Seed quality studies of native shrubs
by Gerhard Peter Weber
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Agronomy
Montana State University
© Copyright by Gerhard Peter Weber (1980)
Abstract:
Three forb end eight shrub species native to the Rocky Mountain and Northern Great Plains regions
were evaluated for seed viability with 2,3,5-triphenyl-2H-tetrazolium chloride (TZ). Specific staining
techniques were developed for each species. Treatments include presoaks, seed coat puncture, or
removal, and seed bisection. Species tested and their viability values were Achillea millefolium L.,
(yarrow) 86%; Linum lewisii (Pursh), (Lewis flax) 93%; RatTbida columnifera (Nutt.) Wooten and
Standley, (prairie coneflower) 72%; Amelanchier alnifolia (Nutt.) Nutt., (serviceberry) 84%; Amorpha
fruticosa L., (indigobush) 94%; Artemisia tridentata Nutt., (big sage-brush) 66%; Ceretojdes lanata
(Pursh) Howell, (winterfat) 91%; Prunus virginiana L., (chokecherry) 98%; Purshia tridentata (Pursh)
DC., (antelope bitterbrush) 100%; Rhus trilobata Nutt., (skunkbush sumac) 91%; and Symphoricarpos
albus (L.) Blake, (snowberry) 68%.
Skunkbush sumac (Rhus trilobata Nutt.) and serviceberry (Amelanchier alnifolia Nutt.) are native
shrubs extensively distributed in the western United States, and have achieved importance for their use
in revegetation of disturbed lands. Standard germination tests were performed on each species
according to methods outlined in the literature. Seed viability was determined with triphenyl
tetrazolium chloride (TZ). Results indicated special techniques would be required to affect the rapid
germination which is needed in current seed testing programs. Both species have hard or impermeable
seed coats and embryo dormancy normally overcome by cold stratification or fall sowing. Results
confirm that skunkbush sumac germination is promoted by 75 minutes acid scarification and that
KNO3 or GA produce no additional response. Acid scarification for 30 minutes and a mixture of
thiourea (TU) and benzyladenine (BA) as a moistening agent for the media was beneficial to
serviceberry germination. Analysis predicted maximum germination to occur at 100 ppm BA and 100
mM TU. An interaction of BA and TU on germination was observed at the lower concentrations
tested. STATEMENT OF PERMISSION TO COPY
In presenting th is thesis in p a rtia l fu lfillm e n t o f the require­
ments fo r an advanced degree a t Montana State U n iv e rs ity , I agree th at
the Library shall make i t fre e ly a v a ila b le fo r inspection.
I fu rth e r
agree th a t permission fo r extensive copying o f th is thesis fo r
scholarly purposes may be granted by my major professor, o r, in his
absence, by the D irecto r o f L ib ra rie s .
I t is understood th a t any
copying or pu blication o f th is thesis fo r fin a n c ia l gain shall not be
allowed without my w ritte n permission.
Signature
Date
s
SEED QUALITY STUDIES OF NATIVE SHRUBS
AND FORBS
by
GERHARD PETER WEBER
A thesis submitted in p a rtia l fu lfillm e n t
o f the requirements fo r the degree
of
MASTER OF SCIENCE
in
Agronomy
Approved:
Graduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
August, 1980
iii
ACKNOWLEDGMENTS
I wish to express my sincere g ra titu d e to Dr. Loren E. Wiesner
fo r serving as my major professor and fo r giving fre e ly o f his advice,
guidance and frien d sh ip ; Dr. Ronald Lockerman fo r his candid advice
and fo r help with the preparation o f th is th esis; Dr. Jarvis Brown and
Mr. Lee Hart fo r t h e ir help and cooperation with the completion o f my
degree program; and Dr. Richard Lund fo r helping with the s t a tis t ic a l
analysis o f the data presented in Chapter I I o f th is th e s is .
I also thank my w ife , Debby, fo r giving her support and fo r showing
the patience and perseverance necessary when one is married to a
graduate student.
TABLE OF CONTENTS
Page
VITA ..................... .... . . .............................. .....................................................
ACKNOWLEDGMENTS..................................... ................................ ....
LIST "OF TABLES..................................................................
LIST OF FIGURES
....................................................................................................
ABSTRACT......................................................................................
REVIEW OF LITERATURE ......................................................................
Seed V ia b ilit y as Determined byTetrazolium ... ............................
The S e e d .........................................................
G e rm in a tio n ............................ ......... .................................................
D o rm a n c y ......................... f .......................................................... ....
CHAPTER I:
TETRAZOLIUM VIABILITY PROCEDURES
FOR NATIVE SHRUBS AND FO R B S ................................. : . .
Introduction ...................................................................................................
M aterials and Methods ...............................................................................
Results and D is c u s s io n .............................................................
Suggested Tetrazolium Test Procedures . ; ......................................
CHAPTER I I :
ii
Hi
v
vi
v ii
I
T
5
7
7
21
22
24
26
36
IMPROVING GERMINATION OF SKUNKBUSH
SUMAC ANDSERVICEBERRYSEED..................................................
42
In tr o d u c tio n ................................ ..................................................................
M aterials and Methods . . . . .
..........................................................
Results and D is c u s s io n ..............................................................................
43
46
51
APPENDIX........................
66
LITERATURE CITED . ...........................................................................................
69
V
LIST OF TABLES
Table
1.
2.
3.
4.
5.
6.
7.
8.
9.
10 .
A l.
Page
Percentage v i a b i l i t y , e f f e c t o f la c to p h e n o l , e f f e c t
o f seed coat p rep a ra tio n on S ta in in g , and optimum
te tra z o liu m s ta in in g tim e fo r e ig h t shrubs and
three forbs .....................................
27
Suggested tetrazo liu m v ia b i lit y procedures fo r
e ig h t shrubs and three f o r b s ..........................................................
37
Comparison o f mean percentage germination of
skunkbush sumac as affected by acid s c a r if i­
c a tio n , GAg and K N O g .................................
52
Comparison o f mean percentage germination of
skunkbush sumac as a ffected by acid s c a r if i­
cation and four le v e ls ‘o f K N O g ............................. ' .......................
52
E ffe c t of acid s c a rific a tio n fo r 0 , 60, 75, or
90 minutes and 0 or 24 hour soaks in HgO or 0.2%
KNO3 on speed o f emergence o f skunkbush sumac
a fte r 17 days in the greenhouse ......................................................
53
E ffe c t o f acid s c a r ific a tio n .fo r 0 , 60, 75, or
90 minutes and 0 or 24 hour soaks in HgO or 0.2%
KNO3 on to ta l emergence percent o f skunkbush
sumac a fte r 3 weeks in the greenhouse................................. .... .
53
Comparison o f mean percentage germination of
serviceberry as affected by acid s c a rific a tio n
and a benzyl adenine/thiourea m ixture . . . . . . . . . . . .
55
Comparison o f mean percentage germination of
serviceberry as affected by leaching f o r .24 hours
and GA3 . . .......................................................... ....................................
56
Analysis o f variance fo r the germination response
o f serviceberry to mixtures of BA and TU ............................. ..
61
Tetrazolium v i a b i lit y and germination o f serviceberry as affected by seed size .................................................. .
62
Common and s c ie n t if ic names o f plants referred
to in the lit e r a t u r e review ..................... . . . . . . . . . .
67
vi
LIST OF FIGURES
Figure
1.
2.
3.
4.
5.
6.
Page
TetratzolIum stained and unstained embryos of:
yarrow showing pericarp and puncture p o in t,
Lewis f la x , and p r a ir ie coneflower .........................
. . . . .
28
Tetrazolium stained and unstained embryos of:
serviceberry, indigobush showing chipping
p o in t, w in te rfa t showing in ta c t seed, seed
coat removed and puncture p o in t, and chokecherry showing stony endocarp and highly
stained embryonic a x i s ......................... . . . .............................
31
Tetrazolium stained and unstained embryos of:
antelope bitterbush and snowberry . . . . . . . . . . . .
34
E ffe c t o f low concentration BA/TU mixtures
on the germination o f serviceberry ..............................................
58
E ffe c t o f high concentration BA/TU mixtures
on the germination o f s e rv ic e b e r r y ..............................................
59
E ffe c t o f BA/TU mixtures a t various concen­
tra tio n s on the germination of serviceberry
as predicted by regression analysis . . ..................................
60
v ii
ABSTRACT
Three forb end e ig h t shrub species native to the Rocky Mountain
and Northern Great Plains regions were evaluated fo r seed v ia b ilit y
with 2 ,3 ,5 - t r i p h en yl-2H -te tra zo lium chloride (T Z ). S pecific staining
techniques were developed fo r each species. Treatments include pre­
soaks, seed coat puncture, or removal, and seed b ise c tio n , Species
tested and t h e ir v ia b i lit y values were A ch ille a m ille fo liu m L .,
(yarrow) 86%; Linum le w js ii (Pursh), (Lewis fla x ) 93%; Ratibjda
egIumnife r a ( N u t t .) Wooten and Standley, (p r a ir ie coneflower) 72%;
Amelanchier a ln if o lia (N u tt.) N u t t ., (serviceberry) 84%; Amdrpha
fru tic o s a L ., (indigobush) 94%; Artem isia trid e n ta ta N u t t ., (big sagebrush) 66%; Ceretdjdes lanata (Pursh) Howell, (w in te rfa t) 91%; Prunus
y irg in ia n a L ., (chokecherry) 98%; Purshia trid e n ta ta (Pursh) DC.,
(antelope b itte rb ru s h ) 100%; Rhus tfilo b 'a ta N u tt., (skunkbush sumac)
91%; and Symphdricarpos albus ( L . ) Blake, (snowberr.y) 68%.
Skunkbush sumac ( Rhus tr ilo b a te N u tt.) and serviceberry
(Amelanchier a ln if o lia N u tt.) are native shrubs extensively d is trib u te d
in the western United S ta te s , and have achieved importance fo r th e ir
use in revegetation o f disturbed lands. Standard germination tests
were performed on each species according to methods o u tlined in the
lit e r a t u r e . Seed v i a b i lit y was determined with triphenyl te tr a z o lium
chloride (T Z ). Results indicated special techniques would be required
to a ffe c t the rapid germination which is needed in current seed te s tin g
programs. Both species have hard or impermeable seed coats and embryo
dormancy normally overcome by cold s t r a t if ic a t io n or f a l l sowing.
Results confirm th a t skunkbush sumac germination is promoted by 75 min­
utes acid s c a rific a tio n and th a t KNOg or GA produce no additional
response. Acid s c a rific a tio n fo r 30 minutes and a mixture o f thiourea
(TU) and benzyl adenine (BA) as a moistening agent fo r the media was
b e n e fic ial to serviceberry germination. Analysis predicted maximum
germination to occur a t 100 ppm BA and 100 mM TU. An in te ra c tio n o f BA
and TU on germination was observed a t the lower concentrations tested.
LITERATURE REVIEW
(
Seed V ia b ilit y as Determined by Tetrazolium
H is to ric a l
Seed production and marketing depends upon the accurate assessment
o f seed q u a lity , s p e c ific a lly p u rity and germination.
Whereas, the
former may be evaluated in a few minutes, the la t t e r may require a time
range o f two weeks to several months.
Decisions regarding the process­
ing and marketing o f a p a rtic u la r seed lo t may often be delayed pending
the resu lts o f germination te s ts .
For th is reason rapid methods of
assessing seed v i a b i lit y have been studied since the e a rly 1900's (17,
35).
These e a rly in vestig ations concentrated on chemically analyzing
ground and pulverized
groups o f seeds.
U nfortunately, the lo calized
defects of in divid ual seeds such as fractu res or dead tissues escaped
detection with these methods (6 9 ).
One method was based on the theory
th a t nonviable seeds were more permeable than liv e seeds and would
re a d ily lose e le c tro ly te s to a water solution (4 3 ).
