Photophysiology of Turion Germination in Spirodela polyrhiza (L.) SCHLEIDEN. II. Influence of After-ripening on Germination Kinetics

j. PlantPhysiol. Vol. 135. pp. 274-279(1989)

Photophysiology of Turion Germination in Spirodela polyrhiza (L.) SCHLEIDEN. II. Influence of After-ripening on Germination Kinetics K.-J. ApPENROTH, J. OPFERMANN*, W. HERTEL, and H. AUGSTEN Dept. ofBiology - Plant Physiology - and" Dept. of Chemistry, University of Jena, DDR-6900 J en a, von-Hase-Weg 3, GDR Received April 19, 1989 . Accepted June 30,1989

Summary To evaluate the influence of cold (278 K) and warm (298 K) after-ripening conditions on germination kinetics of dark-grown and light-grown, phosphate-limited as well as light-grown, sulfate- and nitratelimited turions of Spirodela polyrhiza four non-linear approximation functions were tested. Although slightly inferior to the Wei bull function, the Mitscherlich function was chosen because the parameters characterizing germination, i.e. lag (z), rate of increase (k) and maximal germination response (M) are of physiological relevance. Without after-ripening, turions show very low M and k, which means they are highly dormant. Red light irradiation alone is unable to break dormancy. The only exceptions are nitrate-limited turions. During cold after-ripening responsiveness to red light develops and results in an increase of M and k and a decrease of z. After an extended period of cold after-ripening (6 weeks), M also rises in dark germination. After-ripening is described as a gradual response. Dark-grown, phosphate-limited, cold after-ripened turions show higher differences between the parameters of dark and red-light-induced germination than light-grown ones. Consequently, dark-grown turions are more suitable photophysiological objects. Warm after-ripening is less effective to break dormancy than cold after-ripening, especially with lightgrown turions. This stresses the importance of low temperature for the survival strategy under natural conditions.

Key words: Spirodela polyrhiza (L.) Schleiden, after-ripening, germination, non-linear approximation. Introduction Immediately after their formation turions of Spirodela polyrhiza show very low germination percentages, even if favourable environmental conditions are applied (Augsten et al., 1988). This is the result of an evolutionary adaptation to natural conditions and part of the survival strategy of the plants (Kandeler, 1988). The dormant state of turions has variable length depending on the kind of turion-inducing factors and cultivation conditions during turion development (Landolt and Kandeler, 1987). Chilling or cold afterripening is a very common method to break dormancy and is routinely applied by most investigators in the turion field Gacobs, 1947; Henssen, 1954; Czopek, 1964; Newton et aI., 1978). However, none of them has systematically investigat© 1989 by Gustav Fischer Verlag, StUttgart

ed the influence of after-ripening on germination. In contrast, investigations on after-ripening in seeds are very numerous. Chilling is commonly required by seeds which have embryo dormancy. Herbaceous and woody species with coat-imposed dormancy also respond to chilling (Bewly and Black, 1982). The aim of the present paper is to investigate the influence of warm and cold after-ripening on red-light-induced and dark germination of light-grown and dark-grown turions formed under phosphate-limited conditions. Moreover, we have compared the results with light-grown turions formed under sulfate- and nitrate-limited conditions. Details about these different turion types were given by Appenroth et al. (1989). We had to transfer the essential aspects of germination into statistical parameters. In this way it is possible to distinguish

After-ripening and germination of turions

between superior and inferior germination. There are two different approaches to solve this problem. Brown and Mayer (1988 a) tested different single-value germination indices and they found that none of them could be recommended as a way of fully characterizing germination. Since powerful computers are common there is little reason not to opt for an alternative approach, which is to fit a non-linear function to the data. Very popular is the use of probit analysis (Hsu et aI., 1984; Schimpf et aI., 1977). Besides the logistic function we have used the Mitscherlich function, a Gaussian approximation (three-parameter functions) as well as the Weibull function (four-parameter function).

