Ann. appl. Bwl. (1976), 84,415-424 Printed in Great Britain

Synchronization of germination of celery seeds

BY P. J. SALTER AND R. J. DARBY National Vegetable Research Station, Wellesbourne, Warwick

(Accepted 9 'June 1976)

S U M M A R Y

The effects of osmotic pre-treatments on the germination response of celery seeds were studied in a series of experiments designed to optimize that treatment. The experimental variables were different osmotica (polyethylene glycol (PEG) or a mixture of potassium nitrate and tri-potassium orthophosphate), levels of osmotic potential, temperature and duration of treatment. The rate and synchronization of germination were very significantly improved by many of these pre-treatments but the maxi- mum effects on cv. Lathom Blanching were obtained by treating the seeds with a solution of the salt mixture of c. -10 bars potential at 15 "C for 21 days. This treatment resulted in 50 % germination of the viable seeds within 1-4 days at a temperature of 20 "C compared with 13.7 days from the untreated seeds. There were indications that the optimum pre- treatment may vary somewhat between varieties and seed stocks. The results showed that the effects of the two osmotica on germination response were similar, thus enabling a technique to be developed for treating, with the salt solution, larger quantities of seed and germinating them prior to fluid drilling in the field.

INTRODUCTION

There are considerable economic advantages in drilling celery (Apium graveolens L. var. duke (Mill.)) seed directly into the field but germination is slow and unpre- dictable and often only a small proportion of the viable seeds produce seedlings. Consequently crops are normally grown from transplants raised in glasshouses. With the objective of improving seed germination in celery, Thomas, Palevitch, Biddington & Austin (1975) studied the phytochrome-mediated dormancy mechanism involved and have described the beneficial effects of applying growth regulators to the seeds followed by drying back.

An alternative approach has been to provide optimum conditions for germination in the laboratory and then to mix the germinating celery seeds into a carrier gel before sowing in the field with a fluid drill. Using this system successful establishment andgoodyieldsof celery havebeenobtained(Currah, Gray&Thomas,1974; Biddington, Thomas & Whitlock, 1975). To obtain the maximum advantage from this technique germination of the seeds needs to be as uniform and as rapid as possible, characteris- tics not normally present in celery. Studies were made therefore of the effect of seed 'priming' (Heydecker, 1974) on the synchronization of germination of celery seed. The technique developed by Heydecker involves the placing of seeds in contact with a solution of a high molecular weight fraction of polyethylene glycol (PEG). By

416 P. J. SALTER AND R. J. DARBY controlling the concentration of this solution, the temperature and the treatment duration, it is possible to allow partial imbibition of the seed and the initial germina- tion processes to take place but to prevent radicle emergence (germination). With celery, Heydecker, Higgins & Gulliver (1974) reported that germination was very rapid after seeds had been treated with a PEG '6000' solution of - 12.5 bars potential at 15 "C for 14 days.

MATERIALS A N D M E T H O D S

For the first experiment Heydecker's treatment, and variants of it, were employed and the PEG solutions were made up as described by Williams & Shaykewich (1969). In successive experiments the treatments were altered to try to maximize synchronous germination. Solutions of PEG with osmotic potentials approximating to - 10 and - I 5 bars were made up by dissolving 230 and 270 g, respectively, of Carbowax 20 M , made up to I 1 with distilled water; the pH of these solutions was 643.

In addition to PEG a mixture of laboratory grade potassium nitrate (KNO,) and tri-potassium orthophosphate (K,PO,. H,O) 'salt mixture' was used as the osmoticum and an attempt was made to obtain solutions with comparable potentials to those of PEG. For the nominal - 10 bars potential solution 10.60 g of KNO, and 11-50 g of &PO,. H,O were made up to I 1 with distilled water; for the - 12-5 bars solutionsthe weights were 13-14 and 1497 g, and for the - 15 bar solution 15-68 and 18-42 g respectively. The pH of each of the salt solutions was 12-1.

Measurements of the actual potential of the solutions were made using a thermo- couple psychrometer (Campbell, Campbell & Barlow, 1973) and the measured poten- tials are given in Table I.

Seed of cv. Slow-bolting Green No. 12 was used for the first experiment and seed of a single stock of cv. Lathom Blanching for the later experiments.

