The effect of water stress on the germination of Citrullus lanatus seeds

F.C. Botha, N. Grobbelaar and J.G.C. Small Margaretha Mes Institute for Seed Research, University of Pretoria, Pretoria

The germination of Citru/lus lanatus seeds is extremely sensitive to water stress. A decrease of 13,6% in the total water content is sufficient for the complete inhibition of germination. Exposure of seeds to water stress at a very late stage of germination after normal incubation in water prevents radicle emergence. Prolonged water stress treatment does not induce secondary dormancy in these seeds. Water stress which completely inhibits germination does not appear to affect the conversion of phytochrome intermediates to Pfr or the photoreversibility of phytochrome during light treatments. S. Afr. J. Bot. 1984,3: 111-114

Die kieming van Citrullus lanatus-sade is besonder gevoelig vir waterspanning . 'n Verlaging van 13,6% in die waterkonsentrasie van die sade is voldoende om kieming volledig te rem. Wanneer sade aan waterspanning blootgestel word op 'n laat stadium van kieming, na normale inkubering in water, word kiemwortel-verskyning nog steeds verhoed. Langdurige blootstelling van die sade aan waterspanning induseer nie 'n sekondere rustoestand nie. Waterspanning wat kieming volledig rem het skynbaar min invloed op die vorming van Pfr uit intermediere fitochroomverbindings sowel as op die foto-omskakeling van fitochroom tydens ligbehandelings. S.-Afr. Tydskr. Plantk. 1984,3: 111-114

Keywords: Citrullus lanatus, phytochrome, secondary dormancy, water content, water stress

F.C. Botha and J.G.C. Small Present address: Department of Botany, University of the Orange Free State, Bloemfontein, 9300 Republic of South Africa

N. Grobbelaar* Department of Botany, University of Pretoria, Pretoria, 0002 Republic of South Africa *To whom correspondence should be addressed

Accepted 15 December 1983

Introduction

Although water stress is probably one of the most important factors preventing germination of seeds in the soil, the sensitivity to water stress of only a few species has been investigated (Hegarthy & Ross 1980/81; Evensen & Loy 1978; Khan 1960).

Knowledge regarding the effect of water stress on the germination of the Cucurbitaceae is limited to studies on Cucumis sativus L. (Hegarthy & Ross 1980/81) and two watermelon varieties (Sachs 1977; Evensen & Loy 1978). Nothing is known about the hydration level required by the seeds of this plant fami ly for germination.

According to Khan (1960) and Khan & Karssen (1980) exposure of seeds to water stress could cause the induction of secondary dormancy. The general occurrence of this phenomenon is still much in doubt (Hegarthy 1977).

The seeds of Citrullus lanatus used in this study are typically negatively photoblastic (Botha et al. 1982a & b). It is possible that water stress can inhibit germination by pre­venting suitable levels of Pfr to be attained, since it is known that complete conversion of phytochrome to Pfr is only possible in hydrated tissue (Kendrick 1976).

The aim of this study was to determine the sensitivity of C. lanatus seeds to water stress and to ascertain whether water stress induces secondary dormancy. The opportunity to test the effect of water stress on phytochrome conversions in these seeds also arose.

Materials and Methods

Seeds of Citrullus lanatus (Thunb.) Matsumura & Nakai were obtained from plants growing in the wild as described by Botha et al. (1982a). Seeds were air-dried at room temperature and stored in airtight glass containers in the dark at 0-5°C.

Germination tests were conducted at 27°C in the dark. Seeds were germinated in closed airtight glass containers (Botha et al. 1982a) . In all cases 30 seeds were used per replicate. Seeds in each container were watered only once with 2,5 cm3 of either distilled water , a mannitol solution or a poly-ethylene glycol (PEG) solution at the beginning of the experiments. At least six replicates per treatment were used throughout. The water potential (1JI) of the mannitol solutions was determined by using the equation

1J1 = -miRT (Salisbury & Ross 1978)

112

where m = molality of the solution , i = ionization constant of the solute, R = the gas constant and T = absolute temperature (K) .

The method of Michel & Kaufmann (1973) was used to calculate the '1/J of the PEG 6000 solutions.

The water content of the seeds was determined after oven drying at 70°C to constant mass. Red and far-red light were obtained as described by Both a et al. (1982b).

To investigate the role of the structures surrounding the embryo on the sensitivity to water stress of the seeds, the testa and underlying thin membrane were removed (Botha et al. 1982a). Germination is taken to be the series of steps normally occurring prior to the emergence of the radicle from the seed coat (Mayer & Shain 1974) and is terminated by the emergence of the radicle. A seed was considered to have germinated when the radicle had emerged. Germinated seeds were counted under a dim green safe light (Smith 1975). Least significant differences (LSD) were calculated by using the Tukey procedure (Steel & Torrie 1980).

