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Plant Science

Categories
+ Phycology
+ Plant biochemistry
+ Plant breeding
+ Plant cell biology
+ Plant developmental biology
+ Plant immunity
+ Plant metabolism
- Plant molecular biology
Chromatin
- DNA
DNA extraction
DNA modification
DNA recombination
DNA sequencing
DNA structure
Gene expression
Genotyping
Mutagenesis
Genetic analysis
Protein
+ RNA
+ Plant physiology
+ Plant transformation
Protocols in Past Issues

Optimized Protocol for DNA Extraction in Three Theobroma Species
AR Angie F. Riascos-España
BC Brayan A. Toro Cuastumal
MZ María I. Castro Zambrano
JA Juan C. Zambrano Arteaga
Pedro A. Velasquez-Vasconez Pedro A. Velasquez-Vasconez
Q&A 0 Q&A
2283 Views
May 5, 2025

DNA extraction is a crucial step in molecular biology research, particularly for genetic and genomic analyses. These studies require a high concentration of high-quality DNA, which is often a challenge for underexplored species or when the available plant material consists of aged tissue. To address these challenges, the cetyltrimethylammonium bromide (CTAB)-based DNA extraction method has been optimized to improve efficiency and yield. The process begins with an overnight incubation of plant tissue macerated with liquid nitrogen in a solution containing a high concentration of CTAB (4%). Subsequently, the mixture undergoes two washes with chloroform: isoamyl alcohol. The nucleic acids are then precipitated using isopropanol, followed by a wash with 70% ethanol to ensure purity. Finally, the purified DNA is resuspended in ultrapure water. This optimized procedure produces high-quality DNA suitable for various downstream applications, including PCR and sequencing, even from older leaves of the three Theobroma species: T. cacao, T. bicolor, and T. grandiflorum. Additionally, this protocol significantly enhances throughput and allows for the parallel processing of a substantially larger number of samples compared to conventional techniques.

Quantification of Botrytis cinerea Growth in Arabidopsis thaliana
PS Patricia Scholz
KC Kent D. Chapman
TI Till Ischebeck
AG Athanas Guzha
Q&A 1 Q&A
2994 Views
Aug 20, 2023

Yield losses attributed to plant pathogens pose a serious threat to plant productivity and food security. Botrytis cinerea is one of the most devastating plant pathogens, infecting a wide array of plant species; it has also been established as a model organism to study plant–pathogen interactions. In this context, development of different assays to follow the relative success of B. cinerea infections is required. Here, we describe two methods to quantify B. cinerea development in Arabidopsis thaliana genotypes through measurements of lesion development and quantification of fungal genomic DNA in infected tissues. This provides two independent techniques that are useful in assessing the susceptibility or tolerance of different Arabidopsis genotypes to B. cinerea.


Key features

• Protocol for the propagation of the necrotrophic plant pathogen fungus Botrytis cinerea and spore production.

• Two methods of Arabidopsis thaliana infection with the pathogen using droplet and spray inoculation.

• Two readouts, either by measuring lesion size or by the quantification of fungal DNA using quantitative PCR.

• The two methods are applicable across plant species susceptible the B. cinerea.


Graphical overview



A simplified overview of the droplet and spray infection methods used for the determination of B. cinerea growth in different Arabidopsis genotypes

Safe DNA-extraction Protocol Suitable for Studying Tree-fungus Interactions
SK Susanna Keriö
ET Eeva Terhonen
JL Jared M. LeBoldus
Q&A 0 Q&A
8173 Views
Jun 5, 2020
We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host material (Picea abies, Populus trichocarpa, Pseudotsuga menziesii) infected with different pathogens (Heterobasidion annosum, Heterobasidion parviporum, Leptographium wagenerii, Sphaerulina musiva). Additionally, DNA was extracted from pure cultures of the pathogens and several endophytic fungi. PCR success rate was 100% for young poplar material and fungal samples, and 48-72% for conifer and mature broadleaved plant samples. We recommend using 10-50 mg of fresh sample for the best results. The method offers a safe and low-cost DNA extraction alternative to study tree-fungus interactions, and is a potential resource for teaching purposes.

