- Email: [email protected]
Roles of different seed components in controlling tomato seed germination at low temperature
Scientia Horticulturae, 56 (1994) 197-206
197
Elsevier Science Publishers B.V., Amsterdam
Roles of different seed components in controlling tomato seed germination at low temperature Shai L e v i a t o v a, O d e d Shoseyovb, Shmuel Wolff 'a
aDepartment of Field and VegetableCrops, Faculty of Agriculture, P.O. Box 12, Rehovot 76-100, Israel bDepartment of Horticulture, Faculty of Agriculture, Rehovot 76-100, Israel (Accepted 23 July 1993)
Abstract The role of different seed components in controlling the germination of tomato (Lycopersicon esculentum) seeds under low temperature conditions was studied in cold-resistant plant line PI341988 and cold-sensitive commercial cultivar UC82B, as well as in eight progeny lines of these two parents. At 12°C, PI341988 and UC82B seeds had a mean time of germination (MTG) of 8.7 days and 19.7 days, respectively. Removal of seed testa by NaOC1 treatment shortened the time required for germination by 3 days in both plant lines, but did not reduce the significant differences in germination rate between them. Removal of the seed coat and the endosperm layer in front of the embryo's radicle tip eliminated the differences in germination rate under low temperature conditions between the ten tested lines. All lines exhibited similar radicle elongation rates, both at 25°C and at 12°C. These results indicate that the main barrier to germination of tomato seed at low temperatures is imposed by the endosperm layer. At 12°C, plant line PI341988 exhibited a higher respiration rate than UC82B, indicating its higher metabolic activity under low temperature conditions. Key words: Embryo; Endosperm; Seed coat Abbreviation: MTG = mean time of germination
Introduction T h e ability t o g e r m i n a t e r a p i d l y is essential for seeds to o v e r c o m e stresses s u c h as w a t e r o r o x y g e n deficits, salinity, soil crusting, p a t h o g e n s a n d insects ( H e g a r t y , 1978; W o l k a n d H e r n e r , 1 9 8 2 ) . T h e g e r m i n a t i o n rate o f t o m a t o (Lycopersicon e s c u l e n t u m ) d e c r e a s e s p r o g r e s s i v e l y as t h e soil t e m p e r a t u r e is d e c r e a s e d f r o m 25 to 1 0 ° C , a n d is c o m p l e t e l y i n h i b i t e d b e l o w 1 0 ° C ( S c o t t a n d J o n e s , 1985; D a h a l et al., 1 9 9 0 ) . H o w e v e r , the g e r m i n a t i o n rate differs a m o n g L. esculenturn g e n o t y p e s , a n d r a p i d g e r m i n a t i o n a b o v e 1 0 ° C is heritable (E1-Sayed a n d J o h n , 1973; C a n n o n et al., 1973; W o l f et al., 1 9 8 6 ) . F o r *Corresponding author.
© 1994 Elsevier Science Publishers B.V. All rights reserved 0304-4238/94/$07.00
198
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
example, seeds of the cold-tolerant tomato plant introduction accessions PI341988, PI 120256 and PI 174263 germinate more rapidly than seeds of the cold-sensitive tomato line T5 at low temperatures (Dahal et al., 1990). PI341988 seeds also germinate more rapidly than those of the cold-sensitive line st-24 at 10°C, and are more responsive to priming (Liptay and Schopfer, 1983). The time of germination has been found to be correlated with seed weight in L. esculentum and Lycopersicon pimpinellifolium F 1 and F2 progenies. The paternal effect of L. pimpinellifolium in decreasing germination time was assumed to be related to reduced seed size (Whittington and Fierlinger, 1972 ). Seed coats may influence the seed's germination ability by interfering with water uptake, gas exchange or the diffusion of endogenous inhibitors, or by mechanically restricting embryo growth (Ikuma and Thimann, 1963 ). Pavlista and Haber (1970) proposed that both the mechanical force of the growing embryo pushing against the endosperm and the enzymatic weakening of the endosperm are necessary for lettuce seed germination. Weakening of the mechanical restraint imposed by the endosperm opposing the top of the radicle is also a prerequisite for germination in pepper (Watkins and Cantliffe, 1983) and tomato (Groot and Karssen, 1987) seeds. The base temperature for germination was reduced by 5 oC for tomato plant lines PI341988 and T5, when the endosperm/testa cap opposite the radicle tip was removed. The germination rate was also significantly increased by this treatment (Dahal et al., 1990). These results indicate that germination-related processes can occur at temperatures lower than the base temperature for germination. Low temperature may affect the rate of embryo growth, and its ability to overcome the mechanical restraint imposed by either the endosperm or the seed coat surrounding it. No concrete information is available with regard to the effect of low temperature on the activities of different tomato seed tissues, or the relationship between these activities and the ability to germinate under low temperature conditions. The aim of this research was to determine the role played by the testa and endosperm layer of the tomato seed in controlling germination ability at low temperature. Materials and methods
