beads to produce artificial seeds (ASs). The ASs were stored at 4, 15, and 24 °C temperatures up to 180 days. The ASs showed 57.8 % germinability even after 4 months of storage at 15 °C. Plantlets were acclimatized with a survival rate of 96.0 % and after 13–14 months, 92.4 % of these plants produced flowers. Chromosomal studies revealed cytological stability of the regenerants containing 2n = 30 chromosomes in root tips and n = 15 chromosomes in pollen grains, same as parental plants. The meiotic behaviour of the regenerants is also similar to that of parental plants. Randomly amplified polymorphic DNA analysis revealed that there is no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirm the very reliable method for short-term conservation and large-scale production of true-to-type plantlets of L. revoluta, which can be applied in pharmaceutical industries.

Micropropagation of a giant ornamental bromeliad Puya berteroniana through adventitious shoots and assessment of their genetic stability through ISSR primers and flow cytometry

Abstract: An efficient micropropagation protocol has been developed for Puya berteroniana, a giant Chilean bromeliad with attractive turquoise flowers and great horticultural potential. Plant material for the experiment was multiplied via repeated sub-cultures of adventitious shoots, originally derived from a single in vitro germinated seedling, on a half-strength Murashige and Skoog (MS) medium (Murashige and Skoog in Physiol Plant 15:473–497, 1962) containing 0.44 µM 6-benzyladenine (BA). For the in vitro propagation experiment, the shoots were cultured on a half-strength MS medium supplemented with BA (0.04–2.22 µM) or zeatin (0.05–2.28 µM) alone or in combination with α-naphthaleneacetic acid (NAA) (0.54 µM). The maximum shoots per explant (5.5), was obtained on a medium containing 0.44 µM BA. Rooting of the shoots was tested on a medium supplemented with NAA (0.54–2.69 µM) or indole-3-acetic acid (0.57–2.85 µM). The best rooting was achieved on a medium containing 2.69 or 1.61 µM NAA. The rooted plantlets were transferred ex vitro, with 98.3 % survival rate. Inter simple sequence repeat (ISSR), flow cytometry, and karyological analysis were used to evaluate true-to-type of the in vitro regenerants. Ten randomly chosen plants and control plant were used. Twenty ISSR primers produced 95 clear, distinct, and reproducible bands per analysed sample. All amplified products were monomorfic, and no polymorphism was detected. Similarly, flow cytometric analysis confirmed that the ploidy level in all plantlets was stable. Karyological analysis revealed number of somatic chromosomes 2n = 50. As no somaclonal variation was detected in culture, this micropropagation protocol can be used for the mass production of P. berteroniana plants.

Lignan accumulation in callus and Agrobacterium rhizogenes-mediated hairy root cultures of flax (Linum usitatissimum)

Abstract: Agrobacterium rhizogenes is gaining intensity in research to develop and produce a large number of commercially important secondary metabolites, such as lignans. Worldwide, lignans are receiving great attention because of their putative beneficial effects on human health. In this study, we investigated the potential of callus and A. rhizogenes, hairy roots, of flax Linum usitatissimum in accumulating lignans, namely, secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO) and matairesinol (MAT). Callus cultures were established on Gamborg B5 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzyladenine or gibberellic acid (GA3). These callus cultures were transformed with A. rhizogenes, which were initiated on 1.0 mg l−1 2,4-D + 0.5 mg l−1 GA3 or 1.0 mg l−1 GA3 media. Both hairy root culture 1 and hairy root culture 2 accumulated SDG, SECO and MAT with a total lignan concentration of 1.227 and 1.057 µmol g−1, respectively. However, non-transformed cultures did not accumulate MAT but accumulated SECO. Determination of total phenolic content by high-performance liquid chromatography revealed higher phenolic acid content in hairy root cultures compared with that in non-transformed cultures. Antioxidant activity, as measured by 2,2-diphenyl-1-picryl-hydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid-free radical scavenging assays, was significantly higher in hairy root cultures than in non-transformed cultures. Furthermore, the extract of hairy root culture showed inhibition of proliferation of human breast cancer cell line (MCF-7).

Expression analysis of MusaNAC68 transcription factor and its functional analysis by overexpression in transgenic banana plants

Abstract: NAC (NAM, ATAF and CUC) proteins are plant-specific transcription factors regulating development and responses to stress. One of NAC proteins from banana is NAC68, and expression analysis indicated its positive association to stress conditions. The 5′-proximal region of MusaNAC68 was isolated and sequence analysis indicated presence of stress related cis-elements and cis-elements involved in auxin-signaling. Expression of MusaNAC68 was maximum in roots and positively correlated with application of α-naphthaleneacetic acid. Nuclear localization of MusaNAC68 was determined by fusion of green-fluorescent protein with MusaNAC68 and transiently overexpressing in banana embryogenic cells. Transgenic lines were marginally taller and displayed more abundant roots than control along with altered expression of auxin-responsive genes like auxin-responsive factors and IAA/Aux (indoleacetic acid-induced protein) genes. Transgenic plants overexpressing MusaNAC68 displayed higher activity of polyphenol oxidase and rapid browning of plant extract. Transgenic lines showed better tolerance to stress induced by NaCl and mannitol and produced more shoot biomass. Leaf disc assay showed that transgenic lines retain more chlorophyll and lower malondialdehyde than control under salinity and drought. Transgenic line constitutively overexpressing MusaNAC68 showed elevated expression of many stress-responsive genes indicating its involvement in salinity and drought tolerance in banana.

Influence of plant growth regulators and spermidine on somatic embryogenesis and plant regeneration in four Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn)

Abstract: Effect of plant growth regulators and spermidine on somatic embryogenesis and regeneration was investigated in finger millet. Mature embryos, and 3 days old seedling-derived shoot apical meristems were cultured on Murashige and Skoog (MS) medium containing picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid. Improved embryogenesis (84.7 %) was found on MS medium containing 4.0 mg L−1 2,4-D and 0.5 mg L−1 kinetin in both explants. MS medium containing 1.5 mM spermidine along with 4.0 mg L−1 2,4-D and 0.5 mg L−1 kinetin produced highest frequency (90.4 %) of somatic embryogenesis from mature embryo derived callus in genotype ‘CO(Ra)-14’. On same medium somatic embryogenesis frequencies of ‘GPU-25’, ‘Try-1’ and ‘Piyur-2’ genotypes were 55.5, 85.3 and 58.7 %, respectively after 4 weeks of incubation in dark. MS medium containing 4.0 mg L−1 6-benzylaminopurine, 0.2 mg L−1 2,4-D and 1.5 mM spermidine was found to be optimum for shoot regeneration or somatic embryos in all four genotypes of finger millet. We also studied the influence of exogenous spermidine (2.0–4.0 mM) on regeneration of ‘CO(Ra)-14’ mature embryo-derived new and long-term (20–180 days old) calluses. Highest regeneration frequency 93.1 % and mean number of shoots 25.5 were produced on MS medium containing 3.0 mM spermidine, 4.0 mg L−1 BAP and 0.2 mg L−1 2,4-D using 60 days old callus. Regenerated shoots effectively rooted on half-strength MS medium and successfully acclimatized in soil with 100 % survival rate and they grew normally without showing any morphological variation. Genetic variation of in vitro derived plants and control plants were analyzed by RAPD markers.

In vitro tetraploid plants regeneration from leaf explants of multiple genotypes in Populus

Abstract: The present study develops a protocol for tetraploids of multiple genotypes induced from in vitro leaf explants of diploid full-sib progeny [(P. pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis)] after a colchicine treatment. Leaf explants from a ten genotypes full-sib progeny were cultured in MS basal medium containing with 1.78 μM BA and 0.27 μM NAA for 4, 5, and 6 days and transferred to the same liquid MS medium containing different concentrations of colchicine (50, 75, or 100 μM) for 2, 3, and 4 days, respectively. The results indicate that preculture duration, colchicine concentration, and exposure time had significative impacts in tetraploid induction rate but no significant correlation with the genotype. The feasible protocol involves treating a leaf explants pre-culture for 5 days followed by their transfer to liquid MS with 75 μM colchicine for 3 days, for most genotypes of full-sib progeny which could regenerate from leaf explants in the shoot regeneration medium. Tetraploid plantlets are identified using flow cytometric analysis and further confirmed after chromosome counts. Size and frequency of leaf stomata was significantly distinguish the tetraploid from the diploid plants.

