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- Author or Editor: Sandip Mukhopadhyay x
Transient expression of electroporation-mediated DNA uptake was monitored in callus-derived protoplasts of two asparagus (Asparagus offcinalis L.) genotypes by measuring the GUS activity. The level of expression and the viability of the protoplasts were influenced by the voltage and duration of the electric pulse. An increased plasmid DNA concentration and the presence of polyethylene glycol (PEG) in the electroporation medium enhanced the transient expression level. A considerable increase in GUS activity was observed in the presence of both PEG and heat-shock treatments than with PEG treatment alone. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 94 ms duration. The two genotypes differed in their responses in vitro and also showed variable levels of transient expression. The present technique was suitable to obtain transgenic plants, as histochemical GUS assay revealed GUS activity in the protoplast-derived microcolonies as well as in callus tissues.
The effects of culture media, culture modes, and carbon sources on plating efficiencies of protoplasts of two genotypes of Asparagus officinalis L. were investigated. Protoplasts grew best in a semisolid culture system containing half-strength MS medium with 1 mg NAA/liter, 0.5 mg zeatin/liter, 0.6 M glucose, and 0.1% Gelrite. The plating efficiencies were 12.5% and 8.1% for genotypes G 203 and G 171, respectively. Embryogenic calli were produced from protoplast-derived microcalli after culturing on MS medium with 1 mg 2,4-D, 3% sucrose, and 0.2% Gelrite. The somatic embryos were initiated, matured, and then germinated to plantlets in MS medium containing 0.1 mg NAA/liter, 0.3 mg 2-iP/liter (EMM), and different levels of carbohydrates. Transfer of somatic embryos from EMM with 10% glucose to EMM containing 2% sucrose produced the highest number of bipolar embryos and plantlets. The plantlets regenerated shoots and roots in MS medium with 3% sucrose, 0.1 mg NAA/liter, 0.1 mg kinetin/liter, and 1.28 mg ancymidol/liter. Cytological analysis of these plants revealed 2n = 20 chromosomes.