Showing papers in "Plant Cell Tissue and Organ Culture in 2012"
TL;DR: Evidence from various studies indicate the rising popularity and advantages of topolins compared to other CKs, and emphasis on their metabolism, structure–activity relations and effect on morphogenesis in vitro is placed.
Abstract: Since the discovery of topolins as naturally occurring aromatic cytokinins (CKs), they have emerged as genuine alternatives to the long serving CKs such as benzyladenine, zeatin and kinetin in plant tissue culture (PTC). Globally, the past 15 years has witnessed a surge in the use of topolins and their derivatives in research laboratories. Topolins, especially the meta-topolin and its derivatives have been employed for culture initiation, protocol optimization and for counteracting various in vitro induced physiological disorders in many species. Evidence from various studies indicate the rising popularity and advantages (although not universal for all species) of topolins compared to other CKs. In this review, we assess the use of topolins in PTC with emphasis on their metabolism, structure–activity relations and effect on morphogenesis in vitro. In addition, the review provides a detailed list of species that have been used to study the effect of topolins in comparison with other CKs, the growth parameters affected and recommended concentrations are also provided.
156 citations
TL;DR: It is shown that the choice of cytokinin type and concentration exogenously supplied during tissue culture markedly influences not only shoot proliferation but also the in vitro production of bioactive secondary metabolites.
Abstract: Aloe species are highly-prized for their ornamental value and have been utilized for centuries in traditional medicine. Due to their habitat loss and exploitation for medicinal and ornamental plant trade, many species in this genus have become threatened. One of the most important globally rated medicinal species in Aloe genus is A. arborescens. The current study evaluated the roles of different aromatic cytokinin types and concentrations on direct organogenesis, in vitro bioactive secondary metabolite production and antioxidant activity of regenerated shoots of A. arborescens. There was an increase in the number of adventitious shoots produced per explant with an increase in concentration in cultures treated with meta-topolin (mT), meta-topolin riboside (mTR), meta-methoxytopolin (MemT) and benzyladenine riboside (BAR), reaching an optimum at either 5.0 or 7.5 μM. Overall, the treatment with 5.0 μM mT gave the largest number of transplantable shoots (regenerated shoots with length greater than 10 mm). Rooted shoots were successfully acclimatized after 8 weeks with a survival frequency above 90 % and no observable morphological abnormalities. Variable amounts of total iridoids, phenolics, flavonoids and condensed tannins were detected in regenerated shoots from all the cytokinin treatments. An increased free-radical scavenging activity with an increase in concentration was recorded in regenerated shoots from mT and mTR treatments, reaching an optimum at 7.5 μM concentration. The present study shows that the choice of cytokinin type and concentration exogenously supplied during tissue culture markedly influences not only shoot proliferation but also the in vitro production of bioactive secondary metabolites.
114 citations
TL;DR: It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control, and there was no substantial influence of elicitors on rosmarinic acid secretion into the culture media.
Abstract: The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g−1 DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g−1 DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.
113 citations
TL;DR: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.
Abstract: The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.
102 citations
TL;DR: In this article, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids, and the results showed that tetraploid plants could produce 200% higher scopolamine than their diploid counterparts.
Abstract: In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30 months after transformation Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions The highest yields of scopolamine (1387 mg l−1) and hyoscyamine (1077 mg 1−1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60 g/l sucrose or Gamborg’s B5 with 40 g/l sucrose, respectively Although the hyoscyamine is the main alkaloid in the H muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine
92 citations
TL;DR: The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of the authors' in vitro propagation system for guava.
Abstract: To evaluate genetic homogeneity of 1-year-old guava (Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300 bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200 bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava.
82 citations
TL;DR: An effective pistachio (Pistacia vera L.) micropropagation system was developed involving rapid axillary bud proliferation and ex vitro rooting, which generated an optimal number of shoots with suitable morphological features.
