Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna

Owing to the presence of higher amount of polyphenolic and polysaccharide compounds, Terminalia arjuna (Roxburgh) is a significant medicinal plant in the Indian primeval medicine system. Polyphenolic and polysaccharide compounds also acts as inhibitors during Genomic DNA isolation from young leaves of T. arjuna, resulting in recovery of low quality genomic DNA, which affects downstream applications like PCR, restriction digestion’s, etc. In this study, nine different methods of genomic DNA isolation were used, out of which two methods were modified CTAB based methods, third one was HEPES based method and remaining six methods was FTA Plant Saver Card based. Out of the six FTA card based methods, in the first method, leaves were directly pressed inside the circle of FTA card. In the second and third methods, the leaves were homogenized with PBS and DNase RNase free water and the sample was applied on the FTA card. In the fourth and fifth methods: finally recovered DNA from two modified CTAB based methods was directly applied to the FTA card. In the sixth method, DNA precipitated after first phenol:chloroform:isoamyl alcohol precipitation of modified CTAB based methods dissolved in DNase RNase free water and applied to FTA Card. To optimize the PCR conditions, BSA (400 ng/μl), formamide (2.5%), DMSO (5% and 10%) and glycerol (5%, 10%, 15%, and 20%) was added into the PCR mix as enhancement agents for amplification of low quality genomic DNA (A₂₆₀/A₂₈₀ – 1.27 ± 0.090) of T. arjuna recovered using the HEPES Based method. It was found that the BSA was the best among them followed by 10% glycerol. In addition of BSA to the PCR mixture, it specifically enhances the amplification of the low quality DNA. It reduces the noise in-between the amplified bands and increases the intensity of PCR product.     

Safer DNA extraction from plant tissues using sucrose buffer and glass fiber filter

For some plant species, DNA extraction and downstream experiments are inhibited by various chemicals such as polysaccharides and polyphenols. This short communication proposed an organic-solvent free (except for ethanol) extraction method. This method consists of an initial washing step with STE buffer (0.25?M sucrose, 0.03?M Tris, 0.05?M EDTA), followed by DNA extraction using a piece of glass fiber filter. The advantages of this method are its safety and low cost. The purity of the DNA solution obtained using this method is not necessarily as high as that obtained using the STE/CTAB method, but it is sufficient for PCR experiments. These points were demonstrated empirically with two species, Japanese speedwell and common dandelion, for which DNA has proven difficult to amplify via PCR in past studies.     

An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.     

A simple protocol for isolating genomic DNA from chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> tratt) for RFLP and PCR analyses

Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols, polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analyses.     

A high-throughput DNA extraction protocol for tropical molecular breeding programs

Liquid handling robotics and capillary electrophoresis genetic analyzers now offer high-throughput solutions for 2 of the 4 key steps in PCR-based DNA marker-assisted fingerprinting (DNA extraction, PCR amplification, electrophoresis, data analysis). Thus, DNA extraction remains the most significant bottleneck at the bench for large-scale applications in plant breeding and germplasm characterization. We report on a rapid and low-cost method for relatively high-throughput extraction of high-quality DNA from young and mature leaves of sorghum, pearl millet, chickpea, groundnut, and pigeonpea. The procedure uses a modified CTAB/β-mercaptoethanol method for DNA extraction in a 96-well plate. The quantity and quality of the DNA extracted per sample is adequate for more than 1000 PCR reactions. A relatively high throughput of 96–384 samples per person per day can be achieved, depending on the crop. A major timesaving aspect of the protocol is the absence of a manual sample-grinding step. Finally, the cost is a magnitude lower than commercial plate-based kits, and, as such, is likely to have substantial application in tropical molecular breeding programs.     

Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.     

