Showing papers in "Plant Cell Tissue and Organ Culture in 2013"
TL;DR: A higher production of withanolide A, withanone and withaferin A from the elicited-hairy roots of W. somnifera under optimal inoculum mass, harvest time, elicitor exposure time and its concentration is reported.
Abstract: Withania somnifera, an important medicinal plant that possesses a variety of bioactive secondary metabolites collectively known as withanolides. Hairy roots with an initial inoculum mass of 5 g FW were elicited separately with methyl jasmonate (MeJ) and salicylic acid (SA) at various concentrations for different exposure times after 30 days of culture. Enhanced production of biomass (32.68 g FW and 5.54 g DW; 1.23-fold higher), withanolide A (132.44 mg/g DW; 58-fold higher), withanone (84.35 mg/g DW; 46-fold higher), and withaferin A (70.72 mg/g DW; 42-fold higher) were achieved from 40 day-old harvested hairy roots elicited with 150 μM SA for 4 h exposure time. The present study reports a higher production of withanolide A, withanone and withaferin A from the elicited-hairy roots of W. somnifera under optimal inoculum mass, harvest time, elicitor exposure time and its concentration. These results will be useful for biochemical and bioprocess engineering for the viable production of withanolides in hairy root culture.
115 citations
TL;DR: The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures, implying that salcylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin.
Abstract: Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.
105 citations
TL;DR: The development and regulatory approval of ‘HoneySweet’ demonstrate the ability of RNAi technology to contribute to the sustainability of stone fruit production in regions impacted by PPV.
Abstract: Sharka disease, caused by Plum pox virus (PPV) was first recorded in Bulgaria during the early twentieth century and since that first report, the disease has progressively spread throughout Europe and more recently to Asia, Africa, North and South America. Few PPV resistance genes have been found to naturally occur in Prunus and this has led to the investigation of biotech approaches to the development of resistance through genetic engineering (GE). A notable example of the utility of this approach is 'HoneySweet' plum. PPV protection in this case is based on RNA interference (RNAi) and resis- tance has been shown to be highly effective, stable, durable, and heritable as a dominant trait. Extensive testing and risk assessment of 'HoneySweet' in laboratory, greenhouse and in the field for over 20 years has demon- strated not only the effectiveness but also the safety of the technology. 'HoneySweet' has been cleared for cultivation in the USA. By the appropriate regulatory agencies. The development and regulatory approval of 'HoneySweet' demonstrate the ability of RNAi technology to contribute to the sustainability of stone fruit production in regions impacted by PPV. Although it has taken almost 100 years since the identification of sharka, we are now able to effectively protect stone fruit species against this disease through the application of GE.
102 citations
TL;DR: Meta-topolin (mT) was more effective in improving shoot proliferation and phenolic production compared to BA and supplementation with low NAA concentrations resulted in reduced in vitro flavonoid production in most cases, when compared to treatments with cytokinin only.
Abstract: Although the effectiveness of topolins in plant tissue culture systems has recently been highlighted, there is a dearth of information on their interactions with auxins in relation to shoot organogenesis and secondary metabolite production. The current study evaluated the role of topolins singly or in combination with an auxin in comparison to 6-benzyladenine (BA) on shoot proliferation and secondary metabolite production of Huernia hystrix, a medicinal and ornamental stem-succulent of the endemic flora of southern Africa. Meta-topolin (mT) was more effective in improving shoot proliferation and phenolic production compared to BA. In general, the exogenous addition of α-naphthalene acetic acid (NAA) significantly increased shoot proliferation. The highest number of regenerated shoots (12.2 ± 0.98 shoots per explant) was recorded with medium containing 20 μM mT supplemented with 10 μM NAA and was three-times higher when compared to the treatments with cytokinin only. This suggests a synergistic interaction of auxin with cytokinin. On the other hand, supplementation with low NAA concentrations resulted in reduced in vitro flavonoid production in most cases, when compared to treatments with cytokinin only. Moreover, differences in cytokinin concentrations (even when used in combination with NAA equimolar concentrations) significantly affected secondary metabolite production in some cases. The current findings highlighted the differential effects of auxin-cytokinin interactions on shoot proliferation and the production of secondary metabolites in H. hystrix.
