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The phosphate requirement of the ferns Notholaena cochisensis and Notholaena sinuata Item Type text; Thesis-Reproduction (electronic) Authors Ferrell, Cleon Duane, 1941- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 13/05/2018 01:07:30 Link to Item http://hdl.handle.net/10150/551800
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The phosphate requirement of the fernsNotholaena cochisensis and Notholaena sinuata
Item Type text; Thesis-Reproduction (electronic)
Authors Ferrell, Cleon Duane, 1941-
Publisher The University of Arizona.
Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.
Download date 13/05/2018 01:07:30
Link to Item http://hdl.handle.net/10150/551800
THE PHOSPHATE REQUIREMENT OF THE FERNS
NOTHOLAENA COCHISENSIS AND NOTHOLAENA SINUATA
by
Cleon Duene Ferrell
A T h e s is S u b m itte d t o th e
COMMITTEE ON AGRICULTURAL BIOCHEMISTRY AND NUTRITION
I n P a r t i a l F u l f i l l m e n t o f th e R e q u ire m e n ts
F o r th e D eg ree o f
MASTER OF SCIENCE
I n th e G ra d u a te C o l le g e
THE UNIVERSITY OF ARIZONA
1965
V
STATEMENT BY AUTHOR
T h is t h e s i s h a s b e e n s u b m it te d i n p a r t i a l f u l f i l l m e n t o f r e q u i r e m e n ts f o r an ad v an ced d e g re e a t The U n i v e r s i t y o f A r iz o n a and i s d e p o s i t e d i n th e U n i v e r s i t y L ib r a r y t o b e made a v a i l a b l e t o b o r ro w e rs u n d e r r u l e s o f t h e L ib r a r y .
B r i e f q u o ta t io n s from t h i s t h e s i s a r e a l lo w a b le w i th o u t s p e c i a l p e r m is s io n , p r o v id e d t h a t a c c u r a te acknow ledgm ent o f s o u rc e i s m ade. R e q u e s ts f o r p e r m is s io n f o r e x te n d e d q u o ta t io n from o r r e p r o d u c t io n o f t h i s m a n u s c r ip t i n w ho le o r i n p a r t may b e g r a n te d b y th e h e a d o f th e m a jo r d e p a r tm e n t o r t h e Dean o f t h e G ra d u a te C o l le g e when i n h i s ju d g m en t t h e p ro p o s e d u s e o f t h e m a t e r i a l ife i n t h e i n t e r e s t s o f s c h o la r s h i p . I n a l l o th e r i n s t a n c e s , h o w ev er, p e rm is s io n m u st b e o b ta in e d from t h e a u th o r .
SIGNED:
APPROVAL BY THESIS DIRECTOR
T h is t h e s i s h a s b e e n a p p ro v e d on th e d a te shown b e lo w :
~ A r th u r r . KemmererP r o f e s s o r o f A g r i c u l t u r a l B io c h e m is try
ii
ACKNOWLEDGMENTS
The a u th o r e x p re s s e s s in c e r e g r a t i t u d e :
To D r. E . B. K u r tz , J r . f o r th e t o p i c o f t h i s t h e s i s .
' To D rs . A. R. Kem m erer, K. M a tsu d a , and M. Z a i t l i n f o r
c o u n s e l and g u id a n c e d u r in g th e c o u rs e o f t h i s s tu d y and i n
p r e p a r a t i o n o f t h i s t h e s i s .
iii
TABLE OF CONTENTS
Page
INTRODUCTION .................................................................................................................... 1
REVIEW OF LITERATURE.......................................................... 2
EXPERIMENTAL METHODS................................................................................................. 4
RESULTS AND DISCUSSION ............................................................................................ 18
SUMMARY.............................................................................................................................. 31
LITERATURE C IT E D ...................................... 32
iv
LIST OF TABLES
T a b le Page
I . C om parison o f t o t a l s a l t and io n c o n c e n t r a t io n s o fth e n u t r i e n t m ed ia ( m m o l e s / l i t e r ) ............................................ .... . 11
I I P h o sp h a te c o n te n t o f n u t r i e n t m ed ia (m M )....................................... l 4
v
LIST OF FIGURES
F ig u re Page
1 S ta n d a rd c u rv e f o r p h o s p h a te a n a ly s e s b y th e K i t s o n -M ello n m e t h o d ............................................................................................... 13
2 The f r e s h w e ig h t o f N. c o c h i s e n s i s grow n on v a r io u sam ounts o f p h o s p h a te ............................................................................................. 2b
3 The f r e s h w e ig h t o f N. s i n u a t a grow n on v a r io u sam ounts o f p h o s p h a te . . . . . ................................................................... 25
4 The f r e s h w e ig h t o f N. c o c h i s e n s i s grown on m ed ia c o n ta in in g a d e f i c i e n c y o r an e x c e s s o f p h o s p h a teo r s u l f a t e ......................................................................................................... 26
5 The f r e s h w e ig h t o f N. s i n u a t a grown on m ed ia c o n ta in in ga d e f i c i e n c y o r an e x c e s s o f p h o s p h a te o r s u l f a t e . . . . 27
6 P l a t e s o f N. c o c h i s e n s i s and N. s i n u a t a p r o t h a l l i agrow n on d i f f e r e n t a g a r m e d i a ........................................................... . 28-30
vi
ABSTRACT
I n 1963 i t was r e p o r t e d t h a t N. c o c h is e n s i s w ould grow
e q u a l ly w e l l on b o th K n o p 's and P r a n t l ' s a g a r m ed ia w h i le Eh s in u a t a
w ould grow o n ly on th e fo rm e r medium. S in c e P r a n t l ' s medium la c k s
th e p h o s p h a te io n and c o n ta in s an e x c e s s o f t h e s u l f a t e i o n , i t was
p o s t u l a t e d t h a t N. c o c h i s e n s i s h a s a v e ry low p h o s p h a te r e q u ir e m e n t
o r i s a b le t o s u b s t i t u t e th e s u l f a t e io n f o r th e p h o s p h a te io n .
The p u rp o se o f t h i s t h e s i s was t o show th e lo w e s t l e v e l o f p h o s p h a te
r e q u i r e d by b o th f e r n s and t o show th e i n f lu e n c e o f th e s u l f a t e io n
on th e am ount o f g r o w th .
E x p e r im e n ta l ly , p r o t h a l l i a o f b o th s p e c ie s w ere c u l t u r e d on
a g a r m ed ia t h a t c o n ta in e d v a r io u s am ounts o f p h o s p h a te and s u l f a t e .
E ach a g a r medium was a n a ly z e d f o r p h o s p h a te , and th e g ro w th re s p o n s e s
w ere d e te rm in e d by p h o to g ra p h ic and f r e s h w e ig h t s t u d i e s .
When th e r e s u l t s - o f t h e s e s t u d i e s w ere o b t a in e d , i t was found
t h a t K. s i n u a t a , a s w e l l a s N. c o c h i s e n s i s , w ould grow on P r a n t l ' s
m edium . I n b o th c a s e s th e f e r n s grown on P r a n t l ' s medium h ad a lo w er
f r e s h w e ig h t th a n th o s e grown on K n o p 's m edium. U nder th e e x p e r im e n ta l
c o n d i t io n s u s e d , b o th f e r n s r e q u i r e d 0 .8 mM HPO^ and 2 .7 mM 80^ p lu s t h e
o th e r io n s o f K n o p 's s o l u t i o n f o r m ax im al g ro w th . No p r o t h a l l i a ! g ro w th
was e v id e n t on m ed ia c o n ta in in g no p h o s p h a te and s u l f a t e . B o th f e r n
s p e c ie s showed some g ro w th on m ed ia c o n ta in in g no p h o s p h a te b u t h ig h
am ounts o f s u l f a t e . On th e s e m ed ia b o th f e r n s h ad l a r g e r c e l l s th a n th o s e
vii
viiigrown on m ed ia l a c k in g p h o s p h a te and s u l f a t e . When b o th f e r n s w ere
■grown on a d e q u a te am ounts o f p h o s p h a te , t h e r e was an i n c r e a s e i n
c e l l s i z e and num ber.
