Showing papers in "Plant Cell Tissue and Organ Culture in 2015"

Tomato (Solanum lycopersicum L.) in the service of biotechnology

Abstract: Originating in the Andes, the tomato ( Solanum lycopersicum L.) was imported to Europe in the 16th century. At present, it is an important crop plant cultivated all over the world, and its production and consumption continue to increase. This popular vegetable is known as a major source of important nutrients including lycopene, β-carotene, flavonoids and vitamin C as well as hydroxycinnamic acid derivatives. Since the discovery that lycopene has anti-oxidative, anti-cancer properties, interest in tomatoes has grown rapidly. The development of genetic engineering tools and plant biotechnology has opened great opportunities for engineering tomato plants. This review presents examples of successful tissue culture and genetically modified tomatoes which resistance to a range of environmental stresses improved, along with fruit quality. Additionally, a successful molecular farming model was established.

Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f.niedzwetzkyana)

Abstract: We have induced callus tissues from one R6/R6 homozygous genotype red-fleshed apple individual which was the hybrid offspring of Malus sieversii f.niedzwetzkyana and ‘Fuji’, and investigated the effect of auxin alone and auxin combined with cytokinin or nitrogen deficiency on anthocyanin synthesis. In callus culture, auxin alone significantly inhibited anthocyanin biosynthesis with the increase of auxin concentration. The inhibitory effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on anthocyanin accumulation was about tenfold stronger than naphthalene acetic acid. Anthocyanin regulatory genes (MdMYB10 and MdbHLH3) and structural genes were dramatically suppressed by 0.6 mg/L 2,4-D. The inhibitory effect of auxin on anthocyanin biosynthesis was influenced by cytokinins 6-benzylaminopurine (BAP) and thidiazuron (TDZ) as well as nitrogen deficiency. Auxin and cytokinin displayed the interaction in controlling anthocyanin biosynthesis. Co-treatment of auxin and cytokinin (BAP or TDZ) significantly enhanced the cytokinin-induced increase in anthocyanin levels but too high auxin concentration strongly inhibited anthocyanin synthesis even in the presence of cytokinin. Nitrogen deficiency could reverse the inhibition of anthocyanin accumulation by auxin.

In vitro culture of Achillea millefolium L.: quality and intensity of light on growth and production of volatiles

Abstract: The influence of different light spectra and intensities was evaluated in an in vitro culture of Achillea millefolium L. (yarrow). The treatments were: use of light emitting diode (LED) lamps in the blue, red, green and white wavelengths, and the intensities of 13; 27; 35; 47 and 69 µmol m−2 s−1, obtained with a cool fluorescent lamp. At 45 days of culture in hormone-free MS medium, the production of dry matter, survival, rooting, length of shoots and roots, numbers of roots, pigments, as well as volatile constituents, were evaluated. The quality and intensity of light significantly influenced the in vitro growth of yarrow. In the experiment with LEDs, the blue spectrum provided the highest dry matter accumulation, number of roots, percentage of rooting and survival. In different light intensities, 27 µmol m−2 s−1 showed the highest values for the variables analyzed. Thus, blue LED spectrum or cool fluorescent lamp with 27 µmol m−2 s−1 benefits the in vitro growth of yarrow. A variation in number, content and profile of volatile constituents under the influence of quality and light intensity was also observed. The major constituents identified were sabinene, 1,8 cineole, borneol, β-caryophyllene and β-cubebene, independent of the light treatments. The amount and composition of the volatile compounds ranged with the intensity and quality of light. Thus, it is possible to adjust the ambient light in order to yield the compounds of interest.

Effects of ultraviolet C, methyl jasmonate and salicylic acid, alone or in combination, on stilbene biosynthesis in cell suspension cultures of Vitis vinifera L. cv. Cabernet Sauvignon

Abstract: Vitis vinifera L. cv. Cabernet Sauvignon cell suspension cultures were treated with ultraviolet C (UV-C), methyl jasmonate (MeJA) and salicylic acid (SA), alone or in combination, to investigate the effects on stilbene biosynthesis. The application of elicitors at the proper dosage or concentration did not exert a negative effect on cell growth. All treatments enhanced both stilbene production inside the cells and trans-resveratrol accumulation in the culture medium. UV-C irradiation for 20 min or MeJA at 100 μM was efficient in promoting stilbene accumulation. The combined treatment of UV-C and MeJA highly induced total intracellular stilbene production to the maximum of 2005.05 ± 63.03 μg g−1 DW, and showed a synergistic effect on extracellular trans-resveratrol accumulation to 3.96 ± 0.2 mg l−1. SA at 100 μM was less efficient than UV-C and MeJA in promoting stilbene production. However, the combined elicitation of UV-C and SA further promoted intracellular stilbene production to the maximum of 1630.93 ± 44.17 μg g−1 DW, and markedly increased extracellular trans-resveratrol accumulation to 2.33 ± 0.15 mg l−1. Intracellular total phenolics and total flavonoids contents also significantly increased after elicitations. Relative expression of genes involved in stilbene and flavonoid biosynthesis was up-regulated, and there was a synergistic effect of UV-C together with MeJA or SA on STS expression. The results suggested that the combined treatment of UV-C together with MeJA or SA can be used as an efficient method to enhance stilbene production in V. vinifera cell cultures.