Measurements of
the e le c tr ic a l conductivity o f the leachate would vary d ire c tly with
seed v i a b i l i t y .
This technique has recen tly been more successfully
applied to determinations o f seed vigor (3 5 ).
Another method (1 6 ),
involved measurements o f the r e la tiv e q u a n titie s o f heat produced by a
seed sample.
High heat production was re la te d to extensive microbial
a
a c t iv it y , whereas low heat was thought to in dicate poor v ia b i lit y and
vigo r.
V ia b ilit y assessment based upon the evaluation o f individual seeds
was eventually recognized as being superior to group evaluations (6 9 ).
The techniques used were based on v it a l s ta in in g ; th a t is , the a b ilit y
o f some m aterials to d if f e r e n t ia lly stain liv e and dead tis s u e .
Moore
(69) c red its Dr. Turina o f Yugoslavia with the development o f v ita l
stain in g techniques and Dr.' Neljubow o f Russia fo r applying the use o f
indigo carmine, a tontoxic dye, to th is technique.
He found th a t th is
dye would penetrate dead tissue more re a d ily than healthy tissues.
Evaluations o f v i a b i lit y were based on the r e la tiv e proportions of
colored and uncolored tissues.
However, v it a l stainin g based on dyes
was found to be o f lim ite d value.
Hasagawa is credited (69) with the development o f a v it a l staining
procedure based on the seed's metabolic processes.
With the application
o f selenium and te llu riu m s a lts , enzymatic a c t iv it y w ith in the seed
would a ffe c t a color change.
As described by others (83, 17, 69, 12)
work with these techniques was taken up by the German s c ie n tis t, Lakon,
who developed the topographical v ia b i lit y te s t.
This te s t stressed the
reaction o f s p e c ific seed tissues and resulted in considerable accuracy
in seed v i a b i lit y assessment.
The to x ic selenium s a lts were soon
abandoned by Lakon in favor o f nontoxic tetrazolium s a lts which func­
tioned in the same manner.
He found th a t when the colorless tetrazolium
3
solution came in contact with liv e seed tissue i t was reduced to an
insoluble red pigment; whereas, nonliving tissue did not s ta in .
V ia b ility was then estimated by evaluation o f the extent and location
o f stained embryo tis s u e .
S pecific Mode of Action
Of the several types o f tetrazolium s a lts , the 2 ,3 ,5 -trip h e n y l
te tr a z o li urn chloride d e riv a tiv e , now commonly known as TTC or TZ, is
best suited fo r use in the topographical te s t (17, 3 5 ).
Tetrazolium
chloride occurs as a white to pale yellow c ry s ta llin e powder, re ad ily
water soluble and darkens on exposure to lig h t .
In the presence of
via b le tissue the colorless TZ solutions forms the insoluble red
triph en yl formazan according to the follow ing reaction (8 3 ):
N = N - C 6 hS
Colorless
Red Color
D etailed studies (51, 83, 84) have indicated th a t one or more of
the dehydrogenase enzyme systems appears to be involved in the reduction
re a tio n .
S p e c ific a lly , the reduction o f TZ in corn embryo tissues is
4
c a ta lized by diphosphopyridine nucleotide (DPN)-Tinked dehydrogenases,
p a r tic u la r ly the malic and alcohol systems, and is mediated by
diaphorase (8 4 ).
This sequence may be summarized in the follow ing way
(85):
Food Reserves
digestive & other
enzymes
>V
Respiratory
Intermediates
(H) dehydrogenases
r
i
Coenzyme
Z
Reduced Tetrazolium
Energy fo r maintenance
\
Oxygen
The a b il it y to measure dehydrogenase a c t iv it y is o f p a rtic u la r
s ig n ific a n c e .
These enzymes are o f a d e lic a te nature and are responsi­
ble fo r the maintenance o f energy without which the embryo could not
remain a liv e (8 5 ).
Therefore, the loss o f active dehydrogenases
probably indicates loss o f germinating a b il it y (5 1 ).
Measurement of
5
other enzymes have not provided consistent re s u lts .
For example, the
h yd ro lytic enzymes responsible fo r food.m obilization and other re s p ira ­
to ry enzymes such as catalase and peroxidase are o f a more stable
nature and have been shown to e x is t in dead seeds (8 5 ).
Other advantages o f tetrazolium include it s n o ntoxicity and th a t
i t is one o f the few organic compounds which is colored in the reduced
s ta te (8 3 ).
The in s o lu b ility o f the reduction product is important.
Since the reaction occurs w ith in c e lls and the pigment is nondiffusable,
there is a sharp d e lin eatio n between viable and nonviable tissue (1 7 ).
F in a lly , i t has been shown through extensive te s tin g th a t loss o f
dehydrogenase enzyme a c t iv it y tends to p a ra lle l loss in seed v ia b ilit y
(8 5 ).
This c h a ra c te ris tic coupled with a thorough knowlege o f seed
s tru c tu re , makes possible accurate v i a b i lit y assessment in much shorter
times than s im ila r re su lts obtained with germination te s ts .
The Seed
A tru e seed is a f e r t i liz e d mature ovule containing the embryonic
p la n t, stored nu trien ts and the integument(s) d iffe re n tia te d as the
p ro te c tiv e seed coat or te s ta (25, 57, 6 2 ).
applied to the u n it o f dissem ination.
The term "seed" is usually
Many dispersal units which are
often re fe rre d to as seeds are not tru e seeds but s in g le , sometimes two
to several seeded f r u it s .
The pericarp remains and may even fuse to
the te s ta , as in cereal grains (57, 8 ) .
P hysio logically and
6
biochem ically, these dispersal units should be considered as seeds
( 8 , 9 5 ).
The seeds o f Angiosperrns develop as a re s u lt o f a process called
"double f e r t i liz a t io n " (6 2 ).
The embryo is derived from the f e r t i l i ­
zation o f the egg c e ll by one o f the male nuclei from the pollen tube.
The "second" f e r t i liz a t io n is the fusion o f a male pollen nuclei with
two mother plant polar n u c le i.
This f e r t i liz a t io n resu lts in the
development o f the endosperm which may p e rs is t as a storage organ, or
degenerate and remain rudimentary, possibly fused to the seed or f r u i t
coat (6 2 ).
The te s ta or true seed coat comprising the embryo envelope
is derived from one or both integuments o f the ovule.
At times the
te s ta may be derived from tissues other than th a t o f the integuments,
e .g ., the nucellus, the endosperm or ra re ly the chalaza ( 8 , 6 2 ).
te s ta is usually a hard coat.
The
Its physiological importance is derived
from an often f a t t y or waxy c u tic le , and one or more layers o f thickened
p ro te c tiv e c e lls ( 8 , 6 2 ).
These features are responsible fo r variab le
degrees o f im perm eability to water and/or gases and consequently exert
some regulatory influence over the re s p ira tio n o f the embryo ( 8 , 6 2 ).
The features o f the te s ta in some species are apparently lacking , the
outer covering being derived from e x tra -o v u la r tissue is c a lle d a p e ri­
carp.
Seeds with th is stru ctu re are a c tu a lly f r u it s , fo r example
sunflowers (6 2 ).
7
Germination
Germination, as defined by Bewley and Black ( 8 ) consists o f those
processes which begin with water uptake and successfully term inate with
the emergence o f the ra d ic le or hypocotyl through the seed coverings.
This d e fin itio n is deceptively simple, as the physiological events
leading to the protrusion o f some p art o f the embryo through the seed
coat are not well understood.
A d d itio n a lly , where germination ends and
growth begins is d i f f i c u l t to d e lin e a te.
Emergence o f the ra d ic le may
in dicate th a t germination has occurred but th is may already be con­
sidered a p art o f growth (6 2 ).
Whether th is growth is a re s u lt o f c e ll
expansion, c e ll d ivis io n or both is s t i l l unresolved (7 ) .
Probably the
fundamental processes which cause germination are d iffe r e n t from those
o f growth ( 8 , 62, 8 0 ).
A seed must be in a favorable environment fo r germination to occur.
S p e c ific a lly , there must be adequate moisture, s u itab le temperatures,
a correct ambient gas composition, and in c ertain photoblastic species,
a requirement fo r lig h t (62, 8 ) .
A seed which is unable to germinate
due to a lack o f one or a ll o f these environmental factors is termed
quiescent (95, 62, 8 0 ).
Dormancy
Dormancy describes the condition in which an otherwise viable seed
f a ils to germinate under environmental conditions su ita b le fo r germi­
nation (95, 6 2 ), and is considered advantageous in adopting the growth
.8
cycles o f the plant to variatio n s in the environment.
Chances o f sur­
v iv a l are increased because dormant tissues have a great resistance to
adverse environmental conditions (95, 5 5 ).
is usually a disadvantage.
AgronomicalI y , seed dormancy
Delayed seedling emergence may re s u lt in
poor stand establishment or require the use o f la rg e r seed qu an tities
■
v ■
than would be required i f a ll seeds germinated equally (6 1 ).
Vegis
(94) defines true dormancy as a s ta te in which normal growth cannot be
resumed regardless o f the external conditions.
Vegis1'd e fin itio n seems
overly r e s t r ic t iv e i f taken l i t e r a l l y and has been subject to c ritic is m
(95, 7 0 ).
The ap p lic a tio n o f proper treatments may stim ulate the germi­
nation o f dormant seeds o f many species.
For example, the removal of
the seed coat o f antelope bitterbrush (71) or peach (30) allows rapid
germination o f normally dormant seeds.
The s c ie n tific lit e r a t u r e on dormancy is vast (95) and the c la s s i­
fic a tio n schemes too numerous and complex to describe in great d e ta il.
This indicates th a t the nature of seed dormancy i t s e l f is a variab le
and complex condition.
Nikolaeva (70) has devised a d e ta ile d and com­
plex dormancy c la s s ific a tio n scheme.
He defines dormancy as arising
from four major causes, (A) due to properties of the outer coverings,
(B) underdeveloped embryos, (C) physiological condition o f the embryo
and it s inner coverings, and (D) types o f combined dormancy.
The
numerous, sub classificatio n s o f these main categories are o f value
mainly in providing a means o f comparing dormancy mechanisms with
9
methods used to break dormancy (9 5 ).
A sim pler and more commonly used c la s s ific a tio n was published by
Crocker in 1916 (1 5 ).
He describes dormancy as re s u ltin g from (A)
immaturity o f the embryo, (B) im perm eability o f the seed coats to w ater,
(C) mechanical resistance o f the seed coat to embryo growth, (D) low
perm eability o f the seed coats to gases, (E) dormancy re s u ltin g from a
metabolic block w ith in the embryo i t s e l f , (F) a combination o f factors
A-E, or (G) secondary dormancy.
The term secondary dormancy applies to
the condition in which quiescent Seeds have lo s t th e ir a b il it y to
germinate as a re s u lt o f being imbibed under conditions unfavorable fo r
germination.
Primary dormancy is sp ecified when seeds are dormant a t
the time o f dispersal or harvest (5 4 ).
Crocker's seven facto rs contrib uting to dormancy remain adequate
in summarizing current knowledge o f the subject with the exception of
embryo dormancy.
in 1916.
Endogenous mechanisms are b e tte r understood now than
Id e n tific a tio n and ch a rac te riza tio n o f plan t growth substances
in recent years is la rg e ly responsible fo r th is increase in knowledge.
Coat Imposed Dormancy
The various layers o f the seed coat provide protection fo r the
embryo from the environment.
The seed coat may exert a profound
influence by providing a b u ffe r between the embryo and.the environment.
The morphological features o f the seed coat may present a b a rrie r to
water uptake and prevent gaseous exchange.
The seed coat may also
10
prevent germination by mechanical r e s tr ic tio n and by containing growth
in h ib ito rs .
An e a rly publication by Crocker (14) reported th a t seed
dormancy o f many species was due to seed coat im perm eability to water.