Abstract: An effective pistachio (Pistacia vera L.) micropropagation system was developed involving rapid axillary bud proliferation and ex vitro rooting. The highest shoot proliferation frequency was obtained from nodal explants cultured on Murashige and Skoog (MS) basal salts containing Gamborg (B5) vitamins and supplemented with 4 mg l−1 6-benzyladenine (BA). The addition of 2 mg l−1 meta-topolin (mT) generated an optimal number of shoots with suitable morphological features, while kinetin (KIN) was found to be unsuitable for pistachio shoot proliferation. Microcuttings were rooted ex vitro after being dipped in rooting powder. The peak ex vitro rooting response was achieved after shoot explants were treated with Rhizopon® 2% indole-3-butyric acid (IBA). Rooted plantlets were transplanted in plastic pots containing a peat–perlite–vermiculite (1:1:1) mixture and then transferred to the greenhouse. After 2 months, 81.5% survival of rooted microshoots was achieved.
75 citations
TL;DR: A system that could lower the cost of in vitro propagation by using liquid medium, as well as to evaluate the secondary metabolism in the systems tested, was established.
Abstract: Hypericum perforatum L. is a medicinal plant that has been extensively studied because of its bioactive properties. The objective of this study was to establish a system that could lower the cost of in vitro propagation by using liquid medium, as well as to evaluate the secondary metabolism in the systems tested. Nodal segments of H. perforatum were obtained from in vitro shoots and grown in three liquid culture systems: total immersion (TI), partial immersion (PI), and paper bridge support (PB). Semi-solid medium (3 g L−1 Phytagel™) was used as control (SS). The organogenic responses were evaluated, and phenolic compounds, hypericin, and the activity of polyphenol oxidases (PPO) and peroxidases (POX) were quantified. After 80 days of culture, induction and proliferation of adventitious shoots were similar in the PI and SS systems (65.3 and 71.3 shoots, respectively), whereas PB resulted in the fewest shoots per explant (29.5 shoots). Longer shoots were obtained under the PI conditions. Hyperhydricity was observed in the shoots from the TI system. Browning was visible in shoots from the TI and PB systems. The highest concentrations of phenolic compounds and hypericin were observed in shoots derived from PI and PB, at 80 days of culture. POX activity was higher in shoots cultured in PI at 40 days, whereas PPO was significantly more active at 80 days of culture. Likely, POX was more related to shoot growth, whereas PPO played a later role in response to the culture environment and medium stress.
71 citations
TL;DR: High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA, and these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis.
Abstract: Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.
70 citations
TL;DR: The increase of 20E levels by the photoautotrophic system offers new prospects for increasing the commercial production of this species, and for studies that could elucidate the biosynthetic pathway of phytoecdysteroids in plants.
Abstract: Pfaffia glomerata (Spreng.) Pedersen is a medicinal species of great interest because it produces the phytoecdysteroid 20-hydroxyecdysone (20E). Generally, because of atypical growing conditions, in vitro propagated plants function less efficiently as autotrophs and have poorly developed morphological structures. This study analyzed the autotrophic potential of P. glomerata propagated in vitro and evaluated the influence that this has on 20E biosynthesis. Physiological and structural parameters of plants subjected to heterotrophic, photomixotrophic and photoautotrophic growth conditions were evaluated. Levels of 20E were measured by HPLC. Plants were acclimatized in a mixture of soil, sand and substrate, in a greenhouse. Conditions that provided higher carbon input led to an increase in plant growth, and the presence of sucrose was critical, in closure systems without a gas permeable membrane, for normal anatomical development of the micropropagated plants. The absence of sucrose increased photosynthesis and conditions that enhanced photoautotrophy induced greater levels of 20E. The increase of 20E levels by the photoautotrophic system offers new prospects for increasing the commercial production of this species, and for studies that could elucidate the biosynthetic pathway of phytoecdysteroids in plants.
68 citations
TL;DR: An efficient plant regeneration protocol has been developed from embryogenic callus derived from cotyledonary explants of Clitoria ternatea Linn.