In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae)

Abstract: Thymus persicus, which grows in the northwest area of Iran, is a valuable natural source of medicinal pentacyclic triterpenoids (PTs), i.e. betulinic acid, oleanolic acid and ursolic acid. An in vitro approach to polyploidy induction in the plant was examined for the first time. Polyploidization was induced using in vitro-grown T. persicus shoot-tips immersed in colchicine (0, 0.05, 0.1, 0.3 and 0.5 %) for 12–48 h. Ploidy levels of regenerates were determined by either flow cytometry or chromosome counting at the metaphase. The results confirmed that the mother diploid plant contained a chromosome number of 2n = 2x = 28, 2C DNA = 1.20 pg, whereas 2n = 4x = 56, 2C DNA = 2.39 pg was detected in the induced autotetraploids (CV% < 4 %). Out of 960 shoot tip segments exposed to colchicine, 7.80 and 1.04 % were tetraploid and mixoploid, respectively. The most efficient conditions for inducing polyploidy were the treatment with 0.3 % colchicine for 12 h, followed by 0.3 % for 24 h. The effect of polyploidization on growth and anatomical characteristics as well as PTs production was also measured. Tetraploids differed markedly from diploids, showing lower plantlet height, shorter roots, thicker stems and darker leaves, as well as longer and wider stomata and reduced stomatal density on the abaxial and adaxial leaf surfaces. Furthermore, a positive trend in triterpenoid production was obtained with the tetraploid and mixoploid T. persicus plants. These findings indicate that tetraploids can potentially be used in further breeding efforts to obtain a wide range of triterpenoids with improved medicinal properties.

Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers

Abstract: A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l−1) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator’s type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l−1), IAA (0.1 mg l−1) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l−1 IBA + 100 mg l−1 activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l−1 each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD 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 A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum.

Epigenetic regulation and gene markers as signals of early somatic embryogenesis

Abstract: Identification and characterization of gene markers for somatic embryogenesis offer the possibility of determining the embryogenic potential of somatic cells before any morphological changes appear and to provide information on molecular regulation of early somatic embryogenesis. In this review, based on evidence reported in the literature, we assess the reliability of the established SERK, LEC and WUS marker genes as well as the usefulness of mediating epigenetic regulation by DNA methylation, chromatin remodeling and microRNAs as markers of the induction of somatic embryogenesis. The evidence suggests that the SERK gene marker may be less reliable than has been thought while the germin-like protein-encoding genes may, like their protein products, be useful markers of early somatic embryogenesis.

SpWRKY1 mediates resistance to Phytophthora infestans and tolerance to salt and drought stress by modulating reactive oxygen species homeostasis and expression of defense-related genes in tomato

Abstract: WRKY transcription factors play essential roles in diverse signaling pathways related to plant defense responses. However, research focusing on the WRKY family in tomato is fairly limited. In this work, a pathogen-induced SpWRKY1 gene from the wild tomato Solanum pimpinellifolium L3708 showing that its overexpression in cultivated tomato Solanum lycopersicum cv. Zaofen No. 2 results in markedly increased resistance to Phytophthora infestans than untransformed wild-type plant, mainly demonstrated by less severe cell death, lower reactive oxygen species (ROS) production, malonaldehyde (MDA) content, relative electrolyte leakage (REL) and stomatal conductance; and higher peroxidase (POD), superoxide dismutase (SOD), phenylalanine ammonia-lyase, chlorophyll content and photosynthetic rate. This resistance was also coupled with enhanced the expression of ROS scavenging-related genes, SA/JA-responsive genes and SA/JA biosynthesis-related genes. This overexpression was accompanied by regulating the expression of an ABA biosynthetic gene, reveals a potentially positive role of SpWRKY1 in ABA-mediated stomatal closure. Furthermore, transgenic tomato also displayed an enhanced tolerance to salt and drought stress by decreasing ROS generation, reducing MDA content and REL, improving POD, SOD and proline content, keeping leaf relative water content, preventing chlorophyll loss, and protecting photosynthetic rate and stomatal conductance, accompanied by not only enhanced expression of some ROS scavenging-related and stress-related genes, but also directly up-regulated the expressions of PR genes in response to salt and drought stress. These findings broaden our knowledge about the functions of SpWRKY1 in diverse signalling pathways and may be useful in improving tomato plants tolerance to biotic and abiotic stress.