INTRODUCTION
In 1959 H ev ly (6 ) r e p o r t e d t h a t th e x e r o p h y t ic ro c k f e r n ,
N o th o la e n a c o c h i s e n s i s G oodd ing , w ould grow e q u a l ly w e l l on b o th K n o p 's
(2 ) and P r a n t l ' s (2 ) m e d ia ; h o w ev er, a s i s t e r s p e c ie s i n t h e N o th o la e n a
co m p lex , N o th o la e n a s i n u a t a (L a g a sc a ex S w a r tz ) K a u l f u s s , w ould d ie i f
grow n on th e l a t t e r m edium . K n o p 's s o l u t i o n i s a b a la n c e d n u t r i e n t
s o l u t i o n w h i le P r a n t l ' s s o l u t i o n l a c k s t h e p h o s p h a te io n and h a s an
e x c e s s o f s u l f a t e i o n ( ( 2 ) . The r e s u l t s o f H e v ly ' s e x p e r im e n t i n d i c a t e
t h a t th e p h o s p h a te r e q u i r e m e n t o f N. c o c h i s e n s i s i s l e s s th a n t h a t o f
N. s i n u a t a . S in c e t h e r e a r e no r e p o r t s i n th e l i t e r a t u r e c o n c e rn in g t h e
p h o s p h a te r e q u i r e m e n t o f t h e s e f e r n s , an e x p e r im e n t was d e s ig n e d t o
d e te rm in e th e lo w e s t l e v e l o f p h o s p h a te io n n e c e s s a r y t o s u s t a i n n o rm a l
g ro w th . I n a d d i t i o n , t h e r e i s a s u g g e s t io n t h a t s u l f a t e may s u b s t i t u t e
p a r t i a l l y f o r p h o s p h a te i n p l a n t s ( 9 ) . A s tu d y a l s o was made to
d e te rm in e i f g ro w th o f f e r n s c o u ld o c c u r on p h o s p h a te d e f i c i e n t b u t
s u l f a t e c o n ta in in g m e d ia .
1
REVIEW OF LITERATURE
The M o th o la e n a f e r n s u se d i n t h i s t h e s i s a r e u s u a l l y found
.grow ing on n o r th o r w e s t - f a c in g s lo p e s o f ro c k l e d g e s , c l i f f s , canyon
w a l l s , b o u l d e r s , o r on t h i n , ro c k y s o i l s . N. c o c h i s e n s i s n o rm a lly
grow s on c a lc a r e o u s s a n d s to n e o r l im e s to n e in w h ich th e pH i s n e u t r a l
o r s l i g h t l y b a s i c w h e re a s N. s i n u a t a grow s on ig n e o u s and m etam o rp h ic
ro c k s su ch a s g n e i s s , q u a r t s i t e , o r g r a n i t e i n w h ich th e pH i s n e u t r a l
o r s l i g h t l y a c id i c ( 5 , 6 ) . I n t h e s e h a b i t a t s , th e f e r n s o f t e n a r e
ex p o sed t o e x c e s s e s o r d e f i c i e n c i e s o f w a te r and v a r io u s m in e r a l s ( 6 ) .
S in c e t h e two f e r n s p e c ie s grow w e l l u n d e r w id e ly d i f f e r e n t and
som etim es h a r s h c o n d i t i o n s , t h e i r n u t r i e n t r e q u i r e m e n ts may be v e ry
d i s s i m i l a r . H e v ly 's s t u d i e s w i th n u t r i e n t m ed ia c u l t u r e s s u g g e s t t h a t
t h i s i s , i n f a c t , th e c a s e ; th e p h o sp h a te r e q u ir e m e n t o f N. c o c h i s e n s i s
a p p e a rs t o b e c o n s id e r a b ly l e s s th a n N. s i n u a t a .
S o lu t io n c u l t u r e te c h n iq u e s h av e b e e n u s e d p r e v i o u s ly t o a s s e s s
t h e n u t r i e n t r e q u i r e m e n ts o f f e r n s . I n 1920 Brown (2 ) r e p o r t e d t h a t
B i e j e r n c k 's , B i m e r and Luc a n u s 's , K n o p 's , P r a n t i ' s , and S a c h 's s o l u t i o n s
w ere f a v o r a b le f o r g ro w th o f th e P o ly p o d ia c e a f e r n s . She a l s o s t a t e d
t h a t K n o p 's and P r a n t i ' s s o l u t i o n s w ere b e s t s u i t e d f o r p r o t h a l l i a g ro w th .
S in c e b o th Brown and H ev ly showed t h a t some f e r n s p e c i e s c o u ld grow on
p h o s p h a te d e f i c i e n t b u t s u l f a t e c o n ta in in g m e d ia , i t a p p e a r s l i k e l y t h a t
some s u l f a t e r e p la c e m e n t o f p h o s p h a te can o c c u r , o r t h e r e a r e p h o s p h a te -
s u l f a t e i n t e r a c t i o n s w h ich a r e n o t u n d e r s to o d .
2
3V a r io u s i n t e r a c t i o n s o f p h o s p h a te and s u l f a t e h av e "been shown
t o e x i s t i n p l a n t s . C o ic , L e s a in t , and G ra n d je a n ( 3 ) fo u n d t h a t a l th o u g h
th e g r a i n and s tr a w y i e l d s w ere l e s s i n p h o s p h a te d e f i c i e n t b a r l e y , th e
u p ta k e o f s u l f a t e was g r e a t e r th a n i n p l a n t s grown on p h o s p h a te c o n ta in in g
n u t r i e n t s o l u t i o n s . A ls o , i n h ig h e r p l a n t s , F u j iw a ra and T o r i i (4 )
showed t h a t when s u l f a t e i s l a c k i n g , t h e am ount o f p h o s p h a te i s in c re a s e d ; .
On New Z e a la n d p a s t u r e s d e f i c i e n t i n s u l f a t e and p h o s p h a te L am erink (1 0 )
fo u n d t h a t th e y i e l d o f c o c k s f o o t c o u ld b e i n c r e a s e d b y th e a d d i t i o n o f
p h o s p h a te o r s u l f a t e , b u t t im o th y y i e l d s . c o u l d b e i n c r e a s e d o n ly w i th
s u l f a t e . By a d d in g s u l f a t e and p h o s p h a te to g e th e r , l a r g e i n t e r a c t i o n s
t h a t in c r e a s e d th e h e rb a g e y i e l d o f w h i te c lo v e r and r y e g r a s s w ere ' . . . ; V . i '
o b s e rv e d . A s i m i l a r e f f e c t o f s u l f a t e and p h o s p h a te was a l s o o b s e rv e d b y
T a k a h a sh i e t a l , ( 1 2 ) ) They fo u n d t h a t t h e two io n s en h a n ce d th e
s t im u la to r y e f f e c t o f g i b b e r e l l i n on g e rm in a t io n o f N. tabacum s e e d s .
In v iew o f t h e p h o s p h a te - s u l f a t e i n t e r a c t i o n s , i t a p p e a r s l i k e l y t h a t
s u l f a t e can p a r t i a l l y r e p l a c e p h o s p h a te i n h ig h e r p l a n t s .
To show t h a t t h i s s u b s t i t u t i o n may e x i s t , W ald (1 3 ) s t a t e s t h a t
s u l f a t e and p h o s p h a te are; s i m i l a r i n t h a t th e y h ave w eak , w id e -s p a c e d -
bonds w h ich a r e v u ln e r a b le t o a t t a c k b y o t h e r m o le c u le s t h a t p rom o te
exchange r e a c t i o n s .