In the first two experiments IOO seeds were placed on two layers of filter paper in in 56 x 35 x 22 mm transparent polystyrene boxes. Measured quantities of solutions of the osmotica were applied by pipette. The boxes, which were duplicated for each treatment combination, were placed in illuminated incubators for different experi- mental periods but all treatments ended on the same day. The seeds from each box were then washed and placed on fresh filter paper in a new box in an illuminated incubator at 20 "C. Germination, defined as emergence of the radicle, was recorded daily for ZI days. The final germination percentage was calculated together with the number of days required to achieve 50 % germination of the viable seeds. Because the radicle length must not exceed 5 mm for damage-free fluid drilling of this species, the percentage germination within 48 h of the start of germination was determined. In addition, the maximum percentage germination which occurred during any 48 h period was calculated and was taken as a measure of the efficiency of a treatment in synchronizing germination.

For all experiments most of the germination percentages were in the range where no transformation to angles was required. As a result only minor differences were observed between the statistical analyses on transformed and untransformed data. In order to simplify the presentation of results only the untransformed data are given.

The treatments and results obtained from each experiment are described in chrono- logical order.

Germination of celery seeds 417

RESULTS

Experiment I

The effects on germination of five osmotic treatments given at 10 or 15 "C for 5 , 10,

15 or 20 days were examined: a total of forty treatments. Two osmotica were used (PEG and the salt mixture) each at nominal potentials of - 10 and - 15 bars and, in addition, there was a dry seed control for each temperature and duration of treatment level.

The measured potentials of the different osmotica are given in Table I and show that the potentials of the salt solutions were higher than those nominated whereas those of the PEG solutions were somewhat lower. The mean effects of the main treat- ments had significantly influenced the rate and synchronization of germination as well

Table I . Measured potentials (bars) of the osmotic solutions used in the experiments

Temperature ("C)

Nominal osmotic potential Expt I

PEG -10 bars Salt mixture - 10 bars

Salt mixture - 15 bars

Salt mixture - 10 bars - 12.5 bars - I 5 bars

Salt mixture - 10 bars

PEG - 15 bars

Expt 2

Expt 3

5 I0

-11.2 - 8.8 - 15'5 -11.6

- 8.9 - 8.8 - I01 - I00 -11.8 -11.6

- 8.8

1

I5

-11.3

- 9'7 - 15'9 - 13.2

- 9'7 - 10.5 - 13.2

as the final level of germination. The osmotic treatments significantly improved the uniformity and the rate of germination. The magnitude of the effects increased with the duration of treatment and was greatest at 10 "C. The treatments had small but significant effects, both positive and negative on the final level of germination.

In general, both PEG and the salt mixture at the same nominal potential resulted in similar effects on germination but there were significant interactions with treatment duration and temperature (Table 3). The greatest synchronization of germination was obtained with the 20-day treatment at the nominal -10 bars potential with the treatment a t 10 "C resulting in higher absolute levels of germination than from those treated at 15 "C.

Experiment 2

To confirm and extend the results of the previous experiment to the commonly grown cv. Lathom Blanching the effects of germination of four osmotic treatments of 14, 21, 28 or 35 days duration at 5 , 10 or 15 "C were studied: a total of forty-eight treatments. As experience had indicated that similar effects were obtained with both PEG and the salt mixture only the latter was used to give osmotic treatments of

418 P. J. SALTER AND R. J. DARBY

Table 2. Main treatment eflects on germination of celery seed of cv. Slow-bolting Green No. 12 at 20 "C in Experiment I

Percentage germination Days to A , 50% germin-

Treatments

Osmotic Control (untreated seed) PEG -10 bars' Salt mixture - 10 bars' PEG -15 bars* Salt mixture - 15 bars'

S.E.D. of osmotic treatment means Duration (days)

5

15 I0

20 S.E.D. of duration means Temperature (" C)

I 0

I 5 S.E.D. of temperature means

48 h from start of

germination

29'5 41.0 44.5 39'4 37'6 f 2.99

29'4 29.1 44.0 51.2 k 2.68

42'4 34'4

& 1.89

Maximum in any

48 h perod

40'3 56.1 53.1 51'3 45'1 f 219

45.6 44.7 51'4 55'1

2 1-96

52'0 46.4 f 1.38

ation from start

Total of test

84.6 9'9 88.4 4 6 840 3'8 86.3 5'5 82.4 4'4

+ I * & f0.16

87.0 6.8 84.6 6.0 85.3 5'0 83.6 4'8

f0.95 f0.14

85-3 5'4 85.0 5'9

50.67 ~ O I O

* Nominal potentials. For measured potentials see Table I.