Results and Discussion

The germination of C. lanatus seeds is extremely sensitive to water stress. An osmoticum with a '1/J of -430 kPa completely inhibits germination (Figure 1). Compared with other investigated cucurbits (Sachs 1977; Evensen & Loy 1978; Hegarthy & Ross 1980/81) it appears that C. lanatus seeds are more sensitive to water stress. No differences in the inhibitory effect of PEG and mannitol solutions with equal water potentials on the germination of C. lanatus seeds were observed (data not shown).

100

~ :: \\····· ... · ! ....... ~.. ·f Z e-e Intact seeds i ffi 40 ~ £----• Naked embryos (.!) \ (Testa 3nd underlying # membrane remuved )

20

0 -200 ·-· -400 -600 -800 -1000

'I' l kPa J

Figure 1 The effect of an osmoticum (mannitol) with different water

potentials on the germination of intact seeds and naked embryos of

Citrullus lanatus at 27°C in the dark.

The sensitivity of the seeds to water stress is reduced when the testa and underlying membrane are removed (Figure 1). With naked embryos 50 % germination is obtained at a '1/J of -1000 kPa whereas less than 50 % germination is obtained with intact seeds at a '1/J of -200 kPa. This effect cannot be ascribed to differences in water uptake between intact seeds and the naked embryos (Table 1).

According to Khan (1960) the sensitivity of lettuce seeds to water stress is also reduced by damaging the superficial structures of the diaspore.

Intact C. lanatus seeds show a mass increase of 44 % when incubated in distilled water. In comparison seeds incubated

S.-Afr. Tydskr. Plantk., 1984, 3(2)

Table 1 Water content of the embryos of intact seeds and naked embryos of Citrullus lanatus after incubation in solutions with different water potentials at 27°C in the dark

1/J of incubation medium (kPa)

0 -250 -500 -1000

Percentage water content (relative to dry mass) after a 24-h incubation period

Embryos of intact seeds

38,7 37,0 35 ,3 33,2

Naked embryos

39,1 36,8 35,0 33,0

A difference of more than 1,2 between any two figures is significant at the 5 % level of probability

in a solution with a '1/J of -430 kPa, which completely inhibits germination , show a mass increase of 38 % (Figure 2). Although this represents a difference of only 13,6 % in water content between the stressed and control seeds , it is obvious that this is sufficient to induce changes in the metabolism of the seeds to prevent germination. A decrease in water content of 10- 20 % is sufficient to inhibit the germination of carrot and calabrese seeds (Hegarthy 1977).

A large component of the water is taken up by the seeds as a result of their matrix potential ( '1/Jm). Water taken up owing to the '1/Jm is tightly bound (Harrington 1972). The water component which is important for metabolic processes is probably only that taken up as a result of the osmotic potential ( '1/Jn) of the tissue and will not be as tightly bound as the hydration water.

50

...... . 40 .-• ,..--• .........-

~--+·H--·-·-··· ·· -+---·-- - ----4 I- , __.... .-

z ; .- -w 30 ' ~ <,/ 0 ~ : u f'j . tt: 20 f w '

~ ~ 10 :

0

e--e water

• -- -·• Mannitol <-430 kPa )

10 20 30 40 INCUBATION TIMElh)

50

Figure 2 Increase in water content of Citrullus lanatus seeds incubated

in distilled water or an osmoticum at 27°C in the dark. The percentage

germination of water-treated seeds was 96% and that of the osmotically treated seeds 0% after 96 h.

From the results in Figure 3 it is possible to estimate that the water taken up owing to the seed's '1/Jm represents about 37 % of the seed's dry mass, whereas that owing to the '1/Jn of the seeds represents only about 6 % of the seed's dry mass. Therefore the osmotically active water of the fully hydrated seeds represents only about 14 % of the seed's total water content. This water probably represents the 'metabolically active' component. It is obvious that an osmoticum with a '1/J

of -430 kPa, which completely inhibits germination, appears

S. Afr. J. Bot. , 1984, 3(2)

45

w ~ 42,5 w a: u z

0 -250 -750 -1250 -1750 -2250

"'(kPa)

Figure 3 The effect of mannitol solutions with different water potentials

on the percentage mass increase of Citrullus lanatus seeds at 27°C in

the dark .

to do so by reducing the uptake of 'metabolically active' water. In fact the decrease in water uptake caused by a 1/J of -430 kPa amounts to approximately 70 % of the 'meta­bolically active' water component.