Abstract: An efficient plant regeneration protocol has been developed from embryogenic callus derived from cotyledonary explants of Clitoria ternatea Linn., an important medicinal climber species. Optimum embryogenic callus (75 %) was induced on Murashige and Skoog (MS) medium supplemented with 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D). On subculturing the callus on MS medium supplemented with 2 mg/l 6-benzyladenine (BA) and 0.5 mg/l α-naphthalene acetic acid (NAA), 61 % of cultures responded with a mean number of 22 somatic embryos per gram callus at different stages of development after 45 days of culture. The addition of higher concentrations of sucrose and abscisic acid (ABA) significantly increased the embryogenic response. MS medium supplemented with 2 mg/l BA, 0.2 mg/l NAA and 4 % of sucrose resulted in 76 % of cultures responding with a mean number of 28 embryos per one gram callus. The highest embryogenic response, frequency of 83 % and mean number of 37 embryos per gram callus, was observed when the MS medium was supplemented with 2 mg/l BA, 0.2 mg/l NAA, and 3 mg/l ABA. Synthetic seeds were produced by encapsulating embryos in calcium alginate gel. The gel contained MS medium with 3 % of sucrose, 1.0 mg/l BA and 0.2 mg/l NAA. The synthetic seeds germinated on MS medium supplemented with BA (1.0-4.0 mg/l) alone or in combination with NAA (0.1–0.7 mg/l) or indole-3-butyric acid (IBA; 0.1–0.7 mg/l). The highest synthetic seed germination (92 %) was observed on MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA. The synthetic seeds were stored at 4 °C and lab conditions (25 ± 2 °C) up to 5 months. The synthetic seeds kept at 4 °C showed 86 % viability even after 5 months of storage. Both somatic embryos and synthetic seeds germinated and were transferred to soil successfully.
TL;DR: The results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.
Abstract: Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na+/H+ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na+/H+ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T1 generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T1 transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.
TL;DR: The results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production.
Abstract: Cell suspension cultures of Gymnema sylvestre treated with four different elicitors, methyl jasmonate (MJ), yeast extract, chitin and pectin were studied for the production of gymnemic acid as gymnemagenin equivalent, that was analyzed by high performance liquid chromatography (HPLC). All the four tested elicitors induced gymnemic acid production in cell suspension cultures. Highest gymnemic acid content was achieved following treatment with yeast extract (100.47 ± 0.28 mg/l), this was followed by MJ (70.43 ± 0.26 mg/l), pectin (64.19 ± 0.23 mg/l) and chitin (62.72 ± 0.13 mg/l). The addition of elicitors has shown a significant influence on cell growth that affected cell growth compared to respective controls. The highest gymnemic acid production was obtained after 20 days of elicitation in cultures treated with 0.5 g l−l yeast extract, it was 5.25-folds greater than in control. These results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production. In our present study we could able to overproduce gymnemic acid up to 51.97 ± 0.26 mg l−l (dry weight basis) in yeast extract treated cell suspension cultures.
TL;DR: The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.
Abstract: In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.
TL;DR: Analysis of T1 transgenic tobacco plants overexpressing AhSIPR10 gene showed enhanced tolerance to salt, heavy metal and drought stress through leaf disc senescence, chlorophyll content, seed set and germination assays, thus corroborating a role of salt inducible-PR10 protein in mitigation of abiotic stress-induced damage.
Abstract: Pathogenesis-related proteins are induced in plants in response to stress, pathogen attack or abiotic stimuli, thus playing a cardinal role in plant defense system. A cDNA containing the full-length ORF, AhSIPR10 (474 bp, GenBank acc. no. DQ813661), encoding a novel Salinity-Induced PR class 10 protein was isolated from callus cell lines of peanut (Arachis hypogaea). Real-time quantitative reverse transcription PCR (qRT–PCR) data showed rapid upregulation of AhSIPR10 transcription in peanut callus cultures across salinity, heavy metal, cold and mannitol-induced drought stress environments. Likewise, AhSIPR10 expression was also responsive towards defense/stress signaling molecules salicylic acid (SA), methyl jasmonate, abscisic acid (ABA) and H2O2 treatments. Methyl jasmonate or ABA-induced AhSIPR10 expression was, however, antagonized by SA treatment. A functional role of AhSIPR10 in alleviation of abiotic stress tolerance was further validated through its over-expression in tobacco. Analysis of T1 transgenic tobacco plants overexpressing AhSIPR10 gene showed enhanced tolerance to salt, heavy metal and drought stress through leaf disc senescence, chlorophyll content, seed set and germination assays, thus corroborating a role of salt inducible-PR10 protein in mitigation of abiotic stress-induced damage. Transgenic tobacco lines overexpressing AhSIPR10 displayed adequate photosynthetic CO2 assimilation rates under salt, heavy metal and drought stress environments.