In ta c t seeds o f Russian pigweed would not germinate u n til the seed coat
was broken to allow water im bibition and subsequently 100% germination.
Agronomically , th is condition is termed "hard seededness" and is
e s p e c ia lly prevalent in the legume fa m ily .
The percentage o f hard
seeds may vary among v a rie tie s and is dependent upon the environment in
which the seeds are produced (95, 6 2 ).
Hard seededness o f soybeans is
reported to be the re s u lt o f f a t deposits and lig n ific a tio n in the coat
palisade c e lls (3 ) .
Hard seeds o f white sweet clover can be rendered
permeable with vigorous shaking or by the use o f moderate heat (3 7 ).
These procedures remove the s tro p h io la r plug w ithin the s tro p h io la r
c le f t .
Seed coverings may also prevent gas exchange between the resp iring
embryo and the environment.
Dormancy o f the upper seed o f cocklebur
was shown to be imposed by coat im perm eability to oxygen (1 4 ).
Increasing
I
tensions or removing the te s ta would release dormancy.
These treatments also improved the germination o f w ild oats (5 2 ). The
I
e ffe c t o f COg is usually the reverse o f Og. Most seeds f a i l to germi­
nate i f COg tensions are increased (4 0 ), but dormancy breaking by COg
has been observed in subterranian clover (5 ) and cocklebur seeds (5 6 ),
11
Dormancy due to mechanical re s tric tio n s imposed by the testa are o f
rare occurrence (6 2 ).
This type o f dormancy is d i f f i c u l t to demon­
s tra te as the release o f dormancy by te s ta removal may be c ite d as
evidence fo r other germination in h ib itin g fa c to rs .
However, through a
series o f deductive experiments, Ikuma and Thimann (46) propose that
lig h t s e n s itiv e "Grand Rapids" le ttu c e seeds are unable to germinate
because o f the mechanical re s tric tio n s imposed by the endosperm la y e r.
Exposure to lig h t stim ulates the production o f h yd ro lytic enzymes which
help the ra d ic le free i t s e l f .
(5 3 ).
S im ila r resu lts were obtained fo r li l a c
More d ire c t evidence is supplied by Esashi and Leopold (23) who
measured the th ru s t developed by germinating cocklebur embryos.
Non-
dormant embryos developed more than twice the th ru st o f dormant embryos.
Additional measurements o f the forces required to rupture the testa
indicated th a t these th ru s t differences of dormant and nondormant
embryos were s u ffic ie n t to account fo r the prevention o f germination
(2 3 ).
Dormancy imposed by germination in h ib ito rs contained in the seed
coat is o f ecological sig n ifican ce e s p e cia lly in desert species.
Germination o f these species is prevented u n til adequate r a in f a ll
leaches the in h ib ito rs from the seed.
The amount of moisture necessary
fo r removal o f the in h ib ito r also assures survival o f the seedling (8 0 ).
Seed coat in h ib ito rs are common in members o f the rose fa m ily .
Treating achenes o f antelope bitterbrush w ith thiourea (73) releases
12
dormancy.
Nord (72) postulates th a t thiourea deactivates a seed coat
in h ib ito r .
Attempts to is o la te the substance responsible fo r dormancy
have been unsuccessful (2 0 ).
S im ila r re su lts were obtained by Jackson
and Blundell (48, 49) fo r f ie ld and rugosa rose.
Dormancy o f these
species was believed to be coat imposed by mechanical re s tric tio n s of
growth.
However, when achenes were steeped in various solvents and
analyzed chromato g ra p h ic a lly , fractio n s were found which prevented the
germination o f excised embryos.
Webb and Wareing (99) also found th a t
in h ib ito rs present in the embryo o f sycamore maple are prevented from
leaching outward by the te s ta u n til the seeds were subjected to moist
c h illin g .
There are many ways in which coat imposed dormancy may be removed.
Already mentioned are impaction, moderate heating, leaching with water
or other solvents, s t r a t if ic a t io n , and treatment with th io u rea.
Seed
coats can also be rendered permeable by s c arify in g mechanically in
sandpaper lin e d drums or chemically with concentrated acids.
Thbse
treatments a lt e r seed coat perm eability to water and gases, weaken the
seed coat s tru c tu re , change s e n s itiv ity to lig h t , and possibly remove
chemical in h ib ito rs (62, 8 0 ).
Immaturity of the Embryo
Embryos o f seeds o f some species e s p e cia lly in the .orchis fam ily
and buttercup, may not have completed morphological development or
achieved maximum s ize a t the time of dispersal (6 2 ).
I
Such embryos are
13
termed immature or rudimentary and are unable to germinate u n til d i f ­
fe re n tia tio n and growth are complete.
The embryo o f seeds o f h o lly are
described as a spherical mass of tissue a t the time o f dispersal ( 47 ) .
Germination in nature is achieved a f t e r a period of 8 to 12 months o f
growth and d iffe r e n tia tio n .
Laboratory germination could only be
accomplished by c u ltu rin g embryos in a 5% glucose medium fo r 5 months
a t 25 C.
S im ila rly , rudimentary parsnip embryos w ill d iffe r e n tia te and
increase dry weight by a fa c to r o f 25 before germinating.
occurs only a t temperatures near freezing (8 9 ).
This growth
Work with ash indicates
morphologically d iffe r e n tia te d embryos must grow-in size and assim ilate
food reserves before normal seedlings are produced ( 88 , 9 6 ).
enlarge best when imbibed and kept a t 20 C fo r 2 -htb 3 months.
Embryos
However,
enlarged embryos require an additional cold treatment o f 5 C fo r 2 to 3
months before being returned to an a lte rn a tin g 20-30 C fo r best germi­
nation.
Snowberry seeds require a s im ila r treatment fo r 10 months to
produce embroys which w ill germinate (2 6 ).
In co ntrast, embryos o f
smooth bromegrass are capable o f germination 5 days a f t e r anthesis (3 3 ).
Hormones, Growth Promoting and
In h ib itin g Substances
Abscisic Acid.
The most important n a tu ra lly occurring plant growth
in h ib ito r is undoubtedly abscisic acid CABA)(6 2 ),
be one o f the fiv e classes o f plant hormones.
ABA is considered to
When applied exogenously
14
ABA w ill prevent seed germination and i t has been found to be an endogen­
ous component o f many seeds (11, 9 8 ).
An example o f the effectiveness
o f ABA in c o n tro llin g germination is demonstrated.in cotton f r u it s (4 5 ).
During seed development, the ABA content o f the ovary wall increases.
Iso lated embryos w ill germinate when washed to remove ABA.
The cotton
embryo germination w ill be prevented i f the ABA or ovary e x tra c t is
added back to the washed embryos.
Through evidence obtained with u ltra v io le t absorption spectrum
a n a lysis, chromatography and plan t growth assays, Lipe and Crane (59)
were able to show th a t the germination in h ib ito r in peach is ABA.
A pplication o f e ith e r 10 ppm ABA or peach e x tra c t to normal seedlings
would induce a rosetted growth form, implying th a t the peach e x tra c t
contained ABA.
Further studies (59) indicated th a t the a b il it y Of seeds
to germinate a f t e r 6 weeks o f moist c h illin g correlated with the d is ­
appearance o f the ABA from those seeds.
Gibberel I i n .
Like ABA, g ib b e re llin s (GA) are plant hormones, and
are important in promoting seed germination.
Although more than 50 GA's
have been id e n tifie d , GA3 is most commonly employed in seed germination
studies.
GA's stim ulate germination in seeds whose dormancy is usually
overcome by moist c h illin g , dry storage a f t e r ripening or lig h t (90, 11,
95).
Studies made by Frankland and Wareing (32) in the e a rly 1960's
15
indicated th a t dormancy breaking in hazel and beech requires about 12
weeks o f moist c h illin g .
During th is dormancy breaking period, they
detected no changes in seed in h ib ito r le v e ls .
Measurements o f GA
le v e ls a fte r c h illin g showed only a s lig h t ris e in a c t iv it y .
Several
years la te r Ross and Bradbeer (79) were able to support these results
through th e ir own experiments.
However, due to improved measuring
techniques, GA was shown to be produced in ph ysio lo g ically active
q u a n titie s .
I t was also shown th a t GA synthesis did not take place
during the c h illin g treatm ent but was in it ia t e d once seeds were returned
to temperatures s u ita b le fo r germination.
Work with ash by V il lie r s and Wareing (97) showed th a t germination
and the production o f normal seedlings requires a moist c h illin g t r e a t ­
ment and th a t soaking unchilied embryos in water resulted in the pro­
duction o f stunted seedlings.
Since c h illin g did not change in h ib ito r
le v e ls w ith in the seed, and in h ib ito rs did not leach from the seed
during the soaking treatm ent, i t was postulated th a t the in h ib ito r was
d ilu te d by im bibition which allowed germination in unchilled embryos.
In a d d itio n , they concluded th a t c h illin g produces a growth promoter,
probably GA, which counteracts the e ffe c ts o f growth in h ib ito rs .
A more recent study (2) again with h a ze l, indicated th a t the
presence o f GA synthesis in h ib ito rs , e . g . , phosph.on D and CCC, prevented
GA accumulation and subsequent germination o f p re c h ille d seeds.
re su lts were consistent with the theory th a t GA biosynthesis is
These
16
p re req u is ite fo r c h ille d hazel seed germination.
In the same study
exogenously applied ABA strongly in h ib ite d germination but had l i t t l e
e ffe c t on GA accumulation.
I t was assumed th a t ABA did not a ffe c t GA
synthesis but ra th e r it s action.
The resu lts o f these experiments could be c ite d in support of the
Promoter/In h ib ito r Theory o f seed germination ( I ) which postulates th a t
dormancy onset, control and term ination is regulated by a balance of
growth in h ib ito rs and promoters.
P a rtic u la r emphasis is given g ib b erel-
Iin s in the ro le o f promoter, and abscisic acid the in h ib ito r , although
other growth substances such as the cytokinins are not excluded.
Accordingly, the term ination o f seed dormancy and the onset o f growth
may be accomplished by e ith e r decreasing in h ib ito r content or increasing
promoter le v e ls .
Cytokinins and Thiourea.
Cytokinin (CK) another o f the plant
hormones is the generic term fo r a ll substances displaying k in e tin -lik e
a c t iv it y ; th a t is , the promotion o f c e ll d iv is io n or cytokinesis (8 2 ).
As GA and ABA, the CKls are im plicated in seed dormancy and germination.
They are most e ffe c tiv e in promoting germination when combined with
other dormancy breaking agents such as GA.
CK1s are sometimes more
e ffe c tiv e than GA in counteracting in h ib ito r a c tiv ity (9 5 ).
CK's are a
chemically heterogeneous, group (82) and the s tru c tu ra l s p e c ific ity fo r
a c tiv ity is not very exacting (8 1 ).
synthetic CK is benzyl adenine (BA).
The most commonly u t iliz e d
17
Early in vestig ations concerning the e ffe c ts of CK on seed germina­
tio n were performed using le ttu c e seeds (81)..
BA increased germination
to 59% when controls germinated 9% by overcoming the dormancy imposing
e ffe c ts o f darkness and warm temperatures.
Other in vestig ations with
le ttu c e ( 66 ) showed th a t k in e tin , a CK, not only p a r t ia lly overcomes
dark imposed dormancy but strongly increases the promotive e ffe c t of
lig h t on germination.
Dormant apple seeds normally requirin g 70-80
days o f moist p re c h illin g to break dormancy were stim ulated to germinate
when treated with BA ( 4 ) .
I t was postulated th a t the ro le o f BA was to
overcome the e ffe c ts o f endogenous in h ib ito r .
These e ffe c ts were only
p a r t ia lly removed by BA; embryos germinated in th is manner produced
stunted and abnormal seedlings.
CK a c t iv it y increased progressively
during dormancy breaking and decreased once the buds had opened in
studies (19) of the dormant buds o f deciduous tre e s .