K u rtz and .W aldon ( 9 ) s tu d i e d th e u t i l i z a t i o n o f s u l f a t e i n •
compounds w h ich n o rm a lly c o n ta in p h o s p h a te . U s in g th e o a t p l a n t , th e y
h ave shown t h a t a s u l f a t e d s u g a r ( s ) was fo rm ed when th e o a t was grow n f o r
t h r e e w eeks on a p h o s p h a te d e f i c i e n t m edium . T h e r e f o r e , b e s id e s
p h o s p h o ry la te d s u g a r s , a t l e a s t one s u l f a t e d s u g a r can e x i s t i n h ig h e r p l a n t s .
k
S u l f a t e h a s a l s o b e e n shown t o o c c u r i n h ig h e n e rg y com pounds.
B a n d u r s k i , W ils o n , and S q u ire s ( l ) showed t h a t an enzyme sy s te m e x i s t s ,
i n y e a s t t h a t com bines s u l f a t e w i th ATP^" t o fo rm PAPS. T h is sy s te m was
fo u n d t o c o n ta in two enzym es: ATP s u l f u r a s e , w h ich c a t a ly z e s th e
fo rm a tio n o f APS from ATP and s u l f a t e , and APS k i n a s e , w h ich u s e s ATP
t o c o n v e r t APS t o PAPS. A lth o u g h th e e x i s t e n c e o f h ig h e n e rg y s u l f a t e
compounds h a s b e e n shown i n y e a s t and h ig h e r p l a n t s , s u b s t i t u t i o n f o r
h ig h e n e rg y p h o s p h a te compounds h a s n o t y e t b e e n e s t a b l i s h e d .
I n v ie w o f th e f in d i n g s t h a t s u l f a t e can be u s e d i n p h o s p h o ry la te d
i n te r m e d ia t e s and i n h ig h e n e rg y com pounds, one i s l e d t o s p e c u la te
t h a t s u l f a t e may p a r t i a l l y s u b s t i t u t e f o r t h e p h o s p h a te r e q u i r e m e n t i n
some o rg a n is m s . T h e r e f o r e , i n d e s ig n in g m ed ia 1 t o 6 t o d e te rm in e th e
lo w e s t p h o s p h a te l e v e l r e q u i r e d b y th e f e r n s , am ounts o f KgHPO^ w ere
o m it te d and e q u a l am ounts o f KgSO^ w ere ad d ed t o k eep th e am ount o f K
c o n s ta n t and t o show th e in f lu e n c e o f s u l f a t e .
EXPERIMENTAL METHODS
The m ed ia u s e d i n t h i s s tu d y w ere d e s ig n e d t o d e te rm in e th e lo w e s t
p h o s p h a te l e v e l r e q u i r e d b y th e . f e r n s and t o show th e i n f l u e n c e o f s u l f a t e .
L arg e q u a n t i t i e s o f n u t r i e n t a g a r m ed ia w ere p r e p a r e d , and 10 m l a l i q u o t s
c o n ta in in g 0 .2 g a g a r w ere t r a n s f e r r e d t o 2 x J r 'in c h p e t r i d i s h e s .
A l l s o l u t i o n s e x c e p t Ho. 11 c o n ta in e d , i n e a c h l i t e r , 2 m l o f K u r t z 's m in o r
**■ A b b r e v ia t io n s u s e d a r e : PAPS, a d e n o s in e - 3 ' - p h o s p h a te - 5 ' -p h o s p h o s u l f a te ; APS, a d e n o s in e - 5 ' - p h o s p h o s u l f a t e ; P ^ , o r th o p h o s p h a te ;ATP, a d e n o s in e t r i p h o s p h a t e .
The pH v a lu e s o f e a che le m e n t s o l u t i o n (8 ) and 1 d ro p o f Vj0 F e C lg .
s o l u t i o n w ere a d ju s t e d t o 7 .5 w i th 2N NaOH. E le v e n d i f f e r e n t s o l u t i o n s
w ere u s e d , and th e c o m p o s i t io n s a r e th e f o l l o w in g :
S o lu t io n X (K h o p 's s o l u t i o n )
S a l t C o n e e n t r a t i on o f S a l t
g / l i t e r mM
MgSO^ 0 .2 5 2 .1
CafNOgjg.ltHgO 1 .0 0 4 .2
KC1 0 .1 2 1 .6
KgHPO^ 0 .2 5 1 .4
0 ,6 5 m l 2 N HaOH 1 .2
T o ta l 1 0 .5
S o lu t io n 2
S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgS04 0 .2 5 2 .1
c a ( m 3 )2 .4 p 2o 1 .0 0 4 .2
KC1 0 .1 2 1 .6
K2HP04 0 .2 0 1 .2
KgSC^ 0 .0 5 0 .3
0 .5 0 m l 2 H HaOH 1 .0
T o ta l i o . 4
Solution 3
' S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgSO^ 0 .2 5 2 . 1
Ca(H03 )2 .UH20 1 .00 4 .2
KC1 0 .12 1 . 6
KgHPO^ 0 .1 5 0 . 8
K2S0i+ 0 .10 0 . 6
0 .4 0 m l 2 N MaOH 0 . 8
T o ta l 1 0 .1
S o lu t io n 14-
S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgSO^ 0 .2 5 2 . 1
Ca(N03 ) 2 .4H20 1 .0 0 4 .2
KC1 0 .12 1 . 6
KgHPO^ 0 ,1 0 0 . 6
KgSO^ 0 .1 5 0 . 8
0 .3 0 m l 2 N NaOH 0 . 6
T o ta l 9 -9
Solution 5
S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgS04 0 .2 5 2 .1
Ca($r03 )2 .4H20 1 ,0 0 4 .2
KC1 0 .1 2 1 .6
KgHPO^ 0 .0 5 0 .3
k2 so4 0 .2 0 1 .2
0 .2 0 m l 2 N NaOH 0 . 4
T o ta l
S o lu t io n 6
9 . 8
S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgSO^ 0 .2 5 2 .1
Ca(N03 ) 2 .4H20 1 .0 0 4 .2
KC1 0 ,1 2 1 .6
K2S04 0 .2 5 1 .4
0 .1 0 m l 2 H NaOH 0 .2
T o ta l 9 -5
S o lu t io n 7 ( P r a n t i ' s s o l u t i o n )
S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgSO^ 0 .5 0 4 .2
V O ** 0 .7 0 4 .0
NaCl 0 .2 3 4 .0
CaSO^.PHgO 0 .70 4 .0
0 .0 1 0 . 2
0 .05 m l 2 N NaOH 0 . 1
T o ta l 16 .5
S o lu t io n 8
S a l t C o n c e n t r a t io n o f S a l t
g / l i t e r mM
MgS04 0 .2 5 2 .1
Ca(N03 )2 .4H20 1 .0 0 4 .2
KC1 0 .1 2 1 .6
KgKPO^ 0 .2 5 1 .4
NagSO^ 0 .1 7 . 1 .2
0 .6 0 m l 2 N HaOH 1 .1
T o ta l 1 1 .6
Solution 9
S a l t
MgS04
Ca(N03)2 .4H20
KC1 ■
KgHPO^
KgSOj^
0 .7 5 m l 2 N WaOH
T o ta l
S a l t '
MgClg.SHgO
Ca(N03 )2 .4H20
KC1
0 .1 0 m l 2 N NaOH
T o ta l
C o n c e n t r a t io n o f S a l t
g / l i t e r
0 .2 5
1.00
0 .1 2
0 .2 5
0 .2 5
mM
2.14 .2
1 .6
1 .4
1 .4
1 .5
12.2
S o lu t io n 10
C o n c e n t r a t io n o f S a l t
g / l i t e r
0.68
1.000.22
mM
3 .4
4 .2
2 .9
0.1
10.6
S o lu t io n 11
One l i t e r d e io n iz e d w a te r and 0 .1 m l 2 N HaOH
10T a b le 1 shows a c o m p a riso n o f t h e t o t a l s a l t and io n c o n c e n t r a t io n s
o f th e n u t r i e n t m e d ia . . Medium 1 i s K n o p 's s o lu t i o n p lu s a g a r and i s
u s e d a s t h e f i r s t medium i n a c o n c e n t r a t io n s e r i e s t h a t h a s v a ry in g
am ounts o f p h o s p h a te . S in c e H ev ly (6 ) r e p o r t e d t h a t b o th f e r n s grew
w e l l on K nop’ s m edium , i t s p h o s p h a te c o n c e n t r a t i o n , 1 .4 mM, was th e
maximum u s e d in t h i s s tu d y .
