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KMID : 0903619950360050614
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Journal of the Korean Society for Horticultural Science
1995 Volume.36 No. 5 p.614 ~ p.619
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Protoplast Isolation , Fusion , and Culture in Garlic (Allium Sativum L .
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Abstract
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Protoplasts were isolated from callus derived from the pedicel of ´Namdo´ and from leaf tissue of ´A1maAta´, and fused by polyethylene glycol (PEG) solution. When treated with the enzyme solution of 1% Macerozyme R-10 and 0.4% Cellulase RS, callus from the pedicel of ´Namdo´ yielded the highest number of isolated protoplasts (2.6×10^6 protoplasts/㎖), while, the number of protoplasts obtained from the leaf tissue of ´AlmaAta´ was highest (1.1×10^6 protoplasts/㎖) when the leaf tissue was treated with an enzyme solution of 0.3% Macerozyme R-10, 0.3% Hemicellulase and 3-4% Cellulase R-10 adjusted to pH 5.3. The optimal time required for enzyme solution treatment to produce these results were 5 hours for the callus obtained from the pedicel and 4 hours for the leaf tissue, respectively. The frequency of protoplast fusion was highest when both protoplasts were fused with PEG solution having a molecular weight of 3,350 at 30 for 20 minutes. The fusion solution was gradually diluted with an eluting solution adjusted to 10.5 for 20 min at an interval of 5 min. The fused protoplasts were cultured afterwards in the dark at a temperature of 25℃ in a medium containing 2,4-D, NAA, BAP, kinetin, zeatin or their combination. Cell division began after 3-4 days of culture, but these cells lost their capability to divide after two to three times of division. Budding of cell took place in culture with liquid medium, while an unbalanced division happended in agarose block culture.
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