Material and Methods Cultivation conditions and turion formation Cultivation of Spirodela polyrhiza (L.) Schleiden, strain SJ, and the formation of turions under phosphate-, sulfate- and nitrate-limiting conditions, respectively, were described elsewhere (Appenroth et al., 1989). In short, we used continuous white light (8.5± LOW m -2), constant temperature (298±2K) and mixotrophic conditions (50 mM glucose) for the formation of light-grown turions. Dark-grown turions were formed from mixotrophically precultivated fronds (about 10 d) which were transferred to darkness for a further 18d (298±0.5K). Sterile turions were harvested 28d after frond inoculation. A/ter-ripening Light-grown or dark-grown turions were after-ripened in glucosefree phosphate-, sulfate- or nitrate-limited nutrient solutions, respectively (200ml per 300ml-Erlenmeyer flask) at 298±0.5K (warm after-ripening) or at 278 ± 1 K (cold after-ripening) for different periods in darkness. If light-grown turions were used without afterripening, they received a saturating far-red light pulse and were stored for 2 d at 298 ±0.5 K before germination test. Germination and irradiation Germination tests were carried out in 100 ml-Erlenmeyer flasks containing 75 ml of the following nutrient solution: KH2P0 4

Formation 28 days (298K)

1.5 mM, Ca(N0 3h- 4H 2 0 1 mM, KN0 3 1 mM, MgS0 4 · 7H2 0 ImM, H3B03 5/LM, MnCh·4H 20 13/LM, Na2Mo04 ·2H20 O.4/LM, and Fe (III)-EDT A 25 JLhl. Turions were irradiated 5 min with red light (R-sample) or not irradiated (dark, D-sample) and transferred in amounts smaller than 75 turions per 75 ml nutrient solution. Flasks with more than 75 turions were discarded because these turions showed lower germination responses. Germination was carried out at 298.0±0.5K in darkness up to 21 d and cumulative germination was evaluated periodically in a discontinuous way. Turions were regarded as germinated if the new sprout was visible. With the exception of nitrate-limited turions (about 200 turions per data point) each data point represents the investigation of 400-600 turions. Light sources and irradiation conditions were described in detail by Augsten et al. (1988). The following modifications were used: Red light 656 nm with 180/Lmole quanta m - 2S -1 and far-red light 750 nm with 40/Lmole quanta m -2S-1. 24 V/150W lamps with reflection mirrors were used as light sources. During irradiation interference filters (Carl Zeiss, Jena, GDR) were fixed on small vessels and protected against heat by a 3 ern layer of 1 M aqueous Na2 Cr04 solution. Experimental variables are summarized in Fig. 1.

Fitting the time course 0/ germination To evaluate the kinetics of germination as influenced by nutrientlimitation during turion formation, time and temperature of afterripening, or red light irradiation at the beginning of the germination test, we have used a non-linear curve fitting procedure. Four functions of different parameters were applied as indicated in Table 1. In some cases z (time with 1 % of the maximal response) or h (time with 50 % of the maximal response) were calculated from the parameterized function and are not included in the approximation procedure (Table 2). To investigate the influence of after-ripening the Mitscherlich function was chosen (Tables 3 - 6). Parameters of different data sets were compared (p:5 0.05) by means of the confidence intervals, calculated as mean ± 2 * standard deviation (Horn and Erdmann, 1978).

Mathematical procedure The algorithm used in the computer program consists of a hybrid technique which combines the Levenberg-Marquardt algorithm

~

After-ripening 0-6 weeks (0)

Germinotion ~

0-21 days (298K)

Light conditions IcWLI10d)

I

c WL (Ught-graHn lurions) 0

emp2roture condtflons:

(Dar k·gra.vn turions!

Nutrient COnditionS phosphate-limited

sulfate- limited

Fig. 1: Experimental variables in formation, after-ripening and germination of turions of Spirodela polyrhiza. D = darkness, cWL continuous white light; R = red light.

nitrate -limiled

275

I

278K (cold after-ripening) 298K (warm after-ripening)

Light condtflOns: Smin R+O

r (R

,:dUCed germmatlon)

(dark germination)

..