TL;DR: An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments, which successfully acclimatized and established in field where they grew well without any detectable variation.
Abstract: An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl2·2H2O). A gelling matrix of 4 % Na-alginate and 100 mM CaCl2·2H2O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00 ± 2.09 %) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 30.0 μM adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5 μM α-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.
TL;DR: In this article, a low-cost, simple to manufacture, alternative membrane was developed to promote gas exchange in jars used for in vitro plant tissue culture, using polytetrafluoroethylene film and two or three layers of microporous tape.
Abstract: In vitro propagated plants under conditions of low gas exchange generally show morphological and physiological anomalies that lead to high mortality rates during ex vitro acclimatization. The use of gas-permeable membranes increases natural ventilation in culture vessels, photosynthesis and growth rates. However, commercial membranes are expensive, which limits their application. In this study, low-cost, simple to manufacture, alternative membranes were developed to promote gas exchange in jars used for in vitro plant tissue culture. The membranes were developed using polytetrafluoroethylene film and two or three layers of microporous tape (Missner & Missner®), and were designed to increase the growth of nodal cultures of Pfaffia glomerata (Brazilian ginseng). Conditions that provided higher gas exchange led to an increase in plant growth and content of photosynthetic pigments compared to a closed system without a gas-permeable membrane. The alternative membranes showed similar results for water vapor loss rate and photosynthetic pigments when compared to a commercial membrane. The alternative membranes were also an efficient barrier against contamination and remained intact after being autoclaved multiple times. Among the membranes tested, the traits of the P. glomerata in vitro-derived plants were similar when propagated using the alternative membrane with three layers of microporous tape or the commercial membrane. However, the alternative membrane has a unit cost that is ten times lower than the commercial membrane.
TL;DR: The results obtained confirm the therapeutic potency of Cichorium pumilum used in the traditional medicine and verify that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds.
Abstract: Cichorium pumilum, a member of Asteraceae, is widely used as a traditional medicinal herb An efficient protocol for callus formation and whole plant propagation has been developed Callus cultures were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 15 mg l−1 6-Benzyladenine (BA) and 05 mg l−1 Naphthalene acetic acid (NAA) Maximum numbers of shoots were obtained from calli transferred to shoot regeneration medium containing MS basal medium with 15 mg l−1 BA or Kinetin (Kin) The shoots were effectively rooted on MS medium supplemented with different concentrations of Indole-3-butyric acid In the present study, the antibacterial activity of C pumilum extracts was assayed in vitro by agar disc diffusion and agar well diffusion methods against 10 different bacterial species The results showed effect on the growth of 50 and 70% of the tested bacterial species using methanol and ethanol extracts respectively Klebsiella pneumoniae was susceptible to the ethanolic and methanolic extracts of wild plants and in vitro tissues, whereas Enterococcus faecalis was resistant to all the extracts This study verified that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds The antioxidant activity and total phenol content of callus cultures and in vitro plantlets were lower than those of the wild plants The results obtained confirm the therapeutic potency of Cichorium used in the traditional medicine, in addition, the efficient in vitro production system developed in this study provide sterile and consistent tissues for the investigation of phytochemical and pharmacological effects and germplasm conservation of C pumilum
TL;DR: Different molecular mechanisms of thermotolerance acquisition have been shown to underlie various acclimatization methods and the role of abscisic acid, ethylene, hydrogen peroxide, and salicylic acid in acquired thermot tolerance has been demonstrated by molecular evidence from Arabidopsis mutants and transgenic lines.