CK was absent in -
dormant buds and present once dormancy was broken.
Thiourea (TU) while not considered a plant hormone, is important
fo r it s e ffe c ts in stim ulatin g the germination o f dormant seeds.
TU can
stim ulate dark germination and su b stitu te fo r cold moist treatment in
some species.
R e la tiv e ly high concentrations of TU must be used.
Seeds
are commonly soaked in a 0.5 to 3.0% solu tion o f TU then tran sferred to
water.
When seeds are to be germinated d ire c tly in TU, concentrations
o f IO " 3 to IO " 2 M are usually employed (6 2 ).
18
Thiourea was o r ig in a lly applied to potato tubers to promote
sprouting.
I t was soon being used to stim ulate the germination of
dormant seeds o f sugar and Norway maple, black and red oak (1 8 ),
le ttu c e (9 1 ), and peach (9 2 ).
TU w ill promote the germination of
le ttu c e seeds in the dark, but concentrations s lig h tly above those
optimum fo r germination are in h ib ito ry to growth (9 1 ).
Peach seedlings
produced by the TU stim ulatio n o f unprechilied seeds were dwarfed and
rosetted , ty p ic a l fo r other treatments which a r t i f i c i a l l y stim ulate
peach germination (9 2 ).
F in a lly , as previously mentioned in the discussion o f seed coat
in h ib ito r s , TU seems to in a c tiv a te th is in h ib ito r in antelope b i t t e r ­
brush.
A pplication o f TU to dormant achenes of th is species is as
e ffe c tiv e in promoting germination as removing embryos from th e ir seed
coats (7 3 ).
S tr a tific a tio n
Moist c h illin g has freq uently been mentioned as a method fo r over­
coming seed dormancy.
Seeds of many species, p a r tic u la r ly in the rose
fa m ily , many deciduous tre e s , and some conifers w ill not germinate u n til
exposed to freezing temperatures in the moist condition.
Seeds to be
preconditioned fo r germination by th is method are freq uently layered or
s t r a t if ie d in f la t s containing moist sand or peat.
For effectiveness a
s t r a t ific a t io n period o f weeks or months may be required.
Seeds must be
19
in the imbibed condition to respond to cool temperature treatments (62)
In review, s t r a t if ic a t io n was reported to be advantageous in re ­
moving coat imposed dormancy by changing the properties o f the seed coat
which prevented germination (9 9 ).
S tr a tific a tio n is important in pro­
moting growth and morphological development, in parsnip (8 9 ).
Seeds of
ash ( 88 , 96) and snowberry (26) require warm followed by cold s t r a t i f i ­
cation before embryos are able to germinate.
R elative le vels o f growth
promoting and growth in h ib itin g substances are known to change during
the s t r a t if ic a t io n period.
ABA disappears in peach a f t e r 6 weeks
s t r a t ific a t io n (5 9 ).
S tr a tific a tio n stim ulates GA production in beech, hazel and ash
(32, 79, 97, 2) and th is is a p re req u is ite fo r germination.
I t has
also been shown th a t OK's and TU can s u b s titu te fo r the s t r a t ific a t io n
requirement in some species (4 , 18, 9 2 ).
Many other changes are reported to occur during s t r a t ific a t io n
including increases in a c id ity , water holding capacity, catalase
a c t iv it y , reducing sugars, re sp irato ry r a te , tra n s fe r o f food reserves
to the embryo and changes in enzyme composition (11, 6 2 ).
Although
there is no c le a r evidence as to any one event responsible fo r dormancy
breaking during s t r a t if ic a t io n (6 2 ), a statement by TaylorsPn and
Hendricks (90) summarizes what is generally believed to be the under­
ly in g p rin c ip le :
20
Temperature regimes regulate synchrony o f processes in seeds
by e ffe c ts on reaction rates and changes in the physical
s ta te o f c e llu la r components. In seeds, temperature in f lu ­
ences (th e ) in te g ra tio n of p a rtia l processes as dormancy
continues or is overcome.
I f release o f dormancy is dependent upon a s p e c ific physiological sta te
reached through a complex series o f biochemical changes, then synchrony
and in teg ra tio n o f processes w ill be the only way in which th a t physio­
lo g ic a l condition w ill be a ttain e d .
CHAPTER I :
TETRAZOLIUM VIABILITY PROCEDURES
FOR NATIVE SHRUBS AND FORBS
INTRODUCTION
The increasing disturbance o f large land acreages in recent years
e s p e cia lly due to coal surface mining has increased the commercial sale
o f native species seed fo r reclam ation.
V ia b ilit y te s tin g o f th is seed
has not been p ra c tic a l due to the lack o f known te s tin g procedures and
the extended time periods necessary fo r te s tin g .
Dormancy in some
native species may require long periods o f s t r a t ific a t io n fo r germina­
tio n to proceed.
H is to r ic a lly , the need fo r a rapid method to assess v ia b i lit y led
to the introduction and acceptance o f stain in g seed with 2 ,3 ,5 -trip h e n y l
2H-tetrazolium chloride (TZ) (6 9 ).
Seeds d i f f i c u l t to te s t with con­
ventional germination procedures may successfully be evaluated using
the TZ v ia b i lit y method.
However, th is method does not distinguish
between dormant and nondormant seed.
Our o b jective was to develop TZ
techniques fo r three n ative forb species:
A ch illea m ille fo liu m I .
(yarrow ); Linum le w is jj (Pursh) (Lewis f l a x ) , Ratibida columnifera
( N u t t .) Wooten and Standley (p r a ir ie coneflow er); and e ig h t native
shrub species:
Amelahchier a ln if o lia ( N u t t .) N utt, (s e rv ic e b e rry ),
Amorpha fru tic o s a L. (jndigobush), Artem isia trid e iita ta
N utt, (big
sagebrush), Ceretoides lanata (Pursh) Howell (w in t e r f a t ) , Pruhus
v irg in ia n a L. (chokecherry), Purshia trid e h ta ta (Pursh) DC.
(antelope
b itte rb ru s h ), Rhus tr ilo b a ta N utt, (skunkbush sumac), and Symphorjcargos a!bus (L .) Blake (snowberry).
TZ te s tin g procedures fo r
23
chokecherry (31, 6 0 ), bitterbrush (28, 2 9 ), Amelahchjer sp p ., (4 4 ),
p r a ir ie coneflower (13, 8 7 ), yarrow (87, 6 4 ), and Lewis fla x (13) are
generally nonspecific and d i f f i c u l t to access.
V ia b ilit y tests using
embryo excision have been developed fo r antelope bitterbrush (71, 42)
and skunkbush sumac (4 2 ).
C orrelations between s p e c ific stain in g patterns and actual germi­
nation are obtained a fte r considerable te s tin g experience with a par­
tic u la r species.
inform ation.
I t is not the ob jective o f th is paper to provide th is
TZ v ia b i lit y c o rre la tio n to germination has been reported
fo r the native shrub Stansbury c lif f r o s e (7 5 ).
Research by Grabe (3 4 ),
Delouche (1 7 ), and Moore (68) is invaluable as a guide to the in t e r ­
pretatio n o f staining patterns.
The Suggested Procedures section, including Table 2 is presented
fo r quick reference and guide fo r the seed analyst and researcher.
S pecific procedures such as soaking times are often not c r it ic a l to
successful stainin g and are therefo re adjusted fo r convenience in
laboratory ro u tin e.
Refer to Table I fo r a summarization o f s p e c ific experimental
procedures.
.
MATERIALS AND METHODS
Seed samples were contributed by the Western Energy Company,
Cols t r ip , MT, from lo ts c u rre n tly being used fo r revegetation o f coal
strip-m ine s p o ils .
Two re p lic a te s o f 50 seeds were used fo r the TZ v i a b i lit y te s ts .
Two re p lic a te s o f 100 seeds were used fo r those species which required
minimal preparation, i . e . , yarrow, w in te rfa t, indigobush, and big
sagebrush.
Seeds to be preconditioned by moistening were placed in
small beakers or between b lo tte r papers moistened with tap water.
Preconditioning took place a t room temperature.
Seeds were bisected
and seed coats removed using foreceps, dissecting knives and needles.
A stereoscopic microscope with 10 and 20 power m agnification and with
top and bottom illu m in a tio n was used to aid preparation and in te rp re ­
ta tio n .
A fte r preparation, seeds were placed in Syracuse watch glasses or
small beakers, covered with an ample amount o f TZ s o lu tio n , and covered
with a standard watch glass.
temperature.
Seeds were stained in the dark a t room
TZ solutions were prepared from 2 ,3 ,5 -trip h e n y l-2 H -
tetrazo liu m chloride (3 4 ):
0.1% being used fo r seeds with bisected
embryos and 1% fo r in ta c t seeds and embryos.
Upon completion of
s ta in in g , the seed coats o f some species were cleared using lactophenol
prepared as described in the Tetra^qlium Testing Handbook (3 4 ).
Excess
TZ was removed with p ip e tte and b lo ttin g paper, before covering seeds
25
with lactophenol.
Clearing was conducted in covered watch glasses a t
room temperature fo r a minimum o f I hour.
RESULTS AND DISCUSSION
Yarrow
V ia b ilit y o f the seed lo t tested was 86% (Table I ) .
Lactophenol
induced seed coat c le a rin g , but was not necessary fo r s ta in in te rp re ­
ta tio n as the seed has a translucent pericarp (F ig . TA).
Unpunctured
seeds fo r those punctured through the pericarp only, did not s ta in .
This was probably due to an inner seed coat impermeable to TZ.
optimum time fo r stainin g was 4 hours.
The
Shorter times were inadequate
and longer stainin g times produced l i t t l e additional change.
Lewis flax
V ia b ilit y o f the seed lo t tested was 93% (Table I ) .
Lactophenol
did not c le a r the seed coat, and removal was necessary fo r staining to
proceed.
Dissection o f unimbibed seeds caused extensive embryo damage.
Once imbibed, seed coats were removed by applying pressure to the
d is ta l end (F ig . IB) forcing the embryo from the seed coat.
This
procedure damages cotyledon tip s but did not a ffe c t stain in te rp re ta ­
tio n (3 4 ).
Moistening caused the secretion o f a gel a n ti nous substance
which increased the d i f f i c u lt y of seed manipulation.
occurred a t 4 hours.
strained a t 8.
Optimum staining
Seeds were understained a t 2 hours and over­
Table I .
Percentage v i a b i l i t y , e ffe c t o f lactophenol, e ffe c t o f seed coat preparation on
s ta in in g , and optimum tetrazo liu m stain in g time fo r e ig h t shrubs and three
fo rb s .
V ia b iIi ty
Species
%
Yarrow
Lewis fla x
P ra irie
coneflower
Serviceberry
Indigobush
Big sagebrush
W interfat
Chokecherry
Antelope
bitterbrush
Skunkbush
• sumac
Snowberry
86
93
72
84
94
66
91
98
Lactophenol
e ffe c t
+a
_c
Stained with
seed coat
in ta c t
—
-
-
-
-
+
+
+
slow
+
slow
100
91
68
-
TZ s ta in time in hours
Understain
Optimum
Overstain
2
2
4
4
NCb
8
2
2
12
12
I
2
4
4
18
16
4
6
8
8
NC
NC
8
12
2
4
8
2
2
4
, 4
12
NC
aP ositive (+) e ffe c t.
^NC = l i t t l e ad d itio n al change with increased stainin g times.
cNegative ( - ) e ffe c t.
F ig u re
(p )
and
re m o va l
to
th e
I.
T e tra z o liu m
p u n c tu re
fo r
(B )
d is ta l
p o in t
L e w is
seed
end
s ta in e d
( x ) ,
fla x
(d ).