Enhancement strategies of bioactive compound production in adventitious root cultures of Eleutherococcus koreanum Nakai subjected to methyl jasmonate and salicylic acid elicitation through airlift bioreactors

Abstract: To establish an attractive method for the annual production of valuable Eleutherococcus koreanum-based bioactive compounds, the adventitious roots in airlift bioreactors were subjected to methyl jasmonate (MJ) and salicylic acid (SA) treatment for 1 week before harvest as a chemical elicitor to enhance the productivity of bioactive compounds. After 6 weeks of culture, the addition of 50 µmol MJ was found to be the optimal concentration and elicitor type for biomass and bioactive compound production in the roots, as evidenced by the highest biomass and total production of five target bioactive compounds (i.e., eleutherosides B and E, chlorogenic acid, and total phenolics and flavonoids) without causing any physiological disorders. The total production of the target bioactive compounds at 50 µmol MJ (303.93 mg L−1) was 37.77 % higher than that for the control treatment, and the addition of high concentrations of MJ and SA increased 2,2-diphenyl-1-picrylhydrazyl activity and hydrogen peroxide content in the roots. In addition, the adventitious roots subjected to 50 µmol MJ for 1 week had a competitive edge for commercial use, as evidenced by the high total production of the five target bioactive compounds (per 1 L of medium) when compared to the 3-year field-grown plants.

Micropropagation of Viola uliginosa (Violaceae) for endangered species conservation and for somaclonal variation-enhanced cyclotide biosynthesis

Abstract: Viola uliginosa Besser is a European violet having its main distribution range in the Baltic Sea region. Today it is considered endangered and threatened. Species of Violaceae from different genera and sections are known to produce cyclotides, cyclic polypeptides of much interest due to their medicinal properties and chemical structure. The present study introduced a rare species of violet (V. uliginosa) to in vitro culture for biodiversity protection and as a model for cyclotide biosynthesis research in the Violaceae. Leaf and petiole fragments were cultured on MS medium solidified with agar and supplemented with different concentrations of plant growth regulators: TDZ, KIN and 2,4-D. Direct and indirect (via callus) organogenesis was induced on MS supplemented with TDZ (0.5 or 1 mg l−1) or with equal concentrations (2 mg l−1) of KIN and 2,4-D, followed by callus transfer on 1 mg l−1 TDZ. Shoots were rooted on MS with 2 % sucrose and 0.5 mg l−1 IBA and acclimatized. AFLP marker polymorphism was low but flow cytometry revealed that a large share of the obtained regenerants were tetraploid (2C = 4x = 2.7–2.8 pg), unlike the maternal diploid plants (2C = 2x = 1.4 pg). Eleven different cyclotides were distinguished in the aerial parts of maternal plants. Cyclotide production was significantly higher in tetraploid than in diploid plants regenerated in vitro.

Investigation of the response to salinity and to oxidative stress of interspecific potato somatic hybrids grown in a greenhouse

Abstract: Salinity is one of the major stresses threatening potato plants (Solanum tuberosum L.) by affecting their growth and yield. It leads to oxidative stress by the production of reactive oxygen species responsible for alteration of macromolecules. To improve the tolerance of potato to salt stress, we have used somatic hybridization to produce interspecific potato hybrids by protoplast fusion between the BF15 variety and the wild Solanum berthaultii species. These hybrids showed an improved tolerance to salt stress when cultivated in vitro. The present work aims to analyze the response of the hybrids to salt stress in greenhouse conditions. Thus, the development of plants and their antioxidant capacity in response to salt stress were followed. All hybrids showed better growth and stable chlorophyll content compared to those of the BF15 parent plant. Membrane lipid peroxidation, evaluated by measuring the malondialdehyde accumulation (MDA) in plant organs, showed low levels in the hybrids. Higher antioxidant enzyme activities were measured in the roots of the hybrids when compared to those of the BF15 parent. These hybrids also showed an improved control of Na+ accumulation and a stable K+/Na+ ratio. These results therefore confirm the better tolerance of these hybrids to salt stress when compared to their BF15 parent.

Elicitation of antioxidant secondary metabolites with jasmonates and gibberellic acid in cell suspension cultures of Artemisia absinthium L.

Abstract: Artemisia absinthium L. is a very important species with worldwide traditional medicinal uses. In this study, effects of different concentrations of two important elicitors, methyl jasmonate (MeJA) and jasmonic acid (JA), and a phytohormone gibberellic acid (GA) on growth kinetics, secondary metabolites accumulation and antioxidant activity in cell suspension cultures of Artemisia absinthium L. were investigated. The results showed inhibition in dry biomass accumulation and shorter log phases of cultures growth in response to most treatments, compared to control. Further, we observed enhanced accumulation of total phenolic content (TPC), total flavonoid content (TFC) and highest radical scavenging activity (RSA) in suspension cultures treated with 1.0 mg/l of MeJA, JA and GA, each. The correlation studies between secondary metabolites and antioxidant activity showed TPC-dependent RSA in most cultures. MeJA and JA treated cultures showed no significant variation in the context of total flavonoid contents and 1.0 mg/l of both displayed significantly comparable maximum RSA profiles on day-24 of culture. GA-treated cultures showed minimum accumulation of TPC, but TFC and RSA were found to be significantly comparable to those in MeJA and JA treated cultures. The results showed elicitors-induced enhancement in phenolic and flavonoid accumulation and antioxidant activity in suspension cultures of Artemisia absinthium L.

