Apical 2-cm hypocotyl segments from seedlings of a &quot;short-hypocotyl&quot; cultivar Amsoy 71, like whole seedlings, evolve about twice as much ethylene at 25°C as at 30°C. Segments consisting of two cotyledons and an attached epicotyl evolve ethylene at low rates at both 25°C and 30°C. Hypocotyl segments from seedlings of Cutler 71 also show enhanced ethylene evolution at 25°C. Hypocotyl segments from Corsoy, a &quot;longhypocotyl&quot; cultivar, however, evolve ethylene at low rates at both 25°C and 30°C. Wounding of Amsoy 71 hypocotyl segments does not increase their ethylene evolution. Ethylene evolution at 25°C is reduced and the short-hypocotyl phenomenon is reversed by partial (50%) removal of Amsoy 71 cotyledons at planting time. Key words: Cotyledons -Ethylene -Hypocotyl elongation -Temperature sensitivity. Certain soybean cultivars exhibit poor emergence when planted at a depth of 10 cm and grown at 25°C. The poor emergence is a consequence of reduced hypocotyl elongation (Burris  and Fehr 1971 Abbreviations: H, apical 2-cm hypocotyl segment; HW, wounded hypocotyl segment; C + E, cotyledons+ epicotyl; C + E+H, cotyledons+epicotyl + hypocotyl segment left attached to each other; pt, plant part; PVC, polyvinyl chloride; SD, standard deviation