72 citations
TL;DR: The results suggest that the transcription factor PqWRKY1 is a positive regulator related to osmotic stress and triterpene ginsenoside biosynthesis in P. quinquefolius.
Abstract: Transcription factors (TFs) are important regulating factors that can mediate many life processes. However, no TF genes have previously been reported in Panax quinquefolius (American ginseng), with the exception of a few expressed sequence tags. In this study, 753 unigenes (unique sequences) have been annotated in the plant transcription factor database PlnTFDB (version 3.0) by mining a 454 transcriptome dataset of P. quinquefolius. After classification of the unigenes, 45 unigenes were discovered to be annotated as WRKY transcripts in the public databases. Furthermore, PqWRKY1, one of the WRKY family TF genes that respond to methyl jasmonate, was isolated according to the sequences in the 454 transcriptome dataset. The cDNA of PqWRKY1 (P. quinquefolius WRKY1) encodes a putative protein of 358 amino acids, including a WRKY domain and a zinc finger motif. A subcellular localization assay demonstrated that the protein localizes to the nucleus and has strong transcriptional activation activity in transgenic yeast. In comparison to control lines, the PqWRKY1 transgenic Arabidopsis line exhibited insensitive phenotypes when exposed to high salt or mannitol. Correspondingly, in transgenic Arabidopsis lines, the expression levels of some genes involved in the anti-stress process were relatively higher than those in the control lines. Additionally, genes involved in triterpene biosynthesis were expressed onefold to fivefold higher in the transgenic line compared to the control line. These results suggest that the transcription factor PqWRKY1 is a positive regulator related to osmotic stress and triterpene ginsenoside biosynthesis in P. quinquefolius.
72 citations
TL;DR: Investigation of the effects of salicylic acid and methyl jasmonate on anthocyanin induction, biomass accumulation, and color value (CV) indices for both pigment content (PC) and pigment production (PP) in callus cultures of Rosa hybrida cv.
Abstract: This study was undertaken to investigate the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on anthocyanin induction, biomass accumulation, and color value (CV) indices for both pigment content (PC) and pigment production (PP) in callus cultures of Rosa hybrida cv. Pusa Ajay. A concentration-dependent response was exhibited by cultures on SA and MeJA at different concentrations individually or in combinations to Euphorbia millii medium supplemented with 204.5 mM sucrose, 2.45 μM indole butyric acid and 2.33 μM kinetin. There was positive influence on both callus biomass and anthocyanin accumulation. Treatment with 0.5 μM MeJA was most effective in inducing anthocyanin biosynthesis in callus cultures. Anthocyanin accumulation in callus cultures was enhanced with the addition of SA and MeJA, but these did not differ significantly from control for the number of days required for pigment initiation and for color intensification. Moreover, the addition of 0.5 μM MeJA alone resulted in a higher frequency of color response (97.25 %), PC (3.48 ± 0.07 CV g−1 FW), and PP (1.56 ± 0.03 CV test tube−1) over control. In contrast, the presence of higher levels of SA (400 μM) and MeJA (5.0 μM) reduced frequency of color response, as well as levels of PC and PP. MeJA did not increase biomass accumulation but promoted frequency of color response, PC and PP. Hence, it was suggested that 0.5 μM MeJA promoted anthocyanin production in rose callus cultures. Significant correlation was found between frequency of response and each of the PC (r = 0.988) and PP (r = 0.990). Furthermore, PC and PP were also highly correlated (r = 0.998).
68 citations
TL;DR: This review will provide information on advances and insights on genetic transformation protocols as well as availability of characterized genes involved in resistance to both abiotic and biotic stresses.
Abstract: Citrus is the most important tree fruit crop in the world. However, citrus production is affected by both biotic and abiotic stresses, including drought, extreme temperature, salinity, citrus canker, citrus tristeza virus, and Huanglongbing (or citrus greening), among others. These stresses can severely influence growth and development of both rootstocks and/or scions of citrus trees, thus reducing both fruit production and fruit quality. Modern advances in the tools of plant biotechnology and advances in genomics play important roles in understanding how citrus crops can cope with diseases and adverse environmental conditions. Within the last decades, much progress has been made in identifying and cloning of genes involved in resistance to biotic and abiotic stresses as well in genetic transformation of Citrus and its related genera, such as Poncirus trifoliata and Fortunella spp. In this review, we will provide information on advances and insights on genetic transformation protocols as well as availability of characterized genes involved in resistance to both abiotic and biotic stresses. This will be followed with a discussion on perspectives of future developments in this field.
