12.3@ Light response
Protoplast regeneration in P. patens does not pass through a callus stage as occurs in seed plants. The regeneration comprises three sequential processes; cell wall synthesis, asymmetric protrusion and cell division. Cell wall formation occurs in the dark. By contrast, the subsequent two processes require light(1).
1. Isolate protoplasts and suspend in PRM/T.
2. Pour the suspended protoplasts onto a 9-cm Petri dish containing PRM/B medium overlaid with a cellophane.
3. Incubate at 25˚C for 1-3 days under continuous polarized white light (60 µmolm-2sec-1).@
Phototropic protonemal cell growth is mediated by phytochrome(2) and occurs under unilateral red light. A fluorescent light (lamp: FL20SD; Toshiba Lighting & Technology Corp., Tokyo) through a red plastic plate (Shinkolite A, #102; Mitsubishi Rayon Co., Tokyo)(3) is used as a red light source.
Chloroplast movement is induced by red light as well as blue light in P. patens(3). Chloroplasts move along both microtubules and actin filaments(4). In the gametophyte cells, chloroplasts exhibit dark positioning so that chloroplasts accumulate along cell walls, when the cells are placed in the dark for 1-2 days.@
@@@@@@@ A red acrylic plastic plate (Shinkolite A, #102; Mitsubishi Rayon Co., Tokyo) or a blue plastic film (Ryutate #63; RDS Corp., Tokyo) is used for red and blue light treatment. The colored light is polarized through a linear polarizer (HN22; Polaroid Corp. of Japan, Tokyo) for the analysis of chloroplast photo-movement.
@@@@@@@ Microbeam irradiation is performed with an special inverted microscope (TMD, Nikon, Tokyo) equipped with an epi-fluorescence unit. Monochromatic red light and blue light are obtained through the interference filters (É0 = 658.8 nm,É1/2 = 16.6 nm;É0 = 451.0 nm,É1/2 = 32.0 nm), respectively.
Red light (60 µmolm-2sec-1) grown protonemata without a branch are continuously irradiated by a stimulus white (60 µmolm-2sec-1) light for 1-2 days(5).
(1) Jenkins G.I., and Cove D.J. (1983) Planta 157, 39-45
(2) Mittmann F, Brücker G, Zeidler M, Repp A. et al. (2004) PNAS 101, 13939-13944
(3) Kadota A, Sato Y, Wada, M. (2000) Planta 210, 932-937
(4) Sato Y. Wada M. and Kadota A. (2001) J. Cell Sci. 114, 269-279
(5) Imaizumi T., Kadota A., Hasebe M., and Wada M. (2002) Plant Cell 14, 373-386