Seeds. - Tomato (Lycopersicon esculentum) plant lines PI341988 and UC82B were grown for seed production in a temperature-controlled greenhouse at 2 5 / 1 8 ° C ( d a y / n i g h t ) . Seeds were extracted from mature fruits and left to ferment at r o o m temperature to allow removal of the mucilaginous locular tissue remnants. The extracted seeds were incubated for 3 min in 3% NaOC1, and then for 10 m i n in a Na3PO4 solution to remove pathogens from the seed surface. Seeds were rinsed with tap water, dried and stored in paper bags at
Shai Leviatov et al. / Scientia Horticulturae 56 (1994.) 197-206
199
room temperature. Comparisons between PI341988 and UC82B were always made with seeds from lots having the same harvest date. In order to verify the results obtained with the above two lines, we made crosses for reciprocal F 1. Subsequent to the F 1 generation, we collected F2 seeds and made reciprocal backcrosses of each F1 to either PI341988 or UC82B. Altogether, we obtained eight progeny lines. Five replicates of 30 seeds were sown in 9 cm petri dishes on two layers of W h a t m a n No. 2 filter paper moistened with 5 ml of distilled water. Germination took place in the dark at either 12 + 0.5 °C (adjusted incubator) or 25 + 1 °C (chamber). Germination was defined as the stage at which the seed's radicle had reached 1 m m in length. Germinated seeds were counted daily. Evaluation of germination ability was based on the percentage of germinated seeds or the mean time (in days) to germination ( M T G ) where
Seed germination. -
MTG -
No. of days to germination X no. of seeds germinated Total no. of seeds germinated
T e s t a r e m o v a l - Seeds were stirred in a 10% NaOC1 solution to remove the seed testa. Complete removal of the testa without damage to the endosperm layer was confirmed by light microscope to establish a precise treatment time for each plant line (UC82B, 30 min; PI341988, 15 m i n ) . Control seeds were stirred for the same time in distilled water. o f e n d o s p e r m . - Tissues in front of the radicle tip were carefully removed under aseptic conditions. The scarified seeds were planted in an embryo culture m e d i u m containing 50% Murashige and Skoog ( 1962 ) mixture, 2% sucrose, and 0.8% agar, adjusted to pH 5.7. Plates were kept at 12 or 25 °C. Removal
Five replicates of seeds, 0.2 g in weight, were sown in 25 ml Erlenmeyer flasks on two layers of W h a t m a n No. 2 filter paper moistened with 3 ml of distilled water, and placed in chambers at either 12 or 25 °C. The flasks were sealed for 1 h with a rubber cap, after which 2 ml samples of the atmosphere in the flasks were taken for determination of CO2 concentration by a gas chromatograph (Fisher-Hamilton USA, model 29) equipped with a CO2 detector (Alltech 0.25 × 6.0" 60/80 mash). Respiration. -
Results
Seeds of t o m a t o line PI341988 were lighter than those of UC82B (Table 1 ). Embryo weight was slightly more than 30% of the total seed weight in both plant lines. The embryo weight of PI341988 seeds was found to be 56% of the endosperm layer, compared with 49% in UC82B. Although the embryo weight
200
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
Table 1 Dry weights (mg) of whole seed, embryo and endosperm of tomato lines PI341988 and UC82B Plant line
Seed
Endosperm
Embryo
Embryo/seed ratio
Embryo/ endosperm ratio
UC82B PI341988
3.71 +0.34 2.87+0.30
2.43+0.12 1.81 +0.42
1.19+0.09 1.02+0.03
0.32+0.02 0.35+0.01
0.49+0.05 0.56+0.03
6o
t~
50
30
.
,L
2O
4
6 TIMB
6
10
12
PItOM S O W I ~ G
14
16
18
20
(DAYS)
Fig 1. Germination rate of tomato seeds. Seeds of tomato lines PI341988 (square) and UC82B (circle) at 12°C (full) and 25 °C (empty).
of plant line PI341988 was significantly lower than that of UC82B, there was no significant difference in the embryo/seed weight ratios of both lines. The germination rate of tomato line P I341988 was significantly higher than that of UC82B at both temperatures. At the lower temperature, however, this difference was much more pronounced (Fig. 1 ). Most of the seeds of plant line PI341988 germinated within 10 days under low temperature conditions, with an MTG of 8.7 days. Germination in plant line UC82B started only after 12 days at 12°C, with an MTG of 19.7 days. Removal of the seed testa in NaOC1 solution (yielding decoated seeds) shortened the time to germination by 3 days in both plant lines at 12 °C (Fig. 2). In this particular experiment, germination of plant line UC82B started after 20 days, and testa removal resulted in about 30% germination at that time. Testa removal did not reduce the significant differences in germination rate between the two plant lines. To verify these results we compared the germination rate of intact and decoated seeds from eight progeny families stemming from reciprocal crosses between the two parent lines (Table 2). Despite the maternal influence on the ability to germinate rapidly at low temperatures, removal of the testa did not eliminate the differences in MTG between the various lines. Removal of the seed coat and endosperm in front of the radicle tip also accelerated germination in both lines at low and control temperatures (Fig. 3 ). The effect of this treatment was most pronounced in UC82B, where MTG
201
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206 ~
100
~ E] i ~ II
5O 4O 3O ~°
10 0
~/ 2
4
6
8
10
12
14
1(5
18
20
T/ME FItOM SOWING (OAY~¢)
Fig 2. Effect o f NaOC1 t r e a t m e n t o n g e r m i n a t i o n rate o f t o m a t o seeds at 12 ° C. Seed testae o f t o m a t o p l a n t lines P I 3 4 1 9 8 8 ( s q u a r e ) a n d U C 8 2 B (circle) following t r e a t m e n t with NaOC1 ( e m p t y ) or with water as a control (full).
