University of Montana
ScholarWorks at University of Montana
Theses, Dissertations, Professional Papers
Graduate School
1965
The response of Marchantia polymorpha L.
gemmae in aseptic culture to temperature light and
substrate
Robert J. Thullen
The University of Montana
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Recommended Citation
Thullen, Robert J., "The response of Marchantia polymorpha L. gemmae in aseptic culture to temperature light and substrate" (1965).
Theses, Dissertations, Professional Papers. Paper 6772.
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THE RESPONSE OF MARCHANTIA POLYMORPHA L.
GEMMAE IN AS E PT IC CULTURE TO
T E M P E R A T U R E , LIGHT, AND SUBSTRATE
by
R o b e r t J« Thullen
B . S . F . M ontana State U n i v e r s i ty , I960
P r e s e n t e d in p a r t i a l f u lf il lm e n t of the r e q u i r e m e n t s
f o r the d e g r e e of M a s t e r of Science
MONTANA STATE UNIVERSITY
1965
A p p ro v ed by:
C h a i r m a n , B o a r d of E x a m i n e r s
/’/
/y/
/
________
De^^n, G ra d ua te School
JUL -
Date
1966
UMI Number: EP37573
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ACKNOWLEDGMENTS
My thanks to D r , M e y e r C h e s s i n and to the o t h e r m e m b e r s
of the f a c u l t y and staff of the Botany D e p a r t m e n t f o r a l l the help,
a d v i c e , and p a t ie n c e they e x te nd ed to m e while I was w ritin g this
thesis,
I e s p e c i a l l y want to thank D r , C. C. Gordon f o r his e x t r a
h e lp a n d fo r taking the p h o t o m i c r o g r a p h s ; and m y wife, J u l i e, for
h e r help in typing and o t h er ted io us but n e c e s s a r y j o b s .
TABL E OF CONTENTS
A b s t r a c t ..........................................................................................................................i
I n t r o d u c t i o n ................................................................................................................. 1
M e t h o d s ..........................................................................................................................4
R e s u l t s ..........................................................................................................................8
D i s c u s s i o n ................................................................................................................ 14
Appendix
P l a t e of P h o t o g r a p h s .......................................................................... 19
T a b l e d R e s u l t s .........................................................................................20
B i b l i o g r a p h y .............................................................................................................24
ABSTRACT
T h is e x p e r i m e n t was d e s ig n e d to show the r e s p o n s e of
M a r c h a n t i a p o l y m o r p h a L . g e m m a e grown in a s e p t i c c u l t u r e to t e m ­
p e r a t u r e , lig ht , and s u b s t r a t e .
In the t e m p e r a t u r e e x p e r i m e n t s , the
n u m b e r of r h i z o i d s p r o d u c e d d e c r e a s e d s h a r p l y at 26^C.
Light i n t e n ­
s i t i e s u s e d i n th is e x p e r i m e n t h a d no a p p a r e n t effect on r h i z o i d
i n i ti a t i o n .
Any d e c r e a s e in the n u m b e r of r h i z o i d s was c a u s e d by an
a c c o m p a n y i n g h i g h e r t e m p e r a t u r e and not by light i n te n s i ty ,
A change
in the p o s i t io n of the light f r o m above the g e m m a e to b e n ea th it c a u s e d
a change in the p e r c e n t a g e of r h i z o i d s which a r o s e f r o m the u pp e r and
l o w e r s u r f a c e s , but did not n e c e s s a r i l y change the total n u m b e r .
With
the l ig h t above, g e m m a e on the s u r f a c e and g e m m a e s u b m e r g e d within
a g a r p r o d u c e d 92 and 85% of t h e i r r h i z o i d s , r e s p e c t i v e l y , f r o m the
s u r f a c e op p osi te the light.
When the light was po si tio ne d b enea th the
g e m m a e only 50 and 45% of the r h i z o i d s , r e s p e c t i v e l y , w e r e p r o d u c e d
f r o m the s u r f a c e o pp osite the light.
d uc ed p h e n o m e n a .
This a p p e a r e d to be an auxin i n ­
In the f o r m e r , light and g r a v i t y i n it i a te d r h i zo i d s
f r o m the s a m e side of the g e m m a e .
and g r a v i t y w e r e o p pose d.
In the l a t t e r , the e ff ect s of lig ht
T ota l d a r k n e s s c a u s e d the r h i z o i d s to be
p r o d u c e d only by the l o w e r s u r f a c e r e g a r d l e s s of w he th er the a g a r was
in c o n t a c t with the u p p e r or l o w e r s u r f a c e of the g e m m a e .
s u b s t r a t e t h e r e f o r e ha d no effect on r h i z o i d i n it ia ti on .
The
(The r e s u l t s of
the t o ta l d a r k t r e a t m e n t a r e ad dit io n al evidence that auxin is involved. )
11
The s u b s t r a t e was a p p a r e n t l y only i n s t r u m e n t a l in the s u r v i v a l of the
r h i z o i d s a f t e r o t h e r f a c t o r s h a d c a u s e d t h e i r p ro du ct io n.
