- Email: [email protected]
Effects of Chilling, Light and Nitrogen-containing Compounds on Germination, Rate of Germination and Seed Imbibition ofClematis vitalbaL.
Annals of Botany 79 : 643–650, 1997
Effects of Chilling, Light and Nitrogen-containing Compounds on Germination, Rate of Germination and Seed Imbibition of Clematis vitalba L. R. A. B U N G A RD*†, D. M C N E I L† and J. D. M O R T O N§ † Department of Plant Science and § Animal and Veterinary Sciences Group, Lincoln Uniersity, Canterbury, New Zealand Received : 25 September 1996
Accepted : 28 December 1996
Effects of chilling (5 °C) period, light and applied nitrogen (N) on germination (%), rate of germination (d to 50 % of total germination ; T %) and seed imbibition were examined in Clematis italba L. In the absence of chilling, light &! and N, germination was minimal (3 %). When applied alone, both chilling and N increased germination. Chilling for 12 weeks increased germination to 64 %, and 2±5 m NO− or NH+ increased germination to 10–12 %. Light did not $ % increase germination when applied alone, but did when applied in combination with chilling and}or N. Half the seed − germinated when light was combined with 2±5 m NO or NH+. The influence of chilling, light and}or N on $ % germination was greater when combined, than when either factor was applied alone. Both oxidized (NO−) and $ reduced (NH+) forms of N increased germination, but non-N-containing compounds did not, suggesting the response % was due to N and not ionic or osmotic effects. Without additional N, T % decreased from 16–20 d at zero chilling, to around 5 d at 8 and 12 weeks chilling. &! Although T % was not influenced by an increase in NO− or NH+ from 0±5 to 5±0 m, it did increase with additional &! $ % applied N thereafter. However, the magnitude of the N effect was small compared to that of chilling. Like germination, seed imbibition increased with a longer chilling period, but in contrast imbibition decreased slightly with increased applied NO− or NH+. It is argued that increased imbibition is not directly related to an increase in total $ % germination, but that it may be related to the rate of germination. Possible mechanisms involved in the reduction in dormancy of C. italba seed are discussed. # 1997 Annals of Botany Company Key words : Clematis italba L., germination, dormancy, imbibition, rate of germination, chilling, light, nitrate, ammonium, nitrogen, phytochrome.
INTRODUCTION In New Zealand, Clematis italba L. is an introduced species that has become a weed in native forest remnants. Attempts to control the spread of C. italba are hampered by its prolific production of seed which displays embryo dormancy. Germination of embryo-dormant seed is often stimulated by chilling, light and}or nitrate (NO−). The $ influence of these three factors may have important ecological implications ; a chilling requirement can effectively regulate the timing of seed germination (Bewley and Black, 1985 ; Bouwmeester and Karssen, 1992), while germination in response to NO− and}or light may be important as a gap $ or disturbance detection mechanism (Grime et al., 1981 ; Pons, 1989, 1992). However, the mechanism by which chilling, light and NO− overcome embryo dormancy is not $ well understood. The level of chilling, light or NO− required to reduce $ embryo dormancy varies between species, populations and individual seeds within a population (Probert, 1992). Moreover, in many species, a positive interaction or additive effect on germination of these factors has been demonstrated (Vincent and Roberts, 1977). Many seed that respond positively to light also respond positively to NO−. Studies $ * For correspondence at : Department of Plant and Animal Sciences, University of Sheffield, Sheffield S10 2TN, UK ; email, r.a.bungard!sheffield.ac.uk.
0305-7364}97}06064308 $25.00}0
that show a positive interaction between chilling, NO− $ and}or light are also common. The influence of light, and more specifically the active form of phytochrome (Pfr), on germination is commonly correlated to the presence of NO− and this has led to the $ suggestion that there is a common mechanism for the action of Pfr and NO− in dormancy reduction (Henson, 1970 ; $ Hilton, 1984 ; Hilhorst and Karssen, 1990). Furthermore, the regularity with which a positive interaction between light and}or NO− extends to include chilling (Roberts and $ Benjamin, 1979 ; Van Der Woude and Toole, 1980) has led to the suggestion that all three dormancy-alleviating factors may act through a similar mechanism (Roberts and Benjamin, 1979 ; Bewley and Black, 1985). Hilhorst (1990 a, b) proposed a model that integrated the effects of all three factors. The model suggests that dormancy reduction and the onset of germination are regulated by a plasma membrane-bound protein, and that processes leading to germination can occur when this protein is bound to the active form of phytochrome (Pfr). The model proposes that the binding of the protein to Pfr is enhanced by temperature-induced changes in membrane fluidity that result in the exposure of the protein to NO−, and that this $ exposure increases the affinity of the protein for Pfr. In this study we investigate the influence of chilling, light and a range of nitrogen- (N-containing) and non-nitrogencontaining compounds on the germination characteristics of
bo960391
# 1997 Annals of Botany Company
644
Bungard et al.—Germination of Clematis vitalba
C. italba (a species showing characteristic embryo dormancy) in order to increase the understanding of how these three factors may act, and interact, to reduce seed dormancy.
MATERIALS AND METHODS Ripe seeds used in this study were collected from naturallyoccurring plants near Christchurch, New Zealand and stored dry, at approx. 20 °C until experimentation. There was no change in the dormancy or viability of seed over the period of experimentation. The seed used in this study was subsampled, and the subsample analysed for total-nitrogen (total-N), NO− and NH+ content. Total-N was determined $ % by digesting approx. 1±0 g of seed at 400 °C in 20 ml of H SO containing approx 5 g of K SO : Se (999 : 1 w}w) as # % # % a catalyst. The N content of the digest was then determined by steam distillation of NH using a Tecator 1035 N $ analyser (Ho$ gana$ s, Sweden). The NO− and NH+-N content $ % of seed was determined using methods modified from MacKereth, Heron and Talling (1978), and Baethgen and Alley (1989), respectively. In all experimental treatments, 50 seeds were weighed (around 0±10 g) then placed on one layer of Whatman 182 filter paper within closed, plastic petri dishes. All chilling treatments were at 5 °C and non-chilling}incubation treatments at 20 °C. Treatment solutions were made using deionized water and analytical grade chemicals. For the initial seed moistening 10 ml of appropriate solution was used. Any loss of moisture during the experiments was replaced with deionized water. Seeds were considered to have germinated at the first visible radicle emergence. All experiments were of a completely randomized factorial design, with four replicates of each treatment. All experiments were repeated with similar results. Analysis of results was carried out using analysis of variance. For total germination, analysis was on arcsin transformed data, but the results presented are actual percentage germination for total seed. All other results were analysed without transformation. All differences discussed have a probability (P) value of less than 5 %, and were obtained in repeat experiments. In expt 1, there were four chilling treatments (0, 4, 8, and 12 weeks) and 13 applied nitrogen (N) concentration treatments (0, 0±5, 1±0, 2±5, 5±0, 20±0 and 50±0 m N, applied either as KNO or NH Cl). Each seed lot was moistened $ % with the appropriate N solution and placed in the dark for the duration of each chilling treatment. For zero chilling, seeds were treated in a similar manner, but room temperature (20 °C) for 24 h was substituted for chilling to allow the seed time to imbibe. At the completion of all chilling treatments (including the zero chilling), seeds were removed from dishes, blotted dry on paper towels and re-weighed to calculate water uptake. Seeds were then returned to petri dishes and incubated in the dark. Germinated seeds were counted and removed every 3 d. All germination occurred within 45 d of the start of incubation, but germination counts were continued for a total of 99 d to ensure a comparative duration of imbibition for all chilling periods.
The time (d) taken to reach 50 % of total germination (T %) &! was calculated as an indication of the rate of germination. In expt 2 there were four chilling treatments (0, 4, 8 and 12 weeks), three nitrogen treatments (zero N, and 2±5 m N applied as KNO or NH Cl) and two light treatments $ % (complete darkness and exposure to daylight). Each seed lot was moistened with the appropriate N solution and placed in the dark at either chilling or non-chilling temperatures. To minimize the chances of inadvertent exposure to light, seeds intended for dark treatment were moistened under dimmed laboratory light and then rapidly (within 5 s) wrapped in aluminium foil. Seed in light treatments were not covered with aluminium foil and were exposed to light for around 2 h every 3 d when germination counts were being made. In addition, every other day seed in light treatments was deliberately exposed to daylight for around 1 min. After the appropriate chilling duration, all chilling treatments were transferred to non-chilling temperature, while non-chilling treatments remained at 20 °C. As progressive germination counts could not be carried out on dark treated seeds without the risk of exposure to light, total germination counts for dark treated seeds were carried out when germination was considered to be complete in seeds that had been exposed to light. That is, after there had been no additional germination for 21 consecutive days in any light treated seed. In expt 3 there were two temperature regimes [chilling (5 °C) for 2 weeks followed by non-chilling (20 °C), and continuous non-chilling (20 °C)], and seven applied inorganic salt treatments. The salt treatments were : deionized water ; KNO or NH Cl at 2±5 m ; KCl at 2±5 or 5±0 m ; $ % 2±5 m KNO 2±5 m KCl ; and 2±5 m NH Cl2±5 m $ % KCl. All seed lots were moistened with the appropriate solution and placed in the dark at the appropriate chilling or non-chilling temperature. Imbibition was measured (as for expt 1) on each treatment after 2 d and every following 2– 3 d until the onset of germination. Germinated seeds were counted and removed every 3 d until 21 d had passed with no further germination. In expt 4 there were 13 chemical treatments. These were : deionized water ; CO(NH ) ; CS(NH ) ; KCl ; K SO ; ## ## # % KClO ; KNO ; NaCl ; NaClO ; Na SO ; (NH ) SO ; $ $ $ # % %# % NH Cl ; and NH NO at 2±5 m. Each seed lot was mois% % $ tened with the appropriate chemical solution and incubated. Every 7 d following the initial moistening, the moist filter paper in each petri dish was tested for the presence of NO− $ and NO− using analytical test strips (E. Merck, Darmstadt, # Germany). Only those dishes that had been moistened with NO−-containing salts gave positive and appropriate test $ results. Germinated seeds were counted and removed every 3 d until 21 d had passed with no further germination.