M edia 2 t o 6 r e p r e s e n t th e o t h e r m ed ia i n th e c o n c e n t r a t io n s e r i e s
o f v a ry in g am ounts o f p h o s p h a te ; th e y w ere u s e d t o d e te rm in e th e lo w e s t
l e v e l o f p h o s p h a te r e q u i r e d f o r maximum g ro w th . In t h e i r p r e p a r a t i o n th e
am ounts o f KgHPO^ and KgSO^ w ere r e g u l a t e d t o m a in ta in a c o n s ta n t
p o ta s s iu m c o n c e n t r a t i o n , and th e pH v a lu e s w ere a d ju s t e d t o pH 7 * 5 .
As shown i n T a b le 1 , t h e p h o s p h a te c o n c e n t r a t io n was u l t i m a t e l y lo w e re d
t o 0 .0 mM i n s o l u t i o n 6 , b u t th e s u l f a t e c o n c e n t r a t io n was i n c r e a s e d t o
3 -5 mM.
Medium 7 i s P r a n t i ’ s s o l u t i o n p lu s a g a r . ' T h is medium was p r e p a r e d
t o c o n f irm H e v ly 1s s ta te m e n t ( 5 , 6 ) t h a t N. s in u a t a d i e s and N. c o c h i s e n s i s
l i v e s when grown on i t . I t d i f f e r s from m ed ia 1 t o 6 i n t h a t i t h a s no
p h o s p h a te , b u t i t s s u l f a t e c o n te n t i s a v a lu e 3 t o 4 t im e s h ig h e r th a n i n
t h e fo rm e r s o l u t i o n s .
I n t h i s e x p e r im e n t m ed ia 8', 9? 1 0 , and 11 w ere u s e d a s c o n t r o l
m e d ia . M edia 8 and 9 c o n ta in 3 .5 mM s u l f a t e w i th e x c e s s Na and K.
By a d d in g HagSO^ and KgSO^ t o medium 1 , t h e s e m ed ia w ere p r e p a r e d t o
show t h a t a n y 'p o s s i b l e ch a n g es i n th e am ount o f g ro w th on m ed ia 1 t o 6
was n o t due t o a h a rm fu l e f f e c t o f i n c r e a s i n g am ounts o f s u l f a t e .
11
T a b le 1
C om parison o f T o ta l S a l t and Io n C o n c e n t r a t io n s o f th e N u t r i e n t M edia ( m m o le s / l i t e r )
S o l .No. S a l t Na K Ca Mg N°3 HP04 s o u C l
1 1 0 .5 1 .2 4 .4 4 .2 2 .1 8 .4 1 .4 2 .1 1 .6
2 1 0 .4 1 .0 4 .6 4 .2 2 .1 8 .4 1 .2 2 .4 1 .6
3 1 0 .1 0 .8 4 .4 4 .2 2 .1 8 .4 0 .8 2 .7 1 .6
k 9 -9 0 .6 4 .4 4 .2 2 .1 8 .4 0 .6 2 .9 1 .6
5 9 .8 0 .4 4 .6 4 .2 2 .1 8 .4 0 .3 3 .3 1 .6
6 9 -5 0 .2 4 .4 4 .2 2 .1 8 .4 0 .0 3 .5 1 .6
7 1 6 .5 4 .1 8 .0 4 .0 4 .2 0 .2 0 .0 1 2 .2 4 .0
8 1 1 .6 3 .5 4 .4 4 .2 2 .1 8 .4 1 .4 3 .5 1 .6
9 1 2 .2 1 .5 7 .2 4 .2 2 .1 8 .4 1 .4 3 .5 1 .6
10 1 0 .6 0 .1 2 . 9 4 .2 3 .4 ' 8 .4 0 .0 0 .0 9 -7
11 0 .1 0 .1
12.
Medium 10 was u se d t o d e m o n s tra te g ro w th r e s p o n s e s when b o th s u l f a t e and
p h o s p h a te a r e a b s e n t . S i m i l a r l y , medium 11 was u se d t o show g ro w th
r e s p o n s e s on a g a r when no n u t r i e n t s w ere a d d e d .
U s in g th e m ethod o f K its o n and M ello n ( ? ) , p h o s p h a te a n a ly s e s
w ere p e rfo rm e d on th e m ed ia and th e s p o r e s . T h is m ethod was s e l e c t e d
b e c a u s e th e s e n s i t i v i t y i s 1 ppm, t h e c o lo r r e a c t i o n i s s t a b l e , and
i n t e r f e r e n c e from o th e r d o n s i n th e medium i s n e g l i g i b l e . To p r e p a r e a
n u t r i e n t medium f o r a n a l y s i s , a 25 m l sam ple o f t h e n u t r i e n t s o l u t i o n
c o n ta in in g a g a r was p i p e t t e d i n t o a c r u c i b l e . Ten r e p l i c a t e a n a ly s e s
w ere ru n from e a c h n u t r i e n t m edium ; A f te r c a r e f u l l y d r y in g th e w a te r
from th e sam p les i n t h e c r u c i b l e s b y p l a c i n g them in a d ry in g o v e n , th e
sam p les w ere i g n i t e d and a sh e d a t 5 0 0 ° . The c o n te n t s o f t h e c r u c i b l e s
w ere ta k e n up in 10 m l o f 1 N HKO^ and q u a n t i t a t i v e l y t r a n s f e r r e d t o
100 m l v o lu m e tr ic f l a s k s . The f l a s k s w ere d i l u t e d t o v o lu m e , and 2 5 -35
m l a l i q u o t s c o n ta in in g 10 -2 0 0 jig HPOj ̂ w ere t r a n s f e r r e d t o 50 m l v o lu m e tr ic
f l a s k s . F iv e m i l l i t e r s ea ch o f 7 .5 N HNOg, 0 .0 2 M HH^VOg, and 0 .0 7 M
(nH^)^MQyOg^. 4Hg0 w ere added i n o r d e r , and th e f l a s k s w ere b ro u g h t t o
volum e w i th d e io n iz e d w a te r and s h a k e n . A f t e r o n e - h a l f h o u r , th e
a b s o rb a n c e o f th e sam ple was d e te rm in e d w i th a Beckman DU s p e c t r o p h o to m e te r
a t th e w a v e le n g th o f maximum a b s o r p t io n , 3&5 n y - The r e s u l t s w ere com pared
t o s ta n d a r d p h o s p h a te s o l u t i o n s , p r e p a r e d and a n a ly z e d a t th e same t im e .
An exam ple o f t h e s ta n d a r d p h o s p h a te c u rv e i s shown i n F ig u re 1 ,
The r e s u l t s o f th e p h o s p h a te a n a ly s e s on th e m ed ia c o n ta in in g a g a r
a r e shown i n T a b le 2 . I n a l l c a s e s th e p h o s p h a te fo u n d b y a n a ly s e s was
n e a r l y e q u a l t o th e t h e o r e t i c a l am ount added b y th e n u t r i e n t s o lu t i o n a lo n e
OPT
ICA
L D
EN
SIT
Y
.900-1
.8 0 0 -
.7 0 0 -
,6 0 0 -
.5 0 0 -
4 0 0 -
3 0 0 -
PHOSPHATE— mmoles in reoction mixture
F i g u r e 1 . S t o n d a r d C u r v e f o r P h o s p h a t e A n a l y s e s by t h e K i t s o n - M e I I on M e t h o d .
HU>
Table 2P h o sp h a te C o n te n t o f N u t r i e n t M edia8, (mM)
' M edia No. T h e o r e t i c a l i n medium
(mM)
A n a ly se s o f sam p les^
(mM)
• 1 1 .4 3 1 .4 2
2 1 .1 5 l . l 6
3 0 .86 0 .8 9
4 0 .5 8 0 .52
5 0 .2 9 0 .2 7
6 0 .0 0 0 .05
7 0 .0 0 0 .03
8 1 .4 4 1 .5 5
9 1 .4 4 1 . 4 i
10 0 .0 0 o .o 4
i i 0 .0 0 0 .0 2
a N u t r i e n t m ed ia i n d i c a t e s n u t r i e n t s o l u t i o n p lu s a g a r ,
k I n d i c a t e s a v e ra g e am ount o f 10 r e p l i c a t e s on th e medium.
15F o r exam ple, 1 .4 3 mmoles HPO^ was added t o ea ch l i t e r o f medium 1 ; th e
a v e ra g e am ount fo u n d was 1 .4 2 mM. As shown i n T a b le 2 , m ed ia 6 , 7 , 1 0 ,
and 11 c o n ta in e d no ad d ed p h o s p h a te , b u t s m a l l am ounts w ere fo u n d .
F o r e x a m p le , i n medium 6 , 0 ,0 4 mM was fo u n d . T h is s m a l l am ount o f
p h o s p h a te can p r o b a b ly be a t t r i b u t e d t o e r r o r s i n a n a l y t i c a l p r o c e d u r e s .
S in c e th e s p o re s th e m s e lv e s may h av e h ig h l e v e l s o f endogenous
p h o s p h o ru s , t h e i r c o n t r i b u t i o n t o t h e medium was a l s o d e te rm in e d .
When 4 5 .8 mg o f N. c o c h i s e n s i s s p o r e s , r e p r e s e n t i n g a p p ro x im a te ly 2800
s p o r e s , w ere a n a ly z e d f o r p h o s p h a te , 0 .233 mmoles HPO^ was fo u n d .
By u s in g 58 a s t h e a v e ra g e num ber o f g e rm in a t in g s p o re s p r e s e n t i n e a c h
d i s h , i t was c a l c u l a t e d t h a t 4 .1 x 10*"3 mM HPO^ was c o n t r i b u t e d b y th e
s p o r e s . T h e r e f o r e , t h e am ount o f p h o s p h a te c o n t r i b u t e d b y N. c o c h i s e n s i s
i s i n s i g n i f i c a n t .
F o llo w in g a m ethod d e s c r ib e d b y H evly ( 6 ) , s p o re s o f b o th
N o th o la e n a s p e c i e s w ere o b ta in e d from f e r t i l e f ro n d s t h a t w ere c o l l e c t e d
i n th e T ucson a r e a . The s p o re s w ere p la c e d i n v i a l s and s to r e d i n th e
d a rk t o p r e v e n t p re m a tu re g e m i n a t i o n ( 5 , 6 ) . The f e r n s w ere i d e n t i f i e d
b y D r. H ev ly and a r e on f i l e a t th e U n i v e r s i t y o f A r iz o n a H e rb a riu m .
A m ethod o f s p o re s t e r i l i z a t i o n ( l 4 ) was o b ta in e d from th e
l i t e r a t u r e and m o d if ie d . By u s in g d i s p o s a b le t r a n s f e r p i p e t t e s ,
a p p ro x im a te ly 100 mg o f s p o re s w ere p la c e d on g l a s s w ool i n t h e b o tto m
o f a 7 x ■§- in c h c h ro m a to g ra p h ic colum n t h a t h ad b e e n s t e r i l i z e d p r e v i o u s l y .
The g l a s s w ool a llo w e d l i q u i d s t o d r a i n b u t d id n o t a l lo w th e s p o re s t o
p a s s th r o u g h . To p r e v e n t c lu m p in g , th e s p o re s w ere t r e a t e d b y im m ers in g
th e colum n i n s t e r i l e 0 .1 % .Tween 80 f o r 20 m in u te s . T h is w as fo llo w e d
16
by e x p o s in g th e s p o re s t o 30$ Chlorox"*" f o r 20 m in u te s . The s p o re s
w ere th e n r i n s e d once w i th s t e r i l e , d e i o n i z e d w a te r and f i n a l l y su sp e n d e d
i n 35 m l o f s t e r i l e , d e io n iz e d w a te r . S t e r i l e p e t r i d i s h e s , c o n ta in in g
d i f f e r e n t n u t r i e n t a g a r m e d ia , w ere i n o c u la te d w i th 0 .3 m l o f th e s p o re
s u s p e n s io n s . T h is gave ah a v e ra g e num ber o f 58 and 65 g e rm in a t in g
s p o re s f o r N. c o c h i s e n s i s and N. s i n u a t a , r e s p e c t i v e l y . A f t e r i n o c u l a t i o n
th e p e t r i d i s h e s w ere k e p t i n a c u l t u r e room , m a in ta in e d a t 30° , and
r e c e iv e d c o n tin u o u s l i g h t i n t e n s i t i e s o f 700 -900 f t - c from b an k s o f
c o o l w h i t e , f l u o r e s c e n t l i g h t s . The l i g h t i n t e n s i t i e s w ere m easu red
w i th a W eston 603 p h o to m e te r . S t e r i l e , d e io n iz e d w a te r ( 0 .2 - 0 .3 ) m l ) was
added a t l e a s t once a .w eek t o m a in ta in th e m o is tu re c o n te n t o f th e a g a r
m e d ia . S in c e t h e l i g h t i n t e n s i t i e s v a r i e d from 700 -900 f t - c on th e
g ro w in g t a b l e , t h e d i s h e s w ere r e a r r a n g e d a f t e r ea ch a d d i t i o n o f w a t e r .
The f r e s h w e ig h t o f t h e p r o t h a l l i a l c o n te n t s o f e a c h d i s h was
d e te rm in e d by f i r s t lo o s e n in g th e p r o t h a l l i a from th e a g a r w i th w a te r .
When th e p r o t h a l l i a w ere f r e e d from th e a g a r , th e y w ere c o l l e c t e d on 4 cm
Whatman N o .,1 f i l t e r p a p e r i n a s m a ll B uchner f u n n e l , rem oved w ith a
s p a t u l a o r tw e e z e r s , and p la c e d in a s m a l l , t a r e d v i a l . The s u r f a c e o f
t h e a g a r m ed ia and f i l t e r p a p e r s w ere ch e ck e d w i th a d i s s e c t i n g m ic ro sc o p e
t o be s u re t h a t a l l p r o t h a l l i a and t h e i r r h i z o i d s w ere added t o t h e v i a l .
I f th e p r o t h a l l i a o r th e v i a l h ad d ro p s o f w a te r on t h e i r s u r f a c e , th e
f r e s h w e ig h t was n o t m e a su re d . Once th e i n i t i a l p r o t h a l l i a l w e ig h ts
1 C h lo ro x i s t h e co m m erc ia l name f o r 5 .2 5 $ NaOCl (by w e ig h t ) .
17w ere o b ta in e d , f r e s h w e ig h t m easu rem en ts w ere made e v e ry 5 days u n t i l
t h e t h i r t y - f i f t h d ay a f t e r g e r m in a t io n .
The m o rp h o lo g ic a l c h an g es w h ich o c c u r r e d d u r in g g ro w th w ere
r e c o r d e d on 35 mm s l i d e p i c t u r e s , ta k e n th ro u g h a 10X l e n s o f a Z e is s
208359^ GFL m ic ro s c o p e . The p i c t u r e s w ere ta k e n on Kodachrom e I I c o lo r
s l i d e f i l m , u s in g a Z e is s Ik o n cam era s e t a t l / 2 5 o f a s e c o n d . A Z e is s
LTKL Nr t r a n s f o r m e r s e t a t 8 was u s e d t o c o n t r o l t h e l i g h t s o u r c e , and
th e e m i t te d l i g h t was f i l t e r e d w ith a b lu e f i l t e r .
RESULTS AM) DISCUSSION
To a v o id m ic r o b ia l g ro w th , t h e a g a r was a u to c l a v e d , and th e
s p o re s w ere s t e r i l i z e d w i th a d i s i n f e c t a n t p r i o r t o g e r m in a t io n .
