7. Effects of drugs on moss development
7.1
Hormone treatments
Auxin, cytokinin, and abscisic acid affect
gametophytic differentiation of several mosses. Ethylene has recently reported to have effects on P. patens development (Fujiwara et al. 2003,
BSJ annual meeting). On the other hand,
hormone effects on sporophytic development are mostly unknown.
Materials:
You
should use freshly propagated materials for the following experiments to get
stable results. Never use old materials.
Treatments:
(A) liquid
medium supplemented with hormones
1. Make aliquots of hormone medium into 24-well plate
BCDATG
BCDATG+
1 µM NAA (1-naphtaleneacetic acid)
BCDATG+ 1
µM BA (6-Benzylaminopurine)
BCDATG+
100 µM GA3 (Gibberellin A3)
BCDATG+ 20
µM ABA (Abscisic acid)
BCDATG+
100 µM NPA (1-N-naphthylphthalamic acid: auxin transport inhibitor)
2.
Put protonemata into the solution, seal the plate with parafilm to avoid drying
the solution, and incubate for about a week.
3. Results;
GA3 and
NPA did not show any significant morphological changes.
(B) solid medium supplemented with hormones
1.
Make solid agar plate supplemented with hormones
2.Results;
Auxin
Culture
protonemata in BCD plate with 1uM NAA
à Caulonema-like, or rhizoid-like
protonemata differentiate within a week.
Cytokinin
Culture
protonemata in BCD plate with 0.1uM BA
à Clusters of cells differentiate at
side branches of protonemata within a week.
Combination of auxin and cytokinin
Phenotypes
depend on the ratio of hormones.
à An apical cell of protonemata
becomes enlarged and likely loses polarity.
Polar growth of the apical cells seems to be lost. See pictures below.
Clusters of bud-like cells and apolar apical cells (a magnified picture in the right, and a lower panel) were induced with the supplementation of NAA 0.5µM & BA 50µM.
ABA
Culture
protonemata in a plate with 0.5-50uM BA
à Cell elongation will be
suppressed and brood cell-like cells will be observed. At a treatment of 50uM of ABA, most cells should
cease cell division.