Abstract: Acquired thermotolerance in plants refers to the ability to cope with lethal high temperatures following acclimatization at sublethal high temperatures. Acquired thermotolerance reflects an actual thermotolerance mechanism naturally occurring in plants and has been extensively used in thermotolerant line identification. In recent years, great progress has been achieved in the elucidation of biochemical, physiological, and molecular mechanisms of thermotolerance acquisition by using genomic approaches, including microarray analysis and mutation, knockout, and overexpression of related genes. Heat shock proteins (HSPs), such as Hsp101, BOBBER1, and Hsa32, have been shown to be important for inducement and maintenance of acquired thermotolerance. Downstream target genes and upstream regulation factors of HsfA2, including Hsa32, Apx2, small ubiquitin-like modifier proteins, FK506-binding proteins ROF1 (FKBP62) and ROF2 (FKBP65), and heat shock transcription factor binding protein, have been revealed to be involved in thermotolerance acquisition regulation. Moreover, the role of abscisic acid, ethylene, hydrogen peroxide, and salicylic acid in acquired thermotolerance has been demonstrated by molecular evidence from Arabidopsis mutants and transgenic lines. Most importantly, different molecular mechanisms of thermotolerance acquisition have been shown to underlie various acclimatization methods. Establishment of an experimental system similar to natural conditions is important for further exploration of natural thermotolerance mechanisms.
TL;DR: EV method proved to be most appropriate way to cryopreserve the PLBs of D. nobile and regenerated plantlets showed normal morphology as that of control plants.
Abstract: An efficient protocol for cryopreservation of protocorm like bodies (PLBs) of Dendrobium nobile, based on encapsulation–dehydration (ED) and encapsulation–vitrification (EV), was established. In both cryogenic procedures, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix [comprising 3% (w/v) sodium alginate and 0.1 M CaCl2]. Out of the two methods, EV resulted in higher survival (78.1%) and regrowth (75.9%) than ED (53.3 and 50.2% respectively). Incorporation of 0.4 M sucrose and 2 M glycerol in the encapsulation matrix resulted in higher survival percentage after cryopreservation. In both the cases (ED and EV), shoots regenerated from cryopreserved PLBs with an intermediary PLB formation. Regenerated shoots were successfully rooted in the medium containing 1.5 mg/l Indole-3 butyric acid. Successful acclimatization of plantlets was obtained in the compost containing brick pieces and charcoal chunks (1:1) + a top layer of moss with a maximum survivability (82%). EV method proved to be most appropriate way to cryopreserve the PLBs of D. nobile. Regenerated plantlets showed normal morphology as that of control plants.
TL;DR: The results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.
Abstract: Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.
TL;DR: In this paper, direct and indirect somatic embryogenesis (DSE and ISE) for biolistic transformation of sugarcane by minimal expression cassettes indicated the highest transformation efficiency with ISE (2.2 independent transgenic plants per shot) and the most rapid production of transgenic lines with DSE (12 weeks from explant to plants in soil).
Abstract: The comparison of direct and indirect somatic embryogenesis (DSE and ISE) for biolistic transformation of sugarcane by minimal expression cassettes indicated the highest transformation efficiency with ISE (2.2 independent transgenic plants per shot) and the most rapid production of transgenic plants with DSE (12 weeks from explant to plants in soil). Microprojectiles of 0.3 μm diameter produced 5 times more transgenic lines than 1 μm microprojectiles when used at the same weight basis per shot. A significant reduction of the number of hybridization signals in the Southern blot was also observed with 0.3 μm microprojectiles when compared to 1.0 μm microprojectiles. This suggests that the lower DNA carrying capacity and greater number of the smaller microprojectiles contributes to more transgenic lines with less complex transgene integration. When geneticin sulfate, was used for selection following DSE, significantly more (4.8 times) transgenic plants were produced than with paromomycin sulfate and an equal number of non-expressing plants were produced with both selection agents. In conclusion, optimization of two alternative morphogenic routes for regeneration (DSE and ISE), biolistic and selection parameters generated rooted, transgenic plants of a commercially important sugarcane cultivar with simple transgene integration and within 12 or 19 weeks of culture initiation, respectively. Reducing the complexity of the transgene integration and minimizing the time in tissue culture will likely enhance the performance of the transgenic events.
TL;DR: The results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.
Abstract: The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.
TL;DR: It is demonstrated that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.
Abstract: Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.
TL;DR: An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot and Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture.
Abstract: An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 106 and 106 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds.
TL;DR: The optimized transformation protocol using basal parts of seedling as explants shortened the process by 4–5 weeks, and it has potential use for transformation of other switchgrass cultivars.
