John H. Miller

Fern gametophytes as experimental material

I n t r o d u c t i o n ................................................................................................................................................................................................ 362 C o n d i t i o n s f o r S p o r e G e r m i n a t i o n .................................................................................................................................... 363 C r i t e r i a f o r g e r m i n a t i o n ................................................................................................................................................... 363 W a t e r ...................................................................................................................................................................................................... 363 T e m p e r a t u r e .................................................................................................................................................................................... 364 M i n e r a l n u t r i e n t s ..................................................................................................................................................................... 364 p H ............................................................................................................................................................................................................... 364Effec ts of specif ic c h e m i c a l s .......................................................................................................................................... 364 Bio t i c f a c t o r s ................................................................................................................................................................................. 365 L i g h t ......................................................................................................................................................................................................... 366 Effec t s o f u l t r a v i o l e t a n d i o n i z i n g i r r a d i a t i o n .......................................................................................... 375 S p o r e V i a b i l i t y ....................................................................................................................................................................................... 375 C y t o l o g y of G e r m i n a t i o n .............................................................................................................................................................. 376 D e t e r m i n a t i o n of S p o r e P o l a r i t y .......................................................................................................................................... 376 F a c t o r s I n f l u e n c i n g V e g e t a t i v e G r o w t h ........................................................................................................................ 378 G e n e r a l c u l t u r a l c o n d i t i o n s .......................................................................................................................................... 379 M i n e r a l n u t r i e n t s ..................................................................................................................................................................... 380 G r o w t h on o r g a n i c n i t r o g e n .......................................................................................................................................... 381 Effec ts of specif ic c h e m i c a l s ............................................................................................................................................. 382 Bio t i c f a c t o r s .............................................................. ........... Y. .................................................................................................. 386 L i g h t ......................................................................................................................................................................................................... 386 Ef fec t s of u l t r a v i o l e t a n d i o n i z i n g i r r a d i a t i o n .......................................................................................... 392 T w o d i m e n s i o n a l g r o w t h , p r o t e i n a n d n u c l e i c a c i d m e t a b o l i s m .......................................... 392 P o l a r i t y a n d D o r s i v e n t r a l i t y ................................................................................................................................................... 397 R e g e n e r a t i o n ............................................................................................................................................................................................. 400 T i s s u e C u l t u r e a n d A b n o r m a l G r o w t h ........................................................................................................................ 404 D i f f e r e n t i a t i o n of Sex O r g a n s ................................................................................................................................................ 406 B i s e x u a l i t y .......................................................................................................................................................................................... 406 C u l t u r a l c o n d i t i o n s .................................................................................................................................................................. 407 H o r m o n a l c o n t r o l o f a n t h e r i d i u m d i f f e r e n t i a t i o n ................................................................................. 409 Re lease a n d A c t i v i t y of S p e r m a t o z o i d s ........................................................................................................................ 417 P o l y e m b r y o n y .......................................................................................................................................................................................... 420 Spec ia l A s p e c t s of C y t o l o g y ...................................................................................................................................................... 421 G e n e t i c s ............................................................................................................................................................................................................ 423 A p o g a m y a n d A p o s p o r y .............................................................................................................................................................. 424 A c k n o w l e d g m e n t s ................................................................................................................................................................................. 4.26 R e f e r e n c e s ...................................................................................................................................................................................................... 426

Rhizoid Differentiation in Fern Spores: Experimental Manipulation

Germination in spores of the fern Onoclea sensibilis is initiated by an asymmetric division that partitions the spore into two cells of unequal size. The unequal daughter cells differentiate immediately into distinct types. When spores are germinated on the surface of solutions of methanol, the initial division is symmetrical, and the daughter cells from this equal division develop into the same type of cell. The differentiation of a rhizoid from the smaller cell in untreated spores is suppressed by methanol treatment.

Inhibition of Onoclea sensibilis Spore Germination by Far-red Light and Cis-4-Cyclohexene-1,2-dicarboximide

Summary Red light-induction of Onoclea sensibilis spore germination has been reversed by far-red light, revealing the photoreceptive role of phytochrome. Cis-4-cyclohexene-1,2-dicarboximide, which inhibits processes involved with phytochrome-mediated seed germination, appears mainly to inhibit non-phytochrome-related processes in Onoclea spore germination.

Isolation of rhizoid and prothallial protoplasts from gametophytes of the fern, Onoclea sensibilis

Abstract Methods are described for the selective isolation of protoplasts from rhizoids and prothalial cells of Onoclea sensibilis gametophytes. A cellulase-resistant component in the outer prothallial cell walls necessitated a combined m mechanical-enzymatic procedure to obtain prothallial protoplasts. Rhizoid protoplasts were obtained through the dissolved tips of young rhizoid walls upon brief cellulase treatment of germinated spores. These methods facilitate the independent study of distinct fern cell types which are on divergent morphogenic pathways in vivo.

The use of alizarin red S to detect and localize calcium in gametophyte cells of ferns.

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KCl to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.

Asymmetric cell division and differentiation; fern spore germination as a model. I. Physiological aspects

During germination of Onoclea sensibilis spores, the spore nucleus moves from a central location to one end. Cell division partitions the spore into a small cell which differentiates into a rhizoid, and a larger cell which gives rise, by continued division, to the prothallus. Spore germination is a valuable system in which to study the relationship between asymmetric cell division and the initiation of cell differentiation. It appears that cytoplasmic microtubules and some lipophilic site in the spore are both involved in premitotic nuclear migration. Asymmetric cell division is an obligate step in the initiation of rhizoid differentiation. One hypothesis is that the nucleus must be confined to a small, localised region of the spore for a sufficient time before differentiation can occur, and the confinement is accomplished by the asymmetric cell division. Metal-binding sites are present in the spore coat, specifically on the proximal face. Several types of evidence suggest that the metal-binding region is involved in spore polarity. The sequence of events during germination appears to involve both polarity which is inherent in the spore and polarity which may be imposed by external stimuli. Experiments in which spores are treated with colchicine and polarised red light reveal both types of polarity.