Nuclei Release Methods Comparison for Fresh Leaves of Rice (Oryza sativa) for Efficient High Throughput Flow Cytometry Ploidy Studies (original) (raw)
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An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants
Cytometry Part A, 2009
We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating DNA ploidy using a plate loader on a BD FACS-Canto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone sample handling. In two 96-well plates of samples, the histogram peaks of DNA content from flow cytometry were wellresolved in 189 of 192 samples tested (98.4%), with CV values ranging from 2.98% to 6.20% with an average CV of 4.35% (SD 5 0.68%). This new method is useful in doubled haploid plant breeding programs where early discrimination of haploid and doubled haploid (i.e., diploid) plantlets can confer significantly improved operational efficiencies. We discuss how this method could be further refined including adapting the method to robotic sample processing. ' 2009 International Society for Advancement of Cytometry
A rapid mini-prep DNA extraction method in rice (Oryza sativa
DNA extraction is an important step in molecular assays and plays a vital role in obtaining high-resolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction. Here a rapid mini-prep DNA extraction method, optimized for rice, which was achieved via some modifications in present DNA extraction methods, especially in first step of cell wall lyses and the use of cheap and frequent chemicals found in every laboratory is presented. Normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method. This method is applicable with both dry and fresh samples, does not require liquid nitrogen, and is easy, rapid and applicable in every laboratory.
Research Article A RAPID METHOD FOR RICE GENOMIC DNA EXTRACTION WITHOUT LIQUID NITROGEN AND PHENOL
Introduction Isolation of DNA from plants through efficient, rapid and inexpensive method is necessary for marker assisted selection, diversity assessment, quantitative trait loci analysis, germplasm and transgenic plants identification as well as screening. Several researchers [1-3] described DNA isolation methods, which are extensively used in plant molecular biology, but most of the methods are time consuming, laborious, expensive due to multiple steps, requires liquid nitrogen for sample grinding and Phenol for DNA extraction [4-6]. The Cetyl-trimethylammonium bromide (CTAB) method is one of the most popular methods for rice genomic DNA isolation, including other plants [7], bacteria [8], fungi [9] and animals [10]. Most of the purposed modified methods for DNA extraction method are the modifications of CTAB method described by prior research workers [1,2]. The main cause of diversity and modification in CTAB method is the composition of cell walls and intra-cellular as well as extra-cellular components. Lignification of cereal cell wall makes degradation difficult, which results in restricted DNA extraction [6]. To overcome this problem in the case of rice leaves some researchers has developed methods for DNA extraction [11]. Some other methods have been also reported to minimize the DNA extraction steps but these methods need liquid nitrogen [12] and Phenol. Continuous liquid nitrogen supply is a problem in many developing countries because purchasing time is unpredictable from overseas [7,13]. Phenol is highly corrosive (can cause severe burns) to skin, toxic (absorbed phenol act as a systemic toxin) and mutagen. We have optimized a simple and rapid method for isolating high-quality plant DNA without liquid nitrogen and Phenol at room temperature from the small amount of rice leaves tissues.
Estimation of nuclear DNA content in plants using flow cytometry
Nature Protocols, 2007
Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.
Comparison of four nuclear isolation buffers for plant DNA flow cytometry
Annals of botany, 2006
DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue.
Chromosome Research, 2012
Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using timescale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.
Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species
Annals of botany, 2007
After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry.
Journal of Bioscience and Bioengineering, 2009
Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Münster, Germany, the method of Arumuganathan and Earle, and LB01 buffer) failed, when applied to the hairy roots of red beet (Beta vulgaris). By combining these systems and introducing a centrifugation step, the extraction, staining, and analysis of nuclei from this tissue were performed successfully.
Plant DNA flow cytometry and estimation of nuclear genome size
Annals of botany, 2005
DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, sto...
Efficient DNA Isolation Method fo r Molecular Studies from Leaves and Roots of Rice ( Oryza sativa )
An efficient DNA isolation protocol specifically modified to get pure quality DNA required for the purpose of polymerase chain reaction amplification, Southern blot, RAPD, besides being necessary to study transcription factor genes during the drought/salt stress leaf of rice ( Oryza sativa) has been reported in this paper. In comparison to other known protocols that require expensive equipment and chemical, this protocol seems more economical for getting pure DNA that in turn forms an essential part of routine work of any molecular biology lab where concentration is more towards quality rather than quantity of DNA. Rather than concentrating more on commercially available DNA isolation kits, this protocol is more economical in getting pure quality DNA, free from proteins and polysaccharide compounds. In our case, purity of isolated DNA checked by measuring absorbance (A 260 /A 280) was excellent as the ratio of A 260 /A 280 was 1.75 - 1.92, thereby suggesting that it does not need any additional purification before proceeding for molecular analysis of the isolated DNA samples. From the above study, it becomes quite clear that this protocol being simple and economical finds wide application in genomic study as quality forms an important factor for performing different gene expression studies through PCR.