Overexpression of S-adenosylmethionine synthetase 1 enhances tomato callus tolerance to alkali stress through polyamine and hydrogen peroxide cross-linked networks

Abstract: S-adenosylmethionine synthetase is a member of the stress-induced family genes. Our previous research indicated that overexpression of SlSAMS1 confers alkali stress tolerance to tomato seedlings. However, information regarding the alkali stress tolerance mechanism of SlSAMS1 and the cross-linked network between SlSAMS1 and downstream signal has been limited. To study how SlSAMS1 improves alkali stress tolerance, we manipulated the SlSAMS1 transgenic calluses through a pharmacological approach and found that overexpression of SlSAMS1 was positively correlated with polyamine (PA) and hydrogen peroxide (H2O2) accumulation leading to improve alkali stress tolerance. Additionally, the accumulation of H2O2 in SlSAMS1 overexpression calluses depended on polyamine oxidase activity. The activities of antioxidant system, accumulation of organic acid, Na+ detoxification as well as alkali stress tolerance of the SlSAMS1 transgenic calluses were reversed by PA biosynthesis inhibitors, but not significantly influenced by ethylene biosynthesis inhibitors. These results suggest that overexpression of SlSAMS1 enhances alkali stress tolerance through PA and H2O2 cross-linked networks, which provide new insight into how SlSAMS1 functions as a stress mediatory element in regulating plants tolerance to alkali stress.

DNA methylation and proteome profiles of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures as affected by plant growth regulators supplementation

Abstract: Araucaria angustifolia is a critically endangered conifer native to South America, and somatic embryogenesis (SE) is one of the most promising biotechnological tools for its conservation and mass propagation. In vitro tissue culture and chemical compounds supplemented to culture medium, especially plant growth regulators (PGRs), are known to affect DNA methylation and protein expression profiles, modulating the phenotype and/or the embryogenic potential. Here, we evaluated the global DNA methylation (GDM) levels of A. angustifolia embryogenic cultures (EC) during SE induction and multiplication steps for 1 year subcultures, and identified a wide range of differentially expressed proteins in PGR-free or -supplemented treatments. During long-term subcultures, PGR-supplementation proved to gradually increase the GDM, which may compromise genomic stability and evoke gene expression modifications. Label-free proteomics enabled a robust protein identification and quantification in A. angustifolia EC. Exclusively expression of PIN-like protein in PGR-supplemented treatment indicated a possible differential response of the EC to polar auxin transport, which can generate implications in its morphogenetic response to maturation step. Up-regulation of stress-related proteins in EC from PGR-supplemented treatment suggests its more stressful environment, triggering notable responses to hormonal, osmotic and oxidative stresses. Improved expression of proteins involved with protein folding and stabilization processes in PGR-free treatment could play a protective function in response to stress conditions caused by in vitro culture, and may provide an adaptive advantage to these EC. The expression of several proteins associated to terpenoid biosynthesis suggests that EC from both treatments and cell lines are possibly producing these compounds.

Aspergillus flavus fungus elicitation improves vincristine and vinblastine yield by augmenting callus biomass growth in Catharanthus roseus

Abstract: The influence of fungus elicitor Aspergillus flavus on alkaloid yield was investigated in Catharanthus roseus. The study reveals increased yield of vinblastine and vincristine in cultivated tissues. Different concentrations of extract applied to solid MS medium were: 0.05 % (T1), 0.15 % (T2), 0.25 % (T3), and 0.35 % (T4) along with control (T0). The callus biomass, embryo formation and plant regeneration were studied in response to elicitor treatments. The embryogenic callus was induced from hypocotyls of in vitro germinated seeds and various tissues were exposed to fungal elicitation. The use of A. flavus fungal elicitation improved callus biomass growth, which later differentiated into embryos, maximum somatic embryo induction being in T2 (106.53/callus mass). Biochemical analysis revealed more accumulation of sugar, protein and proline in growing tissues especially amended with elicitor. The somatic embryos germinated into plantlets on 2.24 µM BA added MS medium. The percent germination, shoot-, root length of germinated somatic embryos were high in low doses of elicitation (T1/T2). The quantitative analysis of vinblastine and vincristine yield was conducted in different elicitor treated tissues by the use of HPTLC. Vinblastine yield was maximum in germinating embryos (0.837 µg gm−1 dry weight), A. flavus elicitation at T2 improved vinblastine yield further (0.903 µg gm−1 dry weight). Compared to vinblastine, the yield of vincristine was low and on A. flavus addition, maximum vincristine yield was noted (0.216 µg gm−1 dry weight). The highest 7.88 and 15.50 % increased yield of vinblastine and vincristine respectively was noted on A. flavus elicitated tissues. In order to understand the role of elicitor on plant defense responses various antioxidant enzymes activity were investigated as the addition of elicitor induced cellular stress on tissues. Maturated and germinating somatic embryos had high SOD activity and on elicitation the activity of enzymes was further increased, indicating extra cellular stress on tissues, which yielded enriched level of vinblastine and vincristine at T2/T1.

Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis

Abstract: Somatic embryo (SE) regeneration is an ideal experimental system to realize rapid propagation of excellent clones and genetic improvement for perennial gymnosperms. In the present study, genes encoding the miRNA166 precursor were identified and LamiR166a was successfully transformed into the gymnosperm Larix leptolepis (L. leptolepis) and five LamiR166a over-expressed embryonic cell lines were screened out as stable embryo masses. As expected, the targets of miR166a, LaHDZ31-34, were all down-regulated in transgenic lines according to qRT-PCR results. The results showed that the percentage of normal SEs with 4–7 cotyledons was 77.0 % in wild type (WT) lines, but was reduced to 60.3 % in the pSuper::MIR166a lines with “cup-shaped” embryos comprised 7.0 % of WT and 20.7 % of transgenic embryos. Microscopic observation further showed that the intermediate region surrounded by the cotyledons was larger than in the control, with no upward bulge of the shoot apical meristem (SAM). The expression pattern of the two meristem marker genes CLAVATA (CLV) and WUSCHEL-related homeobox (WOX) were investigated. The results showed that the expression levels of WOX were three times higher in transgenic lines than in WT samples, which suggest that miR166a may indirectly regulate SAM development by directly affecting WOX expression. Besides, overexpression of LamiR166a clearly increased the rooting rate and promoted lateral root formation in L. leptolepis seedlings. These results may provide new insights into the regulatory role of miR166 in gymnosperms, and also new applications for forestry production in practice.

Responses of grass pea seedlings to salinity stress in in vitro culture conditions

Abstract: Physiological and molecular mechanisms of adaptation to abiotic stresses of grass pea (Lathyrus sativus L.) are still poorly understood. Responses of four genotypes of grass pea to salinity stress in tissue culture conditions were investigated at early seedling growth stages. Salinity stress was induced in the agar media by adding 0, 50, 100 and 200 mM of NaCl. Germination and seedling emergence percentage was not significantly affected by 50 and 100 mM of NaCl. However, NaCl in 200 mM concentration lowered level of these parameters. Generally, exposure to NaCl stress significantly reduced length of grass pea seedling organs (root and shoot) but did not influence the content of dry weight in shoots and increased it in the roots in two cases. Increasing salt concentration decreased integrity of cellular membranes both in root and shoot tissues. Higher accumulation of phenolic compounds and significant changes in activity of antioxidant enzymes (peroxidase and catalase) were observed in the roots but not in the shoots. Similarly, the content of proline increased mostly in the roots from moderate (100 mM) salinity conditions. Adverse conditions did not resulted in alterations in photosynthetic pigments content of any tested genotypes. The better performance of shoots than roots may result from in vitro conditions in which experiments were conducted.

Meeting the challenge of stevioside production in the hairy roots of Stevia rebaudiana by probing the underlying process

Abstract: Agrobacterium rhizogenes mediated “hairy root cultures” of the renowned bio-sweetener producing plant—Stevia rebaudiana, were generated to explore their yet undetected biosynthetic potentials concerning the low calorie diterpene glycoside—stevioside. Four stable rhizoclones were studied under light and dark conditions showing better growth under dark. Two of these rhizoclones revealed substantially higher photosynthetic pigment accumulation under light. Evidently, capitalization on the available inter-clonal variability first time showed the stevioside synthesizing exclusivity in the SRA4 rhizoclone under light condition, while the rest failed. The dualities of the glycoside synthesizing power amongst the two photosynthetically active rhizoclones were resolved through quantitative RT-PCR analysis of UGT85C2 gene showing positive expression in the stevioside producing rhizoclone. These findings elucidated the decisive role of UGT85C2 in combination with photosynthetic proficiency of the studied hairy root cultures in regulating the biosynthetic pathway of S. rebaudiana.

