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