65 citations
TL;DR: This is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.
Abstract: Reference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA
POL
II are suitable reference genes for salinity stress-related studies and EF-1α and RNA
POL
II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.
63 citations
TL;DR: Results have shown that overexpression of miR828 in Arabidopsis inhibits transcription of yet another MYB factor, MYB82, indicating that MyB82 is likely to be involved in the anthocyanin biosynthesis pathway.
Abstract: Plant microRNAs (miRNAs) are RNAs of 20–22 nucleotides in length with sequence complementarities to specific mRNAs that are targeted for either cleavage or translational repression. They play critical regulatory roles in plant growth and development. In this study, the functionality of miR828 in the regulation of anthocyanin biosynthesis has been investigated. RNA blotting analysis has confirmed the presence of a conserved 22 nucleotide miR828 in both dicot and monocot plants. Moreover, it has revealed that miR828 is constitutively expressed in different tissues of Arabidopsis. Subsequently, a 35S:pre-miR828 construct has been created and transformed into Arabidopsis. Expression analysis has shown that levels of pre-miR828 and mature miR828 transcripts are increased, while levels of MYB75, MYB90, and MYB113 transcripts, encoding MYB transcription factors that positively regulate anthocyanin biosynthesis, are repressed in transgenic plants. As a result, anthocyanin levels are reduced, as are transcription levels of genes that are directly involved in anthocyanin biosynthesis, including PAL, CHS, CHI, F3H, F3′H, DFR, and LDOX. In addition, overexpression of miR828 in Arabidopsis inhibits transcription of yet another MYB factor, MYB82, indicating that MYB82 is likely to be involved in the anthocyanin biosynthesis pathway.
63 citations
TL;DR: It is concluded that the post-transcriptional regulation of LaMYB33 by miR159 participates in the maintenance of embryogenic or non-embryogenic potential and somatic embryo maturation, providing new insights into the regulatory mechanisms of somatic embryogenesis.
Abstract: During the process of subculture of embryogenic cultures, sometimes they may become non-embryogenic, which is not desirable. However, this offers an opportunity to explore the mechanisms underlying cell fate determination and the maintenance of embryogenic potential of explants during the process of somatic embryogenesis. In a previous study, differential expression of microRNAs (miRNAs) has been detected between embryogenic and non-embryogenic cultures as well as during somatic embryo maturation of Larix kaempferi (Lamb.) Carr. However, little is known about the target genes of these miRNAs during these cellular differentiation processes. In this study, full-length cDNA of the MYB homologue from L. kaempferi, LaMYB33, was cloned. Sequence analysis showed that the miR159 target sequence is present in LaMYB33. The isolation of the miRNA-guided cleavage products of LaMYB33 further suggested that this gene is regulated by miRNA. LaMYB33 transcript levels between embryogenic and non-embryogenic cultures and during the late stage of somatic embryo maturation were measured and the results showed opposite patterns in the expression of LaMYB33 and mature miR159. Based on the relationships between the expression patterns of LaMYB33 and mature miR159, we concluded that the post-transcriptional regulation of LaMYB33 by miR159 participates in the maintenance of embryogenic or non-embryogenic potential and somatic embryo maturation, providing new insights into the regulatory mechanisms of somatic embryogenesis.
61 citations
TL;DR: MrbHLH1, but not Mrb HLH2, was the essential partner of MrMYB1 during anthocyanin biosynthesis regulation in tobacco and bayberry, however, the biosynthetic genes in these two species responded differently to the MrmyB1–MrbHH1 complex.