Table 2 Mean time for germination (days) for seeds of plant lines UC82B, PI341988 and several progeny families at 12 ° C. Seeds were pretreated with distilled water (control), or with 10% NaOC1 until seed testae were completely removed Plant line
H20
NaOC1
UC82B FI: UC82B ×PI341988 F2: UC82B × PI341988 BCI: UC82B× (UC82B×PI341988) BCI: UC82B × (PI341988 × UC82B) PI341988 F 1: PI341988 × UC82B F2: PI341988 × UC82B BCI: PI341988 × ( U C 8 2 B × P I 3 4 1 9 8 8 ) BCI: PI341988 X (PI341988 × U C 8 2 B )
22.5+ 1.4 21.3+ 1.4 15.7 + 0.7 21.7+ 1.0 22.6+1.0 8.4 + 0.2 11.9 + 0.2 12.8 + 0.4 14.2+ 1.2 11.3+0.3
15.3+ 1.6 14.1 +0.8 12.7 + 0.7 14.8+ 1.5 13.8+0.4 6.7 + 0.3 7.2 + 0.5 8.9 + 0.5 8.9+0.4 7.6+0.6
7o 0
F-,
60
5o
3O 20 10 O 0
2
4
6
8
10
12
YZOM 80~NO
14
16
18
20
(DAYS)
Fig 3. G e r m i n a t i o n rate o f scarified seeds, Micropylar e n d o s p e r m was r e m o v e d from seeds o f t o m a t o lines P I 3 4 1 9 8 8 ( s q u a r e ) a n d U C 8 2 B (circle). Scarified seeds were g e r m i n a t e d o n a n e m b r y o culture m e d i u m at t 2 ° G (full) a n d 25 ° G ( e m p t y ) .
202
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206
was reduced from 19.3 to 4.8 days at 12°C and from 2.4 to 1.3 days at 25°C, resulting in similar germination rates of the two plant lines at 25 and 12 ° C. Radicle elongation rates were similar for both plant lines at 25°C (Fig. 4 ( A ) ), following their protrusion from either intact or scarified seeds. At 12 ° C, intact seeds of plant line UC82B did not germinate for the first 10 days; however, when the endosperm layer and seed coat were removed, radicle elongation rates in the two plant lines were similar (Fig. 4 ( B ) ) . After the radicle had emerged from the seed coat, its elongation rate in the intact seeds was similar to that in the scarified seeds at 12 °C as well. Moreover, an analysis of radicle elongation rate at 12 °C in progeny of reciprocal crosses be-
2 1 O
l
/_
4
6
TIMB F/t0M
8
SOWINO (DAYS)
9 8
B
~6
~5 ~3
~2 1 2
4
6
8
10
F i O M ,qOWJQ~G 9 8 ~
"~
7¸ 5
i°
4
~3
~2 1 2
4
6
8
10
12
I~LOM BOWr/NO t.d~Ts)
Fig 4. Embryo growth rate in tomato seeds. Radicle elongation in scarified (empty) and intact (full) seeds of tomato lines PI341988 (square) and UC82B (circle) at (A) 25°C and (B) 12°C. (C) Radicle elongation rate at 12°C of reciprocal FI crosses (full line), UC82B (circle), P1341988 × UC 82B ( square ), and two reciprocal backcrosses (dotted line ) UC82BX (UC82BXPI341988) (circle), and PI341988 X (PI341988×UC82B) (square).
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206
203
T6~
A 0.18
T6 %
0.1;~
~
O.OG
-
0.04
0.00 1
2
TIMB I~tOM 3 0 W I N G (DAY~0 O.22
~5% B
0.18
T
0.13
O.OG
0.6 %
~
0.04
0,00 1
2
3
4
5
6
7
8
g
TIM,~ P R O M ~W'I~TG (l~.=~rl)
Fig. 5. Respiration rate of tomato seeds. Carbon dioxide production in seeds of tomato lines PI341988 (square) and UC82B (circle) at 25°C (A) and 12°C (B). Numbers indicate the percentage of germination.
tween UC82B and PI341988 revealed no significant differences between the various lines (Fig. 4 ( C ) ) . In fact, no significant differences were found in radicle elongation rate between the eight progeny lines and the two parents (data not shown ). Seed respiration rate prior to germination was lower at 12 than at 25 °C in both lines (Fig. 5 ). An increase in respiration rate was associated with the beginning of germination. In this experiment, germination of both lines began on Day 2 at 25 °C and their respiration rates increased significantly on that day. At 12°C, the respiration rate increased sharply in plant line PI341988 after 6 days of imbibition, in parallel with germination. It is interesting to note the significant difference in respiration rates between the two plant lines, before germination started in PI341988 (Fig. 5 (B) ). Discussion The ability of the tomato plant line PI341988 to germinate more rapidly than commercial varieties has been previously reported (Scott and Jones, 1985; Dahal et al., 1990). Although this characteristic is observed at moderate temperatures, it is much more dramatic at sub-optimal temperatures (Fig. 1).