A 20 m in u te t r e a tm e n t w i th 30% C h lo ro x was fo u n d t o be th e maximum
a l lo w a b le t r e a tm e n t o f th e s p o r e s . The t r e a tm e n t rem oved m ost o f th e
m o ld , b a c t e r i a l , and a l g a l c o n ta m in a t io n from th e s p o re s u r f a c e b u t
d id n o t e l i m i n a te t h e im p e r f e c t fu n g i A l t e r n a r i a , D i p l o d ia , and
S p o ro tr ic h u m . The C h lo ro x t r e a tm e n t a l s o h ad a h a rm fu l e f f e c t on
th e s p o re s s in c e some d id n o t g e r m in a te . I n a d d i t i o n , t h e am ount o f
g ro w th o f th e p r o t h a l l i a was l e s s th a n when s p o re s w ere g iv e n s h o r t e r
e x p o s u re s t o C h lo ro x .
When th e s p o re s o f b o th N o th o la e n a s p e c ie s w ere p la c e d on
n u t r i e n t m ed ia and e x p o sed t o c o n tin u o u s l i g h t i n t e n s i t i e s o f 700 t o
900 f t - c , g e rm in a t io n o c c u r r e d on th e s i x t h day a f t e r i n o c u l a t i o n .
I n b o th s p e c i e s th e m o rp h o lo g ic a l ch a n g es w h ich o c c u r r e d a f t e r g e rm in a t io n
w ere s i m i l a r t o th o s e o f t h e b ra c k e n f e r n P te r id iu m a q u il in u m ( l l ) .
A f t e r im b ib in g w a te r , th e s p o rb s r u p tu r e d , and a g r e e n , u n s e p ta t e
f i l a m e n t and one r h i z o i d em erged . W ith in a few d ay s t h e f i l a m e n t
becam e s e p t a t e s and o t h e r r h i z o i d s form ed on i t . I s o l a t e d p r o t h a l l i a
w ould u n d e rg o f u r t h e r d e v e lo p m en t b y p ro d u c in g a p l a t e l e t from th e
f i l a m e n t . T h ese p l a t e l e t s c o n tin u e d t o d e v e lo p i n an i r r e g u l a r sh ap e
b e c a u s e o f c o n t in u o u s , h ig h l i g h t i n t e n s i t i e s ( 9 , 6 ) . When th e
p r o t h a l l i a w ere c ro w d ed , t h e f i l a m e n ts d id n o t d e v e lo p i n t o p l a t e l e t s .
18
19The r e s u l t s o f f r e s h w e ig h t s tu d i e s showed t h a t N. c o c h i s e n s i s
and N. s i n u a t a re s p o n d e d s i m i l a r l y t o d i f f e r e n t n u t r i e n t m e d ia . T h is i s
shown i n F ig u r e s 2 , 3s ^s and 5 . F o r b o th s p e c ie s a f i f t e e n day p e r io d
a f t e r t h e •o n s e t o f g e rm in a t io n was r e q u i r e d b e f o r e p r o t h a l l i a l w e ig h ts
c o u ld be o b ta in e d on m ed ia 1 , 2 , 3s 4 , 7 S 8 , and 9- W ith th e e x c e p t io n
o f medium 7 , P r a n t i ' s m edium , t h e s e m ed ia had a t l e a s t 0 .6 mM p h o s p h a te
and 2 .1 mM s u l f a t e . I n c o n t r a s t a 20 d ay p e r io d was n e e d ed on m ed ia
5 and 6 , w h ich c o n ta in e d 3 -3 and 3*5 mM s u l f a t e and 0 .3 and 0 .0 mM
p h o s p h a te , r e s p e c t i v e l y , b e f o r e e i t h e r s p e c ie s y i e l d e d s u f f i c i e n t
p r o t h a l l i a f o r w e ig h in g . On m ed ia 10 w h ich h a s no s u l f a t e o r p h o s p h a te
and m ed ia 11 w h ich h a s no n u t r i e n t s , t h e p r o t h a l l i a l w e ig h ts w ere
i n s u f f i c i e n t f o r d e te r m in a t io n even a f t e r g ro w in g 35 d a y s .
The r e s u l t s from m ed ia 1 and 7 d i f f e r i n two ways from th o s e o f
H evly ( 6 ) , who r e p o r t e d t h a t W. c o c h i s e n s i s w ould grow e q u a l ly w e l l on
b o th K hop' s and P r a n t l ' s a g a r medium and N. s i n u a t a w ould n o t grow on
P r a n t i ' s m edium . F i r s t , N. c o c h i s e n s i s d id n o t grow e q u a l ly w e l l on
K n o p 's and P r a n t l ' s m e d ia , a n d , s e c o n d ly , N. s i n u a t a d id grow when '
p l a c e d on P r a n t l ' s m edium .
When s p o re s w ere p la c e d on m ed ia 10 and 1 1 , t h e p r o t h a l l i a
g e rm in a te d a t th e same tim e a s th o s e grown on m ore b a la n c e d m e d ia .
The am ount o f g ro w th on medium 11 shows t h a t c e r t a i n n u t r i e n t s r e q u i r e d
f o r n o rm a l g ro w th a r e a b s e n t i n a g a r a lo n e w h ile th e am ount o f g ro w th
on medium 10 shows t h a t p h o s p h a te and s u l f a t e c a n n o t b o th be e x c lu d e d
from th e a g a r medium.
20F ig u r e s 2 and 3 show t h a t N. c o c h i s e n s i s and N. s i n u a t a grew
s i m i l a r l y on n u t r i e n t m ed ia 1 t o 3 . Medium 1 , K n o p 's m edium , i s a
b a la n c e d n u t r i e n t medium w i th p h o s p h a te and s u l f a t e c o n te n t s o f 1 .4 mM
and 2 .1 mM, r e s p e c t i v e l y . Medium 3 i s s i m i l a r t o medium 1 e x c e p t t h a t
th e p h o s p h a te and s u l f a t e c o n c e n t r a t io n s a r e 0 . 8 and 2 . 7 m&% r e s p e c t i v e l y .
The f a c t t h a t th e g ro w th o f p r o t h a l l i a was a p p ro x im a te ly th e same on
t h e s e m ed ia s u g g e s ts th e l e v e l o f p h o s p h a te i n medium 3 i s n o t l i m i t i n g
g ro w th in t h i s s tu d y .
F ig u r e s 2 and 3 a l s o show t h a t th e am ount o f g ro w th o f b o th f e r n s
on medium 4 was l e s s th a n on m ed ia 1 t o 3- I n t h i s medium th e p h o s p h a te
and s u l f a t e c o n te n t s w ere 0 .6 mM and 2 .9 mM, and th e am ounts o f o t h e r
io n s w ere n o t v a r i e d from th o s e on medium 1 . S in c e a p p ro x im a te ly th e
same num ber o f s p o re s o f e a c h s p e c i e s w ere u s e d p e r d i s h , i t may be
c o n c lu d e d t h a t t h e r e q u i r e m e n ts f o r p h o s p h a te o f th e two s p e c ie s a r e t h e
sam e. The f a c t t h a t g ro w th on medium 4 was l e s s th a n on medium 3 shows
t h a t s u l f a t e c a n n o t s t o i c h i o m e t r i c a l l y r e p l a c e p h o s p h a te . Medium 4
c o n ta in s 0 .6 mM p h o s p h a te and 2 .9 mM s u l f a t e w h e re as medium 3 c o n ta in s .
0 .8 mM HPO4 and 2 .7 mM SO4 .