INTRODUCTION
It w as o b s e r v e d that g e m m a e of M a r c h a n t i a p o l y m o r p h a L.
p r o d u c e d r h i z o i d s f r o m the s u r f a c e in c o n ta ct with the soil
( H a b e r l a n d t, 1914 and Smith, 1955).
A g e m m a was c ap ab le of r h i z o i d
p r o d u c t i o n f r o m e i t h e r s u r f a c e when young, but f r o m only one s u r f a c e
when i t b e c a m e o l d e r (Sinnott, I960).
A fter the f i r s t few r h i z o i d s w e r e
p r o d u c e d f r o m the s u r f a c e i n c on ta ct with a s u b s t r a t e , the g e m m a
p r o d u c e d r h i z o i d s f r o m the opposite s u r f a c e when it was r o t a t e d 180^
( Ha b er la n d t) .
Light, g r a v i t y , and o t he r e n v i r o n m e n t a l f a c t o r s (Sachs,
1882 an d Sinnott, I960) i n flu e nc ed the i n it i a l d e velo pm en t of r h i z o i d s .
S u b s t r a t e m o i s t u r e did not s e e m to be a f a c t o r but h u m i d it y was i n ­
f lu e n t ia l ( Ha b er la nd t, 1914).
G e m m a e did not pr od uc e r h i z o i d s f r o m
the side in c o n ta c t with d r y a i r ( Haberlandt, 1914).
D o r m a n c y h a s not been o b s e r v e d in g e m m a e of M. p o l y m o r p h a .
H ow e v er, in t h i r t y y e a r s O p p e n h e i m e r did not o b s e r v e a single g e m m a
g e r m i n a t i n g in a g e m m a e cup (Moewus and S c h ad er , 1952).
There
a p p e a r e d to be a g r ow t h i n h i b i t o r p r e s e n t in the p a r e n t thallus which
p r e v e n t e d g e r m i n a t i o n of an i n t a c t g e m m a .
When a g e m m a was r e ­
m o v e d f r o m the p a r e n t t h a l l u s , and s uitable s u b s t r a t e was found,
germ ination o ccu rred .
F r e e d g e m m a e w e r e light s e n si t iv e (Fitting,
1938 and L i l i e n s t e r n , 1927) but r e a c t i o n to light was m o d if ie d by
t e m p e r a t u r e , s u b s t r a t e , and o t h e r e n v i r o n m e n t a l f a c t o r s (Fitting,
1936; 1937; and 1938).
L ilienstern observed early gemmae m ortality
and she a t t r i b u t e d this m o r t a l i t y to st ro n g light i n t e n s i t y ( L i l i e n s t e r n ,
1927).
S u b s t a n c e s such a s c o u m a r i n and p a r a s o r b i c a c i d ha d been
found to inh ib it r h i z o i d i n i ti a t i o n (Moewus and S c h a d e r , 1952).
H a b e r l a n d t (1914) a t t r i b u t e d the i n it ia ti o n of r h i z o i d s to a b a r o ­
m e t r i c p r e s s u r e s t i m u l u s on p l a s m a and s t a r c h g r a i n a r r a n g e m e n t
which c a m e about t h ro ug h the a c tio n of g r a v i t y .
He f u r t h e r s t a t e d
th at the p o s i t i o n of the n u c le u s p r e d i c t e d the p osi tio n of the r h i z o i d on
the wall of the c e l l .
o u t e r c e l l wall.
The r h i z o i d u s u a l l y a r o s e f r o m the c e n t e r of the
Light, as a s t i m u l u s , was not c l e a r l y u n d e r s to o d ;
but the effect of light was r e c o g n i z e d and light was s aid to have an
influence although not as s t ro n g a s that of g r a v i t y .
H a b e r l a n d t a ls o
i n v e s t i g a t e d the i nf lue nce of the s u b s t r a t e on r h i z o i d i nit i a ti o n and
conclu ded that the s u b s t r a t e a n d m o i s t a i r w e r e p a r t i a l l y r e s p o n s i b l e
for rhizoid production.
The i nfluence of the s u b s t r a t e was v e r y l i m i t ­
ed while the a ction of d r y a i r was effective in e li m in a ti ng r h iz o i d
i n it i a ti o n .
T his c o n c l u s i o n was c o n t r a r y to the w o rk of G e r t z (1926).
In e x p e r i m e n t s with M a r c h a n t i a and L u n u l a r i a G e r t z found that 71%
of the r h i z o i d s f r o m the d r y a i r side of the g e m m a p ro vi de d that this
side was d a r k .
(This was an i n v e r t e d si tu at io n with the light beneath
the g e m m a . )
G e r t z p e r f o r m e d m a n y e x p e r i m e n t s to t e s t the influence of
e n v i r o n m e n t a l f a c t o r s on r h i z o i d i n it ia ti on .
By exposing opposite
s i d e s of a g e m m a to d if f er en t s t i m u l i , he found that the r h i z o i d s w e r e
p r o d u c e d i n v a r y in g p e r c e n t a g e s f r o m e ac h s id e.