204
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
The tomato seed consists of a seed coat enclosing a curved embryo and an endosperm which practically fills the seed lumen not occupied by the embryo (Esau, 1953 ). In order to germinate, the elongating radicle has to puncture the living endosperm as well as the dead seed coat at the placental end of the seed. A negative relationship between tomato seed size and germination ability has been suggested by Whittington and Fierlinger (1972). PI341988 seeds are significantly smaller than those of plant line UC82B, and the endosperm envelope surrounding the embryo is also smaller (Table 1 ). These results suggest that the tissue around the embryo of plant line PI341988 is thinner, and may partially explain the differences in germination ability observed between the lines examined in our study. The mechanical restraint against radicle elongation imposed by the seed coat remains constant during imbibifion (Groot and Karssen, 1987 ), whereas the resistance of the endosperm cap decreases prior to germination (Watkins and Cantliffe, 1983; Groot and Karssen, 1987). Digestion of the seed coat enhanced germination rate in both plant lines. However, this enhancement was relatively small and the significant difference in germination ability between the lines was not affected (Fig. 2, Table 2). Thus, although our data indicate that under low temperature conditions the tomato seed coat imposes a mechanical restraint on radicle elongation, the differences in germination ability of our plant lines at low temperature are not attributable to different characteristics of the seed coats, or to different abilities of the plant lines to digest the seed coat. In pepper seed, removal of a 0.5-mm-thick layer of endosperm directly in front of the radicle tip significantly decreased the MTG at 15 °C, whereas removing the seed coat had no effect at this temperature (Watkins and Cantliffe, 1983 ). These authors suggested that the mechanical barrier formed by the endosperm was the main reason for inhibition of pepper seed germination at low temperatures. The germination rate of seeds in our study increased significantly with removal of the micropylar endosperm. This treatment completely eliminated the difference in germination ability between the two plant lines (Fig. 3 ). Our results indicate that the seed coat does impose a mechanical restraint, but that this restraint is relatively small. The main barrier to germination at low temperatures must therefore be imposed by the endosperm. Differences in embryo vigor have been suggested to account for the different germination abilities of tomato seeds at low temperatures (Liptay and Schopfer, 1983 ). In our study, similar radicle elongation rates were found for scarified seeds of the two plant lines, both at 25 and at 12°C (Fig. 4(B) ), as well as for the eight other lines which differed in their abilities to germinate at low temperatures (Fig. 4 ( C ) ) . These results indicate that the cold sensitivity of plant line UC82B cannot be attributed to low embryonic vigor. A number of studies provide evidence that during the germination process in tomato seeds, endosperm weakening and embryo development are affected differ-
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
205
ently by environmental factors. Groot et al. ( 1988 ) and Karssen et al. (1989) have reported that gibberellin stimulates, but is not essential for embryo growth in t o m a t o seeds, whereas germination will not occur without endogenous or exogenous gibberellin. Abscisic acid and osmotic stress can retard or prevent weakening o f the tissues covering the radicle tip, but are less effective in preventing embryo growth (Liptay and Schopfer, 1983; Groot and Karssen, 1987). The present finding that germination inhibition in seeds o f plant line UC82B can be eliminated by removal of the e n d o s p e r m / t e s t a cap opposite the radicle (Fig. 3 ), and the similar radicle elongation rates observed in all tested lines, are consistent with the hypothesis that weakening o f the endosperm requires a higher temperature than germination processes occurring in the embryo (Dahal et al., 1990). Accepting the hypothesis that gibberellin is involved in controlling e n d o s p e r m weakening, it m a y well be that low temperature inhibits gibberellin release by the embryo. Paralleling germination rates, respiration rates of seeds from both lines were lower at 12°C than at 25 °C. However, whereas the respiration level of UC82B seeds r e m a i n e d low during imbibition, PI341988 seeds produced increased a m o u n t s o f CO2 before the radicle had emerged through the seed coat. It therefore seems that in seeds o f plant line PI341988, metabolic systems become m o r e active earlier at low temperatures than those of UC82B seeds.
Acknowledgments This paper is a contribution from the Uri K.inamon Laboratory. The work was supported by a scholarship from the K i n a m o n F o u n d a t i o n to S.L.
References Cannon, O.S., Gatherum, D.M. and Miles, W.G., 1973. Heritability of low temperature seed germination in tomato. HortScience, 8:404-405. Dahal, P., Bradford, K. and Jones, R.A., 1990. Effect of priming and endosperm integrity on seed germination rate of tomato genotypes I: Germination at suboptimal temperature. J. Exp. Bot., 41:1431-1439. El-Sayed, M.N. and John, C.A., 1973. Heritability studies of tomato emergence at different temperatures. J. Am. Soc. Hortic. Sci., 98:440-443. Esau, K., 1953. Plant Anatomy. Wiley, New York, 735 pp. Groot, S.P.C. and Karssen, C.M., 1987. GibbereUins regulate seed germination in tomato by endosperm weakening:A study with gibberellin-deficientmutants. Planta, 171:525-531. Groot, S.P.C., Kieliszewska-Rokicka,B., Vermeer, E. and Karssen, C.M., 1988. Gibberellininduced hydrolysis of endosperm cell walls in gibbereUin-deficienttomato seeds prior to radicle protrusion. Planta, 174:500-504. Hegarty, T.W., 1978. Seed bed conditions and seedling establishment. Acta Hortic., 83:297307. Ikuma, H. and Thimann, K.V., 1963. The role of the seed coats in germination of photosensitive lettuce seeds. Plant Cell Physiol., 4:169-185.
206
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206
Karssen, C.M., Zagorski, S., Kepczynnski, J. and Groot, S.P.C., 1989. Key role for endogenous gibberellins in the control of seed germination. Ann. Bot., 63:71-80. Liptay, A. and Schopfer, P., 1983. Effect of water stress, seed coat restraint, and abscisic acid upon different germination capabilities of two tomato lines at low temperature. Plant Physiol., 73:935-938. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15:473-497. Pavlista, A.D. and Haber, A.H., 1970. Embryo expansion without protrusion in lettuce seeds. Plant Physiol., 45:636-637. Scott, S.J. and Jones, R.A., 1985. Quantifying seed germination response to low temperature variation among Lycopersicon spp. Exp. Environ. Bot., 25:129-137. Watkins, J.T. and Cantliffe, D.J., 1983. Mechanical resistance of the seed coat and endosperm during germination of C a p s i c u m a n n u m at low temperature. Plant Physiol., 72:146-150. Whittington, W.J. and Fierlinger, P., 1972. The genetic control of time to germination in tomato. Ann. Bot., 36:873-880. Wolf, S., Yakir, D., Stevens, M.A. and Rudich, J., 1986. Cold temperature tolerance of wild tomato species. J. Am. Soc. Hortic. Sci., 111:960-964. Wolk, W.D. and Herner, R.C., 1982. Chilling injury of germinating seeds and seedlings. HortScience, 17:169-173.