F ig u r e s 2 and 3 a l s o show t h a t t h e am ounts o f p r o t h a l l i a l g ro w th
on m ed ia 5 and 6 a r e ev en l e s s th a n on medium 4 and f a r l e s s th a n on
medium 1 . A g a in , b o th s p e c ie s r e s p o n d e d s i m i l a r l y . M edia 5 and 6 d i f f e r
from medium 1 b y h a v in g 0 .3 and 0 .0 mM p h o s p h a te , r e s p e c t i v e l y , i n s t e a d
o f 1 .4 mM p h o s p h i t e ; th e y a l s o d i f f e r b y h a v in g 3 -3 and 3 .5 mM s u l f a t e ,
r e s p e c t i v e l y , i n s t e a d o f 2 .1 mM s u l f a t e . From th e am ount o f g ro w th
o b ta in e d on m ed ia 5 and 6 , i t may b e c o n c lu d e d a g a in t h a t s u l f a t e c a n n o t
21
s t o i c h i o m e t r i c a l l y r e p l a c e p h o s p h a te . H ow ever, t h e r e was some g ro w th
on th e s e m ed ia w h e re as no m e a s u ra b le g ro w th was fo u n d u s in g medium 10
w h ich h ad a l l o t h e r io n s e x c e p t p h o s p h a te and s u l f a t e . S in c e m ed ia 5
and 6 d i f f e r from medium 10 p r i m a r i l y i n t h a t th e y c o n ta in d i f f e r e n t
am ounts o f s u l f a t e f o r g ro w th , t h e r e may be a p a r t i a l r e p la c e m e n t o f .
p h o s p h a te b y s u l f a t e .
F ig u r e s 4 and 5, show b o th f e r n s re s p o n d e d q u i t e w e l l t o medium
7 , P r a n t l ' s m edium . T h is medium d i f f e r s from m ed ia 1 t o 6 i n t h a t
o th e r i o n s , i n a d d i t i o n t o p h o s p h a te and s u l f a t e , w ere v a r i e d . I t i s
somewhat s i m i l a r t o th e c o m p o s i t io n o f medium 6 i n t h a t i t h a s no
p h o s p h a te , b u t t h e s u l f a t e c o n te n t i s c o n s id e r a b ly h ig h e r th a n in th e
l a t t e r m edium. When th e am ount o f g ro w th on P r a n t l ' s medium i s com pared
t o t h a t o f medium 1 0 , t h e c o n c lu s io n i s s i m i l a r t o t h a t o b ta in e d when
m ed ia 6 and 10 a r e com pared : th e a d d i t i o n o f s u l f a t e c a u s e s a l a r g e •
i n c r e a s e i n t h e am ount o f g ro w th .
When th e s p o re s o f b o th f e r n s w ere p la c e d on m e d ia 8 and 9 ,
th e am ount o f g ro w th was e q u a l t o t h a t o b ta in e d from medium 1 , a s shown
i n F ig u r e s 4 and 5 . Medium 8 c o n ta in s t h e same io n c o n te n t a s medium 1
e x c e p t t h a t th e Na c o n te n t i s 3 -5 mM i n s t e a d o f 1 .2 mM and th e s u l f a t e
c o n te n t i s 3 •5 mM i n s t e a d o f 2 .1 mM. . S in c e t h e am ounts o f g ro w th on
medium 8 and 9 w ere th e same a s on medium 1 , e x c e s s s u l f a t e i s n o t h a rm fu l
i n th e p r e s e n c e o f e x c e s s Na o r K. T hese r e s u l t s a l s o show t h a t th e
d e c re a s e i n am ounts o f g ro w th on m ed ia 4 , 6 , and 7 i s due t o o m is s io n
o f am ounts o f p h o s p h a te i n t h e medium an d n o t due t o ch a n g es i n th e Na
o r s u l f a t e c o n te n t s .
22The m o rp h o lo g ic a l c h an g es w h ich o c c u r r e d d u r in g g ro w th a r e
shown in F ig u re 6 . P l a t e 1 shows a N. c o c h is e n s i s p r o th a l l i u m on
medium 1 t w e n ty - f iv e d ay s a f t e r g e r m in a t io n . A f te r c a r e f u l o b s e r v a t io n
w i th a d i s s e c t i n g m ic ro s c o p e , p r o t h a l l i a from m ed ia 2 , 3 , 8 , and 9
w ere fo u n d t o be i n d i s t i n g u i s h a b l e from th o s e on medium 1 . The p r o t h a l l i a
a p p e a r a s b ro a d p l a t e l e t s w i th many r h i z o i d s .
P l a t e s 2 and 3 o f F ig u re 6 show p r o t h a l l i a o f N. c o c h i s e n s i s and
H. s i n u a t a , r e s p e c t i v e l y , on medium 4 tw e n ty - f iv e days a f t e r g e rm in a tio n ..
A lth o u g h th e p l a t e s do n o t show e q u a l g ro w th o f b o th s p e c i e s , m ic ro s c o p ic
e x a m in a tio n r e v e a le d t h a t H. c o c h i s e n s i s was n o t d i s t i n g u i s h a b l e fromY , “ ------------------ ■
N. s in u a t a when t h e i r p r o t h a l l i a w ere com pared . P r o t h a l l i a o f b o th f e r n s
grown on medium 4 , w h ic h ..,c o n ta in s 0 .6 mM HPO4 , h ad a b o u t t h e same c e l l
num ber a s th o s e grow n on medium 1 , b u t t h e c e l l s w ere s m a l le r .
T h is s u g g e s ts t h a t c e l l e n la rg e m e n t r a t h e r th a n c e l l d i v i s i o n was h in d e r e d
b y th e low p h o s p h a te c o n te n t .
P l a t e 4 o f F ig u re 6 shows N. c o c h i s e n s i s p r o t h a l l i a on medium 7 ,
P r a n t l ' s m e d iu m ,tw e n ty - f iv e days a f t e r g e r m in a t io n . The p l a t e l e t s a r e
n o t q u i t e a s w ide a s th o s e on m ed ia 1 and 4 , and th e c e l l num ber i s less../;
th a n th o s e o f p r o t h a l l i a grow n on m ed ia 1 and 4 . The c e l l s i z e o f
p r o t h a l l i a on medium 7 i s s i m i l a r t o th o s e o f p r o t h a l l i a from medium 4 .
S in c e t h e f r e s h w e ig h t s tu d i e s showed t h a t th e d e c r e a s e i n am ount o f
g ro w th on m e d ia .4 and 7 was due t o o m is s io n o f p h o s p h a te , i t may b e
c o n c lu d e d t h a t a d e q u a te am ounts o f p h o s p h a te a l s o i n c r e a s e t h e num ber o f
c e l l s i n a p r o th a l l i u m .
23As shown i n P l a t e 5 o f F ig u re 6 , N. c o c h i s e n s i s p r o t h a l l i a
grown on m ed ia 5 and 6 a r e m ore f i la m e n to u s th a n p l a t e - l i k e tw e n ty - f iv e
d ay s a f t e r g e r m in a t io n . P r o t h a l l i a grow n on th e s e m ed ia h av e
a p p ro x im a te ly t h e same c e l l num ber a s medium 7 , b u t t h e c e l l s a r e
s l i g h t l y s m a l l e r . The d e c re a s e i n am ount o f g ro w th on t h e s e m ed ia from
t h a t on medium 1 was shown t o b e a l a c k o f p h o s p h a te . T h e r e f o r e 5 th e
s m a l le r i n c r e a s e s i n c e l l s i z e and num ber can be a t t r i b u t e d t o t h e l a c k
o f p h o s p h a te . B u t , s in c e t h e p r o t h a l l i a l w e ig h ts f o r b o th f e r n s w ere
g r e a t e r on medium 7 th a n on m ed ia 5 and 6 , i t may b e c o n c lu d e d t h a t s u l f a t e
a l s o can in f lu e n c e c e l l e n la r g e m e n t .
P l a t e 6 o f F ig u re 6 shows W. c o c h i s e n s i s p r o t h a l l i a on medium
10 t h i r t y - f i v e d ay s a f t e r g e r m in a t io n . P r o t h a l l i a from medium 11 w ere
n o t fo u n d t o b e s i g n i f i c a n t l y d i f f e r e n t from th o s e on medium 1 0 .