Abstract: To optimize Agrobacterium tumefaciens-mediated transformation, factors influencing gene delivery, selection of transformed cells, and plant regeneration were investigated using two major switchgrass cultivars, including a lowland tetraploid cultivar Alamo and an upland octoploid cultivar Cave-in-Rock (CIR). Transient expression studies monitored by histochemical β-glucuronidase assay in seedling segments indicated that A. tumefaciens strain EHA105 was more effective in gene delivery than LBA4404 or GV3101. Of three major selectable genes, the bialaphos resistance (bar) gene and the hygromycin phosphotransferase (hpt) allowed effective selection of transformed cells using 2 mg l−1 glufosinate ammonium herbicide and 50 mg l−1 hygromycin, respectively; whereas the neomycin phosphotransferase II gene did not yield effective selection using 100 mg l−1 kanamycin. Herbicide- or hygromycin-resistant calluses were induced from seedling segments after 2–3 months of selection. Transformants of ‘Alamo’ with the bar or hpt were obtained 3–4 weeks after the resistant calluses were transferred onto regeneration medium; in contrast, no regenerant was produced from the calluses of ‘CIR’. Most of transformants showed normal growth in the greenhouse. Low percentages of mature seeds ranging from 1.7 to 8.7% of husks were obtained from open pollinated plants. Southern blot analysis confirmed stable integration of the bar in selected T0 transformants. Reverse transcription PCR and herbicide/hygromycin tolerance tests indicated expression of transgenes. The optimized transformation protocol using basal parts of seedling as explants shortened the process by 4–5 weeks, and it has potential use for transformation of other switchgrass cultivars.
TL;DR: Microprogated oregano plants from WT and they contained similar amount of their TP content and ORAC activity and an increase in luteolin content, which showed significant differences between WT, IN and EX plants.
Abstract: Mexican oregano is composed by a wide range of endemic species which support important economic activities, but with serious limitations. In this work a micropropagation protocol was established to regenerate oregano plants (Poliomintha glabrescens Gray) from apical buds. At the same time we evaluated the impact of this process in the phytochemical profile of the new plants. The optimal induction of apical buds was observed when shoot-tip explants were incubated on solidified Murashige and Skoog (MS) medium supplemented with 0.5 mg ml−1 α-naphthalenacetic acid (NAA) and 0.02 mg ml−1 6-benzyladenine. Also, the optimal response to root induction was with MS solid medium added with 0.5 mg ml−1 of NAA. The micropropagation protocol took 17 weeks with an efficiency of 40%, and plants achieved were successfully acclimatized under greenhouse conditions. The total phenolics (TP) concentration, antioxidant capacity (oxygen radical absorbance capacity, ORAC), and luteolin content from methanol extracts of wild type (WT) and in vitro (IN) and ex vitro (EX) plants were determined. The results show that TP content and ORAC were similar for WT and EX plants, but IN plants had the highest TP content and ORAC activity (25 and 28% respectively). Luteolin content showed significant differences between WT, IN and EX, with EX plants having the highest luteolin content (10% more compared to WT). In summary, we successfully microprogated oregano plants from WT and they contained similar amount of their TP content and ORAC activity and an increase in luteolin content.
TL;DR: An Agrobacterium-mediated transformation protocol for grapevine cv.
Abstract: An Agrobacterium-mediated transformation protocol for grapevine cv. Crimson Seedless using sonication and anti-necrotic agents has been optimized, and transgenic lines carrying wheat chitinase and β-1,3-glucanase genes have exhibited enhanced tolerance to downy mildew incited by Plasmopara
viticola. cDNA clones encoding chitinase and β-1,3-glucanase have been isolated from a cDNA library, constructed from scab-infected Sumai-3 wheat, and introduced into a plant cloning vector to generate the plasmids pCAMBAR.chi.11 and pCAMBAR.638. Embryogenic cultures, established from in vitro-derived leaves, of Crimson Seedless were used as explants for Agrobacterium tumefaciens-mediated transformation studies. Sonication of somatic embryos in a bacterial suspension of A. tumefaciens and incorporation of anti-necrotic agents in the co-cultivation medium significantly enhanced transformation efficiency. Transformation efficiency of embryos with either chitinase or β-1,3-glucanase gene was highest when embryos were suspended in a bacterial cell suspension at 0.5 OD600 and sonicated for 2 or 3 s at 60 kHz. Transformation efficiency with chitinase was highest on incorporation of 2 or 3 mg l−1 phenylalanine, 1 or 2 mg l−1 silver nitrate or 400 mg l−1
l-cysteine in co-cultivation medium while incorporation of 20 mg l−1 sodium thiosulphate produced highest transformation efficiency with β-1,3-glucanase. Confirmed transgenic grapevine lines harboring anti-fungal genes exhibited higher levels of chitinase and β-1,3-glucanase transcripts as well as enzymatic activities. Moreover, transgenic lines showed enhanced tolerance to P. viticola infection following detached leaf assays.