Clifford E Lamotte

Irvin C Anderson

Joseph S Burris

Nader Seyedin

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Plant & Cell Physiol. 23(3): 427-431 (1982)
Temperature-dependent Inhibition of Hypocotyl Elongation in
Some Soybean Cultivars:
I. Localization of Ethylene Evolution and Role of Cotyledons1
Nader Seyedin, Joseph S. Burris, Clifford E. LaMotte and
Irvin C. Anderson
Department of Plant Pathology, Seed and Weed Sciences and Department of Botany,
Iowa State University, Ames, Iowa 50011, U.S.A.
Apical 2-cm hypocotyl segments from seedlings of a "short-hypocotyl" cultivar Am-
soy 71, like whole seedlings, evolve about twice as much ethylene at 25°C as at 30°C.
Segments consisting of two cotyledons and an attached epicotyl evolve ethylene at low
rates at both 25°C and 30°C. Hypocotyl segments from seedlings of Cutler 71 also show
enhanced ethylene evolution at 25°C. Hypocotyl segments from Corsoy, a "long-
hypocotyl" cultivar, however, evolve ethylene at low rates at both 25°C and 30°C.
Wounding of Amsoy 71 hypocotyl segments does not increase their ethylene evolution.
Ethylene evolution at 25°C is reduced and the short-hypocotyl phenomenon is reversed
by partial (50%) removal of Amsoy 71 cotyledons at planting time.
Key words: Cotyledons — Ethylene — Hypocotyl elongation — Temperature
sensitivity.
Certain soybean cultivars exhibit poor emergence when planted at a depth of 10 cm and
grown at 25°C. The poor emergence is a consequence of reduced hypocotyl elongation (Burris
and Fehr 1971, Burris and Knittle 1975, Gilman et al. 1973, Grabe and Metzer 1969, Samimy
1978, Samimy and LaMotte 1976). Such "short-hypocotyl" cultivars show normal hypocotyl
elongation at 15, 20 and 30°C; hypocotyls of some other cultivars of "long-hypocotyl" type elon-
gate normally at all four temperatures (Grabe and Metzer 1969). Burris and Knittle (1975)
have demonstrated that removal of 50% of the cotyledonary tissue from anomalous cultivars
transforms hypocotyl elongation at 25°C to normal. The significance of such a cotyledonary
factor also was recognized by Samimy (1978) who demonstrated that more ethylene is evolved
when cotyledons are left attached to the apical 1-cm section of the hypocotyl than when cotyledons
are detached.
This paper reports on 1) the localization of temperature sensitivity in some anomalous
soybean cultivars and 2) the significance of cotyledons in ethylene evolution by hypocotyls of
Amsoy 71 seedlings.
Abbreviations: H, apical 2-cm hypocotyl segment; HW, wounded hypocotyl segment; C + E, cotyledons+
epicotyl; C + E+H, cotyledons+epicotyl + hypocotyl segment left attached to each other; pt, plant part; PVC, poly-
vinyl chloride; SD, standard deviation.
1 Journal Paper No. J-10412 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa,
U.S.A. Project 2191.
427
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428 N. Seyedin et al.
Materials and Methods
Seeds of different cultivars of soybean [Glycine max (L.) Merr.] were preconditioned to ca.
21% moisture. They were surface-sterilized with 5% sodium hypochlorite for 30 sec followed
by several rinses of distilled water and 30 sec of 0.01 N hydrochloric acid. Seeds were soaked in
aerated, running distilled water for 1.5 hr. A rolled towel germination procedure reported by
the Association of Official Seed Analysts (1965) and later modified by Burris and Fehr (1971)
and Samimy and LaMotte (1976) was used to produce seedlings. Twenty-five seeds were
placed 10 cm from the wet paper's top and oriented in a single row with radicles pointing down-
ward. The rolled towels were placed in a plastic container (18x28x30 cm) and covered with
a plastic bag. After 48 hr, 250 ml of water was added to each container.
Seedlings were grown for 4 and 5 days at 30 and 25°C, respectively. These ages were chosen
because seedlings had developed to similar extents as judged by observation of primary root
lengths. Primary root elongation does not show the anomalous temperature sensitivity (Samimy
and LaMotte 1976). All manipulations of plant material were conducted under a dim green
light in a dark room (Samimy 1978).
For determination of ethylene evolution by intact seedlings, groups of 25 seedlings, earlier
grown in rolled paper towels, were sealed into PVC tubes (7.5 cm i.d. X 50 cm) each containing
20 ml of water. A screw-type head allowed easy access to seedlings. Vacuum grease was used
to seal the head. A rubber septum was fitted into the head to permit gas sample withdrawal.
After 18 hr incubation, a 1-ml sample was analyzed for ethylene content. Gas analysis was
accomplished with a Varian model 3700 gas chromatograph equipped with a flame ionization
detector coupled to a Cary model 401 vibrating reed electrometer.
For determination of ethylene from seedling parts, ten etiolated seedling parts were placed
in each 63-ml, black custom-made Plexiglas chamber. Each chamber contained a filter paper
(7-cm diameter) and 7 ml of 50 HIM K-phosphate buffer, pH 7, containing 2% sucrose and 0.05%
chloramphenicol. The air in the sealed chamber was assayed for ethylene content every 3 hr
over a period of 9 hr. After sample withdrawal, the system was flushed with air and sealed for
subsequent ethylene determination. Incubation of seedling parts was at the same temperature
as that at which the seedlings were grown.
To assess wound-induced ethylene evolution, hypocotyl segments were pricked with a fine
needle or were cut into three sections. To assess the role of cotyledons in ethylene evolution half
of each cotyledon was excised from imbibed seeds of Amsoy 71 at planting time. The seeds were
then planted and grown as indicated earlier.
Results
Ethylene evolution by intact seedlings of Amsoy 71 at two temperatures is shown in Table 1.
Ethylene evolution is significantly (p<0.01) greater at 25°C than at 30°C.
Table 1 Rate of ethylene evolution by intact Amsoy 71 soybean seedlings
Rate of ethylene
25°C
2,099°
evolution (Pi seedling"
1 hr-i)
30°C
992
° Significant at 1 % level by Student's t test.
n=4, Rate averaged over 18 hr.
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Cotyledons and temperature sensitivity 429
o o
Fig. 1 Rate of ethylene evolution during each 3-hr interval as affected
by temperature and sampling time in Amsoy 71 soybean seedling parts.
SD is standard deviation in picoliters per plant per hr.
Hypocotyl
Cotyledon* + fpicotyl
3O°C
Cotyledon* +
To localize the temperature-dependent ethylene evolution in Amsoy 71 seedlings, parts
were excised and ethylene evolution was determined. Ethylene evolution (Fig. 1) by C+E is
