SEED GERMINATION OF AMERICAN BASSWOOD IN RELATION TO

SEED MATURITY
D. E. VANSTONE and W. G. RONALD
Research Station, Agriculture Conada, Bor 3001, Morden, Manitoba R}G 1J0.
Received /B NoL,. 1981, acceptetl 2 Feb. 1982.

V,tNsroNE, D. E. nNo RoN,rr-o, W. G. 1982. Seed germination of American

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basswood in relation to seed maturiry. Can. J. Piant Sci. 62: i09-i13.

Seed germination of American basswood collected and sown at weekly intervals
during late summer 1977 increased until the 9 Sept. collection at which germination
was 52Vc; thereafter, germination decreased. The color of the pericarp and the
moisture content of the seed proved reliable indices of maturity rn relation to
germination. All pericarps turned greyish-brown by 9 Sept. and moisture content
of the seed decreased to 16c/c. Germination of excised embryos occurred without
delay at all stages of seed maturity. Examinatton of the structure of the testa by
means of differential staining and light microscopy showed a high degree of cell
orientation, compaction and lignrfication at the advanced stages of maturity.

Le taux de germination de graines de tilleul d'Am6rique r6colt6es et semdes b

Can. J. Plant Sci. 1982.62:709-713.

rntervalles d'une semaine ir la fin de 1'6t6 1977 s'est accru jusqu'd 9,' r6colte en
septembre (moment or) il atteignait 52%) pour ensuite r6gresser. La couleur du
pdricarpe et la teneur en eau de la graine s'avbrent des indices fldbles de maturitd
en rappoft avec la germination. Tous les p6ricarpes ont tourn6 au brun grisatre le
9 septembre et la teneur en eau de la graine a baiss6 >a 16 Vc. Les embryons excis6s
ont germe sans d6lai I tous les stades de maturit6 de 1a graine. L'examen de la
structure des testa par coloration diff6rentielle et microscopie optique r6vdie un haut
degre d'orientation, de compaction et de lignification cellulaires aux stades avancds
de maturit6.

