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PEG-mediated symmetric and asymmetric protoplast fusion in sunflower (Helianthus annuus L.)

Chitpan Kativat and Piyada Alisha Tantasawat

Symmetric and asymmetric protoplast fusions were evaluated with sunflower (Helianthus annuus L.) PI441983 and 10A lines. The optimal cytoplasmic inactivation procedure and conditions for induced fusion of protoplasts by using polyethylene glycol (PEG) were developed. The cell division activities of hypocotyl protoplasts of the 10A line with a cytoplasmic male sterile (CMS) trait were inhibited with different concentrations of iodoacetic acid (IOA; 0, 1.5, 3.0 and 4.5 mM) and incubation periods (15 and 20 min) to generate a recipient parent for a normal cytoplasm trait. The optimal inactivation was achieved with 20 min incubation in 1.5 mM IOA, which was the lowest concentration leading to low levels of both cell division (20.41%) and colony formation (3.70%). When various concentrations of PEG 8000 (0, 10, 20 and 30% (w/v)) and fusion periods (10, 15 and 20 min) were used to induce fusion between hypocotyl protoplasts of the 10A line and mesophyll protoplasts with a normal cytoplasm trait of the PI441983 line (donor parent), 20% (w/v) PEG 8000 for 15 min was found to be optimal for induced fusion, giving a high frequency of binary fusion (26.16%) and lowfrequency of multi fusion (12.96%). When both symmetric and asymmetric protoplast fusion procedures were performed, fusion products could develop, divide and form colonies in the culture medium, and also have a tendency to generate microcalli. However, the densities of protoplast-derived colonies in asymmetric fusion were lower than those in symmetric fusion. The efficient procedures developed in this study will be beneficial for future sunflower breeding programs for hybrid production

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