scholarly journals A simplified method to isolate rice mitochondria

2020 ◽  
Author(s):  
Yanghong Xu ◽  
Xiaoyi Li ◽  
Jishuai Huang ◽  
Leilei Peng ◽  
Dinghui Luo ◽  
...  
Keyword(s):  
Genome Structure ◽  
Plant Mitochondria ◽  
Extraction Methods ◽  
Protoplast Isolation ◽  
Physiological Status ◽  
Time Saving ◽  
Plant Mitochondrial Genome ◽  
Protein Levels ◽  
Modified Method ◽  
Simplified Method

Abstract Background: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies.Results: To isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies.Conclusion: The combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

scholarly journals A simplified method to isolate rice mitochondria

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Yanghong Xu ◽  
Xiaoyi Li ◽  
Jishuai Huang ◽  
Leilei Peng ◽  
Dinghui Luo ◽  
...  
Keyword(s):  
Genome Structure ◽  
Plant Mitochondria ◽  
Extraction Methods ◽  
Protoplast Isolation ◽  
Physiological Status ◽  
Time Saving ◽  
Plant Mitochondrial Genome ◽  
Protein Levels ◽  
Modified Method ◽  
Simplified Method

Abstract Background Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies. Results To isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies. Conclusion The combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

scholarly journals A Simplified Method to Isolate Rice Mitochondria

2020 ◽  
Author(s):  
Yanghong Xu ◽  
Xiaoyi Li ◽  
Jishuai Huang ◽  
Leilei Peng ◽  
Dinghui Luo ◽  
...  
Keyword(s):  
Animal Cell ◽  
Genome Structure ◽  
Plant Mitochondria ◽  
Extraction Methods ◽  
Physiological Status ◽  
Time Saving ◽  
Plant Mitochondrial Genome ◽  
Modified Method ◽  
Simplified Method ◽  
Mitochondria Isolation

Abstract Background: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers participate in the studies of plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, traditional plant mitochondria extraction methods are time-consuming and complicated operation of ultra-centrifuge with the expensive reagent. To develop a more rapid and convenient method for isolation of plant mitochondria, in this study we established a simplified method to isolate rice mitochondria efficiently for further study.Results: To isolate rice mitochondria, the cell wall was first dispelled by enzymolysis to obtain the protoplast which is similar to the animal cell. Then the rice mitochondria were isolated with a modified method basing on the animal mitochondria isolation protocol. The extracted mitochondria were next detected on DNA level and protein level to rule out the contamination of nucleus and chloroplasts. Furthermore, we examined the physiological status and characters of the isolated mitochondria, including the integrity of mitochondria, mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria were remained intact for further studies.Conclusion：The combination of plant protoplasts isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability, and can be broadly applied in the researches on plant mitochondria.

scholarly journals Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector

Plants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2636
Author(s):  
Ganeshan Sivanandhan ◽  
Solhee Bae ◽  
Chaemin Sung ◽  
Su Ryun Choi ◽  
Geung-Joo Lee ◽  
...  
Keyword(s):  
Chinese Cabbage ◽  
Binary Vector ◽  
Transfection Efficiency ◽  
Genome Structure ◽  
Transformation Method ◽  
Protoplast Isolation ◽  
Breeding Cycle ◽  
Functional Studies ◽  
Gfp Reporter Gene ◽  
Protoplast Transfection

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

scholarly journals DNA extraction technique for different rice varieties grown in Sri Lanka

2015 ◽  
Vol 3 (3) ◽  
pp. 398-401
Author(s):  
Ranganathan Kapilan
Keyword(s):  
Sri Lanka ◽  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Rice Varieties ◽  
Modified Method ◽  
Dna Extraction Methods ◽  
Very High ◽  
Different Parts

Extraction of DNA is very important nowadays in bio-molecular researches. Extracted DNA should be purified and the quality of DNA should also be very high. The objective of the study was to develop a simple efficient method to isolate DNA from the rice varieties in an open laboratory environment, and to eliminate the usage of expensive chemicals and tools. The DNA extraction methods developed by the DNeasy plant kit method supplied by QIAGEN, Cheng et al., Doyle et al. and Michiels et al. were applied to five different rice varieties grown in different parts of Sri Lanka. Based on the quantity and quality of the extracted DNA tested by measuring the absorbance of DNA at 260 nm using Nanodrop® ND-1000 spectrophotometer and measuring the ratio of A260 / A280 and gel running on agarose, the efficiency of the extraction method chosen varied among rice varieties. Among the methods used, the methods introduced by DNeasy plant kit method supplied by QIAGEN and Cheng et al, yielded good and amplifiable quality DNA with satisfactory concentration for all the rice varieties tested. Therefore the modified method of Cheng et al, 1987 could be used to extract DNA from rice varieties instead of the commercially available expensive and hazardous DNeasy plant kit method supplied by QIAGEN.Int J Appl Sci Biotechnol, Vol 3(3): 398-401

scholarly journals Effectiveness of Temperature Modification in Decreasing the Bias in Milk Fat Test Results Between the Babcock and Ether Extraction Methods

2003 ◽  
Vol 86 (4) ◽  
pp. 768-774 ◽  
Author(s):  
Joanna M Lynch ◽  
David M Barbano ◽  
Patrick A Healy ◽  
J Richard Fleming
Keyword(s):  
Milk Fat ◽  
Collaborative Study ◽  
Extraction Methods ◽  
Test Results ◽  
Method Performance ◽  
Modified Method ◽  
Temperature Modification ◽  
Ether Extraction ◽  
Aoac Method ◽  
Performance Statistics