Amr:rican basswood (TiLia crmericana L.)
seecL is borne within a tough indehiscent
pericarp. Upon maturity, the testa becomes
harcl and encloses a fleshy endosperm and
a ulell-developed embryo (U.S. Dep.
Agric., Forest Service (USDA) 1914). The
seecl normally matures in mid-September
in l4anitoba, but may persist on the tree
into winter. Since basswood seed persists
on t:he tree, seed collection is ofien post-
ponr:d until long after maturity.
The difficulty in germinating American
basswood seed has been recognized for
many years (Barton 1934; Cumming et al.
1964; Cobb 1932). Despite numerous stud-
ies on pre-germinative treatments, reliable
pror:edures for producing consistent ger-
mination have not been reported (USDA
t91 4).
Can. J. Plant Sci. 62: 709-713 (July 1982)
709
The basis of poor seed germination has
been linked with several factors. It was
shown by Spaeth (1934) over 45 yr ago
that hard seed coats could exclude mois-
ture from the embryo and endosperm. Since
moisture is essential to germination, this
report led investigators to evaluate seed
maturation and moisture as related to
germination.
Hert (1977) stated that seed collection
dates affect the water permeability of the
testa and subsequent germination of seed.
Mohn (1964) indicated a moisture content
of 20-29Vo was associated with good ger-
mination of basswood seed.
The purpose of the present study was
to define seed moisture changes during
maturation and relate such changes to vis-
ual morphological developments associated
0048-12201 821 6203 0709 $2.00
O l9fl2 Agricultural Institute of Canada
 710 CANADIAN JOURNAT- OF PLANT SCIENCE
with optimum seed germination. Ontoge-
netic changes of seed moisture content,
seed dry weight, pericarp color and testa
structure were monitored in relation to
germination.
MATERIALS AND METHODS
Seed was collected from several native and
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cultivated stands over several years of studies.
Al1 experiments reported here utilized seed col-
lected at the Morden Research Station at weekly
intervals from 12 Aug. 7917 to 23 Sept. 1977.
Pericarp Color
The proportion of seeds with green or greyish-
brown pericarps was visually observed and re-
corded for 100 seeds at each collection date.
Seed Moisture Content and Dry Weight
Pericarps were removed from seeds soon after
collection and at each collection date four rep-
Can. J. Plant Sci. 1982.62:709-713.
licates of 15 depericarped seeds were dried to
constant weight at 35"C for 12 h and the per-
cent moisture content was calculated. Data were
analyzed using a randomized complete block
analysis.
Seed Germination
Two germination tests were run on seeds which
were collected and sown weekly.
TEsr l. Seeds were sown in well-prepared
outdoor seed frames to a depth of 15-20 mm.
Treatments were arranged in a randomized
complete block design containing four repli-
cates of 50 seeds. Total germination was re-
corded on 5 June 1978.
TEsr 2. Beginning on 19 Aug., seeds were
sown to a depth of 15 mm in 10-cm plastic
pots in the greenhouse and maintained at 10-15
'C throughout winter. Treatments included in-
tact and depericarped seed with 25 seeds per
pot replicated four times and arranged in a
split-plot design with pericarp treatments as
main plots and dates of collection as subplots.
The pots for the 16 Sept. collection were lost
so that germination data for that date are not
available. Emerged seedlings were counted as
a measure of germination on 10 Apr. 1978.
Prior to analysis, data were transfbrmed using
arc-sine square root transformation.
Growth of Excised Embryos
Ten freshly coilected seeds were depericarped
each week, surface-steriiized in 95Vo ethanol,
and soaked overnight in sterile distilled water.
Seeds fiom 16 and 23 Sept. collections required
scarification with a pin to facilitate imbibition
of water. Following imbibition, embryos were
excised asceptically and cultured on Emsweller
agar medium (Emsweller et al. 1962) in an
incubation chamber for 12 days. The chamber
was maintained at 25"C and was constantiy lit
by cool white fluorescent 1amps. The condition
of the embryos at the end of 12 days was
recorded as transplantable or green but dis-
torted. Transplantable embryos were trans-
planted into soil and their growth was observed
in the greenhouse.
Ontogenetic Development of the Testa
Seeds from weekly collections were deperi-
carped and embedded in paraffin. Ttansverse
sections (20 p.m) were stained with safranin
and fast green using the technique described
by Johansen (1940). The testae were examined
using phase contrast and fluorescent micros-
copy (Carl Zeiss Standard 14 microscope) and
photographed using a 35-mm camera.
RESULTS
Pericarp Color
Pericarp color changed from entirely green
at the beginning of the collection period
to entirely greyish-brown at the end. The
color change was distinctive; prior to 26
Aug. all pericarps were green and on 26
Aug., 2 $ept. and 9 Sept., respectively,
44Vo, 94Vo and 1007o of pericarps were
greyish-brown.
Seed Moisture Content and Dry Weight
There was a significant decrease in seed
moisture content during maturation (Fig.
1). Moisture content of the unripened seed
on 12 Aug. was 62Vo. It remained near
that level for the next 2 wk, then rapidly
decreased the following 3 wk to a level
of '/Vo on 16 Sept. Associated with a de-
crease in moisture content was a change
in the physical consistency of the endo-
sperm. The endosperm was soft and milky
on 12 Aug., gelatinous and white on 9
Sept., and hard, dry and yellowish on 23
Sept.
The dry weight of the seeds did not
change significantly throughout the collec-
 VANSTONE AND RONALD SEED GERMINATION OF AMERICAN BASSWOOD 711
-
tion dates. The overall mean weisht was Moisture
1.1glsample of 15 seeds (70 mg/ieed). -.o Germination

Seed Germination
Spring germination of fall-sown American c
basswood seed was dependent on the date 0)
c
of collection and sowing (Fig. 1). The 9 m"\
Sept. sowing date resulted in the best ger- O
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o C
mination (52Vo). Seed sown durine the 30 :o .9
J
three successive dates in August germi- a o
tr
nated 10, 8 and l4%o, respectively. Seeds m ,o'E
collected in August and early September o
were very succulent and many of them q) roo
decayed within the soil. Seed sown on 16 0)
a
and 23 Sept. exhibited only 6 and 2Vo l2 19 262
germination, respectively. Ungerminated AUG stP

seed was screened from the soil and found
to be firm and impermeable to water.
Germination of greenhouse-sown seed
began in mid-February and continued until
Can. J. Plant Sci. 1982.62:709-713.
Date of Col lection & Sorruing
Fig. 1. Moisture content and spring germina-
tion of American basswood seed collected and
sown at weekly intervals during maturation.