The r e s u l t s of
G e r t z ' s e x p e r i m e n t s left l it t le doubt that g r a v i t y , light, s u b s t r a t e ,
and m o i s t a i r influ e nce d r h i z o i d in itiatio n; but b e c a u s e of a l a c k of
a d e q u a t e h u m i d i t y and light c o n t r o l s the effect of the e n v i r o n m e n t a l
s t i m u l i was not c l e a r l y u n d e r s t o o d .
The following e x p e r i m e n t s w e r e d e s i g n e d in an a t t e m p t to c l a r i f y
the confusing r e s u l t s of p a s t e x p e r i m e n t s and to add a dditio nal i n f o r m a ­
tion about the i n i t ia t i o n of r h i z o i d s during g e r m i n a t i o n of M. polymorpha gemmae.
The o b j e c t i v e s of this study w e r e to d e t e r m i n e the
effect of t e m p e r a t u r e , light and s u b s t r a t e on r h i z o i d i n it i a ti o n of
M a r c h a n t i a p o l y m o r p h a L.
METHODS
B e c a u s e of the e a s e in handling and m a n ip u l a t io n , a g a r was
s e l e c t e d a s the c u l t u r a l m e d i u m .
On a s u g g e s t i o n f r o m D r . C. C.
Gordon^, c o m m e r c i a l o r c h i d a g a r was t r i e d and found s a t i s f a c t o r y .
Both the s t o c k c u l t u r e s and the e x p e r i m e n t a l c u l t u r e s w e r e grown on
this m e d i u m .
The a g a r was m a d e in one l i t e r b a t c h e s a c c o r d i n g to
the m a n u f a c t u r e r ' s d i r e c t i o n s and p o u r e d into s to c k c u l t u r e j a r s and
18 X 150mm t e s t tubes f o r e x p e r i m e n t a l u s e .
G e m m a e to be u s e d in the e x p e r i m e n t s w e r e grown u n d e r s t e r i l e
c on dit io n s.
pha
The s t o c k c u l t u r e s w e r e s t a r t e d with M a r c h a n t i a p o l y m o r -
g e m m a e ( Am m o n s, 1940) f r o m the botany g r e e n h o u s e on the
L i,
Montana State U n i v e r s i t y c a m p u s .
E a r l y e x p e r i m e n t s showed a need
for c o n t a m i n a n t - f r e e plant m a t e r i a l and a way to s t e r i l i z e g e m m a e was
sought.
Although r e f e r e n c e s in the l i t e r a t u r e f r e q u e n t l y gave m e t h o d s
of ke eping the m e d i u m f r e e of c o n t a m i n a n t s , t h e r e was no m e n t i o n of
s t e r i l i z a t i o n of g e m m a e until the w o r k of Kaul, M i t r a , and T r i p a t h i s
(1962),
The s t e r i l i z a t i o n m e t h o d of Kaul et al, was not u s e d a s it was
not d i s c o v e r e d until m y e x p e r i m e n t s w e r e n e a r l y c o m p l e te d .
the following m e t h o d was found s a t i s f a c t o r y .
However,
The g e m m a e u s e d to
s t a r t s t o c k c u l t u r e s w e r e s t e r i l i z e d f o r 3 to 4 m i n u t e s in a solution
^Dr. C. C, Gordon, A s s i s t a n t P r o f e s s o r , Botany and M icro bio lo gy
D e p a r t m e n t , Montana State U n i v e r s i ty ,
of 5% c o m m e r c i a l s od iu m h y p o c h lo r i te b l e a c h plus one d r o p of Tween
detergent.
The s t e r i l i z e d g e m m a e w e r e then t r a n s f e r r e d a s e p t i c a l l y
into s t e r i l e c u l t u r e j a r s containing o r c h i d a g a r .
w e r e k e p t u n d e r 24 h o u r i ll u m i n a t i o n .
T h e s e st oc k c u l t u r e s
The lighting c o n s i s t e d of a 150
watt i n c a n d e s c e n t bulb at a d i s t a n c e of 15 i n ch e s (Anthony, 1962) p r e ­
c ed e d by 4 days of low i n t e n s i t y light ( L i l i e n s t e r n , 1927) f r o m a bulb
of the s a m e w attage at 5 fe et .
B e c a u s e of the l a c k of r o o m t e m p e r a t u r e
c o n t r o l s , the t e m p e r a t u r e was ke pt l e s s than 26°C.
Experience show­
ed that t e m p e r a t u r e s above 26^C often p r o v e d f at al to g e m m a e , and
a l s o to m a t u r e t ha ll i.
E v e n though the g e m m a e w e r e p a l e r in c o l o r
than u s u a l , the c u l t u r e s s e e m e d to be quite h e a l t h y and v i g o r o u s at
25°c,
The l a s t stoc k c u l t u r e was p l a c e d 15 i n c h e s f r o m a 150 watt i n ­
c a n d e s c e n t lig ht s o u r c e with no i n i t i a l subdued light p e r i o d and no
a d v e r s e e ff ec ts w e r e noted.
T h r e e weeks a f t e r the c u l t u r e had been
s t a r t e d , the g e m m a e g r e w into h e a l t h y an d n o r m a l looking thalli.
Unlike the p r e v i o u s s t o c k c u l t u r e s which w e r e kept at 25°C, this
l a t e s t c u l t u r e was p l a c e d in a r o o m at 19 to 20°C.