The effect of cytokinins on shoot proliferation, secondary metabolite production and antioxidant potential in shoot cultures of Scutellaria alpina

Abstract: The present study evaluates the effects of various cytokinins on Scutellaria alpina shoot proliferation and production of polyphenolic metabolites (baicalin, wogonoside, luteolin, luteolin 7-O-glucoside, verbascoside). The shoots were induced from shoot tips on MS medium supplemented with IAA (indole-3-acetic acid, 0.57 µM) and various concentrations of 6-benzylaminopurine (BAP), kinetin, zeatin (1, 2, 4, 8 µM) or tidiazuron (TDZ) (0.2, 0.5, 1 µM). Among the cytokinins tested, BAP was the most effective for shoot induction, and the highest number of shoots (25 per explant) was achieved with 2 and 4 µM BAP. Maximum biomass production was also achieved on these media. Significantly higher baicalin, wogonoside and verbascoside contents were recorded in treatments containing cytokinins combined with 0.57 µM IAA, when compared to cytokinin-free medium. TDZ at a concentration of 0.5 µM was the most effective for polyphenol production. However, supplementation with cytokinins often results in the reduction of luteolin and its 7-O-glucoside production in the shoot culture of S. alpina. ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and ferric reducing antioxidant power assays were used to identify the antioxidant potential of methanolic extracts from shoots cultured in the presence of different types and concentrations of cytokinins. In both tests, the shoots from medium supplemented with 0.5 µM TDZ demonstrated the strongest antioxidant activity. The results indicate that higher polyphenolic content correlated with greater reducing power and antiradical efficiency.

Assessment of genetic and epigenetic changes during cell culture ageing and relations with somaclonal variation in Coffea arabica

Abstract: Long-term cell cultures were used in coffee to study the cytological, genetic and epigenetic changes occurring during cell culture ageing. The objective was to identify the mechanisms associated with somaclonal variation (SV). Three embryogenic cell lines were established in Coffea arabica (2n = 4x = 44) and somatic seedlings were regenerated after 4, 11 and 27 months. Phenotyping and AFLP, MSAP, SSAP molecular markers were performed on 199 and 124 plants, respectively. SV were only observed from the 11 and 27-month-old cultures, affecting 30 and 94 % of regenerated plants, respectively. Chromosome counts performed on 15 plants showed that normal plants systematically displayed normal chromosome numbers and that, conversely, aneuploidy (monosomy) was systematically found in variants. The allopolyploid structure of C. arabica allowed aneuploid cells to survive and regenerate viable plants. No polymorphic fragments were observed between the AFLP and SSAP electrophoretic profiles of mother plants and those of the in vitro progeny. Methylation polymorphism was low and ranged between 0.087 and 0.149 % irrespective of the culture age. The number of methylation changes per plant—normal or variant—was limited and ranged from 0 (55–80 % of the plants) to 4. The three cell lines showed similar SV rate increases during cell culture ageing and produced plants with similar molecular patterns indicating a non random process. The results showed that cell culture ageing is highly mutagenic in coffee and chromosomal rearrangements are directly linked to SV. Conversely, the analysis of methylation and transposable elements changes did not reveal any relation between the epigenetic patterns and SV.

Ectopic expression of SbNHX1 gene in transgenic castor (Ricinus communis L.) enhances salt stress by modulating physiological process

Abstract: The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter that involved in the maintenance of ion homeostasis and compartmentalization of excess Na+ or K+ ions into the vacuole. Transgenic castor plants were developed by an improved method of Agrobacterium mediated genetic transformation using spermidine (1 mM) along with acetosyringone (200 μM), which enhanced the transformation efficiency about twofolds from 2.76 to 5.91 %. Transgenic plants were confirmed by PCR using gene (SbNHX1, hptII and gus) specific primers. The single gene integration event was confirmed by RTqPCR and Southern hybridization. Transgenic lines CL7 and CL13 showed high expression of the SbNHX1 gene compared to CL6 and CL12, therefore selected for physio-biochemical analyses, which were carried out under varying NaCl concentrations. Higher chlorophyll, RWC, K+ content, K+/Na+ ratio and lower electrolytic leakage, proline, MDA, Na+ contents compared to WT confirmed that ectopic expression of the SbNHX1 gene enhances salt tolerance of transgenic plants by modulating physiological process under stress condition. Though transgenic lines were affected under stress conditions but performed better compared to WT plants. The present study is the first report of engineering salt tolerance in castor, so far. Transgenic castor may be utilized for the cultivation in marginal salty land and thus open up the possibility of releasing arable land, which is presently under castor cultivation.