In t h e a b se n c e o f p h o s p h a te and s u l f a t e t h e p r o t h a l l i a f a i l e d t o d e v e lo p
a p l a t e l e t o r f i l a m e n t . The c e l l num ber i n p r o t h a l l i a o f b o th s p e c ie s
grow n on m ed ia 10 and 11 i s n o t s i g n i f i c a n t l y d i f f e r e n t from t h a t o f
p r o t h a l l i a grow n on medium 7 , b u t th e c e l l s w ere v e ry s m a l l . T h is a g re e s
w i th a c o n c lu s io n d e r iv e d i n t h e p r e v io u s p a r a g r a p h ; s u l f a t e can in f l u e n c e
c e l l e n la r g e m e n t .
I n summ ary, th e p h o to g ra p h ic s t u d i e s and m ic ro s c o p ic o b s e r v a t io n s
h av e b e e n u s e d t o show t h a t when p r o t h a l l i a o f b o th f e r n s a r e grown on
m ed ia c o n ta in in g a d e q u a te p h o s p h a te , t h e c e l l s i z e and num ber d o es n o t
d e c r e a s e . The a d d i t i o n o f s u l f a t e t o a p h o s p h a te - s u l f a t e d e f i c i e n t
medium a l s o c a u se d th e c e l l s i z e t o b e i n c r e a s e d .
36
32
28
24
2 0
16
12
8
4
2. T
24
A————A
A..........A
MEDIUM123456
20 25I
30
DAYS AFTER GERMINATION
Pr«h weight of N . co ch iten tit grown on vorioue omounti of phoephote.
xxitoin 4 .4 mM K , 4 .2 mM C o , 2.1 mM M g, 8 .4 mM N O and 1 .6 mM C l . The N o , HPO , and SO con- ) of the media ore respectively : Medium I , 1 .2 , 1 .4 , 2 .1 j Medium 7 , 1 .0.1 .2 , 2 .4 ; Medium 3 , 0 .^ , 0 .8 , i, 0 .6 , 0 .6 , 2 .9 ; Medium 5 , 0 .4 , 0 .3 , 3 .3 ; Medium 6 , 0 .2 , 0 .0 , 3 .5 .
25
MEDIUM
DAYS AFTER GERMINATION
Figure 3 . The fresh w i e ^ t o f N . sinuota grown on various amounts of phosphate.
All media contain 4 .4 mM K , 4 .2 mM C o , 2.1 mM Mg , 8 . 4 mM N O and 1 .6 mM C l . The N o , HPO , and SO 4 concentrations (mM) of the medio ore respectively: Medium 1, 1 .2 , 1 .4 , 2 .1 ; Medium 2 , 1.0,1 .2 , 2 .4 ; Medium 3 , 0 . 8 , 0 . 8 , 2 .7 ; Medium 4 , 0 . 6 , 0 . 6 , 2 .9 ; Medium 5 , 0 . 4 , 0 .3 , 3 . 3 . ; Medium 6 , 0 . 2 , 0 . 0 , 3 .3 .
26
MEDIUM
DAYS AFTER GERMINATION
Figure 4 . The fresh weight of hi . cochise n»ii grown on modio contoining o def iciency or on excess of phosphate or sulfate .
Medio 7 , 8 , 9 , and 10 contain the following concentrations of ions (mM) respectively: N o , 4.1 , 3 . 5 , 1 .5 , 0 .1 ; K , 8 .0 , 4 . 4 , 7 .2 ,"2 .9^ C o , 4 . 0 , 4 . 2 , 4 . 2 , 4 .2 ; Mg, 4 .2 , 2.1 , 2 . 1 , 3 .4 ; N O , 0 . 2 , 8 . 4 , 8 . 4 , 8 .4 ; C l , 4 . 0 , 1 .6 , 1 .6 , 9 . 7 . The HFO^ and SO concentrations of the media (mM) ore: 0 . 0 , 1 .4 , 1 .4 , 0 .0 and 12 .2 , 3 . 5 , 3 . 5 , 0 . 0 . Medium contains no nutrients. No growth curves were obtained for medio 10 and 11 .
27
MEDIUM
IS 2 0 2 5 SO 3 5
DAYS AFTER GERMINATION
Figure 5 . The fresh weight o f N . t inuoto grown on medio containing a def iciency or an excew of phosphate or su lfa te .
Media 7 , 8 , 9 , and | 0 contain the following concentrations of ions (mM) respectively: N o , 4 . 1 , 3 . 5 , 1 .5 , 0 .1 ; K , 8 . 0 , 4 . 4 , 7 . 2 , 2 .9 ; C a , 4 . 0 , 4 . 2 , 4 . 2 , 4 .2 ; Mg, 4 . 2 , 2 .1 , 2 . 1 , 3 .4 ; N O , 0 . 2 , 8 . 4 , 8 . 4 , 8 .4 ; C l , 4 . 0 , 1 .6 , 1 .6 , 9 . 7 . The H P O j and SO concentrations of the media (mM) are: 0 . 0 , 1 . 4 , 1 0 . 0 , and 1 2 .2 , 3 . 5 , 3 . 5 , 0 . 0 . MediumII contains no nutr ients . Vio growth curves were obtained for media 10 and 11.
Plate 1 .
Plate 2.
Figure 6.
\
28
N . cochisensis prothallium on medium 1 twenty-five days after germination.
oN . cochisensis prothallium on medium 4 twenty-five days after germination.
Plates of N . cochisensis and N . s inuata prothallia grown on different agar media.
I
29
Plate 3. N . sinuata prothallia on medium 4 twenty-five days after germination.
Plate 4 . N . cochisensis prothal lia on medium 7 twenty-five days after germination.
Figure 6 . — Continued.
30
Plate 5.
Plate 6.
N . cochisensis prothallia on medium 5 twenty-five days after germination.
N . cochisensis prothal lia on medium 1 0 thir ty-f ive days after germination .
Figure 6 . — Continued.
SUMMARY
P r o t h a l l i a o f H o th o la e n a c o c h i s e n s i s and H o th o la e n a s i n u a t a
f e r n s w ere grown from s p o re s and c u l t u r e d on a g a r m ed ia t h a t c o n ta in e d
v a r io u s l e v e l s o f p h o s p h a te and s u l f a t e . P h o to g ra p h ic and f r e s h
w e ig h t s t u d i e s w ere u s e d t o m easu re t h e i r g ro w th r e s p o n s e s .
U s in g th e s e s t u d i e s , i t was p o s s i b l e t o show t h a t t h e lo w e s t
l e v e l o f p h o s p h a te r e q u i r e d b y th e f e r n s i s 0 .8 mM i n t h e p r e s e n c e o f
2 .7 mM s u l f a t e and th e o t h e r io n s o f K n o p 's s o l u t i o n .
When p r o t h a l l i a o f b o th f e r n s w ere grown on m ed ia w i th low
am ounts o f p h o s p h a te , t h e c e l l num ber and s i z e w ould d e c r e a s e .
The a d d i t i o n o f s u l f a t e t o p h o s p h a te - s u l f a t e d e f i c i e n t m ed ia w ould
i n c r e a s e th e c e l l s i z e .
I n c o n c lu d in g , H ev ly (5? 6 ) r e p o r t e d t h a t N. c o c h i s e n s i s w ould
grow e q u a l ly w e l l on K n o p 's and P r a n t i ' s m e d ia ; h o w e v e r, N. s i n u a t a
w ould grow w e l l on K n o p 's medium b u t w ould n o t grow on P r a n t l ' s medium.
T hese r e s u l t s were, n o t v e r i f i e d ; i t was fo u n d t h a t W. c o c h i s e n s i s d id
n o t grow e q u a l ly w e l l on b o th m e d ia , and K. s in u a t a w ould grow f o r a t
l e a s t two m onths i f p l a c e d on P r a n t l ' s m edium . The r e s u l t s o b ta in e d i n
t h i s s tu d y do n o t r u l e o u t th e p o s s i b i l i t y t h a t s u l f a t e can s u b s t i t u t e
f o r p h o s p h a te ; i n f a c t , th e y s u g g e s t t h a t s u l f a t e can s u b s t i t u t e f o r
p h o s p h a te , b u t f u r t h e r w ork i s n e e d ed t o e s t a b l i s h t h i s h y p o th e s i s a s
a f a c t .
31
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