Category: Molecular Botany

Challenges with plants Plant cells contain polysaccharides, phenolics, and cellulose that interfere with DNA quality. Protocol choice depends on tissue type (leaf, seed, woody stem). CTAB method overview Grind frozen tissue in CTAB buffer with high salt. Incubate at 65°C to lyse cells and denature proteins. Extract with chloroform-isoamyl alcohol to separate phases. Precipitate DNA…

Polymerase Chain Reaction PCR amplifies specific DNA regions using template, primers, nucleotides, and heat-stable polymerase. Thermal cycling: denaturation (~95°C), annealing (~55–65°C), extension (~72°C). Primer design Primers flank target sequence (typically 18–25 bp each). Specificity prevents amplifying wrong regions. BLAST primers against genome to check off-target binding. Gel electrophoresis DNA migrates through agarose matrix toward positive…

Light as a signal Phytochromes (red/far-red) and cryptochromes (blue light) trigger massive transcriptional reprogramming when seedlings emerge from soil or when shade is detected. Key regulated processes Chlorophyll biosynthesis genes Photosystem assembly (PSA, PSB genes) Carbon fixation enzymes (Rubisco small subunit) Shade avoidance (elongation genes under low R:FR ratio) Experimental approaches qPCR measures single-gene expression;…

CRISPR-Cas9 mechanism Guide RNA directs Cas9 enzyme to complementary genomic sequence. Cas9 creates double-strand break; cell repair introduces mutations (knockouts) or templated edits. Applications in crop science Knockout susceptibility genes for disease resistance Reduced polyphenol oxidase to limit browning Modified flowering time for climate adaptation Oil composition changes in oilseed crops Regulatory landscape Some jurisdictions…

From RNA to insight RNA-seq captures snapshot of expressed genes. Useful for comparing healthy vs stressed plants, or tissue types (root vs leaf). Workflow summary Extract high-quality RNA (RNase-free environment). Library preparation and sequencing on NGS platform. Align reads to reference genome or assemble de novo. Statistical analysis for differential expression (fold-change, p-values). Functional follow-up…

Proteins as phenotype While DNA is potential, proteins execute function. SDS-PAGE separates proteins by size; Western blot detects specific proteins with antibodies. Sample preparation Grind tissue in buffer with protease inhibitors. Clear extracts by centrifugation. Quantify protein concentration before loading equal amounts per lane. Applications in botany Stress-induced enzyme levels (e.g., antioxidant enzymes) Storage protein…