Virus elimination from in vitro apple by thermotherapy combined with chemotherapy

Abstract: We evaluated the effectiveness of thermotherapy at different temperatures, chemotherapy with different concentrations of ribavirin, and combinations of these two methods on virus elimination from apple We used Malus cv ‘Xinhongjiangjun’, an apple cultivar widely grown in China, infected with Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) The survival and regeneration of plants were evaluated, and the efficiency of virus eradication was determined by reverse-transcription polymerase chain reaction with two primer pairs for each virus All of the plants treated with 15 and 25 μg/ml ribavirin survived, although their proliferation was slightly inhibited Ribavirin treatments at 15 and 25 μg/ml resulted in virus elimination rates of 744 and 750 %, respectively Higher temperatures significantly affected the growth and proliferation of plants, and almost no axillary shoots regenerated under the highest temperature (38 ± 05 °C) The average virus elimination rate after 34–36 °C treatments for 20 days was no more than 45 % The virus elimination efficiency was enhanced by combining thermotherapy and chemotherapy treatments A treatment combining ribavirin (25 μg/ml) and thermotherapy at 36 °C (R25 + T36) resulted in high virus eradication efficiency (950 %) Across all of the treatments, the average survival rate of apical shoots (176/273) was 12 % lower than that of axillary shoots (266/349); the average virus elimination efficiency was 653 % for apical shoots and 727 % for axillary shoots The results also demonstrated that it was easier to eliminate ASPV than to eliminate ACLSV and ASGV

A Scutellaria baicalensis R2R3-MYB gene, SbMYB8 , regulates flavonoid biosynthesis and improves drought stress tolerance in transgenic tobacco

Abstract: R2R3-MYB proteins are involved in the primary and secondary metabolism, developmental processes and the responses to biotic and abiotic stresses. Little is known about the functions of R2R3-MYB proteins in Scutellaria baicalensis Georgi which is a traditional Chinese medicinal plants. In this study, the function of a S. baicalensis R2R3-MYB protein, SbMYB8, was investigated. SbMYB8 had similar expression pattern with SbC4H and SbCHS in ABA-treated S. baicalensis, indicating that SbMYB8 might be involved in the flavonoid metabolism. SbMYB8 protein could bind to the GmMYB92 BS3 sequence of SbCHS promoter region, regulating the expression of SbCHS. The SbMYB8 protein was localized to the nucleus where it activated transcription. The transgenic tobacco plants over-expressing SbMYB8 had higher caffeoylquinic acid contents, compared to that in wild type plants. Overexpression of SbMYB8 also changed the expression level of some flavonoid biosynthesis-related genes. It was found that overexpression of SbMYB8 can improve stress tolerance of transgenic plants, and can alter the activity and expression levels of some antioxidant enzymes. These results indicate that SbMYB8 plays important roles in flavonoid biosynthesis and stress tolerance of plant.

NaCl amendment improves vinblastine and vincristine synthesis in Catharanthus roseus : a case of stress signalling as evidenced by antioxidant enzymes activities

Abstract: An improved synthesis of vinblastine and vincristine was observed in Catharanthus roseus L. (G). Don by using NaCl as an elicitor. Various in vitro grown embryogenic tissues were cultivated under salinity stress for enhanced synthesis of alkaloids. Different levels of salt [control (0 mM), NT1 (25 mM), NT2 (50 mM), NT3 (75 mM), NT4 (100 mM), and NT5 (125 mM)] were amended in MS and callus biomass growth (fresh- and dry-weight) and biochemical attributes at various embryogenic stages were studied. Maximum callus biomass reduction was observed in 125 mM NaCl amended medium. Antioxidant enzymes i.e. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase and glutathione reductase activities were assayed as in vitro grown tissues were elicitated with NaCl, causing cellular stress. The antioxidant enzymes activity increased linearly with increasing NaCl level in medium, 4.97 EU min−1 mg−1 SOD and 3.14 EU min−1 mg−1 CAT, both being maximum in proliferating embryos at NT5. Quantitative estimation and comparative yield of alkaloids were made in response to NaCl stress in different cultivated tissues by using HPTLC method. Vinblastine content was observed to be maximum in regenerated leaves (14.17 mg/g dry wt) on 25 mM NaCl amended medium, followed by in vitro raised shoots. Similarly, better accumulation of vincristine (5.12 mg/g dry wt) was also noted in NaCl amended medium especially at low level (NT1). The data presented indicate that the synthesis of Catharanthus alkaloids was growth specific and was influenced by NaCl levels.

Overexpression of maize SDD1 ( ZmSDD1 ) improves drought resistance in Zea mays L. by reducing stomatal density