Abstract: Anthocyanins, being important for both plant functions and human health, were transcriptionally regulated by the MYB–bHLH–WD40 transcription complex. The key MYB regulator for Chinese bayberry (Myrica rubra), MrMYB1, has been characterized in previous studies, while the specific bHLH partner(s) are unknown. In this study, MrbHLH1 and MrbHLH2 were isolated based on their homology to known plant bHLHs involved in anthocyanin biosynthesis regulation. Coordinate expression of MrbHLH1 with MrMYB1 and the anthocyanin biosynthetic genes was observed during fruit development, while MrbHLH2 showed a weaker correlation. Further transient assays in tobacco leaves suggested that MrbHLH1, but not MrbHLH2, was associated with MrMYB1 and triggered significant anthocyanin production. The lack of function of the MrbHLH2 in anthocyanin biosynthesis regulation suggested that different MrbHLH genes within the same phylogenic subfamily have different functions. Overexpression of MrMYB1 and MrbHLH1 in tobacco confirmed the crucial role of MrMYB1–MrbHLH1 in anthocyanin biosynthesis and all of the structural genes from NtCHS were up-regulated by the complex. Dual luciferase assays, however, indicated that MrMYB1 and MrbHLH1 selectively activated five of the eight promoters of biosynthetic genes from bayberry (MrCHI, MrF3′H, MrDFR1, MrANS, MrUFGT), although expression levels of all eight biosynthetic genes including MrCHS and downstream genes were coordinately increased during fruit ripening. Moreover, the interaction between MrbHLH1 and MrMYB1 was confirmed by yeast two-hybrid assay. In conclusion, MrbHLH1, but not MrbHLH2, was the essential partner of MrMYB1 during anthocyanin biosynthesis regulation in tobacco and bayberry, however, the biosynthetic genes in these two species responded differently to the MrMYB1–MrbHLH1 complex.
TL;DR: It is suggested that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var.
Abstract: Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L−1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L−1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.
TL;DR: This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways inpopulus.
Abstract: Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a >80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein–protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.
TL;DR: Transgenic expression of LcDREB2 and LcSAMDC2 in Arabidopsis can improve the salt stress tolerance of transgenic lines and cooperate with Lc SAMDC2 contributes to resistance to abiotic stress.
Abstract: S-Adenosyl-methionine decarboxylase (SAMDC) and dehydration responsive element-binding proteins (DREBs) can improve plant resistance to abiotic stresses. These proteins have been extensively studied, but the mechanism for transcriptional regulation of SAMDC remains unclear. In this paper, the LcSAMDC2 gene and its promoter were isolated from Leymus chinensis. Two DRE cis-elements were identified from the promoter of LcSAMDC2 and shown to bind with LcDREB2. Subcellular localization and yeast one-hybrid assay revealed that LcDREB2 is a transcription factor. An electrophoretic mobility shift assay (EMSA) showed that LcDREB2 can bind to the LcSAMDC2 promoter probe containing a DRE element. Over-expression of LcDREB2 in L. chinensis callus increased expression of LcSAMDC2. Co-expression of LcDREB2 and the promoter of LcSAMDC2 fused with GUS in tobacco activated GUS activity. These results indicate that LcSAMDC2 is the downstream gene of LcDREB2. In addition, transgenic expression of LcDREB2 and LcSAMDC2 in Arabidopsis can improve the salt stress tolerance of transgenic lines. These results indicate that LcDREB2 cooperating with LcSAMDC2 contributes to resistance to abiotic stress.
TL;DR: Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays.
Abstract: To introduce genetic resistance against alfalfa weevil (Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production.
TL;DR: An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated transformation and growth rate of hairy root.
Abstract: An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated transformation and growth rate of hairy root. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, and three explants types, hypocotyls, leaves and excised shoots were examined. The highest frequency of transformation was achieved using LBA9402 strain in the excised shoots. Several inoculation and co-cultivation media and different concentration of arginine were evaluated using LBA9402 strain and the excised shoots as explant. Interestingly, a drastic increase in the frequency of transformation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH4NO3 KH2PO4, KNO3 and CaCl2 was used. The effect of sucrose concentration and the ratio of NH4
+: NO3
− on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH4
+ to NO3
− on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization.
TL;DR: The system described in this study can be used for practical application of the trehalose-6-phosphate synthase gene in crop improvement and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants.