compared with that evolved by H. Results, the average of two replications, show that ethylene
evolution by H is significantly greater than that by C + E at 25°C. At 30°C, ethylene evolution
by H and C + E is low and similar.
Ethylene evolution by apical 2-cm segments from hypocotyls of three different cultivars is
shown in Fig. 2. In segments from seedlings of Amsoy 71 and Cutler 71, two "short-hypo-
cotyl" cultivars, greater rates of ethylene evolution are observed at 25°C than at 30°C. In
segments from seedlings of Corsoy, a "long-hypocotyl" cultivar, ethylene evolution is low and
similar at 25 and 30°C.
Ethylene evolution by hypocotyl segments from Amsoy 71 seedlings with 50% cotyledon
7 8
Z O
2 3 *
AMSOY 71 CUH.tR 71 CORSOY
Fig. 2
Fig. 2 Rate of ethylene evolution during each 3-hr interval as affected by temperature in hypocotyl segments from
soybean seedlings of three cultivars. SD is standard deviation in picoliters per gram fresh weight per hr.
Fig. 3 Effects of temperature, 50% cotyledon excision at planting time, and small seed size on rate of ethylene evolu-
tion during each 3-hr interval in excised hypocotyl segments from Amsoy 71 seedlings. SD is standard deviation
in picoliters per gram fresh weight per hr.
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430
Table 2 Rate of ethylene
wounding at 25 and 30cC
Sampling interval
(hr)
Exp. I"
0-3
3-6
6-9
Exp. II»
0-3
3-6
6-9
evolution during
N. Seyedin et ;
each 3-hr interval
il.
by Amsoy 71 soybean seedling
Rate of ethylene evolution (pi pt"1 hr"1)
25°C
H
1,641
1,484
890
C + E+H
1,886
3,663
1,969
HW
1,146
1,160
750
C+E+HW
1,572
2,256
1,221
30°C
H
344
265
289
C+E+H
600
1,681
2,083
parts as affected by
H W
487
344
197
C+E+HW
658
1,220
891
" In Exp. I, H = apical 2-cm hypocotyl segment; HW=hypocotyl segment pricked 10 times with a fine needle;
standard deviation = 216 pi p t - 1 hr"1. n = 2
* In Exp. II, C + E + H = cotyledons+epicotyl+apical 2-cm hypocotyl segment; C + E + H W = C + E + H having
hypocotyl pricked 10 times with a fine needle; standard deviation=237 pi pt"1 hr-1. n=2
excision at planting time is significantly less (p<0.01) than that by excised hypocotyl segments
from seedlings with intact cotyledons (Fig. 3). Cotyledon excision does not significantly affect
ethylene evolution by hypocotyl segments at 30°C (Fig. 3). The effect of cotyledon removal on
ethylene evolution at 25°G can be mimicked (Fig. 3) by selecting for a small seed size (12 g per
100 seeds), each about half the size of a large seed (24.9 g per 100 seeds). Ethylene evolution in
these small seeds is low and similar to that of controls at 30°C.
Mean ethylene evolution generally decreased due to wounding, but differences were not
statistically significant (Table 2). Similar findings resulted when hypocotyl segments were cut
into three sections. At 25°C, the rate of ethylene evolution by H over a 15-min interval (1778
pi pt"1 hr-1) was similar to those measured at first and second sampling times in Fig. 1.
Discussion
Samimy and LaMotte (1976) have shown that 5-day-old etiolated seedlings of Clark, which
exhibit an inhibition of hypocotyl elongation, evolve twice as much ethylene at 25°C as do
seedlings of Mandarin, which grow normally. Results presented here (Table 1) reveal that
Amsoy 71 is similar to Clark in its response to temperature. Moreover, apical 2-cm segments
from hypocotyls of Amsoy 71 and Cutler 71 seedlings show a similar temperature dependence
(Fig. 2). Segments, each consisting of two cotyledons and an attached epicotyl, evolve ethylene
at similar rates at 25 and 30°C (Fig. 1). These findings show that the temperature-dependent
enhancement of ethylene evolution is localized in the hypocotyl with a high rate of ethylene
evolution immediately below the hypocotyl arch. These findings are consistent with those of
other investigators. Goeschl et al. (1967) reported that ethylene evolution by etiolated pea
epicotyls was confined to the plumule and plumular hook. Sakai and Imaseki (1971) reported
that ethylene production by mung bean was most rapid in the segments immediately below the
hypocotyl hook. Ethylene evolution by hypocotyl segments of Corsoy, a "long-hypocotyl"
cultivar, is low at both temperatures. We believe that our results could not have been compli-
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Cotyledons and temperature sensitivity 431
cated by wound ethylene evolution since early, short term ethylene measurements agreed with
those made later.
Burris and Knittle (1975) have demonstrated that partial removal of cotyledons results in
normal hypocotyl elongation in Amsoy seedlings grown at 25°C. The present research indicates
that 50% cotyledon removal reduces ethylene evolution by ca. 50% at 25°C but not at 30°C.
Results presented here further implicate ethylene in the hypocotyl growth anomaly and
indicate that the temperature-dependent enhancement of ethylene evolution is localized in the
hypocotyl tissue. Some unknown factor supplied by the cotyledons may regulate ethylene
evolution in the hypocotyl at 25°C.
The authors are thankful for the assistance of David F. Cox, Hollys E. Heer, John F. Heer and Robbie J. Kerkove.
References
Association of Official Seed Analysts (1965) Rules for testing seeds. Proc. Assoc. Off. Seed Anal.
BurrisJ. S. and W. R. Fehr (1971) Methods for evaluation of soybean hypocotyl length. Crop Sci. 11: 116-117.
Burris, J. S. and K. H. Knittle (1975) Partial reversal of temperature-dependent inhibition of soybean hypocotyl
elongation by cotyledon excision. Crop Sci. 15: 461-462.
Gilman, D. F., W. R. Fehr and J. S. Burris (1973) Temperature effects on hypotyl elongation of soybeans. Crop
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Goeschl, J. D., H. K. Pratt and B. A. Bonner (1967) An effect of light on production of ethylene and the growth of
the plumular portion of etiolated pea seedlings. Plant Physiol. 42: 1077-1080.
Grabe, D. F. and R. B. Metzer (1969) Temperature-induced inhibition of soybean hypocotyl elongation and seed-
ling emergence. Crop Sci. 9: 331-333.
Sakai, S. and H. Imaseki (1971) Auxin-induced ethylene production by mung bean hypocotyl segments. Plant &
Cell Physiol. 12:349-359.
Samimy, C. (1978) Effects of light on ethylene production and hypocotyl growth of soybean seedlings. Plant
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Samimy, C. and C. E. LaMotte (1976) Anomalous temperature dependence of seedling development in some
soybean [Glycine max (L.) Merr.] cultivars: Role of ethylene. Plant Physiol. 58: 786-789.
(Received October 5, 1981; Accepted January 28, 1982)
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