Table 3. Treatment interaction effects on germination percentage of celery seeds 48 h from start of germination at 20 "C in Experiment I

Temperature ("C)

I 0 15

A I

.A f \ f \

Duration (days) . . . 5 I 0 15 20 5 I 0 I5 20

Control (untreated seed) 25.5 27.0 33.5 40.0 11-5 34'0 42.5 22.0 PEG - 10 bars' 25.5 30.0 46.0 68.0 45.5 31.5 333 48.0 Salt mixture - 10 bars+ 22.0 47.5 62.5 69.0 25-0 12.5 57.5 60.0 PEG -15 bars* 30.0 48.0 47.0 55.0 45.5 14.0 27.0 48.5 Salt mixture - 15 barsf 35.0 27'5 46.5 61.5 28.0 18.5 44.0 40.0

Osmotic treatment

S.E.D. 5 5'24

* Nominal potentials. For measured potentials see Table I.

nominally - 10, - 12.5 and - 15 bars potential; the measured potentials are given in Table I. In addition, there was a dry seed control.

The osmotic treatment greatly improved the uniformity of germination, increased the total germination and resulted in considerably faster germination compared with the control seed (Table 4). With minor exceptions there were no significant differences between the effects of the different osmotic potentials.

Treatment duration means showed that lengthening the time reduced the total germination percentage but increased the rate, whilst the 21-day treatment gave the

Germination of celery seeds 4'9

Table 4. Main treatment eflects on germanation c j celery seed at 20 "C in Experiment 2

Treatment Osmotic

Control (untreated seed) Salt mixture - I o bars* - 12-5 bars* - I 5 bars*

S.E.D. of treatment means Duration (days)

14

28 35

21

S.E.D. of duration means Temperature ("C)

5

15 I 0

S.E.D. of temperature means

Percentage germination Days to I 50%tF-- 48 h from Maximum ation

germination 48 h period Total of test start of in any from start

3'2

29.0 29.1 29'9 f 1-30

18.5 25'9 24' I 22-7

f 1.30

6.8 25'2

36.3 f 1-13

6.3 19.8 13.7

31.7 52.0 4'0 32'9 50'4 4'0 32'5 53.8 4'4 f 1.17 f1.31 fo.15

22'0 48.4 7'1 29.5 48.0 6.7 26.7 41 '0 6 2 25'3 38.7 6 2 f 1.17 f1.31 f 0 . 1 5

12.9 36.1 9'1 26.5 44'2 5 6 38.1 51.7 4'9 f 1'02 +I-13 f 0 . 1 3

* Nominal potentii-. For measurec potentials see Table I,

Table 5 . Treatment interaction effects on germination of celery seed at 20 "C in Experiment 2

Temperature A , Untreated Duration of salt mixture treatment (days)'

("C) 14 21 28 35 control

(a) Germination percentage 48 h from start of germination

5 6.0 7'3 9'3 9'7 3'1 I0 18.0 34'8 39'0 37.8 3'6 I5 47'0 58.7 44.3 4 0 0 2.9 S.E.D. of salt-treated means = f 2.92. S.E.D. for comparisons between untreated and treated means = f 2.61.

(b) Total germination percentage 5 5 0 2 46.0 38.7 33'3 18.5

I 0 58.8 5 5 ' 0 49'2 48.5 18.3 I5 65'3 67'7 54'7 57'7 22.6 S.E.D. of salt-treated means = f 259. S.E.D. for comparisons between untreated and treated means = k 2.44.

(c) Days to 5 0 yo germination from start of test 5 8.5 8.6 8.2 6.8 13.0

10 4'5 2.9 1.8 1'9 14.2 ' 5 2'3 1'4 1'7 2'2 14.0 S.E.D. of salt-treated means = f 0.30 S.E.D. for comparison between untreated and treated means = 0.28

* Mean effects of the temperature x duration treatments averaged over the nominal - 10, - 12.5 and - 15 bars potential salt treatments.

420 P. J. SALTER AND R. J. DARBY most uniform germination. The data also showed that the beneficial effects of the osmotic treatments increased significantly as temperature was raised.

A series of statistical analyses were carried out on the germination response data both including and excluding the control treatment because the germination of the untreated seed was so much less than that of the treated seed. The results indicated that there were no significant differences in the effects of the nominal - 10, - 12.5 and - I 5 bars potential treatments on germination. Therefore, the data were averaged over these three treatments for comparisons with the untreated control. There were sig- nificant interactions between treatments and the duration and temperature variables. The results (Table 5 ) indicate that synchronization of germination was greatly improved by the osmotic treatment, the effect being progressively increased as the treatment temperature was raised. The treatment duration had no significant effect at 5 "C; at 10 "C, 14 days was insufficient to have the maximum effect obtainable at that temperature, whilst at 15 "C the 21-day treatment gave the greatest effect in synchronizing germination of all the treatment combinations examined.