Abstract: Drought is one of major factors limiting the high and stable yields of maize, and thus, improvement of water use efficiency (WUE) and cultivation of drought-resistant maize cultivars have become very urgent. Previous research shows that SDD1 from Arabidopsis thaliana, a key gene during stomatal development, negatively regulates stomatal density, reduces transpiration, and improves WUE and drought resistance without reducing biomass. In this study, maize SDD1 (ZmSDD1) containing intron was cloned, and the phylogenetic tree was constructed, revealing a close genetic relationship with the SDD1 of Oryza sativa. ZmSDD1 was expressed in the leaves, shoots, and roots of maize. Under drought, cold, and salt stress conditions, the expression of ZmSDD1 in B73 maize leaves is reduced. To reveal the functions of ZmSDD1, we built a plant expression vector pGM626-Ubi-SDD1-ABt containing ZmSDD1 which was driven by the maize Ubiquitin promoter. Transgenic plants were obtained via shoot-tip transformation. The stomata number on leaf surfaces of the plants within Ubiquitin::ZmSDD1 overexpression plants was 30 % lower than that in the wildtype ones. After repeated drought treatments the transgenic maizes demonstrated a much higher survival rate with 6.68-fold ZmSDD1 overexpression, which indicated the expression of the gene negatively regulating stomata density and caused the reduction of stomatal conductance and transpiration rate after drought-treatment. In addition, ZmSDD1 enhanced the drought resistance in the transgenic maizes through improving WUE and photosynthetic rate which was considered as the result of the higher PEPCase activity, CO2 utilization rate and the lower CO2 compensation point.

Expression of the chickpea CarNAC3 gene enhances salinity and drought tolerance in transgenic poplars

Abstract: CarNAC3 contains 285 amino acids and a conserved NAC domain. NAC genes, including NAM, ATAF1, ATAF2, and CUC2, are members of one of the largest transcription factor families in plants. CarNAC3, a member of the NAP group, plays an important role in plant development and responses to abiotic stresses. In this study, CarNAC3 was transformed into hybrid poplar plants (Populus deltoides × P. euramericana ‘Nanlin895’) using Agrobacterium tumefaciens. PCR analysis confirmed integration of the introduced T-DNA into the target genome. Reverse transcription PCR confirmed the transformation, and Southern and northern blotting verified the transgene copy number and gene expression, respectively. Fourteen lines of positive transformants were transplanted into a greenhouse to verify their drought and salt tolerances. Under normal conditions, transgenic poplar plants were shorter than the wild-type, but under drought and salt stresses, they maintained their normal rooting and stem growth rates, while those of the wild-type plants were suppressed. Under stress conditions, CarNAC3 expression caused increases in proline and photosynthetic pigment levels and in antioxidant enzyme activities. Furthermore, the expression of CarNAC3 lowered malondialdehyde concentrations compared with the wild-type control. Overall, our results indicated that the CarNAC3 transgene enhanced drought and salt tolerance in transgenic poplar plants.

Silver nanoparticles affect ACS expression in Tecomella undulata in vitro culture

Abstract: ACC synthase, a key enzyme for the production of the simplest olefin ethylene, can regulate many aspects of the plant life cycle and is important in in vitro culture conditions. This study was carried out to shed light on the gene expression pattern of ACS in the leaves of in vitro regenerated Tecomella undulata (Roxb.) Seem. which are affected by silver nanoparticles (AgNPs). This led to a better understanding of the correlation between AgNP and regeneration. We identified a 1,216 bp fragment of the TuACS gene (JQ582835.1) containing three exons (986) and two introns (365 bp) as the first nuclear gene identified from T. undulata, along with β-tubulin (JQ776639.1) as a house keeping gene, aimed at considering expression level of TuACS in MS media supplemented with AgNPs. Bioinformatics showed that TuACS shares a modest level of sequence similarity with other members of the PLP family, such as aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase). Visual inspection of explants grown in AgNPs media versus AgNPs free MS media revealed an increase in the mean number of fresh shoots per explants, the percentage of explants producing shoots, and plant survival. In AgNP media, TuACS expression was reduced, which may be partially responsible for the observed delayed explant senescence and increased survival under in vitro conditions. ACS promoter analysis revealed cis-acting regulatory elements such as light-responsive, hormone-responsive, and stress-responsive elements. The reduction of ACS mRNA may change the hormone balance in explants in AgNPs media. These observations suggest that AgNPs may serve to not only improve explant lifespan but increase multiplication as well.

Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research.

Abstract: As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world’s climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture (Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S.palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research. Electronic supplementary material The online version of this article (doi:10.1007/s11240-014-0658-2) contains supplementary material, which is available to authorized users.

Ozone-elicited secondary metabolites in shoot cultures of Melissa officinalis L.

Abstract: The effects of ozone treatment (200 ppb, 3 h) on the accumulation of main secondary metabolites have been investigated in Melissa officinalis (lemon balm) aseptic shoot cultures in order to evaluate the biotechnological application of this gas for improving the yield of secondary metabolites of medicinal plants During the treatment, we found (1) an activation of enzymes involved in phenolic metabolism [as confirmed by the increase of shikimate dehydrogenase, phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase activities (about twofold higher than controls)], (2) a development of cellular barriers with a higher degree of polymerization of monolignols [as indicated by the increase of lignin (+23 % compared to controls)], (3) an accumulation of phenolic compounds, in particular rosmarinic acid (about fourfold compared to control plants cultivated in filtered air) and (4) an increase of antioxidant capacity (as documented by the improved 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity) The effect of ozone as elicitor of the production of secondary metabolites in lemon balm shoot cultures was dependent on the specific regime, the time of exposure and the concentration of the stressor After the end of the treatment, we found cell death and hydrogen peroxide (H2O2) deposition concomitant with a prolonged superoxide anion-generation, suggesting that a transient oxidative burst had occurred

Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato ( Solanum lycopersicum Mill.)

Abstract: Cold is a major constraint for tomato growth and productivity; as it is a cold sensitive crop. DREB1A plays a key role in generating cold tolerance in tomato by regulating the response of multiple genes under chilling stress. In this study, cold tolerant gene (DREB1A) driven by Lip9 promoter, was transformed in three tomato genotypes through Agrobacterium tumefaciens, employing tissue culture independent transformation strategy. Overnight imbibed seeds of tomato were surface sterilized, 3-day old shoot apical meristem of the developing seedling were pierced and incubated for twenty minutes with A. tumefaciens strain EHA-105 having OD600 nm of 1.0. The treated seeds were co-cultivated with Agrobacterium for 2 days. The regenerated shoots were subjected to 35 mg/l hygromycin as a selection for a period of 2–3 weeks. The presence of DREB1A and hpt genes in the hygromycin resistant (T0) transgenic plants was evaluated by PCR analysis. The transgene activity was detected in T1 plants by Reverse Transcriptase Polymerase Chain Reaction and Southern blotting that showed the stable integration of the transgene to the next generation. Physiological analysis of T2 transgenic plants depicted that increased expression of DREB1A induced only during chilling stress. After various chilling stresses, stomatal conductance, transpiration rate and relative water contents of transgenic lines were significantly higher than those of NT plants. While leaf osmotic potential of transgenic plants was lower as compared to NT plants. The established procedure is novel and can produce stable transgenic tomato plants in efficient manner by saving potential resources in terms of cost and time.

Methyl jasmonate and salicylic acid synergism enhances bacoside A content in shoot cultures of Bacopa monnieri (L.)

Abstract: This study has been carried out with the aim of enhancing the production of bacoside A in plant cultures of Bacopa monnieri through the process of elicitation. Optimisation of suitable concentrations of plant growth regulators and harvest time has been accomplished for the highest biomass production and bacoside A accumulation. Plant cultures were elicited with methyl jasmonate (MJ) and salicylic acid (SA) separately and in combinations in different concentrations for a period of 1–4 weeks. Three weeks of elicitation with 50 µM MJ or 50 µM SA produced about two and threefold higher quantity of bacoside A respectively when compared to unelicited controls. Highest accumulation was noticed in a combination of 25 µM MJ + 25 µM SA treated for 3 weeks duration. It has produced about 59.18 mg/g DW (dry weight) of bacoside A3 (3.5-fold higher), 95.30 mg/g DW of Bacopaside II (5.2-fold higher), 46.11 mg/g DW of Jujubogenin isomer of Bacopasaponin C (4.8-fold higher) and 68.16 mg/g DW of Bacopasaponin C (5.9-fold higher), altogether accounting for the production of 269.71 mg/g DW (almost fivefold higher) of bacoside A. Hence the protocol formulated provides an efficient alternative for mass production of whole plants with increased metabolite content.

Improvements in Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) for large-scale production of transgenic plants

Abstract: Cassava (Manihot esculenta Crantz) is a major staple food crop of the humid tropics. As a heterozygous, vegetatively propagated crop, robust transformation protocols must be developed for elite cultivars that allow predictable production of large numbers of independent transgenic plant lines. A high throughput Agrobacterium-mediated transformation system was developed for the elite East African farmer-preferred cassava cultivar TME 204 using the GFP visual marker gene. Inclusion of the antibiotic moxalactam in culture medium used to produce embryogenic target tissues prior to inoculation with Agrobacterium increased recovery of independent GFP-expressing transgenic callus lines by up to 113-fold compared to the control. Enhanced transformation was also observed when TME 204 tissues were pretreated with other cephalosporins, namely cefoperazone, cefoxitin, cefmetazole and cefotaxime. Similar but less dramatic increases in transformation efficiencies were seen for the West African cultivars Oko-iyawo and 60444 when pre-treated with moxalactam. Dilution of Agrobacterium suspensions used for co-culture was found to increase transformation efficiencies, resulting in regeneration at an average of 33 GFP-expressing TME 204 plants per cc settled cell volume at OD600 0.05, compared to 15 plants at the more commonly used OD600 0.5. The optimized transformation systems were successfully utilized for the integration of genetic constructs for disease resistance and nutritional enhancement into more than 750 plants of TME 204.