TL;DR: The Southern blot and quantitative reverse transcription-polymerase chain reaction (QRT–PCR) analyses demonstrated that the PpTLP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines.
Abstract: Thaumatin-like proteins (TLPs) belong to the pathogenesis-related proteins 5 family, and members of TLP gene family accumulate in plants in response to pathogen infection or environmental stress. The biological functions of plant TLPs are believed to be diverse, and they are involved in host defense against both the biotic and abiotic stresses as well as the other physiological processes. Lots of TLPs were well studied as antifungal proteins in vivo or in vitro. To isolate TLP genes involved in self-defense during fruit development, a novel gene PpTLP was isolated from the fruit of Pyrus pyrifolia Nakai cv Huobali which is a kind of local sand pear with high resistance to biotic and abiotic stresses in Yunnan province of China. The nucleotide sequence of PpTLP was highly homologous with TLPs from other plant species, and the protein encoded by PpTLP has 16 conserved cysteine residues and 5 amino acid residues related with antifungal activity of plant TLPs, an arginine residue, a glutamic acid residue, and 3 aspartic acid residues. What is more, the putative protein PpTLP has the highly conserved main-chain conformation and the folding patterns as other plant TLPs with antifungal activity. PpTLP was abundantly expressed in pericarp and leaf of P. pyrifolia Nakai cv Huobali. Furthermore, in order to verify the function of PpTLP, the constitutive plant expression vector of PpTLP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and quantitative reverse transcription-polymerase chain reaction (QRT–PCR) analyses demonstrated that the PpTLP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpTLP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Sclerotinia sclerotiorum, Phomopsis sp., Phytophthora parasitica var. nicotianae, and Alternaria sp. in different degrees.
TL;DR: Both white light and blue light were equally beneficial for the total production of phenolic acids in cultures of both plants, whereas thetotal production of furanocoumarins was clearly better stimulated by blue light in R. graveolens and by darkness in the subspecies.
Abstract: Extracts from the biomass of Ruta graveolens and Ruta graveolens ssp. divaricata cultured in vitro under different light conditions (far-red, red and blue light, UV-A irradiation, in darkness and white light) were tested for the amounts of free phenolic acids and cinnamic acid (twelve compounds) as well as furanocoumarins and umbelliferone (seven compounds) using HPLC methods. Total amounts of the investigated groups of compounds in the cultures of both plants increased from 2.6 to 6.7 times, depending on light quality, and the maximum values reached were 106.50 and 1,276.74 mg 100 g−1 DW (in R. graveolens), and 106.97 and 262.54 mg 100 g−1 DW (in the subspecies), respectively. Both white light and blue light were equally beneficial for the total production of phenolic acids in cultures of both plants, whereas the total production of furanocoumarins was clearly better stimulated by blue light in R. graveolens and by darkness in the subspecies (i.e. the amounts were respectively 1.44 and 1.7 times higher than in the biomass cultivated under white light). The amounts of individual compounds in both plant cultures increased from about 2.2 to 26.3 times depending on light quality. The following bioactive compounds were obtained in quantities which are of interest from a practical perspective: in R. graveolens culture—protocatechuic acid (45 mg 100 g−1 DW), isopimpinellin (about 500 mg 100 g−1 DW) and bergapten (about 270 mg 100 g−1 DW), and in the subspecies culture: p-coumaric acid (70 mg 100 g−1 DW) and isopimpinellin (about 210 mg 100 g−1 DW).