I d e a l ly one g e m m a was p l a c e d in a t e s t tube, but s o m e t i m e s two
o r m o r e g e m m a e would be i n a d v e r t e n t l y p l a c e d on the a g a r .
If two o r
m o r e g e m m a e w e r e p l a c e d in a t e s t tube, they w e r e s e p a r a t e d to
l e s s e n any p o s s i b l e i n t e r a c t i o n .
B e c a u s e of the s h o r t growing p e r i o d
involved in the e x p e r i m e n t , t h e r e should have be en no c om p eti tio n
b e t w e e n g e m m a e i n the s a m e t e s t tube.
Su rf ac e g r ow n g e m m a e w e r e
p l a c e d on the a g a r s u r f a c e with a s m a l l p l at in u m s p a t u l a .
Submerged
g e m m a e w e r e p l a c e d e i t h e r in s l i t s m a d e i n the a g a r with the s p a t u la
or w e r e c o v e r e d with s m a l l p i e c e s of a g a r tak en f r o m the edge of the
m edium .
S o m e t i m e s it was n e c e s s a r y to c o v e r the s l i t s with p i e c e s
of a g a r to i n s u r e that the g e m m a e w e r e s u b m e r g e d .
The e x p e r i m e n t c o m p r i s e d 15 t r e a t m e n t s .
Light i n t e n s i t y and
p ositio n, t e m p e r a t u r e , and g e m m a e s u b s t r a t e r e l a t i o n s h i p w e r e v a r i e d
to d e t e r m i n e the effe ct on r h i z o i d i nit i at i o n and p r o du ct io n.
Illu m ina­
tion with lig ht of t h r e e d i f f e r e n t i n t e n s i t y r a n g e s ; high, m e d i u m , and
low i nte n si ty ; was u s e d in the e x p e r i m e n t (See Table 1),
A complete
s u m m a r y of the e x p e r i m e n t a l conditions can be found in the appendix.
When counting r h i z o i d s , a p o l a r i z e d light m i c r o s c o p e was u s e d
and the d e n s e r h i z o i d tips w e r e c ounted to avoid confusion.
It was
p o s s i b l e to count a c c u r a t e l y both the u p p e r s u r f a c e and the total
n u m b e r of r h i z o i d s without tu rn in g o v e r the g e m m a .
However, when
it was n e c e s s a r y to i n v e r t the g e m m a , the g e m m a was f i r s t m oun te d
be tw e e n two c o v e r g l a s s e s .
In this way, the unit was t u r n e d over w i t h ­
out d i s t u r b i n g the m o u n t e d g e m m a .
Although m o s t m i c r o s c o p i c w ork was done with living s p e c i m e n s ,
f ix ed m a t e r i a l was e x am in e d .
The m i c r o t e c h n i q u e p r o c e d u r e s w e r e
p r e d o m i n a n t l y those of S a s s (1958) and Johanns on (1940).
TABLE 1
LIGHT INTENSITY RANGES
2 day growing
period
5 day growing
period
High
High
Me dium
90 f t - c
45 f t - c
Low
ist
2 4 hr
2nd
24hr
Remain­
der
45 f t - c *
90
120
120
Continuous i l l u m i n a t i o n
*Foot c a n d l e s .
60
10 f t - c
25
RESULTS
At a p p r o x i m a t e l y 26°C, t h e r e was a r a t h e r s h a r p d e c r e a s e in
the n u m b e r of r h i z o i d s p r o d u c e d (See F i g u r e 1),
The d e c r e a s e was
s e e n i n the s u b m e r g e d g e m m a e a s well a s those gro wn on the s u r f a c e
of the a g a r .
How ev er, a d e c r e a s e in the n u m b e r of r h i z o i d s was not
the only effe ct of high t e m p e r a t u r e .
At 28°C those g e m m a e which did
p r o d u c e r h i z o i d s had only a t y p i c a l o r a b n o r m a l l y shaped r h i z o i d s .
At t e m p e r a t u r e s below 26^C the r h i z o i d s w e r e long, s t r a i g h t ,
t a p e r in g s li g h tl y o v e r t h e i r e n t i r e l en gth to the r e g io n of the tip, and
the tip r o u n d e d off in what a p p e a r e d to be a h e m i s p h e r e . Above 2 6^ 0
the r h i z o i d s w e r e s h o r t , 1/4 n o r m a l le n g th o r l e s s ; with g r e a t e r t a p e r
than n o r m a l ; and the tip was b ulbous.
The u p p e r s u r f a c e r h i z o i d s
grown at the h i g h e r t e m p e r a t u r e s showed a v e r y s h a r p c u r v a t u r e in
r e s p o n s e to g r a v i t y .
At 28°C, t h e s e u p p e r s u r f a c e r h i z o i d s g r e w
downward i m m e d i a t e l y a f t e r c l e a r i n g the s u r f a c e of the g e m m a (See
F i g u r e 2).
H o w eve r, at t e m p e r a t u r e s l e s s than 2 6^ 0 the u p p e r
s u r f a c e r h i z o i d s a r c h e d m o r e gently, and u s u a l ly s t a r t e d a downward
p a th s o m e d i s t a n c e f r o m the g e m m a .