Abstract: Trehalose is a non-reducing disaccharide of glucose that confers tolerance against abiotic stresses in many diverse organisms, including higher plants. It was previously reported that overexpression of the yeast trehalose-6-phosphate synthase gene in tomato results in improved tolerance against abiotic stresses. However, these transgenic tomato plants had stunted growth and pleiotropic changes in appearance. In this study, transgenic tomato plants were generated by the introduction of a gene encoding a bifunctional fusion of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes from Escherichia coli under the control of the CaMV35S promoter. Transgenic plants accumulated higher levels of trehalose in their leaves and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants. All of the transgenic plants had normal growth patterns and appearances. Therefore, the system described in this study can be used for practical application of the gene in crop improvement.
TL;DR: Thin-layer chromatography analysis indicated that multiple shoots and roots from in vitro-derived plants produced high amounts of phenolic acids, primarily of rosmarinic acid (RA).
Abstract: A protocol for in vitro production of genetically uniform populations of the medicinal plant Eryngium planum, rich in selected phenolic acids, has been established. Shoot-tips were collected from axenic seedlings and grown on a Murashige and Skoog basal medium supplemented with 6-Benzyladenine (BA) and Indole-3-acetic acid (IAA). The highest shoot proliferation efficiency (17 shoots per explant) was obtained when 1.0 mg L−1 BA and 0.1 mg L−1 were added. Proliferating shoots were rooted and transferred to soil (89 % frequency of survival). Flow cytometric analysis of intact (field-grown) and microrpropagated plants revealed that all plants were uniform in genome size and had similar DNA contents. Thin-layer chromatography (TLC) analysis indicated that multiple shoots and roots from in vitro-derived plants produced high amounts of phenolic acids, primarily of rosmarinic acid (RA). Levels of phenolic acids in in vitro-derived plants were similar to those of intact plants. Furthermore, high-performance liquid chromatography revealed that root cultures in liquid medium accumulated substantial levels of RA. Thus, rapid establishment of in vitro-grown organ cultures of E. planum can also serve as reliable sources for bioactive compounds.
TL;DR: In-depth protocols that will allow for the generation of PLBs using TCLs are provided and these will be useful for large-scale mass propagation in bioreactors or for long-term storage as synthetic seeds.
Abstract: Thin cell layers (TCLs) offer a simple yet effective protocol that has contributed to major advances in clonal micropropagation of orchids. TLCs have been successfully used for protocorm-like body (PLB) and callus induction in Aranda, Coelogyne cristata, Cymbidium spp., Dendrobium spp., Doritaenopsis, Paphiopedilum, Renanthera, Rhynchostylis, Spathoglottis, and Xenikophyton. TCLs have also been a bulwark for genetic transformation studies of select genera. This review takes an in-depth look at how TCLs have been employed in orchid biotechnology and provides in-depth protocols that will allow for the generation of PLBs using TCLs. As PLBs in orchids are deemed somatic embryos, these will be useful for large-scale mass propagation in bioreactors or for long-term storage as synthetic seeds.
TL;DR: The salt tolerance in these transgenic OE lines indicates that the improvement of photorespiration stimulated the antioxidant system to scavenge ROS and also suggests the role of serine during salt stress.
Abstract: Salt stress has attracted increasing attention due to its toxic ability to restrict plant growth, and the photorespiration pathway has been shown to develop improved plant tolerance to abiotic stress. In this study, an Arabidopsis photorespiratory pathway gene serine: glyoxylate aminotransferase (SGAT), named as AtAGT1, was successfully overexpressed in duckweed (Lemna minor) to investigate the salinity defense capability in three transgenic overexpressed (OE) lines. Increased SGAT activity and decreased endogenous serine levels in these transgenic plant lines under salt stress resulted in enhanced protection against root abscission, higher maximum quantum yield of photosystem II (Fv/Fm), increased defense from cell damage as a result of improved cell membrane integrity, a decrease of reactive oxygen species (ROS) accumulation, and a strengthened antioxidant system. The salt tolerance in these transgenic OE lines indicates that the improvement of photorespiration stimulated the antioxidant system to scavenge ROS. The change of serine level also suggests the role of serine during salt stress. This transgenic engineering in duckweed not only introduced salt tolerance to this aquatic plant but also reveals a significant role of photorespiration during salinity stress.
