7.1 Hormone
treatments
Auxin, cytokinin, and abscisic acid affect gametophytic
differentiation on several mosses.
Ethylene is 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 lose polarity. Polar growth
of the apical cells seems to be lost.
See pictures below.
Cluster of bud-like cells and apolar apical cell (a
magnified picture in the right, and a lower panel) were induced with the
supplementation of NAA0.5 µM&BA50 µM.
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 of cells should cease cell division.