The effect o c c u r r e d r e g a r d l e s s
of w h e t h e r the g e m m a was s u r f a c e g r ow n or s u b m e r g e d in the a g a r .
During the s a m e se t of e x p e r i m e n t s , an i n v e r t e d t r e a t m e n t
( T r e a t m e n t L) p r o d u c e d no r h i z o i d s .
While the r o o m t e m p e r a t u r e was
28°C, the t e m p e r a t u r e n e a r the g e m m a e was s u s p e c t e d to be h i g h er
t ha n 28*^C due to a b l ac k cloth which was u s e d to e li m in a te r e f l e c t i o n .
8
F IG U R E 1
T E M P E R A T U R E E F F E C T ON THE
PRODUCTION OF RHIZOIDS
70
Average
Number
of Rhizoid s
per Gemma
60
50
40
30
20
10
0
20
22
24
26
Tem perature
28
30
C
A 5 day growing p e r i o d with continuous
i l l u m i n a t i o n f r o m above. (See Table 1)
G e m m a e gro w n b e n e a th a g a r
surface.
G e m m a e grown on the a g a r
surface.
F IG U R E 2
CURVATURE OF RHIZOIDS AS E F F E C T E D
BY T E M P E R A T U R E
Rhizoid
Gemma
T e m p e r a t u r e l e s s than 26 C
Rhizoid
Gemma
T e m p e r a t u r e g r e a t e r than 26*^C
10
11
The effect of s u b s t r a t e was t e s t e d to a l i m i t e d ex te nt.
O bser­
v a ti on s m a d e on g e r m i n a t i n g g e m m a e by the a u th or b e f o r e this
e x p e r i m e n t showed that so m e r h i z o i d s f o r m e d f r o m the side of the
g e m m a e not in c o n ta ct with the s u b s t r a t e .
The l o g i c a l q u e s tio n was
w h e t h e r the n u m b e r of r h i z o i d s would i n c r e a s e if the g e m m a e w e r e
s u r r o u n d e d by s u b s t r a t e .
T h e r e was a n i n c r e a s e of l e s s than 10%
c a u s e d by the s u b s t r a t e when the g e m m a e w e r e s u b m e r g e d within the
a g a r (See F i g u r e 3).
In t r e a t m e n t Q, w h e r e no light was u s e d and the
a g a r was in c o n ta c t with the u p p e r s u r f a c e only, 100% of the r h i z o i d s
w e r e p r o d u c e d f r o m the l o w e r s u r f a c e .
H e r e s u b s t r a t e con ta ct had
no effect.
When the p o si tio n of the light was ch ang ed so that the i ll u m i n a t io n
c a m e f r o m b e n e a t h the g e m m a e , a change in the n u m b e r of r h i z o i d s
coming f r o m e ac h s u r f a c e was o b s e r v e d .
When the g e m m a e w e r e
s u r f a c e g r ow n with the lig ht coming f r o m above, the g e m m a e had n e a r ­
l y a l l the r h i z o i d s coming f r o m the s u r f a c e not facing the light.
On the
a v e r a g e 92% of the r h i z o i d s w e r e found to a p p e a r f r o m the side of the
g e m m a e facing downward and f a r t h e s t f r o m the light (See f i g u r e 3).
The 8% of the r h i z o i d s growing f r o m the light facing s u r f a c e m i gh t
have been i n du ce d by light being r e f l e c t e d f r o m the s u r f a c e of the g l a s s
t e s t tube and f r o m the a g a r .
Such a c on cl usi on was s u g g e s t e d by the
d a r k t r e a t m e n t s ( T r e a t m e n t s P a n d Q) w h e r e 100% of the r h i z o i d s
g r e w f r o m the downward facing s u r f a c e .
By changing the p osi tio n of
F IG U R E 3
E F F E C T OF LIGHT, GRAVITY, AND
MOISTURE
100
90
80
Percent
of
Rhizoids
70
60
(Sonfocf
50
40
30
20
10
0
I P o s i t i o n of the light
Above
^ Beneath
Above ' B en ea th
Grown on s u r f a c e
Grown s u b m e r g e d
The light a r e a r e p r e s e n t s the p e r c e n t
of the r h i z o i d s coming f r o m the s u r f a c e of
the g e m m a facing u p w a r d s .
The d a r k a r e a r e p r e s e n t s the p e r c e n t
of the r h i z o i d s coming f r o m the s u r f a c e of
the g e m m a facing downward.
12
13
the light to b e n e a t h the g e m m a , about 50% of the r h i z o i d s now a r o s e
f r o m the side of the g e m m a facing downward and the light, and 50% of
the r h i z o i d s a r o s e f r o m the s u r f a c e of the g e m m a e facing u p w a r d and
opp osite the light s o u r c e .
When the g e m m a e w e r e s u b m e r g e d within
the a g a r a like change o c c u r r e d .
With the light above the s u b m e r g e d
g e m m a e , about 85% of the r h i z o i d s w e r e p r o d u c e d f r o m the s u r f a c e of
the g e m m a e facing downw ard and opposite the light (See F i g u r e 3),
When the light was p l a c e d b e n e a t h the s u b m e r g e d g e m m a e , about 45%
of the r h i z o i d s a r o s e f r o m the downward and light facing s u r f a c e , and
55% of the r h i z o i d s a p p e a r e d f r o m the s u r f a c e facing u p w a r d and
opposite the light.
