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1.
Abolfazl Barzegari Sepideh Zununi Vahed Sina Atashpaz Sajjad Khani Yadollah Omidi 《Molecular biology reports》2010,37(2):833-837
Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted
intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant
tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions
associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid,
easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly
efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA
contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from
leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP.
Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues. 相似文献
2.
Genomic DNA of high quality and quantity is needed to analyze genetic diversity with AFLP.Carpobrotus plant species, like most succulents, contain high amounts of polysaccharides and polyphenols, making PCR amplification difficult.
Our protocol eliminates contaminants before DNA isolation by using leaf callus as plant material. This simple and inexpensive
technique gives an average DNA yield of 1800 ng/g of callus and high reproducible profiles in AFLP. Our results indicate that
no genetic variability is associated with callus culture conditions. This technique is suitable for studying genomic polymorphism
in succulents and other plants when classic DNA extraction procedures fail. 相似文献
3.
Monique van Wordragen Roshani Shakya Ruud Verkerk Regis Peytavis Ab van Kammen Pim Zabel 《Plant Molecular Biology Reporter》1997,15(2):170-178
This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery
of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed
a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer
of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts
of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used
an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective. 相似文献
4.
Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that
in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the
micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three
media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism
(AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability
of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from
a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic
variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns
occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites
representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to
concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA
methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant. 相似文献
5.
Antonio Andrade-Torres Carlos Oropeza Luis Sáenz Tomás González-Estrada José Efraín Ramírez-Benítez Karen Becerril José Luis Chan Luis Carlos Rodríguez-Zapata 《Biologia》2011,66(5):790-800
Coconut palm (Cocos nucifera) is a plant species recalcitrant to in vitro morphogenesis and no protocols for the genetic transformation of coconut tissues have been published. The present study aimed
to develop a protocol for genetic transformation of this palm species; evaluating reporter genes, transformation methods,
and conditions for the use of antibiotics to select transformed plant cells. The gene gusA was first used for Agrobacterium tumefaciens mediated transformation of coconut embryogenic calli. However, endogenous GUS-like activity was found in calli not co-cultured
with bacteria. Then essays for Agrobacterium-mediated transformation were developed using green and red fluorescent genes. Both genes are suitable as reporter genes for
coconut transformation. In order to establish a protocol for coconut genetic transformation, an approach was used that combined
biobalistics to generate micro-wounds in explants, vacuum infiltration and co-culture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis related gene WUSCHEL). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated
with either biobalistics or infiltration, followed by bacteria co-culture. PCR amplification of DNA extracts from transformed
embryogenic callus produced a band with the expected size using WUSCHEL primers (862 bp). No band was obtained using the VirE2 primers. This is the first report of transient genetic transformation of C. nucifera and it is the first step toward a protocol that will be useful for the study of the role of genes of interest and for practical
applications, such as the improvement of coconut micropropagation via somatic embryogenesis. 相似文献
6.
RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the
authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with
PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along
with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric
analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g)
and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at
126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream
molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely
to be widely applicable. 相似文献
7.
Summary Factors influencing the Agrobacterium-mediated transformation of both monocotyledonous and dicotyledonous plant species have been widely investigated. These factors
include manipulating Agrobacterium strains and plasmids, growth conditions for vir gene induction, plant genotype, inoculation and co-culture conditions, and the selection agents and their application regime.
We report here a novel physical parameter during co-culture, desiccation of plant cells or tissues post-Agrobacterium infection, which greatly enhances transfer DNA (T-DNA) delivery and increases stable transformation efficiency in wheat.
Desiccation during co-culture dramatically suppressed Agrobacterium growth, which is one of the factors known to favor plant cell recovery. Osmotic and abscisic acid treatments and desiccation
prior to inoculation did not have the same enhancement effect as desiccation during co-culture on T-DNA delivery in wheat.
An efficient transformation protocol has been developed based on desiccation and is suitable for both paromomycin and glyphosate
selection. Southern analysis showed approximately 67% of transgenic wheat plants received a single copy of the transgene. 相似文献
8.
9.
Preparing high-quality DNA from moss (Physcomitrella patens) 总被引:1,自引:0,他引:1
Physcomitrella patens, a moss, is the only land plant that performs high rates of homologous recombination, making it a valuable tool for functional
genomics. Unfortunately, commercially available plant DNA preparation kits are ineffective withPhyscomitrella. Furthermore, labor-intensive CTAB preparations produce low yields, and the DNA is degraded and contaminated. We present
a protocol that is faster and doubles the DNA yield obtained from standard procedures. The high-quality DNA prepared is suitable
for PCR reactions and Southern blot analysis. 相似文献
10.
Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100 总被引:1,自引:0,他引:1
Kwon HwangBo Su Hyun Son Jong Suk Lee Sung Ran Min Suk Min Ko Jang R. Liu Dongsu Choi Won Joong Jeong 《Plant biotechnology reports》2010,4(1):49-52
A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps.
We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient
PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing
plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging
the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic
tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines. 相似文献
11.
Petr Cápal Nicolas Blavet Jan Vrána Marie Kubaláková Jaroslav Doležel 《The Plant journal : for cell and molecular biology》2015,84(4):838-844
A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants. 相似文献
12.