197
Elsevier Science Publishers B.V., Amsterdam
Roles of different seed components in controlling tomato seed germination at low temperature Shai L e v i a t o v a, O d e d Shoseyovb, Shmuel Wolff 'a
aDepartment of Field and VegetableCrops, Faculty of Agriculture, P.O. Box 12, Rehovot 76-100, Israel bDepartment of Horticulture, Faculty of Agriculture, Rehovot 76-100, Israel (Accepted 23 July 1993)
Abstract The role of different seed components in controlling the germination of tomato (Lycopersicon esculentum) seeds under low temperature conditions was studied in cold-resistant plant line PI341988 and cold-sensitive commercial cultivar UC82B, as well as in eight progeny lines of these two parents. At 12°C, PI341988 and UC82B seeds had a mean time of germination (MTG) of 8.7 days and 19.7 days, respectively. Removal of seed testa by NaOC1 treatment shortened the time required for germination by 3 days in both plant lines, but did not reduce the significant differences in germination rate between them. Removal of the seed coat and the endosperm layer in front of the embryo's radicle tip eliminated the differences in germination rate under low temperature conditions between the ten tested lines. All lines exhibited similar radicle elongation rates, both at 25°C and at 12°C. These results indicate that the main barrier to germination of tomato seed at low temperatures is imposed by the endosperm layer. At 12°C, plant line PI341988 exhibited a higher respiration rate than UC82B, indicating its higher metabolic activity under low temperature conditions. Key words: Embryo; Endosperm; Seed coat Abbreviation: MTG = mean time of germination
Introduction T h e ability t o g e r m i n a t e r a p i d l y is essential for seeds to o v e r c o m e stresses s u c h as w a t e r o r o x y g e n deficits, salinity, soil crusting, p a t h o g e n s a n d insects ( H e g a r t y , 1978; W o l k a n d H e r n e r , 1 9 8 2 ) . T h e g e r m i n a t i o n rate o f t o m a t o (Lycopersicon e s c u l e n t u m ) d e c r e a s e s p r o g r e s s i v e l y as t h e soil t e m p e r a t u r e is d e c r e a s e d f r o m 25 to 1 0 ° C , a n d is c o m p l e t e l y i n h i b i t e d b e l o w 1 0 ° C ( S c o t t a n d J o n e s , 1985; D a h a l et al., 1 9 9 0 ) . H o w e v e r , the g e r m i n a t i o n rate differs a m o n g L. esculenturn g e n o t y p e s , a n d r a p i d g e r m i n a t i o n a b o v e 1 0 ° C is heritable (E1-Sayed a n d J o h n , 1973; C a n n o n et al., 1973; W o l f et al., 1 9 8 6 ) . F o r *Corresponding author.
© 1994 Elsevier Science Publishers B.V. All rights reserved 0304-4238/94/$07.00
198
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
example, seeds of the cold-tolerant tomato plant introduction accessions PI341988, PI 120256 and PI 174263 germinate more rapidly than seeds of the cold-sensitive tomato line T5 at low temperatures (Dahal et al., 1990). PI341988 seeds also germinate more rapidly than those of the cold-sensitive line st-24 at 10°C, and are more responsive to priming (Liptay and Schopfer, 1983). The time of germination has been found to be correlated with seed weight in L. esculentum and Lycopersicon pimpinellifolium F 1 and F2 progenies. The paternal effect of L. pimpinellifolium in decreasing germination time was assumed to be related to reduced seed size (Whittington and Fierlinger, 1972 ). Seed coats may influence the seed's germination ability by interfering with water uptake, gas exchange or the diffusion of endogenous inhibitors, or by mechanically restricting embryo growth (Ikuma and Thimann, 1963 ). Pavlista and Haber (1970) proposed that both the mechanical force of the growing embryo pushing against the endosperm and the enzymatic weakening of the endosperm are necessary for lettuce seed germination. Weakening of the mechanical restraint imposed by the endosperm opposing the top of the radicle is also a prerequisite for germination in pepper (Watkins and Cantliffe, 1983) and tomato (Groot and Karssen, 1987) seeds. The base temperature for germination was reduced by 5 oC for tomato plant lines PI341988 and T5, when the endosperm/testa cap opposite the radicle tip was removed. The germination rate was also significantly increased by this treatment (Dahal et al., 1990). These results indicate that germination-related processes can occur at temperatures lower than the base temperature for germination. Low temperature may affect the rate of embryo growth, and its ability to overcome the mechanical restraint imposed by either the endosperm or the seed coat surrounding it. No concrete information is available with regard to the effect of low temperature on the activities of different tomato seed tissues, or the relationship between these activities and the ability to germinate under low temperature conditions. The aim of this research was to determine the role played by the testa and endosperm layer of the tomato seed in controlling germination ability at low temperature. Materials and methods
Seeds. - Tomato (Lycopersicon esculentum) plant lines PI341988 and UC82B were grown for seed production in a temperature-controlled greenhouse at 2 5 / 1 8 ° C ( d a y / n i g h t ) . Seeds were extracted from mature fruits and left to ferment at r o o m temperature to allow removal of the mucilaginous locular tissue remnants. The extracted seeds were incubated for 3 min in 3% NaOC1, and then for 10 m i n in a Na3PO4 solution to remove pathogens from the seed surface. Seeds were rinsed with tap water, dried and stored in paper bags at