TL;DR: In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog medium containing different levels of α-naphthaleneacetic acid and 6-benzyladenine to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method.
Abstract: Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l−1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g−1 dry weight (DW), and between 50.23 and 81.56 mg 100 g−1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g−1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g−1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g−1 DW) and syringic acid (41.20 mg 100 g−1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g−1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g−1 DW) were predominant.
TL;DR: This is the first report of a multi-step acclimation conferring tolerance to 150 mM NaCl in leaf-derived calli of M. truncatula, and suggests a Na+/K+ homeostasis.
Abstract: Salinity is a major abiotic stress that limits plant productivity. Plants respond to salinity by switching on a coordinated set of physiological and molecular responses that can result in acclimation. Medicago truncatula is an important model legume species, thus understanding salt stress responses and acclimation in this species is of both fundamental and applied interest. The aim of this work was to test whether acclimation could enhance NaCl tolerance in calli of M. truncatula. A new protocol is described incorporating multi-step up acclimation over 0–350 mM exogenous NaCl. By the end of the experiment, calli were tolerant to 150 mM and competent for embryogenesis at 100 mM NaCl. Positive and negative linear relationships between Na+ and K+ uptake and exogenous NaCl concentration intercepted at 160 mM suggesting a Na+/K+ homeostasis. Proline level peaked at 100/150 mM whilst highest osmolarity and lowest water content occurred at 250/350 mM NaCl. The concentration of water soluble sugars was positively related to 0–250 mM NaCl whilst callus growth and embryogenesis occurred regardless of endoreduplication. Expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1), proline synthesis (P5CS) and ploidy level (CCS52 and WEE1) peaked at 100/150 mM NaCl. Hence, these genes and various physiological traits except sugar levels, served as useful markers of NaCl tolerance. To our knowledge, this is the first report of a multi-step acclimation conferring tolerance to 150 mM NaCl in leaf-derived calli of M. truncatula.
TL;DR: This study is the first comparative proteome analysis between the embryogeniccallus and the non-embryogenic callus of the H99 inbred line and reveals some key proteins that may have significant roles in molecular events during somatic embryogenesis in this species.
Abstract: A proteomic approach based on two-dimensional electrophoresis (2-DE) and mass spectrometry was performed to investigate somatic embryogenesis in the H99 inbred maize line by comparing embryogenic and non-embryogenic callus. Protein spots (n = 42) were differentially expressed between embryogenic calli and non-embryogenic calli according to our image analysis. Among them, 33 proteins were differentially expressed by at least threefold, with 15 up-regulated and 18 down-regulated in the embryogenic callus versus the non-embryogenic callus. However, only nine proteins were expressed in either of the calli. Twenty-nine protein spots were identified using mass spectrometry analysis and classified into several categories based on the matrix-science MASCOT and NCBI databases. These categories included cell proliferation (10.34 %), transcription and protein processing (17.24 %), stress response (10.34 %), signal transduction (3.45 %), metabolism and energy (48.28 %) and hypothetical function (10.34 %). Their putative roles are discussed according to their relevance in somatic embryogenesis. Real-time reverse transcription polymerase chain reaction analysis revealed that the expression levels of five selected genes were consistent with the profiles detected in the 2-DE gels, further confirming the proteomic analysis. This study is the first comparative proteome analysis between the embryogenic callus and the non-embryogenic callus of the H99 inbred line. Our results show the differentially expressed proteins between the two callus types and reveal some key proteins that may have significant roles in molecular events during somatic embryogenesis in this species.
TL;DR: The new approach based on heat-induced flowering could be a refinement of the fast breeding program using the possibility of turning-on-turning-off flowering in physiological well developed plants.