DISCUSSION
The m o r t a l i t y of g e m m a e o b s e r v e d u n d e r e x p e r i m e n t a l c o n d i ­
t ions by L i l i e n s t e r n (1927), which she a t t r i b u t e d to high light i n ­
t e n s i t y , was q u e s t io n a b l e .
The light, in L i l i e n s t e r n ' s e x p e r i m e n t s ,
w as sunlight at a south window in A p r i l ,
The i n t e n s i t y of this s u n ­
light m a y have b e e n l e s s than that found in n a t u r e during the s u m m e r
in s o m e a r e a s in h a b it ed by M a r c h a n t i a p o ly m o r p h a.
A fter a f o r e s t
f i r e M, p o l y m o r p h a would g r o w if the a r e a had sufficient m o i s t u r e
(Wilson and L o o m i s , 1952),
The d i f f e re n c e b e tw e e n the f o r e s t c o n d i ­
t io n s, a f t e r a f i r e ha d denuded the land, and L i li e n s t e r n * s e x p e r i m e n t s
was p r o b a b l y t e m p e r a t u r e . L i l i e n s t e r n gave no t e m p e r a t u r e data. The
t e s t tubes u s e d in h e r e x p e r i m e n t s , when p la c e d in the sun, p r o b a b l y
h e a t e d m o r e than the cool m o i s t h a b it at w h er e M, p o l y m o r p h a is
found in n a t u r e .
T em perature,
as the c a u s e of g e m m a e m o r t a l i t y , was a ls o
s u g g e s t e d by the c o m p l e t e l a c k of s u r v i v a l of the g e m m a e o c c u r r i n g
in T r e a t m e n t L w h e r e the t e m p e r a t u r e was s u s p e c t e d to be above
28^C,
In T r e a t m e n t L two c o n s e c u t i v e 24 hour p e r i o d s of r e d u c e d
light i n t e n s i t y , 45 and 90 f t - c , w e r e u s e d , followed by 3 days of 120 f t - c .
O t h e r g e m m a e which g r e w u n d e r the s a m e light s o u r c e but at a s l i g h t ­
ly l o w e r t e m p e r a t u r e , h e r e the r e c o r d e d t e m p e r a t u r e was 28°C, did
not die but p r o d u c e d so m e r h i z o i d s . The l a s t s to c k c u l t u r e , as noted
in the m e t h o d s , was p l a c e d 15 i n c h e s f r o m a 150 watt bulb yielding
14
15
132 f t - c at a t e m p e r a t u r e of 19 to 20°C with no p e r i o d of r e d u c e d light
i n t e n s i t y and it did not show a b n o r m a l de velo pm ent.
E x p e r i m e n t a t i o n by H a b e r l a n d t (1914) a t t r i b u t e d r h i z o i d i n it i at i o n
to e n v i r o n m e n t a l i n flu e n c e s upon the n u cle us and s t a r c h g r a i n s of the
cell.
H a b e r l a n d t p r o p o s e d that r h i z o i d i n it ia ti o n was e s t a b l i s h e d by
the a c tio n of the nu c le u s and s ta to li th ic s t a r c h in c e l ls p r e d e s t i n e d to
p r o d u c e r h i z o i d s . Smith (1955) a l s o s t a t e d that som e c e l l s w e r e p r e ­
d e s t i n e d to p r o d u c e r h i z o i d s .
" G e m m a e lodging on soil develop
r h i z o i d s f r o m c o l o r l e s s c e l l s on the face next to the soil. " Such
c o l o r l e s s c e l l s (it was a s s u m e d that c o l o r l e s s m e a n t the l a c k of
c h l o r o p l a s t s ) ha d not b ee n o b s e r v e d during the a u t h o r ' s e x p e r i m e n t .
Although s om e c e l l s looked c o l o r l e s s , upon c l o s e r e x am i n a tio n the
c e l l s did c o n ta in c h l o r o p l a s t s . No s e c t i o n e d g e m m a showed c e l l s which
w e r e f r e e of c h l o r o p l a s t s in the cup o r b e f o r e g e r m i n a t i o n .
Some
r h i z o i d s w e r e o b s e r v e d with c h l o r o p l a s t s , and it was s u s p e c t e d t h e s e
c h l o r o p l a s t s m i g r a t e d f r o m the c e l l which p r o d u c e d the rh i z o i d . How­
ever,
r h i z o i d - p r o d u c i n g c e l l s w e r e p r e d e t e r m i n e d to a l i m i t e d extent;
r h i z o i d s w e r e p r o d u c e d only by the c e l l s c e n t r a l l y l o ca te d on the
s u r f a c e of the g e m m a .
Only a few r h i z o i d s w e r e o b s e r v e d growing
f r o m the edge of the g e m m a and then n e v e r f r o m the vici ni ty of the
apical initials.