Advances in breeding and biotechnology of legume crops

Abstract: Legume crops are relevant globally to the feeding and the nutrition of humans and animals because of their relatively high seed content of protein and essential amino acids. Additionally, they are related to sustainable agriculture, considering their ability to associate with atmospheric nitrogen fixing bacteria (Rhizobia). Despite this, several technical constraints of legumes crops have maintained their worldwide production far behind from cereals. This review article focuses in current information about recent advances in breeding and biotechnology of the major leguminous crops. Conventional breeding has mainly focused in improving multiple vegetative and reproductive traits that have associated to distinct heritability values, which reflects how amenable each character is for genetic improvement. Legumes have strongly entered into the genomics era through the complete genome sequencing of several species in the last decade. Moreover, a wealth of tools and techniques of Fabaceae genomics are now available and discussed throughout this article. In addition, there is an increasing amount of quantitative trait loci, candidate genes, and genes associated to abiotic and biotic resistance and to agronomic traits that have been reported, which will potentially allow more rapid progress of legume genetic improvement. Two successful examples of genetically modified legume crops are examined in this paper: glyphosate-resistant transgenic soybean and transgenic common bean resistant to Bean golden mosaic virus. Finally, legumes genomics and breeding programs, using classical breeding methods, marker-assisted selection, and biotechnological tools face a promising momentum for further application of technology and information that could boost their global production.

Expression of a grape bZIP transcription factor, VqbZIP39, in transgenic Arabidopsis thaliana confers tolerance of multiple abiotic stresses

Abstract: The basic region/leucine zipper (bZIP) transcription factors are known to play key roles in response to abiotic stress. In this study, a bZIP gene (VqbZIP39) was isolated from grape (Vitis quinquangularis) and constitutively expressed in Arabidopsis under control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis thaliana plants showed enhance salt and drought stress tolerance during seed germination and in the seedling and mature plant stages. Various physiological parameters related to stress responses were analyzed to gain further insight into the role of VqbZIP39 and it was found that osmotic stress caused less damage to the transgenic seedlings than to the corresponding wild type plants. This correlated with an increase in endogenous ABA content as a consequence of the constitutive over-expression of VqbZIP39, and the up-regulated expression of stress-inducible target genes associated with tolerance of drought, high-salt, and oxidative stresses. Our results suggest that the expression of VqbZIP39 in A. thaliana likely enhances the tolerance to multiple abiotic stresses through the ABA signaling pathway, and may therefore have a similar function in the response to abiotic stresses in grape.

Sucrose induced osmotic stress and photoperiod regimes enhanced the biomass and production of antioxidant secondary metabolites in shake-flask suspension cultures of Prunella vulgaris L.

Abstract: Prunella vulgaris L. is a very important species with worldwide traditional medicinal uses. Despite of its medicinal importance, there is lack of research efforts on cultivation and phytochemicals production of this plant. In the present study, an effort has been made to optimize various culture conditions for establishment of cell suspension culture and production of desired secondary metabolites. Initially, callus was induced from petiole explants on Murashige and Skoog medium supplemented with 2.0 mg/l naphthalene acetic acid (NAA) in dark. For establishment of suspension culture, friable calli were shifted to liquid medium fortified with 2.0 mg/l NAA. The differential effects of sucrose (5–50 g/l), pH (5–6) and different photoperiod regimes on suspension biomass, secondary metabolites accumulation and antioxidant activities were investigated. Maximum levels of biomass, phenolics, flavonoids, protein content and antioxidant activities were displayed by cultures treated with 20–25 g/l of sucrose. Similarly, a lower pH of 5.3 resulted in enhanced biomass, phenolics, flavonoids, protein content and antioxidant activities. Furthermore, the cultures grown under 12D/18L, 14D/16L and 16D/14L photoperiods exhibited higher levels of biomass and secondary metabolites, compared to control (16L/8D). These investigations conclude that sucrose, different pH levels and photoperiod regimes modulate cell growth and the production of bioactive compounds in cell suspension cultures of P. vulgaris.