RESULTS For the seed used in these experiments, the total-N content was in the range 2±8 to 3±0 % on a dry weight basis, NO−$ content was negligible (not detectable at levels at least as low as 0±1 µmol g−" f. wt) and NH+-content was less than % 0±3 µmol g−" f. wt.
Bungard et al.—Germination of Clematis vitalba
645
T 1. Summarized analysis of ariance table for Fig. 1 (A) and Table 2 (B)
N level (A) N form (B) Chilling period (C) A¬B A¬C B¬C A¬B¬C Error Total
6 1 3 6 18 3 18 168 223
Source
d.f.
Light treatment (A) Chilling period (B) Applied N (C) A¬B A¬C B¬C A¬B¬C Error Total
1 3 2 3 2 6 6 72 95
!0±001 !0±001 !0±001 !0±001 !0±001 0±091 0±006
Total germination (P value) !0±001 !0±001 !0±001 !0±001 !0±001 !0±001 !0±001
% of total SS
T % &! (P value)
% of total SS
Imbibition (P value)
% of total SS
13±1 2±5 56±0 2±8 12±8 0±4 2±4 10±4
!0±001 !0±001 !0±001 !0±001 !0±001 0±006 0±335
3±8 0±4 86±9 1±1 4±3 0±2 0±4 3±0
!0±001 !0±001 !0±001 0±134 0±480 0±101 0±884
10±9 2±4 56±3 1±4 2±5 0±9 1±6 24±0
% of total SS 14±9 62±9 11±6 3±3 0±6 1±8 3±0 1±7
Experiment 1 The summarized analysis of variance table is presented in Table 1. In the absence of applied N or chilling, only 6 % of seed germinated. There was a significant interaction between the influence of applied NO− or NH+ and chilling on $ % germination (Fig. 1). Without N, germination increased with increased chilling period to around 75 % at 8 weeks chilling, and then changed little with additional chilling up to 12 weeks. In the absence of chilling, germination increased with increased applied NO− to around 50 % at 2±5 m and $ then changed little with additional applied N up to 50±0 m. At 4 weeks chilling, germination increased with increased applied NO− from around 32 % at zero N to around 75 % $ at 2±5 m, and then changed little with additional N thereafter. Nitrate did not affect germination at 8 or 12 weeks chilling where values were in the range 70 to 90 % regardless of applied N level (Fig. 1). In the concentration range 0±5–2±5 m applied N, NH+ was similar to NO− in its % $ effect on germination at all chilling periods. However, at + concentrations from 5±0 to 50±0 m, NH was less effective % at stimulating germination at 0 and 4 weeks chilling compared to NO−, and slightly depressed total germination $ at 8 and 12 weeks chilling (Fig. 1). The rate of germination and seed imbibition were affected by N supply and chilling period regardless of N form (Fig. 1). There was no interaction effect of chilling and N supply on either the rate of germination or the extent of seed imbibition ; both increased with increased chilling period at all applied N levels. At zero N, increased chilling duration decreased the time to 50 % total germination (T %) from &! around 16 d to around 5 d with increased chilling from zero
A
Nitrate Total germination (%)
d.f.
B
Ammonium
100 80 60 40 20 0
0
5
10 15 20 50
0
5
10 15 20 50
0
5
10 15 20 50
0
5
10 15 20 50
5 10 15 20 50 Applied NO–3 (mM)
0
20 T50% (d)
(B)
Source
15 10 5 0
C
Imbibition –1 (g H2O g seed f. wt.)
(A)
Total germination (P value)
1.4 1.2 1.0 0
5 10 15 20 50 Applied NO–4 (mM)
F. 1. Effect of different concentrations of nitrate and ammonium, and different chilling durations (E, 0 ; +, 4 ; _, 8 ; and U, 12 weeks) on (A) total germination, (B) rate of germination (T %) and (C) &! imbibition of C. italba seeds. Error bars are one s.e.m. (n ¯ 4).
646
Bungard et al.—Germination of Clematis vitalba
T 2. The effect of chilling duration, applied nitrate and ammonium, and light on the total germination (%) of C. vitalba 0 m N Light treatment
2±5 m NO−
$
Chilling period (weeks)
Dark
%
Germination (%)
0 4 8 12 0 4 8 12
Light
2±5 m NH+
3³2 16³1 42³1 64³1 4³2 35³2 70³1 82³1
10³1 26³2 64³2 80³1 47³1 75³2 82³2 85³2
12³1 25³1 66³2 85³2 45³2 77³2 87³2 83³2
Values are means³s.e. of four replicates.
T 3. Effect of temperature regime and a range of salt forms and concentrations on imbibition after 9 d, imbibition just prior to germination and total germination of C. vitalba seed Applied salts HO
#
Total ion concentration (m) N ion concentration (m) Continuous 20 °C Imbibition after 9 d (g H O g−" seed f. wt) # Imbibition just prior to germination (g H O g−" seed f. wt) # Total germination (%)
0 0
1±17a ³0±01 1±21a,b ³0±01 5a ³1 2 weeks at 5 °C followed by continuous 20 °C Imbibition after 9 d 0±99a (g H O g−" seed f. wt) ³0±02 # Imbibition just prior to germination 1±28a,b (g H O g−" seed f. wt) ³0±01 # Total germination (%) 24a ³1
KNO
NH Cl
KCl
5±0 2±5
5±0 2±5
5±0 0
1±16a ³0±01 1±23a ³0±02 50b ³2
1±07b ³0±01 1±15b,c ³0±02 53b ³2
1±06b ³0±02 1±14c,d ³0±01 7a ³2
0±97a ³0±01 1±30a ³0±01 69b ³1
0±95a ³0±01 1±24b ³0±01 71b ³2
0±94a,b ³0±02 1±25a,b ³0±01 22a ³2
$
%
KNO NH Cl $ % KCl KCl 10±0 2±5
KCl
10±0 2±5
10±0 0
0±96c ³0±01 1±10c,d ³0±01 48b ³3
0±92c ³0±02 1±08d ³0±02 49b ³3
0±94c ³0±01 1±08d ³0±01 4a ³1
0±88b ³0±01 1±17c ³0±02 68b ³2
0±86b,c ³0±01 1±15c ³0±01 66b ³1
0±87b,c ³0±02 1±13c ³0±01 20a ³1
Values with a common letter within rows are not significantly different based on 95 % confidence intervals calculated using the pooled s.d. from the analysis of variance. Values are means³s.e. of four replicates.
to 8 weeks, and then decreased T % to around 4 d with &! additional chilling up to 12 weeks. At zero N, increased chilling over the range 0 to 12 weeks increased seed imbibition from an average of 1±12 g g−" to 1±36 g g−". In contrast to the influence of increased chilling duration, the rate of germination and extent of seed imbibition decreased with increased NO− and NH+ supply (Fig. 1). In general, at $ % all chilling levels, T % remained unchanged with increased &! applied NO− in the range 0–20±0 m, and then increased $ slightly with additional applied NO− to 50±0 m. In the N $ concentration range 0±5–2±5 m, NH+ was similar to NO− in % $ its effect on T %, but at 20±0 and 50±0 m, T % was greater &! &! with NH+ than NO−. In general, at all chilling levels, seed % $ imbibition was not influenced by applied NO− in the range $ up to 5±0 m, but decreased by 0±10 to 0±14 g g−" with the addition of 20±0 or 50±0 m NO−. In comparison, at all $ chilling levels, seed imbibition decreased with increased
applied NH+ over the entire concentration range used. % Compared to zero N, imbibition at 50±0 m applied NH+ % was reduced by between 0±12 and 0±19 g g−". Experiment 2 The summarized analysis of variance table is presented in Table 1. Moist chilling (4, 8 and 12 weeks), additional N (2±5 m NO− or NH+) and light all influenced germination $ % (Table 2). In the presence of light, both chilling and N increased germination to a similar extent as that shown in expt 1. Chilling and applied N also increased germination in the dark but to a lesser extent than in the presence of light. In the dark without chilling, addition of NO− or NH+ $ % increased germination from 3 to around 10 % while, with 4 weeks chilling, addition of NO− or NH+ increased ger$ % mination from 16 to around 25 %. Light enhanced the effect
Bungard et al.—Germination of Clematis vitalba T 4. Effect of a range of nitrogen and non-nitrogen containing chemicals on total germination of C. vitalba
Chemical
Total germination %
Chemical
Total germination % 6c³1
HO
#
N-containing KNO $ NH Cl % NH NO % $ (NH ) SO %# % CO(NH ) ## CS(NH )
##
56a³3 48a³3 38b³2 40a,b³1 38b³1 39b³2
Non N-containing KCl NaCl K SO # % KClO $ NaClO $ Na SO
#
%
4c³2 4c³1 8c³2 6c³2 4c³2 6c³2
Values with a common letter are not significantly different based on 95 % confidence intervals calculated using the pooled s.d. from the analysis of variance. Values are means³s.e. of four replicates.
of NO−, NH+ and chilling, but did not increase germination $ % when applied alone. Experiment 3 After 9 d, imbibition at each N treatment was greater in seed at non-chilling temperature compared to seed at chilling temperature (Table 3). However, prior to the onset of germination the reverse was true and imbibition was less in seed that had been maintained at non-chilling temperatures compared to seed that had been transferred from chilling to non-chilling temperatures. As in expts 1 and 2, germination, independent of applied N level, was greater in seed that had undergone chilling compared to non-chilled seed. Furthermore, regardless of the total ion concentration, germination was greater in treatment solutions that contained N (either as NO− or NH+) compared to solutions that $ % did not contain N (Table 3). In both chilling and nonchilling treatments, imbibition was reduced in seed where treatment solutions contained a total ion concentration of 10 m compared to treatment solutions where the ion concentration was either 5±0 or 0 m, but this was not reflected in germination (Table 3). Experiment 4 The ability of a range of N- and non-N-containing compounds to stimulate germination was examined in expt 4. All N-containing compounds used [NO− and NH+ salts, $ % and CO(NH ) and CS(NH ) ] stimulated germination, ## ## whereas all non-N-containing salts with a similar range of non-N-containing ions, did not stimulate germination (Table 4). In general, N-containing compounds where the N-ion concentration was 2±5 m (KNO , NH Cl) generally $ % stimulated around 50 % germination, whereas those compounds where the N-ion concentration was 5±0 m (CO(NH ) , CS(NH ) , (NH ) SO , NH NO ) were slightly ## ## %# % % $ less effective, stimulating around 40 % germination. For all non-N-containing compounds, germination was similar to the control (deionized water) and was always less than 10 %.
647
DISCUSSION Seed dormancy is a common characteristic of many successful weed species such as Clematis italba L., and this dormancy can be reduced by a range of factors. In this paper, the influence of chilling, N and light on seed dormancy and germination of C. italba was investigated. The influence of these factors is discussed, firstly, with respect to the germination of C. italba specifically, then with respect to their possible mode of action in embryodormant seed in general.
Chilling, light and N, and the dormancy and germination of C. vitalba Clematis italba seed collected from naturally occurring plants had a high proportion of dormancy. Germination, in the absence of any dormancy reducing factors was minimal, and often zero, even after 3 months under conditions that were suitable for the germination of non-dormant seeds. Chilling, N (KNO , NH Cl) and light all influenced seed $ % germination (Table 2). When applied alone both chilling and N could, to some extent, overcome dormancy, whereas light could not. When chilling, N and light were applied in combinations of two or more factors, the reduction in dormancy was greater than when each factor was applied alone (Table 2). The ability of chilling to increase C. italba seed germination has been reported previously (Rudolf, 1974 ; Van Gardingen, 1986), and the influence of chilling in this study is generally consistent with those results. Previous reports on the influence of light on C. italba germination are comparatively less consistent (McClelland, 1979 ; Van Gardingen, 1986). The ability of light to stimulate germination only when combined with either chilling or N (Table 2), suggests that the inconsistency of previous reports may be related to insufficient control of other factors during experimentation (e.g. chilling and N). There are no previous reports specifically dealing with the influence of applied N on C. italba germination. However, the influence of NO− is $ consistent with a range of temperate woody species where germination is also influenced by chilling and light (Bewley and Black, 1985). With respect to seed germination in the natural environment, a reduction in seed dormancy as a consequence of exposure to chilling, light and N (and more specifically NO−) may have an important influence on when, and where, $ seed germination will occur. Reduced dormancy following chilling, for example, may regulate the seasonal timing of germination (Probert, 1992). The influence of chilling (Fig. 1), combined with the tendency for a flush of germination in spring (Van Gardingen, 1986), would suggest that this is also the case for C. italba. Furthermore, it has been suggested that germination in response to elevated light or NO− may be an effective gap or disturbance detection $ mechanism, and that seedlings that rapidly establish in gaps or following disturbance may gain an advantage over plants that establish later when competition for resources has increased (Pons, 1989, 1992). With this in mind, the ability
648
Bungard et al.—Germination of Clematis vitalba
of C. italba to germinate in response to light and NO− $ (Table 2) is consistent with the establishment of C. italba in recently disturbed forest gaps and margins in New Zealand native forest remnants (Van Gardingen, 1986) and may, to some extent, account for its success as an invasive weed species in that environment.
Chilling, light and N, and the regulation of dormancy in embryo-dormant seed In comparison to NO−, NH+ is often reported to be less $ % effective at stimulating germination (Roberts and Smith, 1977). It has been suggested that dormancy reduction by NO− may result from NO− acting as an electron acceptor $ $ and therefore, the difference in effectiveness between NO− $ + and NH is a consequence of their oxidation states % (Hendricks and Taylorson, 1975 ; Roberts and Smith, 1977). This suggestion has come under some criticism (Hilhorst and Karssen, 1989 ; Karssen and Hilhorst, 1992). Moreover, other studies suggest that the influence of N form may be species-dependent (Hendricks and Taylorson, 1974 ; Singh and Amritphale, 1992 ; Thanos and Rundel, 1995). In this study, the stimulation of C. italba germination occurs to a similar extent with either reduced or oxidized forms of N (NO− and NH+), and does not occur with a range of non$ % nitrogen containing compounds (Table 4). As such, these results support the suggestion that a reduction in dormancy is not dependent on the function of the applied N as an electron acceptor (Hilhorst and Karssen, 1989). In addition, the inability of a range of non-nitrogen containing compounds to stimulate germination (Table 4) suggests that the influence of N-containing compounds is not simply an ionic or osmotic effect, but is dependent on the presence of N. When N was applied in the presence of light, NH+ was as % effective as NO− at stimulating germination at low applied $ N levels (0±5–2±5 m), but less effective at high applied N levels (5±0–50 m) (Fig. 1). The reduction in germination in the presence of high levels of applied NH+ not only occurred % in the absence of chilling, but also occurred at chilling levels that, when applied alone, were capable of overcoming a greater proportion of seed dormancy (8–12 weeks chilling) (Table 4). This suggests that, rather than NH+ having a % decreased ability to overcome dormancy compared to NO−, $ + high NH concentrations had an inhibitory effect on % germination. In addition to an increase in total germination in expt 1, increased chilling duration and increased applied N also influenced seed imbibition and the rate of germination (Fig. 1). It has been reported that changes in water relations and, in particular, changes in the balance between osmotic potential and pressure potential are important in the regulation of seed dormancy (Bewley and Black, 1985). In the present study, although increased chilling duration was associated with both a reduction in seed dormancy and an increase in seed imbibition, a reduction in seed dormancy also occurred with applied N, even though this was not associated with an increase in seed imbibition (Table 3). This does not completely discount a relationship between the influence of chilling duration on seed imbibition and the
reduction in seed dormancy, but it does indicate that the increase in imbibition, as a result of increased chilling, is not an essential part of dormancy reduction in C. italba. In addition to an increase in imbibition, increased chilling duration also increased the rate of germination (Fig. 1). Chilling has been reported to increase the rate of germination in a range of species (Gosling, 1988). In the present study and often in other studies, germination is defined as radicle emergence, whereas the actual physiological point of germination occurs earlier, at the onset of elongation of cells in the radicle (Bewley and Black, 1985). As radicle emergence is primarily due to cell elongation rather than cell division (Bewley and Black, 1985), any factor that influences the rate of cell elongation could be expected to influence radicle emergence and consequently, the apparent rate of germination. Cell elongation is influenced by cell wall extensibility (Schopfer and Plachy, 1985). It has been suggested that cell wall extensibility can be increased by the presence of GA (Karssen, 1995), and that GA can accumulate under chilling conditions (Taylor and Wareing, 1979). Although the present study does not discount a true increase in the rate of germination with increased chilling (that is, a more rapid onset of cell elongation), it is possible that the increased rate of germination in this study may be a consequence of chilling leading to an increase in cell wall extensibility, an increase in the rate of cell expansion, and ultimately, an increase in the rate of radicle emergence. In contrast to the influence of N on total germination, increased applied N influenced imbibition and the rate of germination to a greater extent at high applied N levels (" 5±0 m) rather than low N levels (Fig. 1). A similar decrease in the extent of imbibition when seeds were imbibed in solutions with variable N concentration, but equal total ion concentration (Table 3) suggests that, unlike a reduction in dormancy, a decrease in seed imbibition with applied N is not dependent on the presence of N, but is instead an osmotic effect. As radicle emergence is primarily the result of cell expansion rather than cell division (Bewley and Black, 1985), the decrease in the rate of germination (measured as radicle emergence) in expt 1 (Fig. 1) is also likely to result from an increase in the osmotic potential at high levels of applied N. It is generally accepted that light influences germination through the action of phytochrome (Pons, 1992). The inability of light alone to stimulate germination in C. italba, but the enhanced effect of chilling and N in the presence of light (Table 2), suggests two things : firstly, that either the presence of the active form of phytochrome (Pfr) alone is insufficient to overcome C. italba seed dormancy, or that Pfr is not effective in the absence of chilling and}or N ; and secondly, that there is a common link (involving Pfr) between the action of chilling and applied N. The latter is consistent with a range of reports that suggest a close association between the action of Pfr and NO− (Henson, $ 1970 ; Hilton, 1984 ; Hilhorst and Karssen, 1990) that extends to include the influence of chilling (Roberts and Benjamin, 1979 ; Bewley and Black, 1985). Hilhorst (1990 a, b), recently proposed a model that integrated the influence of chilling, Pfr and NO− in dormancy $ reduction. He proposed the existence of an additional factor
Bungard et al.—Germination of Clematis vitalba in the form of a specific protein bound to the plasma membrane. He argued that processes that lead to germination can occur when Pfr binds to this protein, and suggested that the binding of Pfr and the protein is enhanced by chilling (which exposes additional Pfr-binding sites on the membrane), and by NO− (which acts as a co-factor increasing $ the affinity of Pfr for the protein). For the germination data in our study to fit the model proposed by Hilhorst, two assumptions have to be made : firstly, that Pfr is present in C. italba seeds regardless of the light treatment or that the presence of Pfr is not an absolute requirement for germination ; and secondly, that other N forms (specifically NH+) could also act as a co-factor, equivalent to NO−. We % $ did not determine the initial levels of Pfr in C. italba seed, however, it has been suggested that phytochrome levels can be arrested at photoequilibrium when seeds mature on the plant (Kendrick and Spruit, 1977 ; Pons, 1992). If this is true for C. italba, then the ability of chilling and N to increase germination in the absence of light alone would not discount the model proposed by Hilhorst (1990 a, b). However, it could be expected that the action of a co-factor would be dependent on very specific molecular characteristics. Consequently, it would seem unlikely that a co-factor model would accommodate the similar action of NO− and NH+ on $ % germination that occurs in C. italba (Table 2), and other species (Thanos and Rundel, 1995). We suggest a modification to the model where, instead of NO− acting as a co$ factor, NO− acts through an intermediate process that is $ dependent on the supply of N, but not as dependent on the form of N. SUMMARY A high degree of seed dormancy in C. italba seed is confirmed. It is shown that chilling, N and light can potentially reduce C. italba seed dormancy but, in contrast to chilling and applied N, light is only effective when applied in combination with one or both other factors. The ability of both oxidized and reduced forms of N, but the inability of non-N-containing compounds, to increase germination indicates that the influence of these compounds is N-specific, and not an ionic or osmotic effect. It is shown that chilling and N influence both the rate of germination (T %) and &! imbibition of C. italba seed. However, the influence of chilling is much greater than that of N, and it is suggested that the influence of N-containing compounds on T % and &! imbibition is not N-specific, but is instead an osmotic effect. Hilhorst (1990 a, b) proposed a model that integrated the influence of chilling, light and NO− on seed dormancy. $ We suggest a modification to this model that accounts for the similar influence of NO− and NH+ on germination in $ % C. italba. LITERATURE CITED Baethgen WE, Alley MM. 1989. A manual colorimetric procedure for measuring ammonium nitrogen in soil and plant Kjeldahl digests. Communications in Soil and Plant Analysis 20 : 961–969. Bewley JD, Black M. 1985. Seeds : Physiology of deelopment and germination, 2nd edn. New York : Plenum Press. Bouwmeester HJ, Karssen CM. 1992. The dual role of temperature in the regulation of seasonal changes in dormancy and germination of seed of Polygonum persicaria L. Oecologia 90 : 88–94.
649
Gosling PG. 1988. The effect of moist chilling on the subsequent germination of temperate conifer seeds over a range of temperatures. Journal of Seed Technology 12 : 90–98. Grime JP, Mason G, Curtis AV, Rodman J, Band SR, Mowforth MAG, Neal AM, Shaw S. 1981. A comparative study of germination characteristics in a local flora. Journal of Ecology 69 : 1017–1059. Hendricks SB, Taylorson RB. 1974. Promotion of seed germination by nitrate, nitrite, hydroxylamine, and ammonium salts. Plant Physiology 54 : 304–309. Hendricks SB, Taylorson RB. 1975. Breaking of seed dormancy by catalase inhibition. Proceedings National Academy of Science USA. 72 : 306–309. Henson IE. 1970. The effects of light, potassium nitrate and temperature on the germination of Chenopodium album L. Weed Research 10 : 27–39. Hilhorst HWM. 1990 a. Dose-response analysis of factors involved in germination and secondary dormancy of seed of Sisymbrium officinale. I. Phytochrome. Plant Physiology 94 : 1090–1095. Hilhorst HWM. 1990 b. Dose-response analysis of factors involved in germination and secondary dormancy of seed of Sisymbrium officinale. II. Nitrate. Plant Physiology 94 : 1096–1102. Hilhorst HWM, Karssen CM. 1989. Nitrate reductase independent stimulation of germination in Sysymbrium officinale L. (hedge mustard) by light and nitrate. Annals of Botany 63 : 131–137. Hilhorst HWM, Karssen CM. 1990. The role of light and nitrate in seed germination. In : Taylorson RB, ed. Recent adances in the deelopment and germination of seeds NATO-ASI Series A, Volume 187. New York : Plenum Press, 191–206. Hilton JR. 1984. The influence of light and potassium nitrate on the dormancy and germination of Aena fatua L. (Wild Oat) seed and its ecological significance. New Phytologist 96 : 31–34. Karssen CM. 1995. Hormonal regulation of seed development, dormancy, and germination studied by genetic control. In : Kigel J, Galili G, eds. Seed deelopment and germination. New York : Marcel Dekker Inc., 333–350. Karssen CM, Hilhorst HWM. 1992. Effect of chemical environment on seed germination. In : Fenner M, ed. The ecology of regeneration in plant communities. Wallingford, UK : C.A.B. International, 327–348. Kendrick RE, Spruit CJP. 1977. Phototransformations of phytochrome. Photochemistry and Photobiology 26 : 201–204. McClelland TJ. 1979. Report on research into physiological aspects of growth in Clematis italba. Department of Lands and Surey, File No. 22}260}14, Wellington, New Zealand. MacKereth FJH, Heron J, Talling JF. 1978. Water analysis : some revised methods for limnologists. Scientific Publications of the Freshwater Biological Association 36. Pons TL. 1989. Breaking of seed dormancy by nitrate as a gap detection mechanism. Annals of Botany 63 : 139–143. Pons TL. 1992. Seed responses to light. In : Fenner M, ed. The ecology of regeneration in plant communities. Wallingford, UK : C.A.B. International, 259–284. Probert RJ. 1992. The role of temperature in germination ecophysiology. In : Fenner M, ed. The ecology of regeneration in plant communities. Wallingford, UK : C.A.B. International, 285–325. Roberts EH, Benjamin SK. 1979. The interaction of light, nitrate and alternating temperature on the germination of Chenopodium album, Capsella bursapastoris and Poa annua before and after chilling. Seed Science and Technology 7 : 379–392. Roberts EH, Smith RD. 1977. Dormancy and the pentose phosphate pathway. In : Khan AA, ed. The physiology and biochemistry of seed dormancy and germination. Amsterdam : North-Holland Publishing Company, 385–411. Rudolf PO. 1974. Clematis L. In : Schopmeyer CS, ed. Seeds of woody plants in the United States. United States Department of Agriculture. Agricultural Handbook 450 : 331–334. Schopfer P, Plachy C. 1985. Control of seed germination by abscisic acid. III. Effect of embryo growth potential (minimum turgor pressure) and growth coefficient (cell wall extensibility) in Brasica napus L. Plant Physiology 77 : 676–686. Singh B, Amritphale D. 1992. Effect of light and its interaction with nitrate and ammonium ions in seed germination of Caesulia axillaris. Physiologia Plantarum 85 : 43–48.
650
Bungard et al.—Germination of Clematis vitalba
Taylor JS, Wareing PF. 1979. The effect of stratification on the endogenous levels of gibberellins and cytokinins in seeds of douglas-fir (Pseudostuga menzieii (Mirb.) Franco) and sugar pine (Pinus lambertiana Dougl.). Plant, Cell and Enironment 2 : 165–171. Thanos CA, Rundel PW. 1995. Fire-followers in chaparral : nitrogenous compounds trigger seed germination. Journal of Ecology 83 : 207–216.
Van Der Woude WJ, Toole VK. 1980. Studies on the mechanisms of enhancement of phytochrome-dependent lettuce seed germination by prechilling. Plant Physiology 66 : 220–224. Van Gardingen JR. 1986. The physiological ecology of Clematis vitalba L. M.Sc. Thesis. University of Canterbury, New Zealand. Vincent EM, Roberts EH. 1977. The interaction of light, nitrate and alternating temperature in the germination of dormant seeds of common weed species. Seed Science and Technology 5 : 659–670.
Effects of Chilling, Light and Nitrogen-containing Compounds on Germination, Rate of Germination and Seed Imbibition of Clematis vitalba L. R. A. B U N G A RD*†, D. M C N E I L† and J. D. M O R T O N§ † Department of Plant Science and § Animal and Veterinary Sciences Group, Lincoln Uniersity, Canterbury, New Zealand Received : 25 September 1996
Accepted : 28 December 1996
Effects of chilling (5 °C) period, light and applied nitrogen (N) on germination (%), rate of germination (d to 50 % of total germination ; T %) and seed imbibition were examined in Clematis italba L. In the absence of chilling, light &! and N, germination was minimal (3 %). When applied alone, both chilling and N increased germination. Chilling for 12 weeks increased germination to 64 %, and 2±5 m NO− or NH+ increased germination to 10–12 %. Light did not $ % increase germination when applied alone, but did when applied in combination with chilling and}or N. Half the seed − germinated when light was combined with 2±5 m NO or NH+. The influence of chilling, light and}or N on $ % germination was greater when combined, than when either factor was applied alone. Both oxidized (NO−) and $ reduced (NH+) forms of N increased germination, but non-N-containing compounds did not, suggesting the response % was due to N and not ionic or osmotic effects. Without additional N, T % decreased from 16–20 d at zero chilling, to around 5 d at 8 and 12 weeks chilling. &! Although T % was not influenced by an increase in NO− or NH+ from 0±5 to 5±0 m, it did increase with additional &! $ % applied N thereafter. However, the magnitude of the N effect was small compared to that of chilling. Like germination, seed imbibition increased with a longer chilling period, but in contrast imbibition decreased slightly with increased applied NO− or NH+. It is argued that increased imbibition is not directly related to an increase in total $ % germination, but that it may be related to the rate of germination. Possible mechanisms involved in the reduction in dormancy of C. italba seed are discussed. # 1997 Annals of Botany Company Key words : Clematis italba L., germination, dormancy, imbibition, rate of germination, chilling, light, nitrate, ammonium, nitrogen, phytochrome.
INTRODUCTION In New Zealand, Clematis italba L. is an introduced species that has become a weed in native forest remnants. Attempts to control the spread of C. italba are hampered by its prolific production of seed which displays embryo dormancy. Germination of embryo-dormant seed is often stimulated by chilling, light and}or nitrate (NO−). The $ influence of these three factors may have important ecological implications ; a chilling requirement can effectively regulate the timing of seed germination (Bewley and Black, 1985 ; Bouwmeester and Karssen, 1992), while germination in response to NO− and}or light may be important as a gap $ or disturbance detection mechanism (Grime et al., 1981 ; Pons, 1989, 1992). However, the mechanism by which chilling, light and NO− overcome embryo dormancy is not $ well understood. The level of chilling, light or NO− required to reduce $ embryo dormancy varies between species, populations and individual seeds within a population (Probert, 1992). Moreover, in many species, a positive interaction or additive effect on germination of these factors has been demonstrated (Vincent and Roberts, 1977). Many seed that respond positively to light also respond positively to NO−. Studies $ * For correspondence at : Department of Plant and Animal Sciences, University of Sheffield, Sheffield S10 2TN, UK ; email, r.a.bungard!sheffield.ac.uk.
0305-7364}97}06064308 $25.00}0
that show a positive interaction between chilling, NO− $ and}or light are also common. The influence of light, and more specifically the active form of phytochrome (Pfr), on germination is commonly correlated to the presence of NO− and this has led to the $ suggestion that there is a common mechanism for the action of Pfr and NO− in dormancy reduction (Henson, 1970 ; $ Hilton, 1984 ; Hilhorst and Karssen, 1990). Furthermore, the regularity with which a positive interaction between light and}or NO− extends to include chilling (Roberts and $ Benjamin, 1979 ; Van Der Woude and Toole, 1980) has led to the suggestion that all three dormancy-alleviating factors may act through a similar mechanism (Roberts and Benjamin, 1979 ; Bewley and Black, 1985). Hilhorst (1990 a, b) proposed a model that integrated the effects of all three factors. The model suggests that dormancy reduction and the onset of germination are regulated by a plasma membrane-bound protein, and that processes leading to germination can occur when this protein is bound to the active form of phytochrome (Pfr). The model proposes that the binding of the protein to Pfr is enhanced by temperature-induced changes in membrane fluidity that result in the exposure of the protein to NO−, and that this $ exposure increases the affinity of the protein for Pfr. In this study we investigate the influence of chilling, light and a range of nitrogen- (N-containing) and non-nitrogencontaining compounds on the germination characteristics of
bo960391
# 1997 Annals of Botany Company
644
Bungard et al.—Germination of Clematis vitalba
C. italba (a species showing characteristic embryo dormancy) in order to increase the understanding of how these three factors may act, and interact, to reduce seed dormancy.
MATERIALS AND METHODS Ripe seeds used in this study were collected from naturallyoccurring plants near Christchurch, New Zealand and stored dry, at approx. 20 °C until experimentation. There was no change in the dormancy or viability of seed over the period of experimentation. The seed used in this study was subsampled, and the subsample analysed for total-nitrogen (total-N), NO− and NH+ content. Total-N was determined $ % by digesting approx. 1±0 g of seed at 400 °C in 20 ml of H SO containing approx 5 g of K SO : Se (999 : 1 w}w) as # % # % a catalyst. The N content of the digest was then determined by steam distillation of NH using a Tecator 1035 N $ analyser (Ho$ gana$ s, Sweden). The NO− and NH+-N content $ % of seed was determined using methods modified from MacKereth, Heron and Talling (1978), and Baethgen and Alley (1989), respectively. In all experimental treatments, 50 seeds were weighed (around 0±10 g) then placed on one layer of Whatman 182 filter paper within closed, plastic petri dishes. All chilling treatments were at 5 °C and non-chilling}incubation treatments at 20 °C. Treatment solutions were made using deionized water and analytical grade chemicals. For the initial seed moistening 10 ml of appropriate solution was used. Any loss of moisture during the experiments was replaced with deionized water. Seeds were considered to have germinated at the first visible radicle emergence. All experiments were of a completely randomized factorial design, with four replicates of each treatment. All experiments were repeated with similar results. Analysis of results was carried out using analysis of variance. For total germination, analysis was on arcsin transformed data, but the results presented are actual percentage germination for total seed. All other results were analysed without transformation. All differences discussed have a probability (P) value of less than 5 %, and were obtained in repeat experiments. In expt 1, there were four chilling treatments (0, 4, 8, and 12 weeks) and 13 applied nitrogen (N) concentration treatments (0, 0±5, 1±0, 2±5, 5±0, 20±0 and 50±0 m N, applied either as KNO or NH Cl). Each seed lot was moistened $ % with the appropriate N solution and placed in the dark for the duration of each chilling treatment. For zero chilling, seeds were treated in a similar manner, but room temperature (20 °C) for 24 h was substituted for chilling to allow the seed time to imbibe. At the completion of all chilling treatments (including the zero chilling), seeds were removed from dishes, blotted dry on paper towels and re-weighed to calculate water uptake. Seeds were then returned to petri dishes and incubated in the dark. Germinated seeds were counted and removed every 3 d. All germination occurred within 45 d of the start of incubation, but germination counts were continued for a total of 99 d to ensure a comparative duration of imbibition for all chilling periods.
The time (d) taken to reach 50 % of total germination (T %) &! was calculated as an indication of the rate of germination. In expt 2 there were four chilling treatments (0, 4, 8 and 12 weeks), three nitrogen treatments (zero N, and 2±5 m N applied as KNO or NH Cl) and two light treatments $ % (complete darkness and exposure to daylight). Each seed lot was moistened with the appropriate N solution and placed in the dark at either chilling or non-chilling temperatures. To minimize the chances of inadvertent exposure to light, seeds intended for dark treatment were moistened under dimmed laboratory light and then rapidly (within 5 s) wrapped in aluminium foil. Seed in light treatments were not covered with aluminium foil and were exposed to light for around 2 h every 3 d when germination counts were being made. In addition, every other day seed in light treatments was deliberately exposed to daylight for around 1 min. After the appropriate chilling duration, all chilling treatments were transferred to non-chilling temperature, while non-chilling treatments remained at 20 °C. As progressive germination counts could not be carried out on dark treated seeds without the risk of exposure to light, total germination counts for dark treated seeds were carried out when germination was considered to be complete in seeds that had been exposed to light. That is, after there had been no additional germination for 21 consecutive days in any light treated seed. In expt 3 there were two temperature regimes [chilling (5 °C) for 2 weeks followed by non-chilling (20 °C), and continuous non-chilling (20 °C)], and seven applied inorganic salt treatments. The salt treatments were : deionized water ; KNO or NH Cl at 2±5 m ; KCl at 2±5 or 5±0 m ; $ % 2±5 m KNO 2±5 m KCl ; and 2±5 m NH Cl2±5 m $ % KCl. All seed lots were moistened with the appropriate solution and placed in the dark at the appropriate chilling or non-chilling temperature. Imbibition was measured (as for expt 1) on each treatment after 2 d and every following 2– 3 d until the onset of germination. Germinated seeds were counted and removed every 3 d until 21 d had passed with no further germination. In expt 4 there were 13 chemical treatments. These were : deionized water ; CO(NH ) ; CS(NH ) ; KCl ; K SO ; ## ## # % KClO ; KNO ; NaCl ; NaClO ; Na SO ; (NH ) SO ; $ $ $ # % %# % NH Cl ; and NH NO at 2±5 m. Each seed lot was mois% % $ tened with the appropriate chemical solution and incubated. Every 7 d following the initial moistening, the moist filter paper in each petri dish was tested for the presence of NO− $ and NO− using analytical test strips (E. Merck, Darmstadt, # Germany). Only those dishes that had been moistened with NO−-containing salts gave positive and appropriate test $ results. Germinated seeds were counted and removed every 3 d until 21 d had passed with no further germination.
RESULTS For the seed used in these experiments, the total-N content was in the range 2±8 to 3±0 % on a dry weight basis, NO−$ content was negligible (not detectable at levels at least as low as 0±1 µmol g−" f. wt) and NH+-content was less than % 0±3 µmol g−" f. wt.
Bungard et al.—Germination of Clematis vitalba
645
T 1. Summarized analysis of ariance table for Fig. 1 (A) and Table 2 (B)
N level (A) N form (B) Chilling period (C) A¬B A¬C B¬C A¬B¬C Error Total
6 1 3 6 18 3 18 168 223
Source
d.f.
Light treatment (A) Chilling period (B) Applied N (C) A¬B A¬C B¬C A¬B¬C Error Total
1 3 2 3 2 6 6 72 95
!0±001 !0±001 !0±001 !0±001 !0±001 0±091 0±006
Total germination (P value) !0±001 !0±001 !0±001 !0±001 !0±001 !0±001 !0±001
% of total SS
T % &! (P value)
% of total SS
Imbibition (P value)
% of total SS
13±1 2±5 56±0 2±8 12±8 0±4 2±4 10±4
!0±001 !0±001 !0±001 !0±001 !0±001 0±006 0±335
3±8 0±4 86±9 1±1 4±3 0±2 0±4 3±0
!0±001 !0±001 !0±001 0±134 0±480 0±101 0±884
10±9 2±4 56±3 1±4 2±5 0±9 1±6 24±0
% of total SS 14±9 62±9 11±6 3±3 0±6 1±8 3±0 1±7
Experiment 1 The summarized analysis of variance table is presented in Table 1. In the absence of applied N or chilling, only 6 % of seed germinated. There was a significant interaction between the influence of applied NO− or NH+ and chilling on $ % germination (Fig. 1). Without N, germination increased with increased chilling period to around 75 % at 8 weeks chilling, and then changed little with additional chilling up to 12 weeks. In the absence of chilling, germination increased with increased applied NO− to around 50 % at 2±5 m and $ then changed little with additional applied N up to 50±0 m. At 4 weeks chilling, germination increased with increased applied NO− from around 32 % at zero N to around 75 % $ at 2±5 m, and then changed little with additional N thereafter. Nitrate did not affect germination at 8 or 12 weeks chilling where values were in the range 70 to 90 % regardless of applied N level (Fig. 1). In the concentration range 0±5–2±5 m applied N, NH+ was similar to NO− in its % $ effect on germination at all chilling periods. However, at + concentrations from 5±0 to 50±0 m, NH was less effective % at stimulating germination at 0 and 4 weeks chilling compared to NO−, and slightly depressed total germination $ at 8 and 12 weeks chilling (Fig. 1). The rate of germination and seed imbibition were affected by N supply and chilling period regardless of N form (Fig. 1). There was no interaction effect of chilling and N supply on either the rate of germination or the extent of seed imbibition ; both increased with increased chilling period at all applied N levels. At zero N, increased chilling duration decreased the time to 50 % total germination (T %) from &! around 16 d to around 5 d with increased chilling from zero
A
Nitrate Total germination (%)
d.f.
B
Ammonium
100 80 60 40 20 0
0
5
10 15 20 50
0
5
10 15 20 50
0
5
10 15 20 50
0
5
10 15 20 50
5 10 15 20 50 Applied NO–3 (mM)
0
20 T50% (d)
(B)
Source
15 10 5 0
C
Imbibition –1 (g H2O g seed f. wt.)
(A)
Total germination (P value)
1.4 1.2 1.0 0
5 10 15 20 50 Applied NO–4 (mM)
F. 1. Effect of different concentrations of nitrate and ammonium, and different chilling durations (E, 0 ; +, 4 ; _, 8 ; and U, 12 weeks) on (A) total germination, (B) rate of germination (T %) and (C) &! imbibition of C. italba seeds. Error bars are one s.e.m. (n ¯ 4).
646
Bungard et al.—Germination of Clematis vitalba
T 2. The effect of chilling duration, applied nitrate and ammonium, and light on the total germination (%) of C. vitalba 0 m N Light treatment
2±5 m NO−
$
Chilling period (weeks)
Dark
%
Germination (%)
0 4 8 12 0 4 8 12
Light
2±5 m NH+
3³2 16³1 42³1 64³1 4³2 35³2 70³1 82³1
10³1 26³2 64³2 80³1 47³1 75³2 82³2 85³2
12³1 25³1 66³2 85³2 45³2 77³2 87³2 83³2
Values are means³s.e. of four replicates.
T 3. Effect of temperature regime and a range of salt forms and concentrations on imbibition after 9 d, imbibition just prior to germination and total germination of C. vitalba seed Applied salts HO
#
Total ion concentration (m) N ion concentration (m) Continuous 20 °C Imbibition after 9 d (g H O g−" seed f. wt) # Imbibition just prior to germination (g H O g−" seed f. wt) # Total germination (%)
0 0
1±17a ³0±01 1±21a,b ³0±01 5a ³1 2 weeks at 5 °C followed by continuous 20 °C Imbibition after 9 d 0±99a (g H O g−" seed f. wt) ³0±02 # Imbibition just prior to germination 1±28a,b (g H O g−" seed f. wt) ³0±01 # Total germination (%) 24a ³1
KNO
NH Cl
KCl
5±0 2±5
5±0 2±5
5±0 0
1±16a ³0±01 1±23a ³0±02 50b ³2
1±07b ³0±01 1±15b,c ³0±02 53b ³2
1±06b ³0±02 1±14c,d ³0±01 7a ³2
0±97a ³0±01 1±30a ³0±01 69b ³1
0±95a ³0±01 1±24b ³0±01 71b ³2
0±94a,b ³0±02 1±25a,b ³0±01 22a ³2
$
%
KNO NH Cl $ % KCl KCl 10±0 2±5
KCl
10±0 2±5
10±0 0
0±96c ³0±01 1±10c,d ³0±01 48b ³3
0±92c ³0±02 1±08d ³0±02 49b ³3
0±94c ³0±01 1±08d ³0±01 4a ³1
0±88b ³0±01 1±17c ³0±02 68b ³2
0±86b,c ³0±01 1±15c ³0±01 66b ³1
0±87b,c ³0±02 1±13c ³0±01 20a ³1
Values with a common letter within rows are not significantly different based on 95 % confidence intervals calculated using the pooled s.d. from the analysis of variance. Values are means³s.e. of four replicates.
to 8 weeks, and then decreased T % to around 4 d with &! additional chilling up to 12 weeks. At zero N, increased chilling over the range 0 to 12 weeks increased seed imbibition from an average of 1±12 g g−" to 1±36 g g−". In contrast to the influence of increased chilling duration, the rate of germination and extent of seed imbibition decreased with increased NO− and NH+ supply (Fig. 1). In general, at $ % all chilling levels, T % remained unchanged with increased &! applied NO− in the range 0–20±0 m, and then increased $ slightly with additional applied NO− to 50±0 m. In the N $ concentration range 0±5–2±5 m, NH+ was similar to NO− in % $ its effect on T %, but at 20±0 and 50±0 m, T % was greater &! &! with NH+ than NO−. In general, at all chilling levels, seed % $ imbibition was not influenced by applied NO− in the range $ up to 5±0 m, but decreased by 0±10 to 0±14 g g−" with the addition of 20±0 or 50±0 m NO−. In comparison, at all $ chilling levels, seed imbibition decreased with increased
applied NH+ over the entire concentration range used. % Compared to zero N, imbibition at 50±0 m applied NH+ % was reduced by between 0±12 and 0±19 g g−". Experiment 2 The summarized analysis of variance table is presented in Table 1. Moist chilling (4, 8 and 12 weeks), additional N (2±5 m NO− or NH+) and light all influenced germination $ % (Table 2). In the presence of light, both chilling and N increased germination to a similar extent as that shown in expt 1. Chilling and applied N also increased germination in the dark but to a lesser extent than in the presence of light. In the dark without chilling, addition of NO− or NH+ $ % increased germination from 3 to around 10 % while, with 4 weeks chilling, addition of NO− or NH+ increased ger$ % mination from 16 to around 25 %. Light enhanced the effect
Bungard et al.—Germination of Clematis vitalba T 4. Effect of a range of nitrogen and non-nitrogen containing chemicals on total germination of C. vitalba
Chemical
Total germination %
Chemical
Total germination % 6c³1
HO
#
N-containing KNO $ NH Cl % NH NO % $ (NH ) SO %# % CO(NH ) ## CS(NH )
##
56a³3 48a³3 38b³2 40a,b³1 38b³1 39b³2
Non N-containing KCl NaCl K SO # % KClO $ NaClO $ Na SO
#
%
4c³2 4c³1 8c³2 6c³2 4c³2 6c³2
Values with a common letter are not significantly different based on 95 % confidence intervals calculated using the pooled s.d. from the analysis of variance. Values are means³s.e. of four replicates.
of NO−, NH+ and chilling, but did not increase germination $ % when applied alone. Experiment 3 After 9 d, imbibition at each N treatment was greater in seed at non-chilling temperature compared to seed at chilling temperature (Table 3). However, prior to the onset of germination the reverse was true and imbibition was less in seed that had been maintained at non-chilling temperatures compared to seed that had been transferred from chilling to non-chilling temperatures. As in expts 1 and 2, germination, independent of applied N level, was greater in seed that had undergone chilling compared to non-chilled seed. Furthermore, regardless of the total ion concentration, germination was greater in treatment solutions that contained N (either as NO− or NH+) compared to solutions that $ % did not contain N (Table 3). In both chilling and nonchilling treatments, imbibition was reduced in seed where treatment solutions contained a total ion concentration of 10 m compared to treatment solutions where the ion concentration was either 5±0 or 0 m, but this was not reflected in germination (Table 3). Experiment 4 The ability of a range of N- and non-N-containing compounds to stimulate germination was examined in expt 4. All N-containing compounds used [NO− and NH+ salts, $ % and CO(NH ) and CS(NH ) ] stimulated germination, ## ## whereas all non-N-containing salts with a similar range of non-N-containing ions, did not stimulate germination (Table 4). In general, N-containing compounds where the N-ion concentration was 2±5 m (KNO , NH Cl) generally $ % stimulated around 50 % germination, whereas those compounds where the N-ion concentration was 5±0 m (CO(NH ) , CS(NH ) , (NH ) SO , NH NO ) were slightly ## ## %# % % $ less effective, stimulating around 40 % germination. For all non-N-containing compounds, germination was similar to the control (deionized water) and was always less than 10 %.
647
DISCUSSION Seed dormancy is a common characteristic of many successful weed species such as Clematis italba L., and this dormancy can be reduced by a range of factors. In this paper, the influence of chilling, N and light on seed dormancy and germination of C. italba was investigated. The influence of these factors is discussed, firstly, with respect to the germination of C. italba specifically, then with respect to their possible mode of action in embryodormant seed in general.
Chilling, light and N, and the dormancy and germination of C. vitalba Clematis italba seed collected from naturally occurring plants had a high proportion of dormancy. Germination, in the absence of any dormancy reducing factors was minimal, and often zero, even after 3 months under conditions that were suitable for the germination of non-dormant seeds. Chilling, N (KNO , NH Cl) and light all influenced seed $ % germination (Table 2). When applied alone both chilling and N could, to some extent, overcome dormancy, whereas light could not. When chilling, N and light were applied in combinations of two or more factors, the reduction in dormancy was greater than when each factor was applied alone (Table 2). The ability of chilling to increase C. italba seed germination has been reported previously (Rudolf, 1974 ; Van Gardingen, 1986), and the influence of chilling in this study is generally consistent with those results. Previous reports on the influence of light on C. italba germination are comparatively less consistent (McClelland, 1979 ; Van Gardingen, 1986). The ability of light to stimulate germination only when combined with either chilling or N (Table 2), suggests that the inconsistency of previous reports may be related to insufficient control of other factors during experimentation (e.g. chilling and N). There are no previous reports specifically dealing with the influence of applied N on C. italba germination. However, the influence of NO− is $ consistent with a range of temperate woody species where germination is also influenced by chilling and light (Bewley and Black, 1985). With respect to seed germination in the natural environment, a reduction in seed dormancy as a consequence of exposure to chilling, light and N (and more specifically NO−) may have an important influence on when, and where, $ seed germination will occur. Reduced dormancy following chilling, for example, may regulate the seasonal timing of germination (Probert, 1992). The influence of chilling (Fig. 1), combined with the tendency for a flush of germination in spring (Van Gardingen, 1986), would suggest that this is also the case for C. italba. Furthermore, it has been suggested that germination in response to elevated light or NO− may be an effective gap or disturbance detection $ mechanism, and that seedlings that rapidly establish in gaps or following disturbance may gain an advantage over plants that establish later when competition for resources has increased (Pons, 1989, 1992). With this in mind, the ability
648
Bungard et al.—Germination of Clematis vitalba
of C. italba to germinate in response to light and NO− $ (Table 2) is consistent with the establishment of C. italba in recently disturbed forest gaps and margins in New Zealand native forest remnants (Van Gardingen, 1986) and may, to some extent, account for its success as an invasive weed species in that environment.
Chilling, light and N, and the regulation of dormancy in embryo-dormant seed In comparison to NO−, NH+ is often reported to be less $ % effective at stimulating germination (Roberts and Smith, 1977). It has been suggested that dormancy reduction by NO− may result from NO− acting as an electron acceptor $ $ and therefore, the difference in effectiveness between NO− $ + and NH is a consequence of their oxidation states % (Hendricks and Taylorson, 1975 ; Roberts and Smith, 1977). This suggestion has come under some criticism (Hilhorst and Karssen, 1989 ; Karssen and Hilhorst, 1992). Moreover, other studies suggest that the influence of N form may be species-dependent (Hendricks and Taylorson, 1974 ; Singh and Amritphale, 1992 ; Thanos and Rundel, 1995). In this study, the stimulation of C. italba germination occurs to a similar extent with either reduced or oxidized forms of N (NO− and NH+), and does not occur with a range of non$ % nitrogen containing compounds (Table 4). As such, these results support the suggestion that a reduction in dormancy is not dependent on the function of the applied N as an electron acceptor (Hilhorst and Karssen, 1989). In addition, the inability of a range of non-nitrogen containing compounds to stimulate germination (Table 4) suggests that the influence of N-containing compounds is not simply an ionic or osmotic effect, but is dependent on the presence of N. When N was applied in the presence of light, NH+ was as % effective as NO− at stimulating germination at low applied $ N levels (0±5–2±5 m), but less effective at high applied N levels (5±0–50 m) (Fig. 1). The reduction in germination in the presence of high levels of applied NH+ not only occurred % in the absence of chilling, but also occurred at chilling levels that, when applied alone, were capable of overcoming a greater proportion of seed dormancy (8–12 weeks chilling) (Table 4). This suggests that, rather than NH+ having a % decreased ability to overcome dormancy compared to NO−, $ + high NH concentrations had an inhibitory effect on % germination. In addition to an increase in total germination in expt 1, increased chilling duration and increased applied N also influenced seed imbibition and the rate of germination (Fig. 1). It has been reported that changes in water relations and, in particular, changes in the balance between osmotic potential and pressure potential are important in the regulation of seed dormancy (Bewley and Black, 1985). In the present study, although increased chilling duration was associated with both a reduction in seed dormancy and an increase in seed imbibition, a reduction in seed dormancy also occurred with applied N, even though this was not associated with an increase in seed imbibition (Table 3). This does not completely discount a relationship between the influence of chilling duration on seed imbibition and the
reduction in seed dormancy, but it does indicate that the increase in imbibition, as a result of increased chilling, is not an essential part of dormancy reduction in C. italba. In addition to an increase in imbibition, increased chilling duration also increased the rate of germination (Fig. 1). Chilling has been reported to increase the rate of germination in a range of species (Gosling, 1988). In the present study and often in other studies, germination is defined as radicle emergence, whereas the actual physiological point of germination occurs earlier, at the onset of elongation of cells in the radicle (Bewley and Black, 1985). As radicle emergence is primarily due to cell elongation rather than cell division (Bewley and Black, 1985), any factor that influences the rate of cell elongation could be expected to influence radicle emergence and consequently, the apparent rate of germination. Cell elongation is influenced by cell wall extensibility (Schopfer and Plachy, 1985). It has been suggested that cell wall extensibility can be increased by the presence of GA (Karssen, 1995), and that GA can accumulate under chilling conditions (Taylor and Wareing, 1979). Although the present study does not discount a true increase in the rate of germination with increased chilling (that is, a more rapid onset of cell elongation), it is possible that the increased rate of germination in this study may be a consequence of chilling leading to an increase in cell wall extensibility, an increase in the rate of cell expansion, and ultimately, an increase in the rate of radicle emergence. In contrast to the influence of N on total germination, increased applied N influenced imbibition and the rate of germination to a greater extent at high applied N levels (" 5±0 m) rather than low N levels (Fig. 1). A similar decrease in the extent of imbibition when seeds were imbibed in solutions with variable N concentration, but equal total ion concentration (Table 3) suggests that, unlike a reduction in dormancy, a decrease in seed imbibition with applied N is not dependent on the presence of N, but is instead an osmotic effect. As radicle emergence is primarily the result of cell expansion rather than cell division (Bewley and Black, 1985), the decrease in the rate of germination (measured as radicle emergence) in expt 1 (Fig. 1) is also likely to result from an increase in the osmotic potential at high levels of applied N. It is generally accepted that light influences germination through the action of phytochrome (Pons, 1992). The inability of light alone to stimulate germination in C. italba, but the enhanced effect of chilling and N in the presence of light (Table 2), suggests two things : firstly, that either the presence of the active form of phytochrome (Pfr) alone is insufficient to overcome C. italba seed dormancy, or that Pfr is not effective in the absence of chilling and}or N ; and secondly, that there is a common link (involving Pfr) between the action of chilling and applied N. The latter is consistent with a range of reports that suggest a close association between the action of Pfr and NO− (Henson, $ 1970 ; Hilton, 1984 ; Hilhorst and Karssen, 1990) that extends to include the influence of chilling (Roberts and Benjamin, 1979 ; Bewley and Black, 1985). Hilhorst (1990 a, b), recently proposed a model that integrated the influence of chilling, Pfr and NO− in dormancy $ reduction. He proposed the existence of an additional factor
Bungard et al.—Germination of Clematis vitalba in the form of a specific protein bound to the plasma membrane. He argued that processes that lead to germination can occur when Pfr binds to this protein, and suggested that the binding of Pfr and the protein is enhanced by chilling (which exposes additional Pfr-binding sites on the membrane), and by NO− (which acts as a co-factor increasing $ the affinity of Pfr for the protein). For the germination data in our study to fit the model proposed by Hilhorst, two assumptions have to be made : firstly, that Pfr is present in C. italba seeds regardless of the light treatment or that the presence of Pfr is not an absolute requirement for germination ; and secondly, that other N forms (specifically NH+) could also act as a co-factor, equivalent to NO−. We % $ did not determine the initial levels of Pfr in C. italba seed, however, it has been suggested that phytochrome levels can be arrested at photoequilibrium when seeds mature on the plant (Kendrick and Spruit, 1977 ; Pons, 1992). If this is true for C. italba, then the ability of chilling and N to increase germination in the absence of light alone would not discount the model proposed by Hilhorst (1990 a, b). However, it could be expected that the action of a co-factor would be dependent on very specific molecular characteristics. Consequently, it would seem unlikely that a co-factor model would accommodate the similar action of NO− and NH+ on $ % germination that occurs in C. italba (Table 2), and other species (Thanos and Rundel, 1995). We suggest a modification to the model where, instead of NO− acting as a co$ factor, NO− acts through an intermediate process that is $ dependent on the supply of N, but not as dependent on the form of N. SUMMARY A high degree of seed dormancy in C. italba seed is confirmed. It is shown that chilling, N and light can potentially reduce C. italba seed dormancy but, in contrast to chilling and applied N, light is only effective when applied in combination with one or both other factors. The ability of both oxidized and reduced forms of N, but the inability of non-N-containing compounds, to increase germination indicates that the influence of these compounds is N-specific, and not an ionic or osmotic effect. It is shown that chilling and N influence both the rate of germination (T %) and &! imbibition of C. italba seed. However, the influence of chilling is much greater than that of N, and it is suggested that the influence of N-containing compounds on T % and &! imbibition is not N-specific, but is instead an osmotic effect. Hilhorst (1990 a, b) proposed a model that integrated the influence of chilling, light and NO− on seed dormancy. $ We suggest a modification to this model that accounts for the similar influence of NO− and NH+ on germination in $ % C. italba. LITERATURE CITED Baethgen WE, Alley MM. 1989. A manual colorimetric procedure for measuring ammonium nitrogen in soil and plant Kjeldahl digests. Communications in Soil and Plant Analysis 20 : 961–969. Bewley JD, Black M. 1985. Seeds : Physiology of deelopment and germination, 2nd edn. New York : Plenum Press. Bouwmeester HJ, Karssen CM. 1992. The dual role of temperature in the regulation of seasonal changes in dormancy and germination of seed of Polygonum persicaria L. Oecologia 90 : 88–94.
649
Gosling PG. 1988. The effect of moist chilling on the subsequent germination of temperate conifer seeds over a range of temperatures. Journal of Seed Technology 12 : 90–98. Grime JP, Mason G, Curtis AV, Rodman J, Band SR, Mowforth MAG, Neal AM, Shaw S. 1981. A comparative study of germination characteristics in a local flora. Journal of Ecology 69 : 1017–1059. Hendricks SB, Taylorson RB. 1974. Promotion of seed germination by nitrate, nitrite, hydroxylamine, and ammonium salts. Plant Physiology 54 : 304–309. Hendricks SB, Taylorson RB. 1975. Breaking of seed dormancy by catalase inhibition. Proceedings National Academy of Science USA. 72 : 306–309. Henson IE. 1970. The effects of light, potassium nitrate and temperature on the germination of Chenopodium album L. Weed Research 10 : 27–39. Hilhorst HWM. 1990 a. Dose-response analysis of factors involved in germination and secondary dormancy of seed of Sisymbrium officinale. I. Phytochrome. Plant Physiology 94 : 1090–1095. Hilhorst HWM. 1990 b. Dose-response analysis of factors involved in germination and secondary dormancy of seed of Sisymbrium officinale. II. Nitrate. Plant Physiology 94 : 1096–1102. Hilhorst HWM, Karssen CM. 1989. Nitrate reductase independent stimulation of germination in Sysymbrium officinale L. (hedge mustard) by light and nitrate. Annals of Botany 63 : 131–137. Hilhorst HWM, Karssen CM. 1990. The role of light and nitrate in seed germination. In : Taylorson RB, ed. Recent adances in the deelopment and germination of seeds NATO-ASI Series A, Volume 187. New York : Plenum Press, 191–206. Hilton JR. 1984. The influence of light and potassium nitrate on the dormancy and germination of Aena fatua L. (Wild Oat) seed and its ecological significance. New Phytologist 96 : 31–34. Karssen CM. 1995. Hormonal regulation of seed development, dormancy, and germination studied by genetic control. In : Kigel J, Galili G, eds. Seed deelopment and germination. New York : Marcel Dekker Inc., 333–350. Karssen CM, Hilhorst HWM. 1992. Effect of chemical environment on seed germination. In : Fenner M, ed. The ecology of regeneration in plant communities. Wallingford, UK : C.A.B. International, 327–348. Kendrick RE, Spruit CJP. 1977. Phototransformations of phytochrome. Photochemistry and Photobiology 26 : 201–204. McClelland TJ. 1979. Report on research into physiological aspects of growth in Clematis italba. Department of Lands and Surey, File No. 22}260}14, Wellington, New Zealand. MacKereth FJH, Heron J, Talling JF. 1978. Water analysis : some revised methods for limnologists. Scientific Publications of the Freshwater Biological Association 36. Pons TL. 1989. Breaking of seed dormancy by nitrate as a gap detection mechanism. Annals of Botany 63 : 139–143. Pons TL. 1992. Seed responses to light. In : Fenner M, ed. The ecology of regeneration in plant communities. Wallingford, UK : C.A.B. International, 259–284. Probert RJ. 1992. The role of temperature in germination ecophysiology. In : Fenner M, ed. The ecology of regeneration in plant communities. Wallingford, UK : C.A.B. International, 285–325. Roberts EH, Benjamin SK. 1979. The interaction of light, nitrate and alternating temperature on the germination of Chenopodium album, Capsella bursapastoris and Poa annua before and after chilling. Seed Science and Technology 7 : 379–392. Roberts EH, Smith RD. 1977. Dormancy and the pentose phosphate pathway. In : Khan AA, ed. The physiology and biochemistry of seed dormancy and germination. Amsterdam : North-Holland Publishing Company, 385–411. Rudolf PO. 1974. Clematis L. In : Schopmeyer CS, ed. Seeds of woody plants in the United States. United States Department of Agriculture. Agricultural Handbook 450 : 331–334. Schopfer P, Plachy C. 1985. Control of seed germination by abscisic acid. III. Effect of embryo growth potential (minimum turgor pressure) and growth coefficient (cell wall extensibility) in Brasica napus L. Plant Physiology 77 : 676–686. Singh B, Amritphale D. 1992. Effect of light and its interaction with nitrate and ammonium ions in seed germination of Caesulia axillaris. Physiologia Plantarum 85 : 43–48.
650
Bungard et al.—Germination of Clematis vitalba
Taylor JS, Wareing PF. 1979. The effect of stratification on the endogenous levels of gibberellins and cytokinins in seeds of douglas-fir (Pseudostuga menzieii (Mirb.) Franco) and sugar pine (Pinus lambertiana Dougl.). Plant, Cell and Enironment 2 : 165–171. Thanos CA, Rundel PW. 1995. Fire-followers in chaparral : nitrogenous compounds trigger seed germination. Journal of Ecology 83 : 207–216.
Van Der Woude WJ, Toole VK. 1980. Studies on the mechanisms of enhancement of phytochrome-dependent lettuce seed germination by prechilling. Plant Physiology 66 : 220–224. Van Gardingen JR. 1986. The physiological ecology of Clematis vitalba L. M.Sc. Thesis. University of Canterbury, New Zealand. Vincent EM, Roberts EH. 1977. The interaction of light, nitrate and alternating temperature in the germination of dormant seeds of common weed species. Seed Science and Technology 5 : 659–670.