β(1 → 3)-glucan aerosols in different occupational environments

The aim of this preliminary study was to assess exposure to β(1 → 3)-glucan as well as inhalable dust and viable fungi in different occupational environments. The study was conducted in three different industrial plants: metal plant where metalworking fluids were applied, wastewater treatment plant, and waste composting plant. In selected points simultaneously the stationary air sampling was performed to evaluate the levels of inhalable dust, β(1 → 3)-glucan, and to make a quantitative analysis of airborne fungi. All variables describing the exposure were characterized by a wide range of concentrations. The results were as follows: β(1 → 3)-glucan (1.38–65.1 ng/m³), inhalable dust (0.03–2.93 mg/m³), and fungi (0.16–285 × 10² CFU/m³). The highest concentrations for all parameters were found in the composting plant. In the composting plant, a statistically significant correlation was found between β(1 → 3)-glucan and fungal levels (r = 0.89; p < 0.05). In the metal industry and composting plant, the participation of alkali-soluble fraction was stable, exceeding 90% of all β(1 → 3)-glucan. However, in the wastewater treatment plant, its average amount was much lower—73.6%. The study showed that β(1 → 3)-glucan was present in different occupational environments and it should be taken into consideration as an important part of bioaerosols. However, more studies are required to assess the concentration levels as well as all determinants of exposure.     

Micropropagation and β-ecdysone content of the Brazilian ginsengs <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> and <Emphasis Type="Italic">Pfaffia tuberosa</Emphasis>

The aim of this study was to investigate both a mass in vitro propagation system and the β-ecdysone content in roots and aerial parts of Pfaffia glomerata and Pfaffia tuberosa. Nodal segments of two genotypes (BRA and JB-UFSM) of P. glomerata, originated from aseptically grown plants, were cultivated on hormone-free Murashige and Skoog medium. For the proliferation of P. tuberosa shoots, nodal segments, originated from aseptically grown plants, were either cultivated on hormone-free Murashige and Skoog (MS) medium or were supplemented with 1.0 μM thidiazuron (TDZ); the elongation and rooting of these plants were carried out on MS medium without TDZ. Plantlets of both species were acclimatized and transferred to field conditions. The β-ecdysone content in the plants was determined by high performance liquid chromatography. The BRA genotype showed a greater in vitro proliferation rate and β-ecdysone content than that of the JB-UFSM genotype. The culture of nodal segments of P. tuberosa on medium with 1.0 μM TDZ with subsequent subcultivation of shoots on hormone-free medium was shown to be a suitable method for micropropagation due to the high multiplication rate and good plant development. Both species showed good adaptation to ex vitro conditions. The β-ecdysone content in micropropagated P. tuberosa was similar to that found in field-grown plants. For both species, the aerial parts accumulated higher β-ecdysone content than roots. These results reveal that micropropagation is a successful, alternative method for rapid plant multiplication of both species of Brazilian ginseng. Furthermore, this study demonstrates that these two species have a potential for cultivation that is associated with high β-ecdysone production.     

Regulation of Carotenoid Content in Tomato by Silencing of Lycopene β/ε-Cyclase Genes

To conduct RNAi interference of Lyc-β and Lyc-ε genes, two plant expression vectors were constructed by inserting the intron fragments of the gusA gene into the two target gene fragments, which were designed in anti-sense directions. After the Agrobacterium tumefaciens-mediated transformation, 13 transgenic tomato plants (seven and six for Lyc-β and Lyc-ε, respectively) were obtained, which was further validated by PCR. Real-time PCR revealed that the messenger RNA abundance of Lyc-β gene and Lyc-ε gene in transgenic tomato plants was significantly reduced to 8.95% and 13.16%, respectively, of the level of the wild-type plant. Subsequent high-performance liquid chromatography analysis found that transgenic tomato plant had significantly increased lycopene content, with the highest value of 13.8 μg/g leaf dry weight, which was about 4.2-fold that of wild-type plant. Moreover, Lyc-β and Lyc-ε interference gene effects were observed on downstream products as well. β-Carotene and lutein contents decreased in Lyc-β RNAi lines, ranging from 40.7 to 117.3 μg/g and 4.9 to 23.5 μg/g leaf dry weight, respectively. In Lyc-ε RNAi lines, β-carotene content increased, ranging from 195.8 to 290.2 μg/g, while lutein content decreased, ranging from 3.7 to 11.3 μg/g. For total carotenoids, Lyc-β RNAi lines resulted in 2.9-fold decrease, while Lyc-ε RNAi lines yielded 1.7-fold increase in contents when compared to wild-type control. This study demonstrated that RNAi gene technology is an effective method for enhancing lycopene content in plants.     