276

K.-J. ApPENROTH, J. OPFERMANN, W. HERTEL, and H. AUGSTEN

with a step length optimization. The program is an advanced version of the procedure described by Opfermann (1984, 1985) and is characterized by high iteration velocity as well as numerical stability. The first guess for the parameters is given by a «start calculation» procedure for each type of model function. The experimental errors have a Poisson distribution, i.e. the errors of Gi (fraction of germination) are inversely proportional to the square root of the number of Ni of objects in the experiment i. Therefore the weight of each residue Wi must be Wi = Ni (Christian and Tucker, 1984): N • chi2 = E Wi(Gi - Gif After successful finding of the optimal parame~e;s ihe program provides a large number of statistical tests (F-test, student's t-test, ch?-test, Durbin-Watson-test). The deviations of the parameters as well as regression values are given for the 95 % confidence limits. The goodness-of-fit is characterized by the multiple correlation coefficient R2.

Results

Comparison ojdifferent Junctions The results of approximation using four non-linear functions (Table 1) for dark-grown, phosphate-limited, four weeks cold after-ripened turions are shown in Table 2. In general, all four functions are able to describe the time course of germination. Goodness-of-fit was tested using the coefficient of determination (R2). As shown with results in Table 2 as an example, the following series of goodness-of-fit were obtained with all 28 data sets of the present work : Wei bull > Mitscherlich > Gaussian = Logistic function. This is in accordance with results of Brown and Mayer ( 1988 b). Gaussian approximation and logistic function have the drawback of a perfectly symmetrical distribution. Neither Gaussian approximation nor logistic function provide a direct estimation of the start of germination because these functions begin with an asymptote. Consequently, the start of germination is normally underestimated, i.e. the calculated germination starts earlier than the experimental one (Table 2). Besides the goodness-of-fit it is very desirable that a physiological interpretation is possible using the estimated parameters. This is, however, difficult using the Weibull function. The shape, c, reflects in a mathematical sense the «screwness» of the function or the number of compartments. This is hard to realize from a physiological view. Moreover, in most cases we found a high intercorrelation between the shape parameter, c, and Weibull's k (data not shown). This means that the rate of increase is also difficult to interpret. Conse-

Table 2: Evaluation of cumulative germination of dark-grown, phosphate-limited turions, 4 weeks cold after-ripened using different approximation functions (parameters see Table 1). Values in brackets are recalculated from the parameterized functions. Parameter Weibull Dark germination: M 2.7 ± 1.7 k 0.10±0.16 z 1.9 ±O.s

b

0.83±0.51 (8.1) 0.9495

Mitscherlich

2.3 ±0.3 0.14 ±0.05 1.9 ±0.4 (6.9) 0.9493

Red light pulse-induced germination: M 82.4 ±2.1 80.6 ±1.4 k 1.4 ±0.3 1.2 ±0.2 z 2.0 ± 1.0 1.9 ±0.1 b c 0.61±0.16 (2.4) (2.5) 0.9958 0.9952

Gaussian

2.0 0.28

{
± ±

Logistic

0.1 0.08

(1.5)

±0.1 ±0.19

{
6.4 ± 0.6 0.9448 80.3 68

2.0 0.63

± 1.5 ± 12

2.73 ± 0.07 0.9926

±1.2

(6.4) 0.9432 80.2 3.8

± 1.4 ±0.7

10.4

±2.1

(1.5) (2.7)

0.9944

quently, we have chosen the three-parameter Mitscherlich function, which is only slightly inferior to the four-parameter Wei bull function in respect to goodnes-of-fit but the obtained parameters z, k and M are immediately of physiological significance.

Dark-grown, phosphate-limited turions Cumulative germination kinetics of dark-grown, phosphate-limited turions without after-ripening and with four weeks cold after-ripening is shown in Fig. 2. Without after-ripening turions are highly dormant, whereas afterripening induces highly sensitive, phytochrome-mediated germination (Augsten et al., 1988). The influence of 2,4 and 6 weeks cold after-ripening and 4 weeks warm after-ripening on the Mitscherlich parameters is shown in Table 3. Without a red light pulse the parameter M is very low and shows an increase only after the longest after-ripening period tested. The parameter k also shows only a slight enhancement with time of after-ripening. Lag, however, is shortened significantly. On the other hand, red-light-induced germination shows a very strong increasing maximal response and rate of increase (k) with time of after-ripening, and the lag is shortened as in the dark control. In contrast to cold after-ripening, warm after-ripening shows small effects (Table 3). In complete darkness warm after-ripened turions are highly dor-

Table 1: Functions used to describe cumulative germination (G) as a function of time (t). Parameters: M = asymptotic value of maximal germination response (%); k = rate of germination increase; z = lag in germination (d); c = shape parameter; b = related to the lag; v = variance in the Gaussian distribution, used to calculate k at the point h; h = half-maximal response time. Formula G = M(1-EXP(-k(t-z))<) G = M(1-EXP(-k(t-z))) G = M/(1+EXP(-kt+b)) G = M«C1*D+C2*D2 +C3*D 3)EXP( -(t-h)/(2v))) D = 1/(1+CO*ABS«t-h)/v)) * CO = 0.33267; C1 = 0.17401; C2 = -0.04794; C3 = 0.374

Function Weibull Mitscherlich Logistic Gaussian approximation

Literature Wei bull (1951) Scott et al. (1984) Hsu et al. (1984) Abramowitz and Stegun (1971)

After-ripening and germination of turions G

80

277

o

a

__----------------------------~~R o o

Fig. 2: Cumulative germination G (%) of dark-grown, phosphate-limited turions of Spirodela polyrhiza. a: 4 weeks cold afterripening; b: without after-ripening. D = dark germination, R = red light pulse-induced germination. Table 3: Influence of after-ripening on cumulative germination of dark-grown, phosphate-limited turions using the Mitscherlich function (parameters see Table 1).

Table 4: Influence of after-ripening on cumulative germination of light-grown, phosphate-limited turions using the Mitscherlich function (parameters see Table 1).

After-ripening

Germination

After-ripening

Germination

M(%)

k(d-')

z(d)

Without

D R

1.8± 2.l±

2.1 0.1

0.10± 0.2l±

0.24 0.04

10.4± S.8±

3.5 0.2

Without

D R

26.S± 1.8 29.l±3.4

0.09±0.01 0.14±0.04

3.9±0.2 3.7±0.5

2 weeks, 278 K D R

1.9± 48.8±

0.5 0.1

0.09± 0.93±

0.04 0.01

1.8± 2.4±

0.4 0.1

2 weeks, 278 K

D R

19.4±0.6 41.8±0.2

0.1l±0.01 O.lS±O.Ol

3.0±0.1 3.0±0.1

4 weeks, 278 K D R

2.3± 80.6±

0.3 1.4

0.14± 1.2±

0.05 0.2

1.9± 1.9±

0.4 0.1

4 weeks, 278 K

D R

18.l± 1.5 68.8±2.4

0.17±0.04 0.21 ±0.02

3.0±0.4 2.0±0.1

6 weeks, 278 K D R

12.2± 96.l±

1.3

0.18± 1.5 ±

0.05 0.2

1.9± 2.0±

0.5 0.1

6 weeks, 278 K

D R

28.l± 1.3 89.2± 1.6

0.32±0.09 0.30±0.03

4.6±0.4 2.8±0.1

12 ±1300 0.10± 170 4 weeks, 298 K D 1.2 ± 700 R 3.0± 0.1 66.3± 2.1 0.8S± 0.13 D - dark germination, R = red light pulse-induced germination.

4 weeks, 298 K

D R

S.0±9.6 12.7± 1.1

0.04±0.10 0.46±0.20

1.9± 1.4 2.2±0.5

k(d-')

M(%)

1.4

z(d)

manto The highest germination percentage obtained within 21 d was 0.8 %. As a consequence parameter fitting is inadequate in this case.

D - dark germination, R = red light pulse-induced germination.

Table 5: Influence of after-ripening on cumulative germination of light-grown, sulfate-limited turions using the Mitscherlich function (parameters see Table 1). After-ripening

Germination

M(%)

k(d-')

z(d)

Without

D

S8.0± 200 (7.0± 2.8) 73.0± 1100 1.2) (7.9±

0.006±0.020

3.2±0.7

0.00S±0.080

2.9± 1.8

R

Light-grown, phosphate-limited turions In dark germination without red light application maximal responses show only slight dependence on time of cold afterripening (Table 4). The rate of increase (k) is enhanced, whereas lag shows a complicated relationship with time of cold after-ripening: The lag decreases from 0 to 2 weeks and increases with 6 weeks. Influence of cold after-ripening in red-light-induced germination is represented by a strong increase of the maximal response. Warm after-ripening results in decreased maximal responses. Rate of increase (k) in dark germination is below all other values, but shows an increase after red light induction. The latter effect as well as the very short lag of dark germination after warm after-ripening is difficult to explain.

4 weeks, 278 K

D R

12.l± 0.7 0.21 ±0.04 2.4±0.3 67.0± 1.3 0.47 ±O.OS 2.4±0.1 D - dark germination, R = red light pulse-induced germination. Data in brackets represent highest response values within the interval investigated (21 d).

Light-grown, sulfate-limited turions Fitting of germination kinetics of sulfate-limited turions without after-ripening is obscured by the dormant state. Besides the very low maximal germination value (Table 5) there is a slight second flush just before the end of the germination period investigated. This results in a strong overestimation

K.-J. ApPENROTH, J. OPFERMANN, W. HERTEL, and H. AUGSTEN

278

Table 6: Influence of after-ripening on cumulative germination of light-grown, nitrate-limited turions using the Mitscherlich function (parameters see Table 1). After.ripening

Germination

M(%)

k(d- I )

z(d)

Without

D R

29.5±3.3 6S.6±O.1

O.18±O.O7 O.19±O.Ol

4.2±OA 3.3±O.1

D R dark germination, R

4 weeks, 278 K D =

38.2±2.7 O.19±O.O4 74.7±3.9 O.16±O.O2 = red light pulse.induced germination.

4.6±O.2 2.6±O.2

of the maximal response and in very high standard deviations of all three parameters (Table 5). This also holds true if one of the other functions (Table 1) is used (data not shown). Con~equently, the germination parameters of these dormant turions (Table 5) are not suitable for comparison with those for other turions. Cold after-ripening produces high differences between dark control and red-light-induced germination. This is reflected in the parameters M and k, whereas z is nearly constant.

Light·grown, nitrate·limited turions This type of turion is rather non-dormant, independent of after-ripening (Table 6). Red-light-induced response is also detectable without after-ripening, causing large differences in maximal response. This is also indicated by lag. Rates of increase, however, are about constant, independent of afterripening and irradiation. After-ripening is reflected by an increase in maximal response of dark-germinated as well as redlight-induced turions. The differences between maximal response of red-light-induced and dark germination [M(redlight-induced) - M(dark)], however, are independent of whether turions have after-ripened or not. This means, that the effect of red light, in contrast to all other types of turions, is not increased by after-ripening.

Discussion To compare different germination responses as presented in Fig. 2 and Tables 3-6, curve fitting is indispensable. We chose the Mitscherlich function, although it is slightly inferior to the Wei bull function in relation to goodness-of-fit (Table 2). Richards function is used by Berry et al. (1988). However, as shown by Brown and Mayer (1988 b) the Richards function is inferior concerning goodness-of-fit as well as average relative errors. It is important that the parameters obtained show physiological meaning. This is fulfilled by the Mitscherlich function: once started (lag, z) there is a constant probability of germination for each turion during each time (rate of increase, k), resulting in the ultimate value of maximal response (M). These Mitscherlich parameters in combination with standard deviations enable us to distinguish superior from inferior germination in a specified manner. The Mitscherlich function is very easy to fit, shows good convergence properties and is quite independent of starting values with all 28 data sets tested.

After-ripening is often used to break dormancy of turions of Spirodela polyrhiza, and already Jacobs (1947) stressed the fact that low temperature (i.e. below 283 K) is the most effective environmental factor. However, all investigators used cold after-ripening as a tool for acquiring good germination properties and have not investigated the influence of afterripening on germination kinetics. Moreover, previous investigations were restricted to light-grown, nitrate-limited turions. We have described yield and some properties of turions formed under phosphate-, sulfate- and nitrate-limited conditions, in the presence (light-grown) as well as absence (dark-grown) of light (Appenroth et aI., 1989). Now we are able to compare the germination properties of these different types of turions and to investigate the influence of afterripening on germination behaviour (Tables 3-6). Recently, we showed that red light effects upon turion germination are reversed by immediately applied far-red light irradiation (Augsten et aI., 1988). This means that these red light effects are mediated by phytochrome (see Mohr, 1984).

Turions without after·ripening Employing nitrate-limited conditions, Lacor (1969), Sibasaki and Oda (1979) and Malek and Oda (1980) demonstrated different dormancy states in light-grown turions without after-ripening. Newton et aI. (1978) reported, that the supply of sugar during turion formation removes the requirement of after-ripening to break dormancy. However, nitrate-limited turions represent rather an exception concerning the dormancy state. Phosphate- as well as sulfate-limited turions formed in the presence of sugar are much more dormant than nitrate-limited turions (see Tables 4-6). This holds true especially for dark-grown, phosphate-limited turions. Moreover, the responsiveness to red light is developed to nearly full extent by nitrate-limited turions without afterripening (Table 6). In contrast, the effect of red light in phosphate- and sulfate-limited turions without after-ripening is only very small (Tables 4 and 5). Each of the three Mitscherlich parameters alone is insufficient to account for the red light effects on germination of differently nutrient-limited turions, but the parameter M often shows the most significant change.

Influence of time and temperature on after-ripening In general, red light results in a strong increase of maximal response (M) as well as of the rate of increase (k) in cold afterripened turions. In contrast, germination in darkness without red light shows, in most cases, slightly decreased maximal responses and only a slight rise in the rate of increase. Lag is shortened as influenced by cold after-ripening. This demonstrates, that responsiveness to red light is developed during after-ripening. Neither after-ripening nor phytochrome alone is able to break dormancy completely. Consequently, we have to assume an interaction between the effect of after-ripening and that of phytochrome. This interaction is not an all-or-nothing action, but rather a gradual effect. By extending the time of cold after-ripening to 6 weeks, the lag is not shortened any further, but the maximal re-

After-ripening and germination of turions sponse of dark germination rises markedly (Tables 3 and 4). Because this effect was undesirable in most experiments, conditions were standardized to 4 weeks cold after-ripening for further investigations. Furthermore, the influence of 4 weeks warm after-ripening (298 K) was investigated (Tables 3 and 4). In light-grown turions, this treatment is without positive effects. On the contrary, maximal response is decreased significantly in dark and light-induced germination as well (Table 4). In darkgrown turions, the effect of warm after-ripening in combination with red light irradiation is markedly distinct (Table3). However, the maximal response and the rate of increase are lower and lag is less decreased than with 4 weeks cold afterripening. Consequently, independent of the turion type, warm after-ripening is less effective than cold after-ripening. This clearly shows the importance of low temperature to break dormancy and may reflect the adaptation of the plants to cold winters.

Light-grown and dark-grown turions Whereas dark-grown turions without after-ripening are highly dormant, light-grown turions show higher maximal responses and a shorter lag in dark germination as well as in red-light-induced germination (Tables 3 and 4). These relations are changed after cold after-ripening: Maximal response of dark-grown turions remains very low in dark germination. However, in combination with red light induction, maximal responses and rates of increase rise very strongly with time of cold after-ripening and much more than in light-grown turions. Consequently, the difference between dark germination and red-light-induced germination is greatest in dark-grown turions, and thus these turions are much more suitable for photophysiological studies than lightgrown ones (Augsten et al., 1988). Acknowledgements We thank Dr. H. Gabry~, Univ. Krakow, for proof-reading of the paper and Mrs. Barbara Liebermann for skilful technical assistance.

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SIBASAKl, T. and Y. ODA: Heterogeneity of dormancy in the turions of Spirodela polyrrhiza. Plant Cell Physiol. 20, 563-571 (1979). WEIBULL, W.: A statistical distribution function of wide applicability. J. Appl. Mechanics 18, 293-297 (1951).