(B )
and
(s)
and
L e w is
(C )
u n s ta in e d
fla x ,
p r a ir ie
and
(u s)
(D )
e m bryo s
p r a ir ie
c o n e flo w e r
is
to
o f:
(A )
c o n e flo w e r.
a p p ly
y a rro w
The
p re ssu re
s h o w in g
m e th o d
w ith
a
o f
p e ric a rp
e m bryo
n e e d le
(n )
29
P r a ir ie coneflower
V ia b ilit y of the seed lo t tested was 72% (Table I ) .
(p e ric a rp ) did not respond to the use o f lactophenol.
The seed coat
Staining did not
occur without removing the pericarp and rupturing the impermeable inner
membrane (8 7 ).
Seed coat removal (F ig . TC) was accomplished in the
same manner as fo r Lewis fla x .
moistened b lo tte r paper.
Seeds were preconditioned using
This procedure avoids sampling bias due to
the separation o f f i l l e d and u n fille d seeds placed d ir e c tly in water.
Damage to the inner membrane during preparation allowed TZ penetration.
Staining was optimum a f t e r 4 hours and less than 2 and more than 8 hours
yield ed inadequate re su lts (F ig . ID ).
Serviceberry
V ia b ilit y o f the seed lo t tested was 84% (Table I ) .
was not e ffe c tiv e in c le arin g the seed coat.
seed coats and inner membranes did not s ta in .
Lactophenol
Seeds with in ta c t outer
Seed coats which were
not preconditioned by moistening were d i f f i c u l t to remove.
As with
Lewis f la x , moisture caused the seed coat to secrete a gelatinous
m a te ria l, which made seed manipulation d i f f i c u l t .
c e s s fu lly removed by bisectio n .
Embryos were suc­
The seed coat was cut lo n g itu d in a lly
along the crease s ta rtin g a t the pore which made i t possible to tease
the embryo from the seed coat.
Staining was optimum a f t e r 4 hours.
Two hours provided inadequate staining and embryos were overstained
I
30
a fte r 8 hours.
Stained and unstained embryos are presented in Fig. 2A
Indigobush
V ia b ilit y o f the seed lo t tested was 94% (Table I ) .
Lactophenol
e ffe c tiv e ly cleared the seed coat perm itting stain in te rp re ta tio n with
out seed coat removal.
I t was necessary to chip seed coats fo r s ta in ­
ing to occur (F ig . 2B) due to a high occurrence (68%) o f hard seeds.
Seeds were chipped a t the d is ta l end to avoid damage to the embryonic
a x is .
Staining proceeded slowly and was optimum a t approximately 18
hours.
Extended stainin g times produced l i t t l e change.
Times shorter
than 12 hours were inadequate.
Big sagebrush
V ia b ilit y o f the seed lo t tested was 66% (Table I ) .
Lactophenol
e ffe c tiv e ly cleared the seed coat and f a c ilit a t e d stain evaluation.
Sagebrush seeds (achenes) are s im ila r to those o f yarrow with a p e ri­
carp as an outer covering.
N either the pericarp nor the seed coat
required puncture or removal fo r s ta in in g .
a 1% TZ solution stained in 16 hours.
hours were inadequate.
Seeds placed d ire c tly in to
Staining times less than 12
Few changes in s tain in g c h a ra c te ris tic s were
observed with extended times.
31
F ig u re 2 .
T e tra z o liu m s ta in e d (s ) and u n s ta in e d (u s ) e m b ryo s o f:
b u s h s h o w in g c h ip p in g p o in t ( x ) , (C ) w in t e r f a t s h o w in g i n t a c t s e e d
a n d p u n c tu r e p o in t ( x ) , a n d (D ) c h o k e c h e rry s h o w in g s to n y e n d o c a rp
e m b ry o n ic a x is ( a x ) .
(A ) s e r v ic e b e r r y , (B ) in d ig o ( i ) , se e d c o a t re m o ve d (e )
(e n ) and h ig h ly s ta in e d
32
W interfat
V ia b ilit y o f the seed lo t tested was 91% (Table I ) .
Lactophenol
was e ffe c tiv e in c le arin g the seed coat, but it s use was not necessary
since the seed coat was removed p rio r to s ta in in g .
Seeds placed
d ir e c tly in TZ solution stained slowly and nonuniform ly.
This was
probably due to the non-wetting tendencies o f in ta c t seeds.
Precon­
d itio n in g between moistened b lo tte rs fo r several hours overcame this
tendency.
Seed coats were e a s ily removed once imbibed by puncturing
the central area which is surrounded by the embryo (F ig . 2C).
Naked
embryos were placed d ire c tly in TZ and stain in g proceeded ra p id ly with
4 hours being optimum.
Some staining was evident a f t e r I hour.
Seeds
were overstained a f t e r 8 hours.
Chokecherry
V ia b ilit y o f the seed lo t tested was 98% (Table I ) .
The thickened
stony endocarp does not respond to treatm ent with lactophenol.
A l­
though the endocarp is reported to be permeable to water (3 6 ), seeds
with an in ta c t endocarp did not s ta in .
This may be due to the presence
of an impermeable inner membrane which surrounds the embryo.
techniques fo r endocarp removal are possible.
Many
S p littin g the stone
along the prominent m idrib or suture was an adequate method (F ig . 2D).
This exposed the cotyledons and often l e f t the embryonic axis in ta c t,
allowing accurate v i a b i lit y assessment.
Seeds preconditioned by
33
moistening fo r 24 hours were easier to s p li t than dry seeds.
seed halves stained in 6 hours.
S p lit
Staining fo r 2 hours was inadequate
while 12 hours produced overstain.
Antelope bitterb ru sh
V ia b ilit y o f the seed lo t tested was 100% (Table I ) .
was not e ffe c tiv e in c learin g the seed coat.
coats did not s ta in .
water fo r 16 hours.
Lactophenol
Embryos with in ta c t seed
Seed coat removal was f a c ilit a t e d by soaking in
Cutting the seed coat from end to end along the
th in la te r a l edge followed by c a re fu lly removing the embryo was an
e ffe c tiv e method.
Embryos stained s a tis fa c to r ily in 4 hours.
Staining
fo r 2 hours was in s u ffic ie n t and 8 hours produced overstaining.
Stained and unstained embryos are presented in Fig. 3A.
Skunkbush sumac
V ia b ilit y o f the seed lo t tested was 91% (Table I ) .
was in e ffe c tiv e in clearin g the seed coat (endocarp).
in ta c t endocarp did not s ta in .
Lactophenol
Seeds with an
Soaking in water fo r several hours
softened the endocarp and f a c ilit a t e d b isectio n .
Cutting seeds
la t e r a lly (F ig . 3C) yield ed proximal seed halves with in ta c t embryonic
axes.
Most seeds were extensively damaged during preparation making
stain in te rp re ta tio n d i f f i c u l t .
However, i t was possible to id e n tify
dead embryos, u n fille d seeds, and parasitism .
id e n tify in g stained embryonic parts.
V ia b ilit y was based on
Staining fo r 4 hours was optimum.
34
F ig u re 3.
T e tra z o liu m s ta in e d (s ) and
and (B ) s n o w b e rry ; n o te d a rk , a b n o rm a l
u n s ta in e d (u s ) e m bryo s o f:
s ta in in g o f e nd o sp erm ( d ) .
(A )
(D )
a n te lo p e b itte r b r u s h
S c h e m a tic x - s e c tio n o f
s n o w b e rry c o rre s p o n d in g to B :
e nd o ca rp (a ), m ic ro p ile
( b ) , seed c o a t ( c ) , e n d o sp e rm ( d ) ,
e m b ryo (e ) and c e n t r a l c a v it y ( f ) .
(C ) S c h e m a tic x - s e c t io n o f s k u n k b u s h s u m a c :
b is e c tio n
lin e
( a ) , seed c o a t ( b ) , e n d o c a rp ( c ) , c o ty le d o n (d ), and r a d ic le
( e ) .
35
two hours were in s u ffic ie n t* while 12 hours resulted in oversta in in g .
Snowberry
V ia b ilit y o f the seed lo t tested was 68% (Table I ) .
did not c le a r the seed coat (endocarp).
did not s ta in .
24 hours.
Lactophenol
Seeds with an in ta c t endocarp
Bisection was f a c ilit a t e d by pre-soaking in water fo r
Longitudinal bisection d ir e c tly through the micropore
(F ig . 3 B & D), resulted in embryo exposure.
Embryos are small when
compared with endosperm m a te ria l, and reported, to be rudimentary (2 6 ).
The endosperm o f seeds in the sample evaluated showed abnormal
b ric k -red s ta in in g .
The cause o f th is was unknown, but was postulated
to be the re s u lt o f extensive m icrobial a c t iv it y .
a f t e r 4 hours.
Embryos, were stained
Two hours were found to produce inadequate s tain in g .
Overstaining was not observed.
SUGGESTED TETRAZOLIUM TEST PROCEDURES
Yarrow
Precondition by placing seed between moistened b lo tte r paper over­
night (Table 2 ).
This w ill soften the seed coat fo r puncturing with a
fin e pointed needle.
end.
Puncture in the opaque area a t the broad, d is ta l
Puncturing in the translucent margin (pericarp ) (F ig . TA), when
viewed under a dissecting scope, w ill not be s u ffic ie n t fo r proper
s ta in in g .
Place punctured seeds in 1% TZ fo r 4 hours.
In te rp re t as a
dicotyledonous seed (3 4 ).
Lewis fla x
Precondition by soaking seed in water overnight (Table 2 ).
A fte r
soaking place on b lo tte r paper and use the side o f a curved probe to
apply pressure to the rounded, d is ta l end o f the seed (F ig . IB ).
Press
toward the pointed, ra d ic le end to force the embryo from the seed coat.
Stain in 1% TZ fo r 4 hours.
In te rp re t as a dicotyledonous seed (3 4 ).
The seed secretes a gelatinous substance when moistened, making i t
somewhat d i f f i c u l t to manipulate.
P ra irie coneflower
Precondition seed by placing between moist b lo tte r paper overnight
(Table 2 ).
The seed coat may be removed a fte r preconditioning by
applying pressure to the broad, d is ta l end o f the seed.
Squeeze toward
Table 2.
Suggested t e t r a z o lium v i a b i lit y procedures fo r e ig h t shrubs and three fo rb s •
TZ
cone.
Preparation
Stain time
(h r s .)
@ room temp.
Species
Precondition
Yarrow
Moist b lo tte rs Puncture
overnight
seedcoat ■
I
4
Lewis fla x
Soak in water
overnight
Remove
seedcoat
I
4
P ra ir ie
coneflower
Moist b lo tte rs Remove
seedcoat
overnight
I
4
S e rviceberry
Soak in water
overnight
Remove
seedcoat
I
4
Indigobush
None
Chip
seedcoat
I
16-20
Big sagebrush
None
None
I
16
W in te rfa t
Moist b lo tte rs Puncture
overnight
0.1
4
Chokecherry
Soak in water
24 hours
S p lit
endgcgrp
I
4-6
Antelope
bitterb ru sh
Soak in water
overnight
Remove
seedcoat
I
4
Skunkbush
sumac
Soak in water
24 hours
Bisect
la t e r a lly
0.1
4
Snowberry
Soak in water
24 hours
Bisect
I
lo n g itu d in a lly
4
%
.
Remarks
Gelatinous seedcoat
Gelatinous seedcoat
Remove pod
c le a r with lactophenol
Clear 2 hours with
lactophenol
Remove bracts
in itia lly
38
the pointed end o f the seed which w ill force the embryo, ra d ic le f i r s t ,
from the seed coat (F ig . TC).
and s ta in fo r 4 hours.
Immediately place the embryo in 1% TZ
In te rp re t as a dicotyledonous seed (34)
(F ig . ID ). .
This method o f preparation w ill damage the terminal cotyledon end
and allow more rapid TZ uptake through the impermeable membrane sur­
rounding the embryo.
The damage to the cotyledon must be ignored when
in te rp re tin g the TZ s ta in in g .
Serviceberry
Precondition by soaking seed in water overnight (Table 2 ).
