7.1　 Hormone treatments

Tomomichi Fujita

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.