3.4@ Light response
Protoplast
regeneration in P. patens does not pass the callus stage as that in seed
plants. The regeneration includes three sequential processes; the cell wall
synthesis, the asymmetric protrusion and the cell division. Cell wall formation
occurs in the dark. On the other hand, the subsequent two processes require
light1).
1. Isolate
protoplasts and suspend in PRM/T (see chapter 9).
2. Pour the
suspended protoplasts onto a 9-cm Petri dish containing PRM/B medium overlaid
with a cellophane.
3. Incubate at at
25˚C for 1-3 days under continuous polarized white light (15 Wm-2).@
Phototropic
protonemal cell growth is mediated by phytochrome2) 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 protonemal cells
cultured under red light (0.5 Wm-2), while the effective wavelength
of chloroplast movement is limited to blue light region under white light (5 Wm-2)
likely as other many plant species3). Chloroplasts move along both
microtubules and actin filaments4). In the gametophyte cells,
chloroplasts exhibit dark positioning 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
(0.5 Wm-2) grown protonemata without a branch are continuously
irradiated by a stimulus white (?)light for 1-2 days5).
(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