early-April regardless of sowing date and
pericarp removal. The time between sow-
ing and germination, which was 4.5-6 mo,
must not be considered as a normal strat-
ification period due to the variation in
temperature within the greenhouse. The
presence of the pericarp did not restrict
germination. Overall germination of intact
seeds was 2lqo comparcd with lg%o ger-
mination for depericarped seeds (Table l).
Neither the difference between intact ver-
sus depericarped seed, nor the interaction
between main plots and subplots was sig-
nificant, whereas the difference amons
dates was highly significant. The 9 Septl
mean germination percentage of 33Vo was
Means marked by the same letter do not differ
significantly according to DMR, SVa level.
similar to germination for 26 Aug. seed
and higher than the germination of seed
from the other collection dates. The 26
Aug. and 2 Sept. germination percentages
were not statistically different from each
other.
Growth of Excised Embryos
All excised embryos from every collection
date grew to a transplantable size within
12 days except for a small percentage
which turned green and became distorted
through improper opening of the cotyle-

Table 1. Germination of greenhouse-sown intact and depericarped American basswood seed

Germination (7o;f
Date of collection
and sowing Depericarped
19 Aug. 7 o 8c
26 r',rtg z) 39 3lab
2 Sept 26 18 22b
9 Sept. 42 25 3i a
23 Sept. 7 A
5c
Mean 21 19

I Germination recorded on l0 Aor. 1978.

a-c Means followed by the sarnJ letter do not differ significantly according to DMR, 5Vo level
 7t2 CANADIAN JOURNAI- OF PI-ANT SCIENCE
dons. The distortion of the embryos was
apparently due to injury caused during the
excision process.
Ontogenetic Development of the Testa
The testa was composed of an inner and
outer integument and a thin epidermal layer
(Fig. 2). The inner integument stained red
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with safranin, characteristic of lignin and
the outer integument stained green with
fast green, characteristic of cellulose. The
cells within the inner and outer integu-
ments were arranged radially with a thick-
ness of 10.2 and 1.9 ptm, respectively.
The thickness of the integuments remained
constant throughout maturity. As maturity
progressed, the amount of staining grad-
ually increased and the alignment of cells
became increasingly tight and distinct. The
epidermal layer appeared to be fairly dis-
Can. J. Plant Sci. 1982.62:709-713.
tinct by 26 Aug. By 16 Sept. it became
much thicker and was firmly attached to
the outer integument. It was noted during
the embryo excision procedure that water
would penetrate seeds collected on 16 and
23 Sept. only after the testa was punc-
tured.
Fig. 2. The testa of American basswood seed (TS 160 x magnification): a. unripe seed collected
26 Aug. 1977; b. ripe seed collected 23 Sept. 1977. Note labelling as follows: e, epidermal
layer: o. outer integument: i. inner integument.
DISCUSSION
There were very definite changes in ger-
mination of seed collected at various stages
of maturity. Changes in moisture content,
testa development and pericarp color were
associated with seed maturity. The optimal
stage of maturity for seed collection was
found to be 9 Sept. as indicated by both
greenhouse and outdoor germination tests.
Change of pericarp color on 100% of the
seeds occurred by that date and seed mois-
ture content had decreased rapidly to l6Vo.
Stroempl (1965) accepted through em-
pirical evidence that the testa of American
basswood seed hardens gradually during
maturation and Spaeth (1934) concluded
that the impermeability of the testa of
mature seed results in prolonged delays in
germination. The present study substanti-
ates and explains previous reports through
direct measurements and observations by
means of microscopy. The testa of late-
collected seed which showed lignification,
impregnation with cellulose, and a highly
ordered array of cells in the inner and
outer integuments became impermeable to
 VANSTONE AND RONAI-D
-
water and appeared to contribute
poor germination of such seed.
The greenhouse germination study which
compared intact versus depericarped seed
indicates that the pericarp was not a con-
tributing factor to the complicated seed
dormancy of American basswood. This
conclusion is strengthened by unpublished
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SEED C;ERMINATION OF AMERICAN tsASSWOOD 713
to the Although other researchers (Heit 1977;
Mohn 1964; Stroempl 1965) have indicated
the general importance of seed maturity
as it affects seed germination, the results
we report clearly define the optimal de-
velopmental stage for seed germination.
The color change of the pericarp from
green to greyish-brown can be used as an