Shai Leviatov et al. / Scientia Horticulturae 56 (1994.) 197-206
199
room temperature. Comparisons between PI341988 and UC82B were always made with seeds from lots having the same harvest date. In order to verify the results obtained with the above two lines, we made crosses for reciprocal F 1. Subsequent to the F 1 generation, we collected F2 seeds and made reciprocal backcrosses of each F1 to either PI341988 or UC82B. Altogether, we obtained eight progeny lines. Five replicates of 30 seeds were sown in 9 cm petri dishes on two layers of W h a t m a n No. 2 filter paper moistened with 5 ml of distilled water. Germination took place in the dark at either 12 + 0.5 °C (adjusted incubator) or 25 + 1 °C (chamber). Germination was defined as the stage at which the seed's radicle had reached 1 m m in length. Germinated seeds were counted daily. Evaluation of germination ability was based on the percentage of germinated seeds or the mean time (in days) to germination ( M T G ) where
Seed germination. -
MTG -
No. of days to germination X no. of seeds germinated Total no. of seeds germinated
T e s t a r e m o v a l - Seeds were stirred in a 10% NaOC1 solution to remove the seed testa. Complete removal of the testa without damage to the endosperm layer was confirmed by light microscope to establish a precise treatment time for each plant line (UC82B, 30 min; PI341988, 15 m i n ) . Control seeds were stirred for the same time in distilled water. o f e n d o s p e r m . - Tissues in front of the radicle tip were carefully removed under aseptic conditions. The scarified seeds were planted in an embryo culture m e d i u m containing 50% Murashige and Skoog ( 1962 ) mixture, 2% sucrose, and 0.8% agar, adjusted to pH 5.7. Plates were kept at 12 or 25 °C. Removal
Five replicates of seeds, 0.2 g in weight, were sown in 25 ml Erlenmeyer flasks on two layers of W h a t m a n No. 2 filter paper moistened with 3 ml of distilled water, and placed in chambers at either 12 or 25 °C. The flasks were sealed for 1 h with a rubber cap, after which 2 ml samples of the atmosphere in the flasks were taken for determination of CO2 concentration by a gas chromatograph (Fisher-Hamilton USA, model 29) equipped with a CO2 detector (Alltech 0.25 × 6.0" 60/80 mash). Respiration. -
Results
Seeds of t o m a t o line PI341988 were lighter than those of UC82B (Table 1 ). Embryo weight was slightly more than 30% of the total seed weight in both plant lines. The embryo weight of PI341988 seeds was found to be 56% of the endosperm layer, compared with 49% in UC82B. Although the embryo weight
200
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
Table 1 Dry weights (mg) of whole seed, embryo and endosperm of tomato lines PI341988 and UC82B Plant line
Seed
Endosperm
Embryo
Embryo/seed ratio
Embryo/ endosperm ratio
UC82B PI341988
3.71 +0.34 2.87+0.30
2.43+0.12 1.81 +0.42
1.19+0.09 1.02+0.03
0.32+0.02 0.35+0.01
0.49+0.05 0.56+0.03
6o
t~
50
30
.
,L
2O
4
6 TIMB
6
10
12
PItOM S O W I ~ G
14
16
18
20
(DAYS)
Fig 1. Germination rate of tomato seeds. Seeds of tomato lines PI341988 (square) and UC82B (circle) at 12°C (full) and 25 °C (empty).
of plant line PI341988 was significantly lower than that of UC82B, there was no significant difference in the embryo/seed weight ratios of both lines. The germination rate of tomato line P I341988 was significantly higher than that of UC82B at both temperatures. At the lower temperature, however, this difference was much more pronounced (Fig. 1 ). Most of the seeds of plant line PI341988 germinated within 10 days under low temperature conditions, with an MTG of 8.7 days. Germination in plant line UC82B started only after 12 days at 12°C, with an MTG of 19.7 days. Removal of the seed testa in NaOC1 solution (yielding decoated seeds) shortened the time to germination by 3 days in both plant lines at 12 °C (Fig. 2). In this particular experiment, germination of plant line UC82B started after 20 days, and testa removal resulted in about 30% germination at that time. Testa removal did not reduce the significant differences in germination rate between the two plant lines. To verify these results we compared the germination rate of intact and decoated seeds from eight progeny families stemming from reciprocal crosses between the two parent lines (Table 2). Despite the maternal influence on the ability to germinate rapidly at low temperatures, removal of the testa did not eliminate the differences in MTG between the various lines. Removal of the seed coat and endosperm in front of the radicle tip also accelerated germination in both lines at low and control temperatures (Fig. 3 ). The effect of this treatment was most pronounced in UC82B, where MTG
201
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206 ~
100
~ E] i ~ II
5O 4O 3O ~°
10 0
~/ 2
4
6
8
10
12
14
1(5
18
20
T/ME FItOM SOWING (OAY~¢)
Fig 2. Effect o f NaOC1 t r e a t m e n t o n g e r m i n a t i o n rate o f t o m a t o seeds at 12 ° C. Seed testae o f t o m a t o p l a n t lines P I 3 4 1 9 8 8 ( s q u a r e ) a n d U C 8 2 B (circle) following t r e a t m e n t with NaOC1 ( e m p t y ) or with water as a control (full).