The effects of the combination of treatment variables on the total germination percentage (Table 5 ) were similar to those on synchronization of germination; an increase in the treatment temperature increased the beneficial effect of the osmotic treatment on total germination, the best treatments being 14 or 21 days at 15 "C. The most rapid germination occurred when the osmotic treatment was given for 21 days at I 5 "C although a longer duration at 10 "C and I 5 "C also gave very rapid germination. With these treatments 50% of the final germination had taken place within 48 h of the start of the germination test compared with approximately 14 days for the un- treated controls.

EAperiment 3 Results from the previous experiments indicated that a significant improvement

in the synchronization of germination could be obtained by treating seeds on filter paper with a salt or PEG solution. Using an apparatus made for this purpose (Darby & Salter, 1976) a technique was developed to enable semi-bulk quantities of celery seed to be similarly treated and germinated in the light so that the performance of the treated seeds could be assessed under field conditions.

To make comparisons with similar treatments previously carried out on a filter- paper scale, four sets of apparatus were used to treat 32 g lots of seed with a nominal - 10 bars (measured, - 8.8 bars) potential salt solution for 7, 14, 22 and 27 days at 10 "C. At the end of the treatment the solution was replaced by water and germination took place at 20 "C in the same apparatus. An additional apparatus was set up to germinate untreated seeds and a standard germination test was also carried out on untreated seeds on filter paper. Samples of seeds were removed at intervals, to deter- mine the percentage germination, until a proportion of the seeds had radicles longer than 5 mm.

The effect of the four bulk priming treatments on the rate of germination is shown in Fig. I together with comparable data from untreated seeds. The results confirm those previously obtained on filter paper and show that with increasing duration of the osmotic treatment more rapid and synchronous germination was obtained compared

Germination of celery seeds 421 with untreated seeds. This experiment was terminated when the seeds were in a suitable stage for fluid drilling in the field. The results of the field experiments will be reported elsewhere.

2 4 6 8 10 12 21 Time from start of germination phase (days)

Fig. I . Germination of celery seed at 20 “C after different durations of treatment at 10 “C in a nominal - 10 bars potential salt solution. A, 7 days; A, 14 days; 0, 22 days; 0 , 2 7 days; 0, unprimed control; ., standard germination test on filter paper.

DISCUSSION

Many different seed treatments have been proposed to obtain rapid and uniform germination. These have included the controlled wetting of seeds with water followed by drying back as in the ‘hardening’ (May, Milthorpe & Milthorpe, 1962) and ‘ad- vancing’ (Longden, 1971) techniques, or treatment with solutions of salts (Ells, 1963) or growth regulators (Thomas, Palevitch & Austin, 1972). Studies on the mechanism by which germination of low viability embryos of rye were enhanced following wetting and drying (Sen & Osborne, 1974) showed that the duration of the wetting pre-treatment is critical since there is a balance between the advancement of synthetic processes following imbibition, and the damage to the earliest activated cells incurred by the subsequent drying procedure. These authors suggest that the limited duration of the wetting pre-treatment, beyond which drying causes cell death, may explain why some of the pre-treatment procedures described in the literature were found not to stimulate subsequent germination. Heydecker (1974) postulated that if the amount of water imbibed by seeds was carefully controlled by using an osmotic medium of appropriate potential the preliminary processes of germination could take place without radicle emergence occurring; the seeds could thus be brought to the ‘brink of ger- mination’ without hazard so that when conditions permitted germination would-be

422 P. J. SALTER AND R. J. DARBY rapid and more uniform. The results obtained by Heydecker, Higgins & Gulliver (1973, 1974) support this hypothesis. These workers regarded Carbowax 6000, a polyethylene glycol of high molecular weight, as an ideal osmoticum because the large molecules cannot pass into plant cells, unlike inorganic salts, and because the chemical is inert allowing prolonged treatment without damage to the seed. They acknowledged, however, that commercial application of the technique depended on finding a method of treating the seed in bulk and overcoming the problem of providing aeration to the seeds during treatment. Because this latter problem appeared to be so great, the use of salt solutions, which can be easily aerated in bulk, was examined in the present work.