A simple protocol for DNA isolation from dry roots ofBerberis lycium is described. Four-year-old dry roots are used, and the isolated DNA is suitable for analysis by means of restriction enzyme
digestion and random amplification of polymorphic DNA (RAPD). The method involves a modified CTAB procedure using 1% PVP to
remove polysaccharides and purification using low-melting-temperature agarose. DNA is amplified by means of PCR using 10-mer
random primers from Operon Biotechnologies, Inc. (USA), and DNA samples are digested withTaq I,Hind III andEcoR I and examined on agarose gels. The RAPD reaction is performed according to the 1990 protocol by Williams et al. 相似文献
13.
Miguel Keb-Llanes Gerardo González Bartolomé Chi-Manzanero Diógenes Infante 《Plant Molecular Biology Reporter》2002,20(3):299-299
This protocol permits the simultaneous extraction of clean DNA from many samples with little reagent waste, thus decreasing
the cost of analysis per sample. The procedure is rapid, permitting the processing of 80–100 samples per day. Using this protocol,
we analyzed naturally propagated and micropropagated populations of henequen and otherAgavaceae species using amplified fragment length polymorphism (AFLP). Agaves have succulent leaves with a content that is high in
fiber and chemical compounds. Therefore, this protocol should work for other tropical and subtropical plant species. The protocol
involves precipitation and resuspension of DNA 3 times at the end of the preparation; this increases DNA digestibility and
the sharpness of AFLP bands. 相似文献
14.
K. Balaraju P. Agastian S. Ignacimuthu Kyungseok Park 《Acta Physiologiae Plantarum》2011,33(6):2501-2510
An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used
for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in
vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA3). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on
Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron
(TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot
tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were
transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of
the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based
DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that
there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future. 相似文献
15.
Alejandro Nuño-Ayala Benjamín Rodríguez-Garay Antonia Gutiérrez-Mora 《Plant Cell, Tissue and Organ Culture》2012,109(1):33-39
It is reported for the first time an efficient protocol for indirect somatic embryogenesis and plant regeneration for Jarilla heterophylla (Caricaceae). Initial explants were mature zygotic embryos obtained from seeds collected in two regions of the state of Jalisco,
México (San Luis Soyatlán and Zacoalco de Torres). Two protocols for somatic embryogenesis used previously for Carica papaya were evaluated. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to induction medium A1 without the addition of adenine
hemisulphate yielded the best production of embryogenic callus with the higher total of somatic embryos per explant. With
this protocol the plant regeneration process took about five months. 相似文献
16.
17.
Studies in several plants have shown that Agrobacterium tumefaciens T-DNA can integrate into plant chromosomal DNA by different mechanisms involving single-stranded (ss) or double-stranded
(ds) forms. One mechanism requires sequence homology between plant target and ssT-DNA border sequences and another double-strand-break
repair in which preexisting chromosomal DSBs “capture” dsT-DNAs. To learn more about T-DNA integration in Solanum lycopersicum we characterised 98 T-DNA/plant DNA junction sequences and show that T-DNA left border (LB) and right border transfer is
much more variable than previously reported in Arabidopsis thaliana and Populus tremula. The analysis of seven plant target sequences showed that regions of homology between the T-DNA LB and plant chromosomal
DNA plays an important role in T-DNA integration. One T-DNA insertion generated a target sequence duplication that resulted
from nucleolytic processing of a LB/plant DNA heteroduplex that generated a DSB in plant chromosomal DNA. One broken end contained
a captured T-DNA that served as a template for DNA repair synthesis. We propose that most T-DNA integrations in tomato require
sequence homology between the ssT-DNA LB and plant target DNA which results in the generation of DSBs in plant chromosomal
DNA. 相似文献
18.
A method for isolation and purification of peanut genomic DNA suitable for analytical applications 总被引:2,自引:0,他引:2
Numerous methods are available for isolation of plant genomic DNA, but in practice these procedures are empirical due to variability
in plant tissue composition. Consistent isolation of quality DNA from peanut (Arachis hypogaea L.) is particularly problematic due to the presence of phenolic compounds and polysaccharides. Inconsistencies in extraction
results can be attributed to the age and growth stage of the plant material analyzed. Mature leaves have higher quantities
of polyphenols, tannins, and polysaccharides that can contaminate DNA during isolation. We show that four published protocols
could not be used to isolate peanut DNA of sufficient quality for PCR amplification or Southern hybridization. We have devised
a new protocol that uses DEAE-cellulose purification to isolate peanut DNA useful for downstream applications. 相似文献
19.
Suchita Kamble Hari S. Misra Suresh K. Mahajan Susan Eapen 《Plant Molecular Biology Reporter》2003,21(4):457-458
A protocol for efficient direct gene transfer by using particle gun bombardment was developed for mothbeanVigna aconitifolia L. Jacq. Marechal. Hypocotyl explants from 2 cultivars of mothbean were transformed with 3 plasmids: pBI121, pHS101, and
pHS102. Stable transformants were regenerated on MS medium supplemented with benzyladenine, α-naphthaleneacetic acid, and
kanamycin. The helium pressure, plasmid type, and cultivar that were used determined the stable transformation frequency.
Complete plants were regenerated and transferred to soil. The integration of the stable transgenes and reporter genes in plant
genomes was shown by means of PCR amplification of these genes from plant genomic DNA and Southern blot hybridization with
gene-specific probes. This method allows high-efficiency production of transgenic plants in mothbean.
Suchita Kamble and Hari S. Misra contributed equally to this work. 相似文献