data in which mature depericarped fall- index to determine the proper stage of
sown seed germinated only l%o the first maturity for seed collection and sowing
spring, whereas similar depericarped seed each year. This index can be utilized with-
treated for 45 min. with sulfuric acid ger- out expensive laboratory equipment or
minated 73o/o. technical expertise. Timely collection and
The prompt germination of excised em- sowing of basswood seed is a means by
bryos at each stage of maturity contrasted which commercial propagators can avoid
with the germination results of intact or costly and time-consuming pregerminative
depericarped seed where even seed col- seed treatments required for germination
lected at the optimal stage of maturity of late-collected seed.
showed a germination delay of several
months. Since the Emsweller medium on ACKNOWLEDGMENT
Can. J. Plant Sci. 1982.62:709-713.

which the embryos were placed contained The authors wish to acknowledge the excellent
only sucrose and various minerals and no technical assistance provided by Mr. Henry
germination promoting substances, it seems Hiebert.
that any inhibitors found in the seed must
have been located external to the embryo, BARTON, L. V. 1934. Dormancy in Tilia
perhaps in the endosperm. seeds. Contrib. Boyce Thompson Inst. 6: 69 89.
CUMMING, W. A., MORRISON, J. W. and
Other workers (Barton L934; Heit l9l7:
VITINS, A. 1964. Propagation studies in fruits
Spaeth 1934) have also suggested the pres- and omamentals at Morden Experimental Farm.
ence of inhibitors in basswood seed. Spaeth Agric. Can. Res. Branch, Morden, Man. Con-
(1934), found that when naked seeds con- trib.49.
taining only embryo and endosperm were COtsB, F. E. 1932. Growing trees from seeds.
placed in rnoist petri dishes, the radicle N. S. D. Hortic. 4: 127 128.
and hypocotyl emerged, but normal seed- EMSWELLER, S. L., ASEN, S. and UHR-
ling vigor was lacking. He suggested that lNG, J. 1962. Lilium speciosum x LiLium
an inhibitor was preventing normal ger- aurotum. Lily Yb., N. Am. Lily Soc. 15:7-75.
HEIT, C. E. 197'7. Propagation from seed. Part
mination. The present study advances cur-
27. Am. Nurserymen 145: 1O-11, 100-110.
rent knowledge by showing that the inhib-
JOHANSEN. D. A. 1940. Plant micro tech-
itor is located external to the embrvo. niques. McGraw-Hill, Toronto.
The moisture content of basswood seed MOHN, C. A. 1964. Timing of seed collec-
was studied by Mohn (1964) who indicated tions to increase germination of basswood seed.
that optimal germination occurred at seed- Minn. For. Notes i52.
morsture contents of 2V29Vo, whereas our SPAETH, J. N. 1934. A physiologicai study
studies associated a rnoisture content of of dormancy in Tilia seed. N.Y. Agric. Sta.
167o with optimal germination. This dis- Memoir 169.
crepancy is understandable when one con- STROEMPL, G. 1965. Germination of early
siders that moisture loss from the seed at collected fruit of Tilia americana L. Res. Reo.
this developmental stage occurred at the #64. Ontario Dep. Lands and Forests. Rei.
Rep. 64.
rate of 7-2Vo per day (Fig. l). It must U.S. DEPARTMENT OF AGRICULTURE
also be noted that these two studies were FOREST SERVICE. 1974. Seeds of woody
conducted in different locations with dif- plants in the United States. U.S. Dep. Agric.
ferent provenances. Handb. 450.