O b s e r v a t i o n s on g e m m a e which w e r e allowed to grow l o n ge r than
five days i n d i c a te d that all the r h i z o i d s p r o d u c e d by the g e m m a e t h e m -
16
s e l v e s w e r e f o r m e d by the fifth day.
All r h i z o i d s which a r o s e a f t e r
this t im e a p p e a r e d f r o m the newly f o r m e d thallus t i s s u e and not f r o m
the o r i g i n a l t i s s u e of the g e m m a .
On the b a s i s of d ata f r o m the light p o s i tio n e x p e r i m e n t s , it
a p p e a r e d that r h i z o i d i n i ti a t i o n m a y be m e d i a t e d by auxin.
Light, when
d i r e c t e d a t the g e m m a e f r o m beneath, c a u s e d about 50% of the r h i z o i d s
to be i n i t i a t e d f r o m the s u r f a c e opposite the light and the effect of
gravity.
This was in c o n t r a s t to 92% of the r h i z o i d s which w e r e p r o ­
duced f r o m the s u r f a c e f a r t h e s t f r o m the light when the light was above
the g e m m a e .
In the f o r m e r , the a ction of light and g r a v i t y on the
g e m m a e was opposed.
In the l a t t e r , light and g r a v i t y w e r e both acting
upon the g e m m a to p r o d u ce r h i z o i d s f r o m the s a m e side.
This effect
m a y have b een due to auxin m i g r a t i o n , d e s t r u c t i o n of auxin on the l i g h t ­
ed sid e, a d e c r e a s e in the s e n s i t i v i t y of the t i s s u e on the lig ht ed s id e,
or s o m e c om b in at io n of t h e s e effects (Sinnott, I960).
co nd uct ed in the a b s e n c e of light, all of the
In e x p e r i m e n t s
r h i z o i d s w e r e p r o d u c e d by
the d ownw ard facing s u r f a c e (See T r e a t m e n t s P and Q),
In the above
e x p e r i m e n t s the auxin a p p e a r e d to have been a c te d upon only by g r a v i t y
r e s u l t i n g i n r h i z o i d s f r o m only the l o w e r s u r f a c e .
Although the data on s u b s t r a t e effect was l im i te d , the c o ncl usi on
was that s u b s t r a t e h a d v e r y l i t t le o r no i nfluence.
A gem m a seemed
c a p a b l e of r h i z o i d p r o d u c ti o n with equal s u c c e s s w h e t he r a s u r f a c e
was in c o n ta c t with the a g a r o r not.
E vidence was s e en in T r e a t m e n t
17
Q w h e r e the a g a r c o n t a c te d only with the u p p e r s u r f a c e and the r h i z o i d s
w e r e a l l p r o d u c e d by the l o w e r s u r f a c e . My o b s e r v a t i o n s s u p p o r t e d
the c o n c l u s i o n of H a b e r l a n d t (1914) that something o t h e r than s u b s t r a t e
c o n ta c t w as n e c e s s a r y f o r r h i z o i d f o r m a t i o n . H a b er la n d t s t a t e d that
d r y a i r will stop r h i z o i d i n it i at i o n and s u b s t r a t e c on ta ct was not v e r y
in flu e nti al in r h i z o i d init iat io n .
In n a t u r e the i nit i a ti o n of r h i z o i d s is p r o b a b l y an auxin effect.
The au xin m a y be a c t e d upon by g r a v i t y and light producing r h i z o i d s
f r o m the s u r f a c e w h e r e the auxin is c o n c e n t r a t e d .
F e w o r no r h i z o i d s
a r e p r o d u c e d f r o m the s u r f a c e w he re the auxin is r e d u c e d below s o m e
c r i t i c a l quantity.
Such f a c t o r s a s h u m i d it y and s u b s t r a t e d e t e r m i n e
the s u r v i v a l of r h i z o i d s a r i s i n g u n d e r v a r i o u s e n v i r o n m e n t a l cond itions.
Rhizoids growing into a d r y e n v i r o n m e n t b e c o m e d e s s i c a t e d and die,
but the i n i t i a l s t i m u l u s is p r o b a b l y s t i l l in effect on r h i z o i d - p r o d u c i n g
cells.
Such d e s s i c a t e d r h i zo i d s w e r e o b s e r v e d in s e v e r a l e x p e r i m e n t s .
APPENDIX
PHOTOGRAPHS
Ph oto 1,
A c r o s s s e c ti o n of a g e m m a , h a r v e s t e d 4 days a f t e r
planting, showing 2 u p p e r s u r f a c e r h i z o i d s .
The
s u b s t r a t e , in this and su cceeding photos, was o r c h i d
agar.
Photo 2,
A stoc k c u l t u r e containing an e x c e ll en t gr ow th of
M a r c h a n t i a p o l y m o r p h a , which was s t a r t e d f r o m a
s ingle g e m m a .
Photo 3.
A c r o s s s e c t i o n of a 6 day old g e m m a showing an
u p p e r s u r f a c e r h iz o i d .
Ph oto 4.
A c r o s s s e c t i o n of a 2 day old g e m m a showing a
lower surface rhizoid.
