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#plant culture techniques

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Plant tissue culture 1

Most widely used plant tissue culture medium is:

Murashige and Skoog medium proposed in 1962 for tobacco pith culture remains gold standard and most widely used basal medium for plant tissue culture due to broad applicability and high salt formulation supporting rapid growth across herbaceous and many woody species. Its composition features high concentrations of macronutrients ammonium nitrate 1650 mg per L and potassium nitrate 1900 mg per L providing dual nitrogen source favoring both amino acid synthesis and pH buffering, plus calcium chloride, magnesium sulfate, potassium dihydrogen phosphate; chelated iron as Fe-EDTA, micronutrients manganese, zinc, boron, iodine, molybdenum, copper, cobalt; organic supplements thiamine, nicotinic acid, pyridoxine, glycine, myo-inositol; and sucrose 30 g per L as carbon source. High ammonium to nitrate ratio about 1 to 2 stimulates chlorophyll retention and proliferation rather than rooting. Ionic strength around 90 mM supports vigorous meristematic activity. While alternatives like B5 for protoplasts or WPM for trees exist, full strength MS with modifications half strength for rooting remains default choice referenced in textbooks, commercial laboratories and research protocols worldwide for callus induction, organogenesis, embryogenesis and protoplast culture.

Ref: Murashige & Skoog Physiol Plant 1962; NCBI NBK26873 MS medium composition.

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Plant tissue culture 1

Ideal aseptic condition means:

Ideal aseptic condition means complete absence of viable bacteria, fungi, yeast, algal spores and viruses from all components including culture vessel, nutrient medium, explant surface and environmental air, producing axenic system. Tissue culture media enriched with 30 grams per liter sucrose, vitamins, amino acids and growth regulators represent perfect substrate for microbial proliferation; even few contaminants rapidly multiply outcompeting slow-growing plant cells for nutrients, alter pH through fermentation, release phytotoxic metabolites like hydrogen sulfide, enzymes degrading plant cell walls and kill cultures within days. Achieving asepsis requires rigorous sterilization: medium autoclaving 121 degrees 15 psi 15-20 minutes, tool flaming and glass bead sterilizer, filter sterilization of heat-labile hormones through 0.22 micron membranes, operation inside laminar air flow cabinet providing HEPA filtered ISO Class 5 air, and personnel wearing sterile gloves, lab coats, and following aseptic technique movements. Low bacterial count is not acceptable because residual microbes proliferate, so criterion is zero detectable colony forming units on nutrient agar plates. Maintaining asepsis ensures morphogenetic responses reflect plant physiology rather than microbial interference.

Ref: CDC Guidelines for Aseptic Technique; Bhojwani Plant Tissue Culture manual, Chap. 2.

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Plant tissue culture 1

Surface sterilization of explants commonly uses:

Surface sterilization removes epiphytic and surface-borne microorganisms from explants without killing plant tissues, essential prerequisite for establishing contaminant-free cultures. Most effective protocol uses sequential treatment with 70 percent ethanol for 30-60 seconds followed by sodium hypochlorite solution 0.5-2 percent available chlorine for 10-20 minutes containing surfactant Tween-20 0.1 percent to improve wetting. Ethanol acts as degreasing agent dissolving epicuticular waxes, disrupting microbial lipid membranes and denaturing proteins facilitating penetration of subsequent sterilant. Sodium hypochlorite dissociates in water to hypochlorous acid HOCl strong oxidizer chlorinating amino groups of proteins, oxidizing sulfhydryl groups, nucleotides, and causing lethal damage to bacterial cell walls and fungal spores. Concentration and exposure time optimized for explant type, tender meristems need shorter treatment to avoid phytotoxicity bleaching. Autoclaving at 121 degrees would kill explant cells, dry heat similarly destructive, UV radiation alone has poor penetration into crevices and causes DNA mutations. Repeated sterile distilled water washes after sterilization remove residual chlorine preventing growth inhibition in nutrient medium.

Ref: Bhojwani & Dantu Plant Tissue Culture, Chap. 3 sterilization; NCBI StatPearls Aseptic Technique.

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