Abstract Both the Babcock (AOAC Method 989.04, revised Final Action 2000) and modified Mojonnier ether extraction (AOAC Method 989.05) methods are used in the dairy industry to determine the fat content of milk. Prior to revision in 1997, the Babcock method gave consistently higher fat test results than did the ether extraction. In 1997, a modification of the Babcock method was introduced to bring the results of the Babcock test into closer agreement with the ether extraction. The Babcock method was modified by lowering the temperatures used at various points in the method from about 57.5 to 48°C to increase the density of the material in the Babcock column. A collaborative study of the modification indicated it was successful in bringing the Babcock and ether extraction results into agreement but suggested that performance of the modified method was not as good as that of the unmodified method. In the present study, substantial evidence is presented to validate the success of the Babcock modification in bringing test results into agreement with ether extraction, and to document that temperature modification does not adversely affect method performance. Data were evaluated from an on-going proficiency testing program where 8–15 laboratories tested 7 milk samples in blind duplicate once every 2 months. Laboratories used the unmodified method from 1995 through 1996 and the modified method from 1998 through 1999. Compared with ether extraction, test results from the unmodified Babcock test were consistently higher by an average of 0.022% fat. For the modified Babcock test, average test results were –0.003% fat lower than with ether extraction and not significantly different from zero. AOAC method performance statistics (within-and between-laboratory precision) were equivalent for both the unmodified (Sr = 0.027, SR = 0.041, RSDr = 0.73%, RSDR = 1.08%) and modified (Sr = 0.023, SR = 0.038, RSDr = 0.60%, RSDR = 1.02%) Babcock methods. Modification of the Babcock method was successful in bringing test results into agreement with those of ether extraction.

A modified method for harvesting thoracodorsal artery perforator flaps in a simple and time-saving approach

Microsurgery ◽  
2016 ◽  
Vol 36 (8) ◽  
pp. 642-646 ◽  
Author(s):  
Sang Wha Kim ◽  
Seungki Youn ◽  
Jeong Tae Kim ◽  
Youn Hwan Kim
Keyword(s):  
Perforator Flaps ◽  
Time Saving ◽  
Modified Method ◽  
Thoracodorsal Artery ◽  
Artery Perforator

scholarly journals Modified TCA/acetone precipitation of plant proteins for proteomic analysis

2018 ◽  
Author(s):  
Liangjie Niu ◽  
Hang Zhang ◽  
Zhaokun Wu ◽  
Yibo Wang ◽  
Hui Liu ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Modification ◽  
Protein Extraction ◽  
Extended Period ◽  
Extraction Methods ◽  
Precipitation Method ◽  
Plant Proteins ◽  
Prolonged Incubation ◽  
Acetone Precipitation ◽  
Modified Method

AbstractProtein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

scholarly journals Comparison of genomic DNA extraction methods to obtain high DNA quality from barnyard grass (Echinochloa colona)

2020 ◽  
Author(s):  
Romesh Kumar Salgotra ◽  
Bhagirath Singh Chauhan
Keyword(s):  
Essential Oils ◽  
Dna Extraction ◽  
Leaf Tissue ◽  
Extraction Methods ◽  
High Quality ◽  
Time Saving ◽  
Plant Leaf ◽  
Plant Parts ◽  
Molecular Studies ◽  
Leaf Tissues

Abstract Background: The study of weed genomics is important for the effective management of weeds to enhance crop yield. A rapid, inexpensive and high quality DNA extraction is needed for genomic and other molecular studies. Here, we describe the protocols for DNA extraction from two different parts of the Echinochloa colona plant using modified cetyltrimethylammonium bromide (CTAB) and a commercial kit.Results: In the study, it was observed that the DNA extracted from plant leaf tissues and dry seeds with a modified CTAB protocol was of good quality, with no contaminations of polysaccharides and essential oils. Quality of DNA extracted from dry seeds was comparable with that of plant leaves under both protocols. The extracted DNA from both plant parts was successfully amplified by PCR using the EPSPS microsatellite marker. Compared to the protocol of DNA extracted from leaf tissue, dry seeds will save time and other valuable resources. Moreover, the same protocols can be implemented for the extraction of high-quality DNA for molecular studies in other plant species where a large amount of polysaccharides, secondary metabolites and essential oils are present.Conclusions: Modified methods of DNA extraction from dry seeds are efficient and time-saving which can be used in genotypic and other molecular approaches. High-quality DNA can be isolated from plant leaf tissues using modified CTAB and commercial kits, however, DNA extracted from dry seeds will save time and other valuable resources.

scholarly journals Characterization of Nucleotide Pools as a Function of Physiological State in Escherichia coli

2007 ◽  
Vol 190 (2) ◽  
pp. 718-726 ◽  
Author(s):  
Michael H. Buckstein ◽  
Jian He ◽  
Harvey Rubin
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Growth Curve ◽  
Physiological State ◽  
Extraction Methods ◽  
Modified Method ◽  
New Information ◽  
Deoxynucleoside Triphosphate ◽  
Pool Sizes

ABSTRACT Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a “rebound” of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.

Analysis of the Genome Structure of Plant Mitochondria

1988 ◽  
pp. 275-292
Author(s):  
DAVID M. LONSDALE ◽  
TONY P. HODGE ◽  
PETER J. STOEHR
Keyword(s):  
Genome Structure ◽  
Plant Mitochondria
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