Comparative transcriptome analysis of early somatic embryo formation and seed development in Brazilian pine, Araucaria angustifolia (Bertol.) Kuntze

Abstract: Somatic embryogenesis (SE) is a method for producing embryos in vitro and is considered a highly promising approach for micropropagation and germplasm conservation. However, the application of SE for genetic breeding and ex situ conservation of certain species, such as Brazilian pine, faces several technical challenges, including the difficulty of inducing embryogenic cultures using tissues of mature trees, the loss of embryogenic competence of cell cultures and incomplete development of somatic embryos. In order to understand the genetic factors governing embryogenesis, a comparative transcriptome analysis was performed to elucidate differences between distinct cell cultures, early zygotic and somatic embryos and, unorthodox seed developmental stages. A total of 64 GB of sequence derived from high-throughput Illumina RNA-seq profiling was used for de novo transcriptome assembly. The reference transcriptome resulted in 112,772 predicted unigenes with an average length of 825 bp and an N50 of 1,638 bp. Sequence similarity searches using a public protein database revealed 19,947 unigenes that could be annotated with gene descriptions and gene ontology terms. Analysis of differential gene expression allowed pinpointing of genes whose products are predicted to be involved in cell line embryogenic potential, early somatic embryo formation and unorthodox seed development. The results expand our understanding of the complex molecular events that control embryogenesis suggesting that the regeneration impairment of Araucaria angustifolia cultures is consequence of the auxin signaling failure. The generated data lay the foundation for future functional genomic and evolutionary studies that will advance the understanding of conifer biology and unorthodox seed physiology.

IbSIMT1 , a novel salt-induced methyltransferase gene from Ipomoea batatas , is involved in salt tolerance

Abstract: S-adenosyl-methionine (SAM)-dependent methyltransferase (MTase) genes are a multigene family; however, only a few have been characterized at the functional level. In the present study, a novel salt-induced SAM-dependent MTase gene, named IbSIMT1, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbSIMT1 contains a DUF248 domain of unknown function and an MTase domain. Expression of IbSIMT1 was up-regulated in sweetpotato under salt stress and abscisic acid treatment. The IbSIMT1-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbSIMT1 up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes under salt stress. These findings suggest that the novel IbSIMT1 gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity.

Direct adventitious shoot regeneration, in vitro flowering, fruiting, secondary metabolite content and antioxidant activity of Scrophularia takesimensis Nakai

Abstract: Effects of plant growth regulators, light quality and sucrose on direct adventitious shoot regeneration, in vitro flowering and fruiting of Scrophularia takesimensis were investigated. The highest mean number of shoots per leaf (23.4), petiole (17.2) and stem (20.3) explants was obtained on Murashige and Skoog (MS) medium fortified with 2.0 mg l−1 BA and 1.0 mg l−1 IAA under white fluorescent light (WFL). Red light emitting diodes (LED) gave better shoot growth followed by WFL and blue LED. However, red LED treatment decreased the number of roots and induced callus at the base of shoot. The highest number of roots per shoot (9.2) and a maximum root length (8.9 cm) were obtained when the cultures were maintained under WFL. Somaclonal variation was observed when the shoot buds were cultured on the modified MS medium containing 1.0 mg l−1 IBA. The variant had variegated leaves in comparison with those of normal plantlets. The greatest frequency of flower induction (96.8 %) was obtained when the shoots were cultured on the modified MS medium containing 6.0 % (w/v) sucrose for 45 days under blue LED. The in vitro developed flowers self-fertilized and formed fruits. The culture media and environment had a positive effect on the content of phenolic compounds. Harpagoside content was high in seeds and low in shoots developed in MS medium containing BA. Among the light sources, shoots maintained under blue LED found to be best for harpagoside production (4.9 mg g−1) followed by WFL (3.3 mg g−1) and red LED (2.7 mg g−1). In vitro regenerated shoots had higher capacity to detoxify DPPH free radicals than field-grown plant samples.

Overexpression of PtPCS enhances cadmium tolerance and cadmium accumulation in tobacco

Abstract: Phytochelatins chelate heavy metal ions to decrease their toxicity. The chelates are then transferred to, and stored in, the vacuole. Phytochelatin synthase (PCS), which is involved in phytochelatin synthesis, is thought to be a key enzyme for phytoremediation. In this study, a PCS gene encoding phytochelatin synthase was cloned from poplar (Populus tomentosa Carr.), a widely grown model woody plant that accumulates high levels of heavy metals, especially cadmium. Poplar is considered to have potential applications in phytoremediation. The full-length PtPCS cDNA (1512-bp) encoded a polypeptide of 503 amino acid residues. The PtPCS cDNA was transferred into tobacco by Agrobacterium-mediated leaf disk transformation. The transgenic and wild-type (WT) lines of tobacco were subjected to a one time Cd treatment (90 μmol Cd2+) for 30 days, and then evaluated to determine their Cd tolerance. We evaluated morphological and physiological indices including leaf relative electrolyte leakage, malondialdehyde content, total superoxide dismutase activity, chlorophyll content and root activity. Compared with WT plants, the transgenic plants expressing PtPCS grew better in the Cd treatment and showed significantly higher Cd tolerance. Compared with WT plants, the transgenic lines accumulated higher concentrations of Cd (1.7 to 3.0-fold higher Cd concentration in roots; 1.24 to 2.28-fold higher Cd concentration in leaves). However, the transfer coefficient was lower in the transgenic lines than in wild type. We concluded that PtPCS encodes a functional PCS that may be involved in Cd tolerance and accumulation, but not in Cd transport.