Effects of Progesterone, β-Estradiol,and Androsterone on the Changes of Inorganic Element Content in Barley Leaves

The present study was performed to determine the changes in inorganic element content in barley leaves of mammalian sex hormones (MSH). Barley leaves were sprayed with 10⁻⁴, 10⁻⁶, 10⁻⁹, 10⁻¹², 10⁻¹⁵ M concentrations of progesterone, β-estradiol, and androsterone at 7th day after sowing. The plants were harvested at the end of 18 days after treatment with MSH solutions. The inorganic element concentrations were determined using wavelength dispersive X-ray fluorescence spectroscopy technique. Although the all MSH concentrations significantly (p < 0.05) increased the concentrations of calcium, magnesium, phosphorus, sulfur, copper, manganese, aluminum, zinc, iron, potassium, and chlorine, it decreased those of sodium concentration in barley leaves. The maximum changes in the element concentrations were obtained at 10⁻⁹ M for plant leaves treated with progesterone, 10⁻⁶ M for plant leaves treated with β-estradiol and androsterone. The present study elucidated that MSH significantly (p < 0.05) affected the inorganic element concentrations in barley leaves.     

Ploidy stability of somatic embryogenesis-derived <Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast. plants as assessed by flow cytometry

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.     

Functional analysis of multiple carotenogenic genes from <Emphasis Type="Italic">Lycium barbarum</Emphasis> and <Emphasis Type="Italic">Gentiana lutea</Emphasis> L. for their effects on β-carotene production in transgenic tobacco

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, β-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into β-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and δ-carotene desaturase gene, lycopene β-cyclase, lycopene ε-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced β-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.     

Mammalian sex hormones stimulate antioxidant system and enhance growth of chickpea plants

In the present study, it was aimed to investigate the influence of exogenous mammalian sex hormones (MSH) (progesterone, β-estradiol and androsterone) on the morphological (root and shoot growth) and biochemical parameters (protein and sugar content, antioxidant enzyme activities, and lipid peroxidation and H₂O₂ levels) of chickpea (Cicer arietinum L.) plants growing under control conditions. The solutions of hormones prepared at different concentrations (10⁻⁴, 10⁻⁶, 10⁻⁹, 10⁻¹² and 10⁻¹⁵ M) were sprayed once on the leaves of 7-day plants. The plants were harvested on 18 days after the hormone treatment. Although all of the hormones at the tested concentrations significantly increased plant growth, soluble protein and sugar contents, and antioxidant enzyme activities [superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT)], they decreased H₂O₂ content and lipid peroxidation level when compared with control plants. The activities of SOD, POX and CAT reached to the highest levels at 10⁻⁶ M for progesterone, and 10⁻⁹ M for β-estradiol and androsterone, which maximum growth, protein and sugar contents were determined. The same concentrations also resulted in the lowest levels for H₂O₂ content and lipid peroxidation. It can be interpreted that the MSH improve plant growth and development by affecting some biochemical parameters including antioxidative system.     

Exogenously Treated Mammalian Sex Hormones Affect Inorganic Constituents of Plants

The present study was undertaken to reveal the changes in inorganic constituents of plants exposed to mammalian sex hormones (MSH). Chickpea leaves were sprayed with 10⁻⁴, 10⁻⁶, 10⁻⁹, 10⁻¹², and 10⁻¹⁵ M concentrations of progesterone, β-estradiol, and androsterone at 7th day after sowing. The plants were harvested at the end of 18 days after treatment of MSH solutions and the inorganic components determined using a wavelength-dispersive X-ray fluorescence spectroscopy technique. At all of the concentrations tested, MSH significantly increased the contents of K, S, Na, Ca, Mg, Zn, Fe, P, Cu, and Ni. Interestingly, only Mn and Cl contents decreased. The maximum changes in the inorganic composition were recorded at 10⁻⁶ M for plants treated with progesterone and 10⁻⁹ M for plants treated with β-estradiol and androsterone.     

Plant DNA extraction using silica

Described here is a method that uses silicon dioxide (silica) to extract whole genomic plant DNA of high molecular weight. The protocol is presented in a microcentrifuge format, and yields were approximately 2–4 μg per 200 mg of plant leaf tissue. The method involves fewer steps than many previous extraction protocols and, as shown here for 4 taxonomically distant angiosperms, produces DNA suitable for digestion with restriction endonucleases. The use of commercial kits is not required; the silica costs are comparatively inexpensive (<$0.03 per tube); and CTAB, rather than the more expensive guanidine thiocyanate salt, is used.     

Efficient <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of oilseed mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.] using leaf piece explants

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T ₀ generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T ₁ seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.