18
*■\
*4
y
y--v
&
/
à
.*Ÿ,
-I*'
/
VV
v>
•fv' " " ^
W
/
*o
tv
V
SUM M ARY O F TH E T R E A T M E N T S
G e m m a e on a g a r s u r f a c e
Light f r o m above
% R hizoids f r o m the
% Rhizoids f r o m the
s u r f a c e of the g e m m a
s u r f a c e of the g e m m a
facing the light
not f ac in g the light
Light i nte nsity :
F i r s t 24 h r s .
45 f t - c
Second 24 h r s .
90 f t - c
R emainder
120 f t - c
H a r v e s t e d on fifth day
T reatm ent A
t e m p . 28 ^0
60
40
Treatm ent B & C
t em p . 26°C
16
84
T reatm ent D
t e m p . 20 to
22°c
11
89
Light inte nsi ty :
F i r s t 24 h r s .
Second 24 h r s .
90 f t - c
120 f t - c
H a r v e s t e d a f t e r 48 h r s .
Treatm ent E
t e m p . 16 to
19 ° C
77
93
Light intensity;
F i r s t 24 h r s .
R em ainder
45 f t - c
60 f t - c
H a r v e s t e d a f t e r 48 h r s .
Treatm ent F
t e m p . 25 C
78
22
Light inte nsi ty :
F i r s t 24 h r s .
Rem ainder
10 f t - c
25 f t - c
H a r v e s t e d a f t e r 48 h r s .
Treatm ent G
t e m p . 25 C
85
15
20
21
S u m m a r y C o n 'd
G e m m a e g ro w n s u b m e r g e d
Light f r o m above________________________
% Rhizoids f r o m the
% Rh izo ids f r o m the
s u r f a c e of the g e m m a
s u r f a c e of the g e m m a
facing the light
Light i n te n s it y :
F i r s t 24 h r s .
Second 24 h r s .
R em ainder
45 f t - c
90 f t - c
120 f t - c
H a r v e s t e d on fifth day
Treatm ent H
t em p . 28°C
53
47
Treatm ent I
t e m p . 26°C
18
82
T reatm ent J
t e m p . 20 to
220C
32
68
Light i nte n si ty :
F i r s t 24 h r s .
Second 24 h r s .
90 f t - c
120 f t - c
H a r v e s t e d a f t e r 48 h r s .
T reatm ent K
t em p . 16 to
19°C
15
85
22
S u m m a r y C o n 't
G e m m a e on a g a r s u r f a c e
Light f r o m beneath._______
% Rhizoids f r o m the
% Rh izo id s f r o m the
s u r f a c e of the g e m m a
s u r f a c e of the g e m m a
f ac in g the light________
not facing the light
Light i nte nsity:
F i r s t 24 h r s .
Second 24 h r s .
R em ainder
45 f t - c
90 f t - c
120 f t - c
H a r v e s t e d on fifth day
T reatm ent L
t e m p , 28°C
s u s p e c t e d to
be h ig he r)
T reatm ent M
t e m p . 26°C
0
c o v e r e d with a
b l a c k cloth
0
74
26
Light i n t e n s i t y
F i r s t 24 h r s .
Second 24 h r s .
90 f t - c
120 f t - c
H a r v e s t e d a f t e r 48 h r s .
Treatm ent N
t em p . 16 to
19°C
49
51
23
S u m m a r y C o n ’t
G e m m a e grown s u b m e r g e d
Light b en ea th________________
% Rhizoids f r o m the
% Rh izo id s f r o m the
s u r f a c e of the g e m m a
s u r f a c e of the g e m m a
facing the light
not f acing the light
Light intensity:
F i r s t 24 h r s .
Second 24 h r s .
90 f t - c
120 f t - c
H a r v e s t e d a f t e r 48 h r s
T reatm ent 0
t e m p . 16 to
19^0
42
58
G e m m a e on a g a r s u r f a c e
A ga r below
% R h iz oi ds f r o m the
% Rhizoids f r o m the
s u r f a c e of the g e m m a
s u r f a c e of the g e m m a
f acing u p w a r d
facing downward
Grown in the d a r k
H a r v e s t e d on fifth day
T reatm ent P
t e m p . 25^C
0
100
G e m m a e on a g a r s u r f a c e
A g a r above_____
Grown in the d a r k
H a r v e s t e d on fifth day
Treatm ent Q
t e m p . 25°C
0
100
BIBLIOGRAPHY
A m m o n s , Nelle, 1940. A M a nu al of the L i v e r w o r t s of West V i r g i n i a .
U n i v e r s i t y P r e s s , N o t r e Dame, Indiana.
Anthony, R o b e r t E, 1962. G r e e n h o u s e C u l t u re of M a r c h a n t i a p olym o r p h a and Induction of Sexual R e p r o d u c t iv e S t r u c t u r e s .
T u r t o x News. 40: No. 1, 2-5.
B o n n e r , J a m e s and A r t h u r W . G a l s t o n , 1952. P r i n c i p l e s of P l a n t
P hy s io lo g y . W. H, F r e e m a n and Co. , San F r a n c i s c o .
Buch, H. , 1932. M or ph ol og ie und A n ato mie d e r H e p a t i c a e .
V e r d o o r n , Manual of Bryolo gy, C h a p t e r 2, 41-72.
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