When
bisecting place on moist b lo tte r paper and o rie n t with forceps so the
crease faces upward.
This may be awkward due to a secreted gelatinous
substance which causes the seed to s tic k to the forceps.
Bisect the
seed coat lo n g itu d in a lly s ta rtin g a t the pore and cut along the crease.
Remove the embryo and place in 1% TZ fo r 4 hours.
A short additional
soak, followed by rubbing between fingers w ill a ffe c t removal o f the
inner membrane i f i t did not become detached during dissection .
w ill not stain with the membrane in ta c t.
Staining should be in t e r ­
preted as a dicotyledonous seed (34) (F ig . 2A ).
Indigobush
The seed pod must be removed by hand or with a b e lt thresher
before te s tin g (Table 2 ).
Seeds
This species is a member o f the legume
39
fam ily and may contain a high percentage o f hard seed.
Precondition by
chipping the rounded cotyledon end o f the seed coat with a razor or
scalpel to permit T l im bibition (F ig . 2B).
T l overnight or longer i f necessary.
Place chipped seeds in 1%
Stained seeds should be cleared
with lactophenol fo r approximately I hour.
Clearing w ill permit in t e r ­
p re ta tio n o f s tain in g with the aid o f m agnification, top illu m in a tio n ,
and transm itted lig h t .
Big sagebrush
Separate the sm all, dark colored seed from the usually chaffy
m aterial and place d ire c tly in a 1% T l solution with no preconditioning
(Table 2 ).
Stain 16 hours or overnight and c le a r fo r 2 hours in la c to ­
phenol .
In te rp re t using m agnification and strong illu m in a tio n from below
the seed.
Stained seeds w ill show a uniform red c o lo r, whereas un­
stained seeds w ill remain yellow .
Radicle tip s may sometimes be un­
stained, but these seeds were shown to germinate normally.
W in te rfa t
The outer covering o f the u t r ic le (b racts) must be removed by
hand or with a thresher before in it ia t in g the TZ te s t (Table 2 ).
condition seeds between moist b lo tte r paper overnight.
Pre­
Seeds (n u tle ts )
are hydrophobic and do not imbibe moisture re a d ily i f placed d ire c tly
in to water.
40
The seed is composed o f an embryo embedded in a fibrous m atrix
which is the seed coat and endosperm.
A fte r the presoak, i t is possible
to puncture (F ig . 2C) and p a r t ia lly remove th is m atrix before placing
seeds in a 0.1% TZ s o lu tio n .
Staining proceeds ra p id ly and should be
complete w ith in 4 hours.
Chokecherry
-
Precondition by soaking in water fo r 24 hours (Table 2 ).
The
seeds possess a stony endocarp which may be s p lit by exerting pressure
with a scalpel or razor blade along the m idrib or suture which runs
lo n g itu d in a lly from the attachment scar to the ra d ic le containing end
o f the seed.
This procedure w ill separate the cotyledons, exposing the
inner surface o f each, and the embryonic axis (F ig . 2D).
Place one
seed h a lf with ra d ic le /e p ic o ty l s t i l l attached in 1% TZ and stain 4-6
hours.
Some damage w ill undoubtedly occur during preparation.
Assess­
ment o f damage caused by preparation may be d i f f i c u l t to separate from
natural defects.
Care must be taken not to term a seed abnormal due to
mechanical in ju ry during preparation.
Antelope bitterbrush
Precondition seed by soaking in water overnight (Table 2 ).
When
d issecting , place seed on moist b lo tte r paper, and hold the seed with
forceps to keep the th in edge o f the seed upright.
coat lo n g itu d in a lly .
Bisect the seed
C a re fu lly remove the embryo from the seed coat
41
and immediately place in 1% TZ fo r 4 hours.
The seed should stain
completely and is e a s ily in terp re te d as a dicotyledonous seed (34)
(F ig . 3A).
Skunkbush sumac
Seeds possess an extremely hard endocarp.
hours.
Soak in water fo r 24
Place seed on moist b lo tte r paper, hold with forceps and
bisect la t e r a lly (F ig . 3C).
The embryonic axis which is the broader
seed end should be placed in TZ.
th is portion when b ise c tin g .
during b ise c tio n .
stain fo r 4 hours.
Care should be taken not to in ju re
Often seed parts w ill f l y or be damaged
Place the h a lf containing the axis in 0.1% TZ and
The damage th a t occurs during preparation makes
in te rp re ta tio n d i f f i c u l t .
I t is d i f f i c u l t to id e n tify seed abnormali­
t ie s , however, inform ation on seed f i l l ,
parasitism , and v ia b i lit y can
be determined.
Snowberry
Precondition by soaking seed in water fo r 24 hours (Table 2 ).
Remove and place on b lo tte r paper, fla tte n e d side down.
tu d in a lly , d ir e c tly through the micropore.
in 1% TZ and stain fo r 4 hours.
Bisec lo n g i­
Place one h a lf o f each seed
Embryonic tissue ( r e la t iv e ly small
compared to endosperm) is located near the micropore (F ig . 3B & D).
Endosperm may show abnormal s ta in in g .
CHAPTER I I :
IMPROVING GERMINATION OF SKUNKBUSH
SUMAC AND SERVICEBERRY SEED
INTRODUCTION
Rhus t r ilo b a ta N utt, (skunkbush sumac) and Amelanchier a ln if o lia
N utt, (serviceberry) are native shrubs extensively d is trib u te d in the
western United S tates.
large th ic k e ts .
Skunkbush sumac is a small shrub often forming
I t is valuable as w i ld l if e cover, a c h a ra c te ris tic
which is important when restoring big game ranges even though p a la ta b i l i t y is r e la t iv e ly lo w .(7 7 ).
Skunkbush sumac is unusually per­
s is te n t, w ill endure extreme drought, and is w ell suited fo r revege­
ta tin g areas o f eroded and depleted s o ils (93, 77).
Serviceberry grows as a large shrub or a small tre e and is often
interspersed with other shrubs.
Because o f it s good p ala ta b iI i t y , wide
d is tr ib u tio n , and a v a i la b il it y , i t is recognized as one o f the most
important browse species fo r big game and livesto ck (93, 67, 77).
Both o f these species are increasingly being used fo r range re c la ­
mation and re s to ra tio n .
N either the Association o f O ffic ia l Seed
Analysts (12) nor the In tern a tio n a l Seed Testing Association (60) makes
recommendations fo r laboratory seed germination te s tin g o f these
species.
Seed o f both shrubs e x h ib it varying degrees o f dormancy th a t
is normally overcome by lengthy s t r a t if ic a t io n treatments (9 , 10).
U nfortunately, recommended s t r a t ific a t io n procedures fo r these species
are extremely time consuming and are not p ra c tic al fo r use in seed
te s tin g programs (4 2 ).
44
Germination o f skunkbush sumac is reported to be in h ib ite d by
embryo dormancy and the presence o f a hard, impervious seed coat (42,
10).
Unscarified seeds do not germinate (13, 78) and seeds s t r a t ifie d
fo r 3 months without seed coat removal also remain dormant (2 1 ).
H eit
(42) achieved maximum germination by s c a rify in g seeds fo r 90 minutes
in concentrated sulphuric acid followed by s t r a t ific a t io n fo r I month
a t 3 to 5 C.
These seeds achieved 70% germination in 10 days when
placed in an a lte rn a tin g 20-30 C temperature regime.
Embryo dormancy o f serviceberry seeds is usually overcome by cold
s t r a t ific a t io n ( 9 ) .
Previous research has shown th a t seeds s t r a t if ie d
a t I to 5 C from 3 to 20 months w ill germinate from 84 to 100% (78,
76, 41, 63, 65, 3 8 ).
In other tests (39) seeds have germinated 3% a fte r
3 months s t r a t if ic a t io n .
S c a rific a tio n and s t r a t ific a t io n were neces­
sary to produce a 4% emergence rate fo r seeds which were planted in s o il
(7 4 ).
Acid s c a rific a tio n was o f b e n e fit to the seed germination of
A. Ia evis Mieg.
I t was postulated th a t th is treatment was responsible
fo r the removal o f germination in h ib ito rs (4 4 ).
Seed coat s c a rific a tio n increases germination o f hard seeds of
Fabacae (6 2 ).
Growth promoters such as the g ib b e re llin s , cytokinins,
and thiourea su b s titu te fo r the s t r a t ific a t io n or lig h t requirement in
some species (6 2 ).
The ob jective of th is study was to f a c i l i t a t e rapid
laboratory germination te s tin g by reducing the time required to achieve
germination equal to tetrazo liu m v ia b i lit y fo r skunkbush sumac and
45
serviceberry.
Various dormancy breaking treatments to elim inate the
need fo r the lengthy s t r a t ific a t io n requirement
were evaluated.
MATERIALS AND METHODS
General Procedure
Seed samples o f skunkbush sumac and serviceberry were provided by
the Western Energy Company, Cols t r ip , Montana, from lo ts being used fo r
land revegetation.
Unless otherwise s p e c ifie d , germination o f both
species was conducted a t an a lte rn a tin g 20-30 C12 (9 , 10) with 16 hours
o f lig h t provided by cool white flourescent tubes (standard conditions).
Seeds were placed in p la s tic boxes on b lo tte r paper moistened i n i t i a l l y
with various chemicals and/or plant hormones, and subsequently with tap
water.
Treatments were applied to 3 re p lic a te s o f 50 seeds per box and
germination counts were made weekly fo r 3 weeks.
S tr a tific a tio n pro­
cedures were the same as fo r germination except temperatures were main­
tained a t I to 3 C without lig h t , and fo r durations o f 3 and 5 months.
V ia b ility was determined by a standard te t r a z o li urn (TZ) te s t using
published procedures (1 0 0 ).
M icrobial a c t iv it y was reduced by lig h t ly
dusting seeds with tetrachloro-para-benzoquinone (Spurgon) .
Seeds
1
Temperatures were maintained a t 20 C fo r 16 hours and 30 C for
8 hours. The lig h t treatment was applied continuously fo r 8 hours o f
30 C and 4 hours o f 20 C proceeding and subsequent to the 30 C t r e a t ­
ment fo r a to ta l of 16 hours.
2
Mention o f a trademark or p rop rieto ry product does not constitute
a guarantee or warranty of the product by the Montana A g ric u ltu ra l
Experiment S tation and does not imply it s approval to the exclusion o f
other products th a t may also be s u ita b le .
47
were s c a rifie d with 96% HgSO^, s t ir r in g continuously, then rinsing fo r
30 minutes with flowing tap water.
G ib b e re llin 3 (GA), benzyl adenine
(BA), thiourea (TU), and potassium n itr a te (KNO3 ) were applied aqueously
to the germination b lo tte rs as the i n i t i a l moistening agent.
This
required approximately 25 ml per box.
Main e ffe c ts were tested using fa c to ria l treatment combinations in
a completely randomized design.
Facto rial experiments are ty p ic a lly
analyzed by analysis o f variance techniques which provide tests o f
treatment e ffe c ts a f t e r separation into main e ffe c ts and in te ra c tio n s .
This kind o f analysis was not done because o f the nature o f the response
v a ria b le .
Rates o f germination varied between 0 and 35%, with a very
frequent occurrence o f 0.
Thus, germination counts per re p lic a tio n
were freq u en tly 0 and a t most 20 seeds.
Such counts lik e ly follow a
Poisson p ro b a b ility d is trib u tio n fo r which the variance is equal to the
mean.
The requirement o f a normally d is trib u te d response with homo­
geneous variance fo r the usual analysis o f variance was c le a rly hot
met.
A square root transform ation o f the response followed by the
usual analysis of variance procedure would have been acceptable i f the
zero germination occurrences had been less frequent.
Actual analysis employed two approximately correct techniques.
ordinary Duncan's M u ltip le Range Test was employed i n i t i a l l y on a ll
treatm ent combination mean counts a fte r completing a one-way analysis
o f variance u t iliz in g treatm ent combination c e lls not containing
An
48
exclu sively zero counts.