An Optimized CTAB Method for Genomic DNA Extraction from Freshly-picked Pinnae of Fern, Adiantum capillus-veneris L.
SY Yi Shu
WJ Jin Wan-Ting
YY Yuan Ya-Ning
YF Fang Yu-Han
Q&A 0 Q&A
18192 Views
Jul 5, 2018
As the sister clade of seed plants, ferns are significant materials for plant phylogeny research. However, the genomic DNA extraction protocol for fern samples like modified CTAB method still lacks robustness. Here, we found that the amount and condition of the pinnae samples are critical for gDNA extraction in fern, Adiantum capillus-veneris L. In 500 μl CTAB solution, the recommended amount of pinnae is about 10-20 mg (2-3 pieces). The condition of the pinnae must be instantly-picked from a plant cultivated in a suitable environment. With these factors under control, it is highly reproducible to get the high-quality gDNA with low degradation rate

Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes
MB Moritz Bömer
AT Aliyu A. Turaki
AR Ajith I. Rathnayake
GS Gonçalo Silva
PL P. Lava Kumar
SS Susan E. Seal
Q&A 0 Q&A
10476 Views
Jan 5, 2018
Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses (Bömer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel badnavirus genomes.

Determining Genome Size from Spores of Seedless Vascular Plants
LK Li-Yaung Kuo
YH Yao-Moan Huang
Q&A 0 Q&A
8923 Views
Jun 5, 2017
Seedless vascular plants, including ferns and lycophytes, produce spores to initiate the gametophyte stage and to complete sexual reproduction. Approximately 10% of them are apomictic through the production of genomic unreduced spores. Being able to measure the spore nuclear DNA content is therefore important to infer their reproduction mode. Here we present a protocol of spore flow cytometry that allows an efficient determination of the reproductive modes of seedless vascular plants.

Experimental Pipeline for SNP and SSR Discovery and Genotyping Analysis of Mango (Mangifera indica L.)
MS Michal Sharabi-Schwager
MR Mor Rubinstein
MI Mazal Ish shalom
RE Ravit Eshed
AR Ada Rozen
AS Amir Sherman  [...]
Ron Ophir Ron Ophir
+ 1 Author
Q&A 0 Q&A
11702 Views
Aug 20, 2016
Establishing a reservoir of polymorphic markers is an important key for marker-assisted breeding. Many crops are still lack of such genomic infrastructure. Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are useful as markers because they are widespread over the genome and many technologies were developed for high throughput genotyping. We present here a pipeline for developing a reservoir of SNP and SSR markers for Mangifera indica L. as an example for fruit tree crops having no genomic information available. Our pipeline includes de novo assembly of reference transcriptome with MIRA and CAP3 based on reads produced by 454-GS FLX technology; Polymorphic loci discovery by alignment of Illumina resequencing to the transcriptome reference; Identifying a subset of loci that are polymorphic in the entire germplasm collection for downstream diversity analysis by genotyping with Fluidigm technology.

Genomic DNA Extraction and Genotyping of Dictyochloropsis Green Algae Strains
Francesco Dal Grande Francesco Dal Grande
Carolina Cornejo Carolina Cornejo
CS Christoph Scheidegger
Andreas Beck Andreas Beck
Q&A 0 Q&A
13300 Views
Aug 5, 2015
Dictyochloropsis is an ecologically important genus of free-living and symbiotic green algae. Representatives of this genus are horizontally transmitted among several fungi of the family Lobariaceae, thus forming photobiont-mediated guilds. This protocol is suitable for extracting DNA from algal cultures and lichen samples and for genotyping seven unlinked Dictyochloropsis reticulata microsatellite markers in a single PCR multiplex.


Figure 1. Schematic representation of the analysis pipeline