Isolation and PCR amplification of genomic DNA from dried capsules of cardamon (Elettaria cardamomum M.)

Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage tissue such as cardamom capsules. The yield of DNA was 6–7 μg g⁻¹ tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence Characterised Amplified Regions (SCAR) markers for profiling cardamoms.     

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l⁻¹) or 1-naphthaleneacetic acid (NAA) (5 mg l⁻¹) in combination with benzyladenine (BA) (0.5 mg l⁻¹). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l⁻¹) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l⁻¹), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of DNA from plants high in polyphenolics

DNA suitable for digestion with restriction endonucleases has been isolated fromTheobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper. The method relies on concentrating nuclei away from most of the cytoplasmic components and inhibiting the formation of oxidized polyphenolic compounds and their interaction with the DNA fraction in the remaining steps.     

Isolation of DNA for RAPD analysis from dry leaf material of some<Emphasis Type="Italic">Hesperis</Emphasis> L. specimens

An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated.     

Isolation of the cell-nuclear,mitochondrial, and chloroplast DNA from the ultra-small eukaryoteCyanidioschyzon merolae

Summary The amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) inCyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system.C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×10³kbp, 1.6×10³kbp, and 5.0×10³kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those ofCyanidium caldarium RK-1 andC. caldarium Forma A. The values of cl-DNA and cp-DNA ofC. merolae were about 1.716 and 1.709, respectively. The order in density was different from that ofC. caldarium Forma A but very similar to that ofC. caldarium RK-1. However, the restriction patterns of cp-DNA inC. merolae differed from those ofC. caldarium RK-1.     

Isolation of clean and PCR-amplifiable DNA fromAnthurium andreanum

A DNA extraction procedure that does not require hazardous materials, such as CTAB, phenols, or liquid nitrogen, was optimized forAnthurium andreanum, a plant rich in polysaccharides and polyphenolics. Three DNA isolation techniques were tested. The modified Rowhani protocol (1983), with slight modifications, was found to yield up to milligrams of DNA suitable for RAPD from spathe and leaf tissues. High-quality DNA was obtained readily from spathe tissues, while a spermine precipitation step was found to be essential when DNA was extracted from the leaf tissues.     

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

大花地不容块根生物碱成分的研究

为研究大花地不容块根的生物碱成分,该研究采用酸提碱沉法从大花地不容块根中提取出总生物碱,通过硅胶柱色谱法和制备液相色谱技术对大花地不容块根的总生物碱提取物进行分离纯化,并利用波谱学手段对化合物进行结构鉴定。结果表明:(1)从总生物碱提取物中分离得到11个化合物,分别鉴定为青藤碱(1)、青风藤碱(2)、斯帝酚灵碱(3)、瑞枯灵(4)、异紫堇定(5)、紫堇单酚碱(6)、巴婆碱(7)、sukhodianine(8)、荷包牡丹碱(9)、7-氧代克班宁(10)和巴马汀(11)。(2)体外细胞毒性测试显示,大花地不容总生物碱和其主成分青藤碱对人类肺癌细胞A549的IC₅₀值分别为7.5×10^-4 g·mL^-1和6.59×10^-9 g·mL^-1。化合物2、3、4、7、8、9和10系首次从大花地不容中分离得到。大花地不容块根中含有吗啡烷、原阿朴啡、阿朴啡、苄基四氢异喹啉和原小檗碱5种类型的生物碱。     

Isolation and PCR Amplification of Genomic DNA from Market Samples of Dry Tea

A simple procedure for DNA isolation from processed dried commercial samples of tea is described. The method involves a modified CTAB procedure employing extensive washing, use of 1% PVP to remove polyphenolics and a single phenol:chloroform extraction step. The average yield ranges from 164–494 g/g tea sample for various market samples. The DNA obtained from 11 different brands of tea using this procedure were consistently amplifiable (using both RAPD primers as well as defined sequences as primers) and digestible with restriction endonucleases.     

An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel

An efficient and convenient method is presented for the isolation of nuclear DNA from leaves of wildVitis species that have been dried with silica gel. The nuclear DNA obtained with this method is suitable for both PCR amplification and digestion with restriction endonucleases.     

Factors affecting in vitro microrhizome production in turmeric

In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium –- MS basal medium + 0.88 M BAP (6-benzylaminopurine) + 0.92 M kinetin + 5% coconut water + 2% sucrose + 0.5% agar –- resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium –- MS basal medium + 2.2 M BAP + 0.92 M kinetin + 5% coconut water + 2% sucrose –- at 25±1°C and 16-h light (at 11.7 mol m^–2 s^–1)/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 25±1°C. Half strength MS basal medium supplemented with 80 g l^–1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2 M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1–2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1–0.4 g), medium (0.41–0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.     

DNA Isolation and Amplification from Cacti

The cacti family is a morphologically heterogeneous group comprising 100 genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception of one genus, all members of this family are native to America (Hernandez and Barcenas, 1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gibson and Nobel, 1986). DNA isolation from cacti is notoriously difficult because they contain high amounts of polysaccharides and secondary metabolites which form insoluble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard, 1992). Like in other groups of plants, the secondary metabolites and polysaccharides in cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visually evident by their viscous, glue-like texture and they make the DNA unmanageable when pipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al., 1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We used this method to isolate DNA from 85 species (170 individuals) of 39 genera of the subfamilies Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modification of a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires only a few grams of tissue and does not require destruction of the whole plant to produce high molecular weight genomic DNA. The DNA from this procedure can be amplified consistently by PCR and used for RAPD analysis.     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

Isolation of genomic DNA from the earthworm speciesEisenia fetida

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using³²P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris.     

Isolation of high molecular weight DNA from wholeEuglena cells

Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×10⁷. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp kilobasepairs - Kb kilobases     

Isolation of single-stranded DNA from loop-mediated isothermal amplification products.

Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.     

Effect of Eucalyptus and Mentha leaves and Curcuma rhizomes on Tyrophagus putrescentiae (Schrank) (Acarina: Acaridae) in wheat

Varying concentrations of powdered leaves of Eucalyptus and Mentha and rhizomes of Curcuma were evaluated in controlling Tyrophagus putrescentiae in wheat flour. At higher concentrations, all these plant materials significantly decreased the population build-up of the mite. Eucalyptus and Mentha leaf powders were effective at concentrations ranging from 100 to 5% in bringing about a decrease in the fecundity of the mite and reducing the egg numbers to 51.66 and 25.49 per female, respectively, as compared to 98.16 eggs per female in the controls. Rhizomes of Curcuma were effective even at a concentration of 0.1% reducing the egg laying to 7.66 eggs per female in wheat flour. These materials were more deleterious to immature stages (ova and larvae) than to the mature stages of T. putrescentiae. When tested on whole grains of wheat these plant materials showed more pronounced effects in controlling the mite population compared to that in wheat flour. Curcuma rhizomes were the most promising for possible use against T. putrescentiae.     

cDNA cloning and characterization of a mannose-binding lectin fromZingiber officinaleRoscoe (ginger) rhizomes

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae