ON THE COVER

Vol. 76 No.2 June 2011

Technical note

     Isolation of Intact Gametophytic Protoplasts from Torenia and Lindernia Species

     The embryo sac plays an essential role in plant sexual reproduction. Because the embryo sac is deeply embedded and protected in the sporophytic mother tissues, it is difficult to access the embryo sac and analyze its function. Some plant species in the genus Torenia and Lindernia have unique ovule morphology: half of the embryo sac is protruding from the ovule (see Kawano et al., in this issue). Here we attempted to isolate intact gametophytic cells in the embryo sac of Lindernia micrantha by enzyme treatment. A mature ovary was dissected and the ovules were placed in 300 µl of the cellulase enzyme solution. These were incubated at 28°C for 1 h to generate protoplasts. A cluster of gametophytic protoplasts, which contains one central cell, one egg cell and two synergid cells, was separated from sporophytic tissue of an ovule with no need of further manipulation, making it easy to avoid contamination of other type of cells. Each type of cells were distinguished by their morphology and then successfully collected using a PicoPipet (Altair Co., Japan). This method will be useful to obtain intact gametophytic cells in plants. Scale bar, 20µm.

     (Masahiro M. Kanaoka, Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464.8602, Japan.)

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ON THE COVER

Vol. 76 No.2 June 2011
Technical note

Isolation of Intact Gametophytic Protoplasts from Torenia and Lindernia Species The embryo sac plays an essential role in plant sexual reproduction. Because the embryo sac is deeply embedded and protected in the sporophytic mother tissues, it is difficult to access the embryo sac and analyze its function. Some plant species in the genus Torenia and Lindernia have unique ovule morphology: half of the embryo sac is protruding from the ovule (see Kawano et al., in this issue). Here we attempted to isolate intact gametophytic cells in the embryo sac of Lindernia micrantha by enzyme treatment. A mature ovary was dissected and the ovules were placed in 300 µl of the cellulase enzyme solution. These were incubated at 28°C for 1 h to generate protoplasts. A cluster of gametophytic protoplasts, which contains one central cell, one egg cell and two synergid cells, was separated from sporophytic tissue of an ovule with no need of further manipulation, making it easy to avoid contamination of other type of cells. Each type of cells were distinguished by their morphology and then successfully collected using a PicoPipet (Altair Co., Japan). This method will be useful to obtain intact gametophytic cells in plants. Scale bar, 20µm. (Masahiro M. Kanaoka, Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464.8602, Japan.)
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