The chi-square c alcu latio n proposed by
Snedecor and Cochran (86) fo r te s tin g equal expectation fo r Poisson
variables was also used to te s t various main e ffe c ts as w ell as eq u a lity
o f two or more treatment combinations.
accommodate zero responses.
Such a test.can c o rre c tly
In teractio n s can be tested by a two-way
chi-square contingency ta b le .
Skunkbush sumac
Seeds were s c a rifie d fo r 75 minutes unless otherwise s p e c ifie d .
I n i t i a l l y , s c a rifie d and nonscarified seeds were germinated with HgO,
400 ppm GA or 0.2% KNOg.
To c la r if y responses to GA and KINOg, s c a rifie d
seeds were soaked 0, 8, 16, or 24 hours in GA or HgO and placed in
standard conditions.
KNO3 was tested as a moistening agent o f s c a rifie d
and nonscarified seeds a t concentrations o f 0, 0 .1 , 0 .2 , or 0.4%.
The
e ffe c ts o f s c a rific a tio n fo r 0, 60, 75, or 90 minutes combined with
soaking fo r 0 or 24 hours in 0.2% KNO3 or HgO were evaluated by planting
treated and subsequently a ir dried seeds in f la t s of s o il in the green­
house.
Speed o f emergence indices (SE) were determined using the
formula
where Xn = the number o f seeds emerging on day n.
Means are the
average of 4 re p lic a te s to ta lin g 200 seeds per treatm ent, planted in a
randomized block design.
49
Serviceberry
Seeds were s c a rifie d 30 minutes unless otherwise s p e c ifie d .
Ex­
cised embryos (100) were surface s t e r iliz e d in 0.3% hydrogen peroxide
(HP), a s e p tic a lly placed In s t e r ile p e tri dishes, and maintained under
standard germination conditions (9 ) .
Acid s c a rific a tio n was evaluated
by tre a tin g seeds fo r 0, 10, 30, 45, or 60 minutes and germinating
with HgO or a mixture o f 5 ppm BA and 50 mM TU.
Dormancy breaking
e ffe c ts of GA were tested by germinating s c a rifie d seeds with 0, 250,
500, or 1,000 ppm solutions as i n i t i a l moistening agents fo r the media.
To te s t fo r the presence of water soluble in h ib ito rs , s c a rifie d seeds
wepe held under cold running tap water fo r 24 hours before being placed
in standard conditions with 0 , 200, 400, or 800 ppm GA.
The e ffe c t o f
Q2 enrichment was tested using the hydrogen peroxide barley germination
method (2 7 ).
S c a rifie d and un scarified seeds were placed in 125 ml
Ehrlenmeyer flasks and covered with 0.3% HP or d is t ille d water (DHgO).
The stoppered flasks were kept in standard conditions and solutions were
changed every 24 hours fo r 3 weeks.
Cold soaking with aeration was
tested in the manner described by Barnett (6 ) .
S c a rifie d seeds placed
in 125 ml flasks were covered with 100 ml DHgO and kept a t I to 3 C
(cold) or in an a lte rn a tin g 20-30 C germinater (warm).
Various degrees
o f aeration were obtained by changing the DHgO d a ily or by continuously
bubbling a ir through the solution in the fla s k s .
Seeds were tran s­
ferred to germination boxes a t weekly in te rv a ls fo r 3 weeks and placed
50
under standard conditions.
Various concentrations o f BA and TU were
tested separately and in combination as i n i t i a l moistening agents fo r
b lo tte rs .
S c a rifie d seeds were germinated e ith e r with 0, I , 10, dr
100 mM TU or with 0, 10, 50, or 100 ppm BA.
A th ird experiment u t i ­
liz e d mixtures o f BA and TU in a ll combinations o f the le vels pre­
viously tested .
F in a lly , mixtures of 0 , 1 0 0 , 200, or 400 mM TU and
0 , 100, 200, or 400 ppm BA in a ll possible combinations were evaluated.
S ta tis tic a l analysis was done fo r the square root o f the count o f seeds
germinating per re p lic a tio n .
Such counts tend to have a Poisson
d is trib u tio n when the proportion germinating is small.
Standard r e fe r ­
ences (86) suggest use o f the square root transform ation to bring about
homogeneity of e rro r.
This transform ation also brought about an
improvement in f i t and increased s im p lic ity o f a polynomial model.
The e ffe c ts o f seed size on dormancy were evaluated by using 5/64 x
3/4 inch s lo tte d and 8/64 inch tria n g u la r screens to separate s c a rifie d
seeds in to 3 size classes.
Four 100 seed samples per size class were .
germinated with a mixture o f 100 mM TU and 100 ppm BA.
RESULTS AND DISCUSSION
Skunkbush sumac
The TZ v i a b i lit y o f the seed lo t tested was 91%.
Prelim inary
tests supported other reports (42) th a t acid s c a rific a tio n fo r approxi­
mately 75 minutes is necessary to e l i c i t a s ig n ific a n t germination
response.
I t also appeared th a t combining s c a rific a tio n with GA or
KNO3 could possibly improve germination over th a t o f s c a rific a tio n
alone (Table 3 ) , although the maximum response o f 25% was f a r below
v ia b i lit y .
However, follow -up tests fa ile d to confirm the promotive
e ffe c ts o f KNO3 (Table 4) and GA.
F in a lly , an emergence study was conducted in the greenhouse.
Treatments o f 0, 60, 75, or 90 minutes o f s c a rific a tio n were combined
f a c to r ia lly with soaking treatments o f 0 or 24 hours in 0.2% KNO3 or
DHgO.
Speed o f emergence (SE) (Table 5) fo r un scarified seeds was
slow as compared to s c a rifie d seeds w ith e ith e r soaking treatm ent.
S c a rifie d but unsoaked seeds were interm ediate in speed o f emergence.
Total emergence counts were made a fte r 3 weeks (Table 6 ) .
emergence o f un scarified seeds was poor.
Overall
Regardless o f soaking t r e a t ­
ment, a ll s c a rifie d seeds emerged s im ila r ly and b e tte r than unscarified
seeds.
The s c a rifie d but unsoaked seeds, although slower to emerge,
equalled the to ta l emergence o f s c a rifie d and soaked treatm ents.
52
Table 3.
Comparison* o f mean percentage germination o f skunkbush sumac
as affected by acid s c a r ific a tio n , GA3 and KNO3 .
S c a rific a tio n **
H2O
(min)
- GA3
(400 ppm)
KNO3
. (0.2%)
0
lb
lb
0b
75
15a
22a
25a
*Means followed by the same le t t e r are not s t a t is t ic a lly d iffe r e n t
a t P = 0.05.
**Main e ffe c t o f 0 vs 75 minutes s c a rific a tio n is s ig n ific a n tly d i f ­
fe re n t (R < 0 . 0 1 ) .
Table 4.
Comparison* Qf mean percentage germination o f skunkbush sumac
as a ffected by acid s c a rific a tio n and four le v e ls o f KNO3 .
S c a rific a tio n **
(min)
Percent KNO3* * *
0
0.1
0.2
0.4
0
lb
lb
lb
4b
75
31a
29a
31a
35a
*Means followed by the same l e t t e r are not s t a t is t ic a lly d iffe re n t
a t P = 0.05.
**Main e ffe c t o f 0 VS 75 minutes s c a rific a tio n is s ig n ific a n tly d i f ­
fe re n t (P < 0 .0 1 ).
***Main e ffe c t o f KNQ3 levels is not s ig n ific a n t (P
0 .0 1 ).
53
Table 5.
E ffe c t o f acid s c a rific a tio n fo r 0, 60, 75, or 90 minutes
and 0 or 24 hour soaks in HgO or 0.2% KNO3 on speed of
emergence* (SE)9 o f skunkbush sumac a fte r 17 days in the
greenhouse
S c a rific a tio n time in minutes
60
0
.2c
24 hr H2O
24 hr KNO3
75
90
4.7b
6.5b
5.9b
O
O
0
Soak time
...
11.4a
10.0a
9.7a
.02c
9.7a
10.9a
10.0a
*Means followed by the same le t t e r are not s t a t is t ic a lly d iffe re n t
a t P = 0.05.
X3
a
X;)
9SE = X1 + ^
where X is the number o f seedling
^+ T + n *
emerging on day n,
Table 6.
E ffe c t o f acid s c a rific a tio n fo r 0, 60, 75, or 90 minutes and
0 or 24 hour soaks in H2O csr 0.2% KNO3 on to ta l emergence
percent* o f skunkbush sumac a f t e r 3 weeks in the greenhouse.
S c a rific a tio n time in minutes
Soak time
0
60
75
90
0
2c
37b
48ab
46ab
24 hr H2O
Ic
49ab
55a
52ab
24 hr KNO3
Ic
47ab
54a
56a
*Means followed by the same le t t e r are not s t a t is t ic a lly d i f ­
fe re n t a t P = 0.05.
f
54
These resu lts support the findings o f Hett (42) who reported th at
s c a rific a tio n is b e n e fic ial to the germination of skunkbush sumac.
Acid s c a rific a tio n o f seeds may remove coat-imposed dormancy by changing
perm eability to water or gases, s e n s itiv ity to lig h t , removal of
mechanical re s tr ic tio n or possibly by destroying germination in h ib ito r
substances (6 2 ).
S c a rific a tio n did not completely re lie v e dormancy as.
indicated by the d iffe re n c e between actual germination and TZ v ia ­
b ility .
GA and KNO3 , substances known to break embryo dormancy in some
species by s u b stitu tin g fo r a s t r a t ific a t io n requirement (6 2 ), were
in e ffe c tiv e a t the le vels tested .
E ffo rts to is o la te seed coat e ffec ts
by embryo excision were unsuccessful due to the extreme hardness o f the
endocarp.
How much o f the residual dormancy can be a ttrib u te d to
endogenous mechanisms or to the in e ffic ie n c ie s o f the s c a rific a tio n
method cannot be assessed from these experiments.
E ffo rts directed a t
re lie v in g endogenous dormancy by the use o f KNO3 or GA were unsuccess­
f u l.
S e rviceberry
The TZ v ia b i lit y of the seed lo t tested was 84%.
S tr a tific a tio n
o f un scarified dormant seeds fo r 3 or 5 months produced 34 and 73%
germination, re sp ec tiv e ly .
not g e m in a te .
Untreated seeds and excised embryos did
Prelim inary te s ts showed a s lig h t germination stim ula­
tio n o f dormant seeds due to combining s c a rific a tio n and a
\
55
benzyladenine/thloures ( BA/TU) m ixture.
S c a rific a tio n times o f 10,
30, 45, or 60 minutes, were equally e ffe c tiv e (Table 7 ); however, the
longer s c a rific a tio n times were observed to produce physical damage to
embryos.
As a r e s u lt, 30 minutes o f s c a rific a tio n was used in a ll sub­
sequent experiments and although b e n e fic ia l to germination, th is t r e a t ­
ment reduced the 84% v ia b i lit y o f nonscarified seeds to an average of
60% (Table 10).
G ibberellins su b s titu te fo r the s t r a t ific a t io n or. lig h t requirement
in the seeds of.some species (6 2 ).
Levels o f 250, 500, or 1,000 ppm GA
did not promote the germination o f s c a rifie d u n s tra tifie d seeds.*
Table 7.
Comparison* o f mean percentage germination o f serviceberry
as a ffected by acid s c a rific a tio n and a benzyl adenine/
thiourea (BA/TU) m ixture.
S c a rific a tio n (min)
IvIOiStening agent
0
10
30
45
60
H2O
0
0
0
0
I
BA/TU (5 ppm/50 mM)
lb
2ab
5ab
12a
8a b
*Means followed by the same le t t e r are not s t a t is t ic a lly d iffe re n t
a t P = 0.05.
