Optimization of DNA extraction from seeds and leaf tissues of Chrysanthemum (Chrysanthemum indicum) for polymerase chain reaction

Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.     

Cytoplasmic RNA extraction from fresh and frozen mammalian tissues

The quality of collections of expressed sequence tags andfull-length cDNAs is adversely affected by the presence of "junk" clones derivedfrom unspliced or partially spliced RNAs present in conventional total RNA preparations. One can overcome this problem by using intact cytoplasmic RNA to create cDNA libraries, but the methods in the literature that describe the preparation of RNA only work well for extracting cultured cells. Cell lines are not as diverse as one would like, and to clone comprehensive sets of human and model organism full-length cDNAs, libraries have to be prepared from tissue samples. Thus, we have developed a robust and inexpensive method that allows intact cytoplasmic RNA to be extracted from both fresh and frozen mammalian tissues. A mouse full-length, cap-trapped cDNA library prepared with RNA using this new procedure had excellent characteristics.     

Systematic position of the genusCicer L. (Fabaceae) from data on DNA/DNA hybridization

This investigation was carried out in an attempt to determine the systematic position ofCicer via DNA/DNA hybridization. The data showed that members of the tribeVicieae were related toCicer than toTrifolium andOnonis. It is also showed thatPisum was the nearest species toCicer. Thus, the data presented in this work recommend the classification ofCicer underVicieae rather than a separate tribeCicerideae.     

Rapid and simple procedure for homogenizing leaf tissues suitable for mini-midi-scale DNA extraction in rice

We describe a rapid and simple procedure for homogenizing leaf samples suitable for mini/midi-scale DNA preparation in rice. The methods used tungsten carbide beads and general vortexer for homogenizing leaf samples. In general, two samples can be ground completely within 11.3+/-1.5 sec at one time. Up to 20 samples can be ground at a time using a vortexer attachment. The yields of the DNA ranged from 2.2 to 7.6 microg from 25-150 mg of young fresh leaf tissue. The quality and quantity of DNA was compatible for most of PCR work and RFLP analysis.     

A newAstragalus (Fabaceae) from southern Peloponnisos

Astragalus maniaticus (Fabaceae), a new species endemic to the Mani Peninsula in Peloponnisos, southern Greece, is described and illustrated. Its closest affinities are still uncertain but within Greece, it lacks close allies.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Bi-parental cytoplasmic DNA inheritance in Wisteria (Fabaceae): evidence from a natural experiment

Cytoplasmic inheritance was investigated in interspecific hybrids of Wisteria sinensis and W. floribunda. Species-specific nuclear, mitochondrial and plastid DNA markers were identified from wild-collected plants of each species in its native range. These markers provide evidence for the bi-parental transmission of plastids in hybrid swarms of these two species in the southeastern USA. These population level molecular data corroborate previous cytological evidence of this phenomenon in Wisteria.     

High-efficient extraction of principal medicinal components from fresh Phellodendron bark (cortex phellodendri)

There are three key medicinal components (phellodendrine, berberine and palmatine) in the extracts of Phellodendron bark, as one of the fundamental herbs of traditional Chinese medicine. Different extraction methods and solvent combinations were investigated to obtain the optimal technologies for high-efficient extraction of these medicinal components. Results: The results showed that combined solvents have higher extracting effect of phellodendrine, berberine and palmatine than single solvent, and the effect of ultrasonic extraction is distinctly better than those of distillation and soxhlet extraction. Conclusion: The hydrochloric acid/methanol-ultrasonic extraction has the best effect for three medicinal components of fresh Phellodendron bark, providing an extraction yield of 103.12 mg/g berberine, 24.41 mg/g phellodendrine, 1.25 mg/g palmatine.     

Extraction of DNA from milligram amounts of fresh,herbarium and mummified plant tissues

Summary We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.     

A fully automatable enzymatic method for DNA extraction from plant tissues

Background  

DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA.     

Isolation of endothelial cells from fresh tissues

Here, we present a protocol for the isolation of endothelial cells (ECs) from tissues. ECs make up a minor population of cells in a tissue, but play a major role in tissue homeostasis, as well as in diverse pathologies. To understand the biology of ECs, characterization of this cell population is highly desirable, but requires the availability of purified cells. For this purpose, tissues are mechanically minced and subsequently digested enzymatically with collagenase and dispase. ECs in the resulting single-cell suspension are labeled with Abs against EC surface antigens and separated from the remainder of the cells and debris by capture with magnetic beads or by high-speed cell sorting. Purified ECs are viable and suitable for characterization of diverse cellular properties. This protocol is optimized for human tissues but can also be adapted for use with other species. Depending on the tissue, the procedure can be completed in approximately 6 h.     

Three new species of bauhinia (Fabaceae) from Ecuador

Bauhinia flagelliflora Wunderlin,B. haughtii Wunderlin, andB. pichinchensis Wunderlin, all endemic to Ecuador, are described and illustrated.     

Development of microsatellite markers from Cercis chinensis (Fabaceae)

? Premise of the study: Microsatellite markers were developed to characterize the level of genetic diversity and population genetic structure of Cercis chinensis, a widely cultivated garden plant in China with congeneric species disjunctly distributed in East Asia, North America, and the Mediterranean. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, eight microsatellite markers were developed in C. chinensis. Seven of the markers displayed polymorphism, with the number of alleles ranging from one to four in four populations of C. chinensis. Four to six microsatellite loci exhibited interspecific transferability in C. glabra, C. chuniana, and C. chingii. ? Conclusions: These are the first microsatellite markers developed for C. chinensis, which will be further used in investigation of population genetic structure and phylogeographic pattern of C. chinensis and its congeneric species.     

锦鸡儿属(豆科)一新组合

中间锦鸡儿(Caragana korshinskii var. intermedia), 与柠条锦鸡儿(C. korshinskii)和小叶锦鸡儿(C. microphylla)是近缘种.但以前有不同的分类学名称和处理,本文把它作为柠条锦鸡儿的一个变种(C. korshinskii Kom. var. intermedia (Kuang et H. C. Fu) M. L. Zhang & G. H. Zhu)来处理.     

Chloroplast DNA characters, phylogeny, and classification of Lathyrus (Fabaceae)

Mapped cpDNA restriction site characters were analyzed cladistically and the resulting phylogenetic hypotheses were used to test monophyly and relationships of the infrageneric classification of Lathyrus (Fabaceae) proposed by Kupicha (1983, Notes from the Royal Botanic Garden Edinburgh 41: 209-244). The validity of previously proposed classification systems and questions presented by these classification schemes were explored. Two cpDNA regions, rpoC (rpoC1, its intron, part of rpoC2, and their intergenic spacer) and IR- (psbA, trnH-GUG, part of ndhF, and their intergenic spacers), were analyzed for 42 Lathyrus and two Vicia species. PCR (polymerase chain reaction) amplified rpoC and IR- products digested with 31 and 27 restriction endonucleases, respectively, resulted in 109 potentially informative characters. The strict consensus tree suggests that several of Kupicha's sections may be combined in order to constitute clades. The widespread section Orobus and the South American section Notolathyrus should be combined. Section Lathyrus, characterized by a twisted style, should either include sections Orobon and Orobastrum or be redefined as three sections, one of which is characterized by a 100 base pair deletion in the IR- region. Finally, a weighted parsimony analysis positions sections Clymenum (excluding L. gloeospermus) and Nissolia, both with phyllodic leaves, as sister sections. The affiliation of Lathyrus gloeospermus (section Clymenum) remains problematic.     

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.     

RNA extraction from plant tissues

Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca²⁺, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages.