Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis |
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Authors: | B R Shrestha K Tokuhara M Mii |
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Institution: | (1) Faculty of Horticulture, Chiba University, 684 Matsudo, Chiba 271-0092, Japan;(2) Research and Development Center, Orchid Santuary Dogashima, Nishina, Nishiizu-cho, Kamo-gun Shizuoka, 410-35, Japan |
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Abstract: | Protoplasts isolated from cell suspension culture of Phalaenopsis “Wataboushi” were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing
0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l l-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which
were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the
standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency
of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two
weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible
to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free
NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The
PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to
greenhouse conditions. |
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Keywords: | Beads method Calcium alginate Phalaenopsis Protoplasts Standard method |
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