Table 2 Mean time for germination (days) for seeds of plant lines UC82B, PI341988 and several progeny families at 12 ° C. Seeds were pretreated with distilled water (control), or with 10% NaOC1 until seed testae were completely removed Plant line
H20
NaOC1
UC82B FI: UC82B ×PI341988 F2: UC82B × PI341988 BCI: UC82B× (UC82B×PI341988) BCI: UC82B × (PI341988 × UC82B) PI341988 F 1: PI341988 × UC82B F2: PI341988 × UC82B BCI: PI341988 × ( U C 8 2 B × P I 3 4 1 9 8 8 ) BCI: PI341988 X (PI341988 × U C 8 2 B )
22.5+ 1.4 21.3+ 1.4 15.7 + 0.7 21.7+ 1.0 22.6+1.0 8.4 + 0.2 11.9 + 0.2 12.8 + 0.4 14.2+ 1.2 11.3+0.3
15.3+ 1.6 14.1 +0.8 12.7 + 0.7 14.8+ 1.5 13.8+0.4 6.7 + 0.3 7.2 + 0.5 8.9 + 0.5 8.9+0.4 7.6+0.6
7o 0
F-,
60
5o
3O 20 10 O 0
2
4
6
8
10
12
YZOM 80~NO
14
16
18
20
(DAYS)
Fig 3. G e r m i n a t i o n rate o f scarified seeds, Micropylar e n d o s p e r m was r e m o v e d from seeds o f t o m a t o lines P I 3 4 1 9 8 8 ( s q u a r e ) a n d U C 8 2 B (circle). Scarified seeds were g e r m i n a t e d o n a n e m b r y o culture m e d i u m at t 2 ° G (full) a n d 25 ° G ( e m p t y ) .
202
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206
was reduced from 19.3 to 4.8 days at 12°C and from 2.4 to 1.3 days at 25°C, resulting in similar germination rates of the two plant lines at 25 and 12 ° C. Radicle elongation rates were similar for both plant lines at 25°C (Fig. 4 ( A ) ), following their protrusion from either intact or scarified seeds. At 12 ° C, intact seeds of plant line UC82B did not germinate for the first 10 days; however, when the endosperm layer and seed coat were removed, radicle elongation rates in the two plant lines were similar (Fig. 4 ( B ) ) . After the radicle had emerged from the seed coat, its elongation rate in the intact seeds was similar to that in the scarified seeds at 12 °C as well. Moreover, an analysis of radicle elongation rate at 12 °C in progeny of reciprocal crosses be-
2 1 O
l
/_
4
6
TIMB F/t0M
8
SOWINO (DAYS)
9 8
B
~6
~5 ~3
~2 1 2
4
6
8
10
F i O M ,qOWJQ~G 9 8 ~
"~
7¸ 5
i°
4
~3
~2 1 2
4
6
8
10
12
I~LOM BOWr/NO t.d~Ts)
Fig 4. Embryo growth rate in tomato seeds. Radicle elongation in scarified (empty) and intact (full) seeds of tomato lines PI341988 (square) and UC82B (circle) at (A) 25°C and (B) 12°C. (C) Radicle elongation rate at 12°C of reciprocal FI crosses (full line), UC82B (circle), P1341988 × UC 82B ( square ), and two reciprocal backcrosses (dotted line ) UC82BX (UC82BXPI341988) (circle), and PI341988 X (PI341988×UC82B) (square).
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206
203
T6~
A 0.18
T6 %
0.1;~
~
O.OG
-
0.04
0.00 1
2
TIMB I~tOM 3 0 W I N G (DAY~0 O.22
~5% B
0.18
T
0.13
O.OG
0.6 %
~
0.04
0,00 1
2
3
4
5
6
7
8
g
TIM,~ P R O M ~W'I~TG (l~.=~rl)
Fig. 5. Respiration rate of tomato seeds. Carbon dioxide production in seeds of tomato lines PI341988 (square) and UC82B (circle) at 25°C (A) and 12°C (B). Numbers indicate the percentage of germination.
tween UC82B and PI341988 revealed no significant differences between the various lines (Fig. 4 ( C ) ) . In fact, no significant differences were found in radicle elongation rate between the eight progeny lines and the two parents (data not shown ). Seed respiration rate prior to germination was lower at 12 than at 25 °C in both lines (Fig. 5 ). An increase in respiration rate was associated with the beginning of germination. In this experiment, germination of both lines began on Day 2 at 25 °C and their respiration rates increased significantly on that day. At 12°C, the respiration rate increased sharply in plant line PI341988 after 6 days of imbibition, in parallel with germination. It is interesting to note the significant difference in respiration rates between the two plant lines, before germination started in PI341988 (Fig. 5 (B) ). Discussion The ability of the tomato plant line PI341988 to germinate more rapidly than commercial varieties has been previously reported (Scott and Jones, 1985; Dahal et al., 1990). Although this characteristic is observed at moderate temperatures, it is much more dramatic at sub-optimal temperatures (Fig. 1).