56
S c a rifie d , tap-w ater leached or unleached seeds were germinated with
200, 400, or 800 ppm GA solutions as moistening agents to te s t the
p ro m o te r/in h ib itd r hypothesis o f seed germination ( I ) .
Germination
was not improved over the controls by leaching or GA (Table 8 ).
Fo rty-th ree percent of the embryos from nonscarified and 65% of
the embryos from s c a rifie d seeds were released from t h e ir seed coats
a fte r a continuous 3-week soak in 0.3% HP.
No additional growth was
observed once seeds were tran sferred to germination boxes.
Aerated and cold water soaks have been shown to s u b s titu te fo r or
reduce the s t r a t ific a t io n ,requirement in !dormant seeds o f southern pine
( Pinus p a lu s tris M i l l . )
(6) and sugar maple ( Acer saccharum Marsh.) (50).
Serviceberry seeds did not germinate during or subsequent to cold and
warm water aeration treatm ents. *
Table 8.
Comparison* o f mean percentage germination o f serviceberry as,
a ffected by leaching fo r 24 hours and GAg.a
GAg moisture in ppm
Leach time hours
0
200
400
800
0
I
4
4
I
24
0
5 ................
7
I
*Means are not s t a t i s t ic a l ly d iffe r e n t a t P = 0.05.
aSeeds o f a ll treatments were acid s c a rifie d fo r .30 minutes.
•
57
Experiments were conducted to determine the optimum concentrations
o f BA and TU which would improve germination.
I n i t i a l l y , TU and BA were
tested separately a t concentration ranges found stim ulatory to the
germination o f dormant seeds e f other species (62, 2 4 ).
BA or TU did
not in d iv id u a lly stim ulate germination a t the concentrations tested.
It
had been previously determined th a t a mixture o f 5 ppm BA and 50 mM TU
produced minimal germination o f s c a rifie d seeds (Table 7 ) , therefo re two
experiments were designed to te s t the e ffe c t o f combining various con­
centrations o f BA and TU.
Maximum germination responses were achieved
when 100 mM TU was combined with e ith e r 100, 200, or 400 ppm BA
(Figs. 4 and 5 ).
A model using the o rig in a l count data as the dependent
v a riab le showed a BA lin e a r by TU lin e a r form o f in te ra c tio n to be
present.
When the dependent v a riab le was transformed to the square root
o f the count, the in te ra c tio n was accommodated by a sim pler model which
contained only lin e a r and quadratic terms fo r each main e ffe c t (F ig . 6 ).
Although the regression model is highly s ig n ific a n t (Table 9) and
accounts fo r 83% o f the v a ria tio n in germination (R
= 0 .8 3 ) there is
less confidence in the p re d ic tiv e a b il it y o f the model when concentra­
tions o f TU exceed 100 mM due to fewer germination data points.
A 95%
confidence statement fo r the maximum predicted germination response of
20% is 296 + 57 ppm BA and 100 + 4 mM TU.
S c a rifie d seeds were germinated a f t e r being separated in to large
(1 .3 5 9g /1 0 0), medium (.9530 g /1 0 0 ), small (.6657 g /1 0 0 ), and unsized
58
IOOppm BA
SOppm BA
IO ppm BA
5 ppm BA
H2 O
50
m M
Figure 4.
T u
E ffe c t o f low concentration BA/TU mixtures on the
germination o f serviceberry.
59
G E R M IN A T IO N
— — 4 0 0 ppm BA
—
BOO ppm BA
......... tOO ppm BA
HgO
%
- - -
IO O
Figure 5.
200 300
m MTu
400
E ffe c t of high concentration BA/TU mixtures
on the germination o f serviceberry.
60
MAX AT
BA*2 9 6 ppm
TU • IO O m M
IO O
m M
Figure 6.
T u
E ffe c t o f BA/TU mixtures a t various concentrations
on the germination o f serviceberry as predicted by
regression analysis. Refer to Table 7 fo r regres­
sion model.
61
Table 9.
Analysis o f variance fo r the germination response of
serviceberry to mixtures o f BA and TU.a
Degrees o f
freedom . .
Source
Mean
square
F
2 9 .4 4 **
Regression modelb
4
19.73
Lack o f f i t of
regression model
32
0.50
0.75 NS
Total fo r treatments
in two experiments
36
2.64
3 .9 3 **
Experimental e rro r
74
0.67
-
no
Total
-
-
aData are square root o f count o f germinated seeds per re p lic a te
o f 50 seeds.
2
^Percentage germination =
0.159 + (8.65 x 10"3 )BA - (1.46 x I O"5) BA2
2 X
+ (3.48 x 10"2 )TU - (1.75 x 10"4 )TU2
(1.043 g /10 0 )s ize classes to determine whether dormancy could be a ttr ib u ­
ted to some phenolo gical c h a ra c te ris tic such as stage o f m aturity
(Table 1 0 ).
TZ v i a b i lit y was determined fo r each size class in order
to assess the r e la tiv e dormancy o f the class.
V ia b ilitie s between size
classes did not vary g re a tly ; however, the small size class contained .
a large percentage o f u n fille d seeds.
Germinations were compared using
a germination index (GI = germination * v ia b i lit y x 100) which e lim i­
nates the nonviable seeds from the comparison.
The medium seed GI of
Table 10.
Tetrazolium (TZ) v i a b i lit y and germ ination* o f serviceberry as a ffec te d by seed
s ize .
Average
weight
g/ioo
Size
class
seeds
% of
sample
by
weight
TZ
v ia b ility 3
c la s s **
% empty
seeds
o f size
class
% germ
o f size
c la s s **
GIc
o f size
class
GI o f
sample
Large
1.359
40
63a
0
22b
35b
14
Medium
0.953
41
60a
8
38a
63a
26
Small
0.666
19
56a
19
8c
T4c
3
Unsized
1.043
100
60d
26b
43b
43d
7d l
*Means o f a column followed by the same l e t t e r are not s t a t is t ic a lly d iffe r e n t using
Duncan's MR te s t a t P = 0.05.
t e r m i n a t io n percents and TZ v ia b i lit i e s are s ig n ific a n tly d iffe r e n t w ith in s ize
classes by chi-square analysis a t P = 0.05.
aTZ v i a b i lit y determinations were made on seeds s c a rifie d fo r 30 minutes.
b
Seeds o f a ll treatments were s c a rifie d fo r 30 minutes then germinated with 100 ppm
BA/100 mM TU m ixture.
cGI (germination index) = germination f v i a b i l i t y , to y ie ld percent germination o f
v iab le seeds.
^Calculated data.
63
63% was s ig n ific a n tly greater than the 36% and 14% GI fo r large and
small seeds.
S tr a tif ie d but un scarified seeds germinated norm ally, while excised
embryos o f u n s tra tifie d seeds do not.
S c a rific a tio n plus the addition
o f a BA/TU m ixture to the germination medium was found to have a pro­
nounced promotive e ffe c t on germination.
S c a rific a tio n or the BA/TU
mixture applied in d iv id u a lly were in e ffe c tiv e .
GA had no e ffe c t on promoting germination.
Leaching, soaking, and
S c a rific a tio n appears to
■
induce increased seed coat perm eability to the BA/TU mixture which
stim ulates embryo growth.
TU and BA have been shown to promote the
germination o f other dormant species (62, 2 4 ).
Esashi, e t a l . (24)
showed th a t o f the growth and germination promoting substances tested ,
TU and BA used separately were most e ffe c tiv e in stim ulatin g the axes
and cotyledonary growth o f dormant cocklebur ( Xanthiurn pennsylvanicum
W a llr .) embryos.
Concentrations o f 100 mM TU or approximately 100 ppm
BA (the highest tested ) were found to be optimum fo r germination of
whole dormant seeds.
This correlates with our resu lts fo r service-
berry.
Erez (22) reported th a t in the absence o f purine cytokinins, con­
centrations of 0.1 to I mM TU were optimum fo r promoting growth in
k in e tin requirin g callu s tissues.
Combining TU with z e a tin , k in e tin or
BA produced synerg istic (more than a d d itiv e ) e ffe c ts on the growth
response o f soybean c a llu s .
Concentrations of 0.2 to I ppni BA combined
64
with .01 to I mM TU were optimum.
These concentrations are of con­
siderably lower magnitude than found to be optimum fo r serviceberry;
however, 1t has been shown (62) th a t TU stim ulates germination when the
in te rn a l concentration is r e la t iv e ly low.
The growth stim ulating
e ffe c t o f TU is postulated by Erez (22) to be a re s u lt o f an enhancing
e ffe c t on c e ll d iv is io n and not enlargement.
Zeatin a ffected c e ll
d iv is io n , and the in te ra c tio n between TU and purine cytokinins is
suggested to be due to differences in the modes o f action o f these two
compounds on c e ll d iv is io n .
Eashi, e t a l.
(24) report th a t TU stim ulates growth o f shoot axes,
whereas BA has a greater a ffe c t on growth o f cotyledons.
This cyto-
kinin stim ulatio n o f cotyledon expansion is p rim a rily a function of c e ll
enlargement not c e ll d iv is io n as in other species (5 8 ).
Evidence o f th is nature leads us to suggest th a t the in te ra c tio n
o f BA and TU on the promotion o f germination in dormant serviceberry
seeds may function in one or more ways.
TU may p r e fe re n tia lly promote .
c e ll d ivisio n with BA promoting increases in c e ll s iz e .
A d d itio n a lly ,
or a lte r n a tiv e ly as fo r Xanthiurn, TU may stim ulate a x ia l growth while
BA promotes cotyledon expansion; o r, as postulated by Erez (2 2 ), in t e r ­
action may be due to differences in mode o f action on c e ll d iv is io n .
The occurrence o f empty seeds associated with sm aller seed size
may have p ra c tic a l s ig n ifican ce fo r the seed producer and processor.
65
Seed q u a lity could e a s ily be improved by adjusting cleaning equipment
to discard the small and lig h t seeds.
Germination of the e n tire lo t
would also be improved since the small seed size class produced s ig n if i­
can tly Tower germination than e ith e r large or medium classes.
Dormancy
may be reduced by selecting fo r seeds in the medium weight range.
APPENDIX
Appendix Table I .
Common and s c ie n t if ic names o f plants referred
in the lit e r a t u r e review.
Common Name
S c ie n tific Name
Antelope bitterbrush
Purshia trid e n ta ta (Pursh) DC.
Apple
Malus spp. M il l.
Ash
Fraxinus spp. L.
Beech
Fagus s y lv a tic a L.
Buttercup
Ranunculus spp. L.
Clover, subterranian
T rifo liu m subterraneum L.
Clover, white sweet
M elilotus aIbus Desr .
Cocklebur
Xanthium pennsylvanicum WalI r .
Cotton
Gossypium hirsutum (C u lt.)
Hazel
Corylus avellana L.
Holly
Ile x opaca A it .
Legume fam ily
Fabaceae Reichenb.
Lettuce
Lactuca sativa L.
L ila c
Syringia spp. L.
Maple, Norway
Acer platanoides L.
Maple, sugar
Acer saccharum Marsh.
Maple, sycamore
Acer pseudoplatanus L.
Oak9 black
Quercus nigra L.
Oak, red
Quercus rubra L.
Oats, w ild
Avena fatua L.
Orchid fam ily
Orchidaceae L in d l.
Parsnip
Heracleum sphondylium L.
Peach
Prunus persica Batsch
Potato
Solanum tuberosum L.
Rose fam ily
Rosaceae Juss.
Rose, f ie ld
Rosa arvehsis Huds.
Rose, rugosa
Rdsa rugosa Thunb.
68
Appendix Table I (continued)
Common Name
S c ie n tific Name
Russian pigweed
Axyris amaranthoides L.
Smooth bromegrass
Bromus inermis Leyss.
Snowberry
Symphoricarpos a!bus ( I . ) Blake
Soybean
Glycine max ( L . ) M err.
Sunflower
Helianthus annuus
L
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