Abstract: The Fast-track breeding approach in apple is based on the utilization of the BpMADS4 gene from Betula pendula. However, this approach has several disadvantages which could be solved using other flowering inducing genes and inducible promoters. The FLOWERING LOCUS T genes (PtFT1 and PtFT2) from poplar (Populus trichocarpa) driven by the heat-inducible Gmhsp 17.5-E (HSP)-promoter from soybean (Glycine max) were transferred into apple (Malus × domestica Borkh.) cv. ‘Pinova’ in order to induce flowering. Seven transgenic apple lines were obtained. All transgenic apple lines micrografted onto ‘Golden Delicious’ seedlings used as rootstocks were transferred to the greenhouse. Six out of seven transgenic lines developed flowers after a heat treatment at 42 °C for 1 h daily over a period of 28 days. The transgenic line T836 failed to flower. Flower morphology and pollen vitality of transgenic lines appeared normal. Transgenic plants were successfully used for hybridizations. Pollen from Malus ×robusta 5 applied to flowers of transgenic plants resulted in fruit formation. Heat induced PtFT1, respectively PtFT2 over-expressing rootstocks did not cause flowering in micrografted non-transgenic ‘Pinova’ scions. The mRNA of the PtFT genes was transported from transgenic rootstocks to non-transgenic scions only in one case. As a balance between plant development and flowering is important for the production of early flowering plants usable for a fast-track breeding program the new approach based on heat-induced flowering could be a refinement of the fast breeding program using the possibility of turning-on-turning-off flowering in physiological well developed plants.
TL;DR: Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz were incubated and well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.
Abstract: Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl2·2H2O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA3). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.
TL;DR: Results indicate that theCry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins, which might delay the development of Bt-resistant insects in the field.
Abstract: Although transgenic crops expressing either Cry1Ab or Cry1Ac, both derived from Bacillus thuringiensis (Bt), have been used commercially, the evolution of insects resistance to these CRY proteins has become a challenge. Thus, it has been proposed that co-expression of two Bt proteins with different modes of action may delay the development of resistance to Bt. However, few Bt proteins have been identified as having different modes of action from those of Cry1Ab or Cry1Ac. In this study, transgenic lines of maize over-expressing either Cry1Ie or Cry1Ac gene have been developed. Several independent transgenic lines with one copy of the foreign gene were identified by Southern blot analysis. Bioassays in the laboratory showed that the transgenic plants over-expressing Cry1Ie were highly toxic against the wild-type cotton bollworm (Heliothis armigera), producing mortality levels of 50 % after 6 days of exposure. However, the mortality caused by these plants was lower than that caused by the Cry1Ac transgenic plants (80 %) and MON810 plants expressing Cry1Ab (100 %), which both exhibited low toxicity toward the Cry1Ac-resistant cotton bollworm. In contrast, three transgenic maize lines expressing Cry1Ie induced higher mortality against this pest and were also highly toxic to the Asian corn borer (Ostrinia furnacalis) in the field. These results indicate that the Cry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins. Therefore, the use of transgenic plants expressing Cry1Ie might delay the development of Bt-resistant insects in the field.
TL;DR: It is demonstrated that miR 156 and its target SPL genes are involved in various developmental processes, especially flower development, and miR156 mediates a conserved post-transcriptional regulation pathway in the apple and Arabidopsis plants.
Abstract: The microRNA miR156 is involved in the regulation of plant growth and development by specifically restricting the transcripts of target genes. In this study, the Md-miR156h gene and its target cDNA fragments, specifically, MdSPL2a-b, MdSPL4, MdSPL6a-g, MdSPL9a-b, MdSPL13a-e and MdSPL15, were isolated from the apple cultivar ‘Gala’. Phylogenetic analysis showed that 18 MdSPL genes were putative targets of miR156. Subsequently, the expression construct p35S:Md-miR156h was created and transformed into Arabidopsis plants. Expression analysis showed that Md-miR156h transcripts and mature miR156 accumulation increased, while its target transcripts AtSPL9 and AtSPL15 were downregulated in transgenic Arabidopsis plants. As a result, the transgenic plants exhibited an extended juvenile phase, increased numbers of leaves, short siliques and the partial abortion of seeds compared with the WT control plants. These results demonstrate that miR156 and its target SPL genes are involved in various developmental processes, especially flower development, and miR156 mediates a conserved post-transcriptional regulation pathway in the apple and Arabidopsis.
TL;DR: The encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.