A mixture of potassium nitrate and tri-potassium orthophosphate was used because this mixture has been used by other workers with beneficial results (Ells, 1963; Woodstock, 1969) and nitrogenous salts have been known for over 60 years to enhance germination (Toole, Hendricks, Borthwick & Toole, 1956). In the first experiment the effects of both the salt mixture and the PEG solutions were compared. However, it was not possible to obtain exactly comparable potentials with the two osmotica because the degree of dissociation of the ions of the salts in solution are difficult to predict and only approximations are possible. Furthermore, the osmotic potentials of solutions are affected by temperature (Michel & Kaufmann, 1973) and, in addition, as the treated filter papers tend to dry out during the treatment the osmotic potentials of the remaining solution increase somewhat. Thus it is difficult to obtain osmotic potentials of given levels and to maintain them throughout the treatment period. Measurements of the osmotic potentials indicated that those of the salt solutions were higher than those nominated whereas those of the PEG solutions were somewhat lower (Table I). Therefore the results described need to be examined with these differences in poten- tials in mind which, however, do not invalidate the general conclusions that can be made.

The results show that following a treatment with a near-optimum combination of osmotic potential, duration and temperature level, the speed of germination was very significantly increased and the uniformity of germination was greatly improved com- pared with the untreated seeds. However, these results and those of other experiments suggest that the precise conditions for optimizing the priming treatment are likely to differ from one variety to another and between seed stocks of the same variety. Until these matters have been investigated the optimum combination of variables for the treatment of celery seeds suggested by this work is an osmotic potential of c. - 10 bars for 21 days at I 5 "C, although similar germination responses were obtained with other treatment combinations.

The results also show that the salt mixture used could be equally effective as PEG in improving the germination response of the seeds and this enabled the technique to be developed for treating larger quantities of seed while still providing adequate aeration during the process. Although a quantitative comparison was not made of the germination response of seeds treated in bulk with those treated on filter paper, the results obtained were very similar even though the duration of some treatments were not exactly comparable. For example, by bulk treatment procedure, durations of 7, 14, 22 and 27 days resulted in respectively, 15-4, 17.9, 39-4 and 47-3 % germination within 48 h of the startof germination,compared with 20'5,33.oand37-5 %germination

Germination of celery seeds 423 from 14~21 and 28 days duration of the same osmotic treatment given on filter paper in Expt 2. There are indications that the conditionsprovided for priming and germination of the seed in bulk were as good, if not better, than those given on filter paper. In particular, the unprimed (control) seeds germinated in the semi-bulk equipment (Darby & Salter, 1976) had a higher total germination percentage than seeds of the same stock germinated on filter paper at the same temperature in Expt 2 (Table 5 ) and in Expt 3 (Fig. I). It is possible that during the germination phase the filter papers did not provide an optimum water supply to the seeds although every effort was made to achieve this. Since Expt 3 was carried out, 32 g lots of celery seed have been primed with salt solutions and the germinated seeds fluid drilled in field experiments with promising results.

A n experiment was also carried out to study any interactions between seed size and response to the osmotic treatments. Seeds of cv. Lathom Blanching were size-graded into three sizes of range 0.25 mm and the effects of fifty-six priming treatments on each seed size were examined. The size of seeds had a small, but significant effect on the response to the priming treatments. The small and large seeds were slightly slower ingerminating, were less uniform in their germination and had a lower total germination than the medium grade of seed or the ungraded seed sample. However, because these effects were small and as there were no statistical interactions between seed grades and priming treatments the experiment has not been described here or discussed in detail ; the effects of the priming treatments were similar to those reported for Expts 2 and 3.

The full benefits from fluid drilling germinated seeds can be obtained only if the majority of seeds have germinated simultaneously and are at the same stage of radicle development. With celery seeds the radicles must not exceed 5 mm in length otherwise damage occurs during drilling resulting in reduced seedling stands. Thus, before drilling, maximum germination of the seeds must be achieved over a 48 h period if the full benefits from drilling germinated seeds are to be obtained. It is for this reason that the degree of synchronization of germination has been measured by recording the maximum germination occurring either within 48 h of the start of germination or within any 48 h period.

This work has shown that the rate and synchronization of celery seed germination has been greatly improved by an osmotic pre-treatment immediately followed by con- ditions favourable to germination. Furthermore, Heydecker ( I 974) and his co-workers have found that the effects of their osmotic treatments are much greater when the seeds are merely surface dried before being sown than when the seeds are thoroughly dried back, stored, and then subsequently sown. Indeed, in some instances drying back of the seeds has completely negated the advantages conferred by the pre-treatment. Therefore these two techniques, osmotic pre-treatment of seeds (‘priming’), and fluid drilling of germinated seeds, must not be regarded as alternatives. Rather they should be regarded as complementary techniques to be combined to achieve the common objective of improving seedling establishment in the field, because each may allow the maximum benefit to be obtained from the other.

We thank Mr T. J. Orchard for advice and assistance with the statistical analyses of the data.

424 P. J. SALTER AND R. J. DARBY

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