204
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
The tomato seed consists of a seed coat enclosing a curved embryo and an endosperm which practically fills the seed lumen not occupied by the embryo (Esau, 1953 ). In order to germinate, the elongating radicle has to puncture the living endosperm as well as the dead seed coat at the placental end of the seed. A negative relationship between tomato seed size and germination ability has been suggested by Whittington and Fierlinger (1972). PI341988 seeds are significantly smaller than those of plant line UC82B, and the endosperm envelope surrounding the embryo is also smaller (Table 1 ). These results suggest that the tissue around the embryo of plant line PI341988 is thinner, and may partially explain the differences in germination ability observed between the lines examined in our study. The mechanical restraint against radicle elongation imposed by the seed coat remains constant during imbibifion (Groot and Karssen, 1987 ), whereas the resistance of the endosperm cap decreases prior to germination (Watkins and Cantliffe, 1983; Groot and Karssen, 1987). Digestion of the seed coat enhanced germination rate in both plant lines. However, this enhancement was relatively small and the significant difference in germination ability between the lines was not affected (Fig. 2, Table 2). Thus, although our data indicate that under low temperature conditions the tomato seed coat imposes a mechanical restraint on radicle elongation, the differences in germination ability of our plant lines at low temperature are not attributable to different characteristics of the seed coats, or to different abilities of the plant lines to digest the seed coat. In pepper seed, removal of a 0.5-mm-thick layer of endosperm directly in front of the radicle tip significantly decreased the MTG at 15 °C, whereas removing the seed coat had no effect at this temperature (Watkins and Cantliffe, 1983 ). These authors suggested that the mechanical barrier formed by the endosperm was the main reason for inhibition of pepper seed germination at low temperatures. The germination rate of seeds in our study increased significantly with removal of the micropylar endosperm. This treatment completely eliminated the difference in germination ability between the two plant lines (Fig. 3 ). Our results indicate that the seed coat does impose a mechanical restraint, but that this restraint is relatively small. The main barrier to germination at low temperatures must therefore be imposed by the endosperm. Differences in embryo vigor have been suggested to account for the different germination abilities of tomato seeds at low temperatures (Liptay and Schopfer, 1983 ). In our study, similar radicle elongation rates were found for scarified seeds of the two plant lines, both at 25 and at 12°C (Fig. 4(B) ), as well as for the eight other lines which differed in their abilities to germinate at low temperatures (Fig. 4 ( C ) ) . These results indicate that the cold sensitivity of plant line UC82B cannot be attributed to low embryonic vigor. A number of studies provide evidence that during the germination process in tomato seeds, endosperm weakening and embryo development are affected differ-
Shai Leviatov et al. / Scientia Horticulturae 56 (1994) 197-206
205
ently by environmental factors. Groot et al. ( 1988 ) and Karssen et al. (1989) have reported that gibberellin stimulates, but is not essential for embryo growth in t o m a t o seeds, whereas germination will not occur without endogenous or exogenous gibberellin. Abscisic acid and osmotic stress can retard or prevent weakening o f the tissues covering the radicle tip, but are less effective in preventing embryo growth (Liptay and Schopfer, 1983; Groot and Karssen, 1987). The present finding that germination inhibition in seeds o f plant line UC82B can be eliminated by removal of the e n d o s p e r m / t e s t a cap opposite the radicle (Fig. 3 ), and the similar radicle elongation rates observed in all tested lines, are consistent with the hypothesis that weakening o f the endosperm requires a higher temperature than germination processes occurring in the embryo (Dahal et al., 1990). Accepting the hypothesis that gibberellin is involved in controlling e n d o s p e r m weakening, it m a y well be that low temperature inhibits gibberellin release by the embryo. Paralleling germination rates, respiration rates of seeds from both lines were lower at 12°C than at 25 °C. However, whereas the respiration level of UC82B seeds r e m a i n e d low during imbibition, PI341988 seeds produced increased a m o u n t s o f CO2 before the radicle had emerged through the seed coat. It therefore seems that in seeds o f plant line PI341988, metabolic systems become m o r e active earlier at low temperatures than those of UC82B seeds.
Acknowledgments This paper is a contribution from the Uri K.inamon Laboratory. The work was supported by a scholarship from the K i n a m o n F o u n d a t i o n to S.L.
References Cannon, O.S., Gatherum, D.M. and Miles, W.G., 1973. Heritability of low temperature seed germination in tomato. HortScience, 8:404-405. Dahal, P., Bradford, K. and Jones, R.A., 1990. Effect of priming and endosperm integrity on seed germination rate of tomato genotypes I: Germination at suboptimal temperature. J. Exp. Bot., 41:1431-1439. El-Sayed, M.N. and John, C.A., 1973. Heritability studies of tomato emergence at different temperatures. J. Am. Soc. Hortic. Sci., 98:440-443. Esau, K., 1953. Plant Anatomy. Wiley, New York, 735 pp. Groot, S.P.C. and Karssen, C.M., 1987. GibbereUins regulate seed germination in tomato by endosperm weakening:A study with gibberellin-deficientmutants. Planta, 171:525-531. Groot, S.P.C., Kieliszewska-Rokicka,B., Vermeer, E. and Karssen, C.M., 1988. Gibberellininduced hydrolysis of endosperm cell walls in gibbereUin-deficienttomato seeds prior to radicle protrusion. Planta, 174:500-504. Hegarty, T.W., 1978. Seed bed conditions and seedling establishment. Acta Hortic., 83:297307. Ikuma, H. and Thimann, K.V., 1963. The role of the seed coats in germination of photosensitive lettuce seeds. Plant Cell Physiol., 4:169-185.
206
Shai Leviatov et aL / Scientia Horticulturae 56 (1994) 197-206
Karssen, C.M., Zagorski, S., Kepczynnski, J. and Groot, S.P.C., 1989. Key role for endogenous gibberellins in the control of seed germination. Ann. Bot., 63:71-80. Liptay, A. and Schopfer, P., 1983. Effect of water stress, seed coat restraint, and abscisic acid upon different germination capabilities of two tomato lines at low temperature. Plant Physiol., 73:935-938. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15:473-497. Pavlista, A.D. and Haber, A.H., 1970. Embryo expansion without protrusion in lettuce seeds. Plant Physiol., 45:636-637. Scott, S.J. and Jones, R.A., 1985. Quantifying seed germination response to low temperature variation among Lycopersicon spp. Exp. Environ. Bot., 25:129-137. Watkins, J.T. and Cantliffe, D.J., 1983. Mechanical resistance of the seed coat and endosperm during germination of C a p s i c u m a n n u m at low temperature. Plant Physiol., 72:146-150. Whittington, W.J. and Fierlinger, P., 1972. The genetic control of time to germination in tomato. Ann. Bot., 36:873-880. Wolf, S., Yakir, D., Stevens, M.A. and Rudich, J., 1986. Cold temperature tolerance of wild tomato species. J. Am. Soc. Hortic. Sci., 111:960-964. Wolk, W.D. and Herner, R.C., 1982. Chilling injury of germinating seeds and seedlings. HortScience, 17:169-173.