Abstract: A simple and efficient cryopreservation protocol using encapsulation-dehydration was established for in vitro-grown shoot-tips of apple ‘Gala’ (Malus × domestica Borkh.). Shoot-tips, of 2.0 mm in length and with 5–6 leaf primordia, excised from 4-week-old shoot stock cultures, without cold-hardening, were encapsulated into beads, each being about 5 mm in diameter and containing a single shoot-tip. The beads were precultured on MS medium containing 0.5 M sucrose for 7 days. The precultured beads were dehydrated by air-drying to reduce the water content of the beads to about 22–20 % in 5–7 h, followed by a direct immersion in liquid nitrogen for 1 h. Frozen shoot-tips were re-warmed in a water bath at 38 °C for 2 min and post-cultured on a recovery medium for shoot regrowth. This protocol was successfully applied to four Malus species and one hybrid, among which M. micromalus and M. robusta are wild species native to China. The highest and lowest shoot regeneration rates were found in ‘Gala’ (75 %) and ‘Wangshanhong’ (36 %), with a mean shoot regrowth rate of 61 % attained for the seven Malus genotypes tested. Histological studies revealed that shoots could be regenerated in cryopreserved shoot-tips only when many cells in the leaf primordia and most of the cells in the apical dome survived following cryopreservation. Morphologies of the regenerated plantlets were identical to those from the in vitro stock cultures. Therefore, the encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.
TL;DR: The study was carried out to evaluate the amenability of tropical inbred and hybrid maize lines, using Agrobacterium mediated transformation technique, which can be used to introduce genes of interest into tropical maize lines for genetic improvement.
Abstract: The study was carried out to evaluate the amenability of tropical inbred and hybrid maize lines, using Agrobacterium mediated transformation technique. Agrobacteriumtumefaciens strains EHA101 harbouring a pTF102 binary vector, EHA101, AGL1, and LBA4404 harbouring pBECK2000.4 plasmid, LBA4404, GV and EHA105 harbouring pCAMBIA2301 plasmid, and AGL1 harbouring the pSB223 plasmid were used. Delivery of transgenes into plant tissues was assessed using transient β-glucuronidase (gus) activity on the 3rd and 4th day of co-cultivation of the infected Immature Zygotic Embryos (IZEs) and embryogenic callus. Transient gus expression was influenced by the co-cultivation period, maize genotype and Agrobacterium strain. The expression was highest after the 3rd day of co-culture compared to the 4th day with intense blue staining was detected for IZEs which were infected with Agrobacterium strains EHA105 harbouring pCAMBIA2301 and EHA101 harbouring pTF102 vector. Putative transformants (To) were regenerated from bialaphos resistant callus. Differences were detected on the number of putative transformants regenerated among the maize lines. Polymerase chain reaction (PCR) amplification of Phosphinothricin acetyltransferase (bar) and gus gene confirmed the transfer of the transgenes into the maize cells. Southern blot hybridization confirmed stable integration of gus into PTL02 maize genome and segregation analysis confirmed the inheritance of the gus. A transformation efficiency of 1.4 % was achieved. This transformation system can be used to introduce genes of interest into tropical maize lines for genetic improvement.
TL;DR: This review presents and discusses the development of somatic hybrids in wheat, rapeseed, citrus, and cotton, some of which are already in use or have potential for future commercial use in China.
Abstract: Protoplast fusion offers an opportunity for circumventing barriers to sexual reproduction and allows for gene transfer of nuclear and cytoplasmic genomes to enrich the gene pool of cultivated species. Moreover, protoplast fusion effectively generates novel germplasm for elite breeding of conventional crosses and promotes crop improvement in existing cultivars. Over the past few decades, protoplast fusion in China has considerably progressed not only for food crops but also for economic plants. In this review, we present and discuss the development of somatic hybrids in wheat, rapeseed, citrus, and cotton, some of which are already in use or have potential for future commercial use in China. For example, an elite salt- and drought-resistant line, Shangrong No. 3, has already been registered as a commercial wheat cultivar. Some other hybrids have been found to have disease resistance as well as modified fatty acids, high oil and protein contents, novel cytoplasmic male sterility, and numerous other desirable agronomic characteristics that are useful for further breeding.