Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc) |
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Authors: | Sung Ho Son Sung Mee Choi Kum Boo Choi Yun Hee Lee Dea Sook Lee Myung Suk Choi Young Goo Park |
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Institution: | (1) Division of Biotechnology, Forestry Research Institute, Forestry Administration, 44-3 Omokcheon-dong, 441-350 Suwon, Korea;(2) Plant Biotechnology Institute, Dongbu Research Council, 103-2 Moonjidong, Daeduck Science Town, 305-380 Taejon, Korea;(3) Department of Forestry, Kyungpook National University, 702-701 Taegu, Korea |
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Abstract: | Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14
elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented
with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS
vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed
on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more
than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first
round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent
proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell
and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth
and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV
seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the
tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth. |
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Keywords: | yew tree embryo callus culture visual selection single cell cloning fed batch culture balloon type bubble bioreactor |
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