A secondary dormancy is not induced in C. lanatus seeds during exposure of these seeds to water stress for periods of as long as 144 h (Table 2). When seeds which were exposed to water stress are transferred to pure water , radicle emer­gence starts within 24 h. Although the germination of PEG­treated seeds is higher during the first 48 h than the mannitol-treated seeds, both PEG and mannitol-treated seeds yielded the same germination results from 72 h onwards. According to Khan (1960) and Khan & Karssen (1980) a secondary dormancy develops in lettuce and Chenopodium bonus-henricus seeds during exposure of these seeds to water stress.

Table 2 The effect of pre-treatment in different osmoticums on the subsequent germination of Citrullus lanatus seeds

% germination with water of the pre-treated seeds at 27°C in the

Seeds treated for 144 hat dark after a further :-27°C in the dark with one of the following solutions• Oh 24h 48h 96h

PEG (1JI = - 500kPa) 0 83 ± 8 92±4 96±3

PEG (1JI = -750kPa) 0 51± 15 82±8 96±4

Mannitol(1JI = -500kPa) 0 37 ± 12 56± 10 92 ± 3

Mannitol ( 1JI = -750 kPa) 0 21 ± 10 45 ± 16 96±2

a = solutions applied as in germination tests. None of the seeds

germinated during the pre-treatment

± = standard deviation

When seeds incubated with water for 24 h are transferred to an osmoticum with a 1/J of - 430 kPa, germination is inhibited by 72 % (Table 3). It is therefore obvious that water stress inhibits germination even if 24 h of normal development in the metabolism of C. lanatus seeds has taken place. This appears to be similar to the response shown by lettuce seeds to water stress (Khan 1960) .

Citrullus lanatus seeds are negatively photo blastic (Botha et al. 1982a). The germination of such seeds is taken to be

113

Table 3 The effect of different pre-treatments on the germination of Citrullus lanatus seeds with water and mannitol solutions

Pre-treatment at 27°C in the dark•

24h-water

24h - mannitol

(1JI= -430kPa)

24h-water

24 h- mannitol

(1JI= -430kPa)

24h

0

0

% germination with water at 27°C in the dark after:-

48h 72h

48 ± 5 75 ± 4

25 ± 8 65 ± 9

% germination with mannitol ( 1JI = -430 kPa) at 27°C in the dark after:-

24h 48h 72h

0 2±1 28 ±8

0 0 0

• = solutions applied as in germination tests ± = standard deviation

dependent on the production of Pfr from phytochrome intermediates (Kendrick 1976; Kendrick & Spruit 1977; Smith 1975) . According to Kendrick (1976) the conversion of meta-Rb and meta-Fb to Pfr is possible only in hydrated tissue. Prevention of sufficient phytochrome conversion couid therefore be an important mechanism by which water stress inhibits germination of C. lanatus seeds.

From the results in Table 4 , however, it appears that water stress does not prevent Pfr formation . Exposing osmotically stressed seeds to 1 h of far-red light inhibits germination by 75 % on subsequent transfer to water in the dark. The inhibitory effect of far-red light can be alleviated by a 1 h red light treatment after the far-red light treatment.

It is clear therefore that the seeds are sufficiently hydrated in the osmoticum ( 1/J = -430 kPa) which completely inhibits germination to allow the formation of Pfr from phytochrome intermediates as well as for pigment cycling from Pr ~ Pfr ~ Pr during red and far-red light treatments. Water stress

Table4 The effect of an osmoticum (1/J = -430 kPa) on the red, far-red light sensitivity of Citrullus lanatus seeds. Only 5% germination was obtained when the seeds were kept in the osmoticum in the dark for 98 h

Pre-treatment of the seeds while kept in the osmoticum at27°C

Continuous darkness for 26 h

24 h darkness and 1 h far-red

light and 1 h darkness 24 h darkness and 1 h red light and 1 h far-red light 24 h darkness and 1 h red light and 1 h darkness 24 h darkness and 1 h far-red

light and 1 h red light

± = standard deviation

% germination with water after

a further 72 h in the dark

95 ±4

25 ± 10

20±6

88 ± 6

65 ± 10

114

which inhibits the germination of Rumex crispus and Lactuca sativa seeds also apparently does not prevent phytochrome cycling during light treatments (Berrie & Drennan 1974; Duke 1978) .

From the present results it is concluded that the inhibitory effect of water stress on germination can not be attributed solely to the prevention of Pfr formation from phytochrome intermediates.

It has been proposed that for germination to occur a hypothetical component X is required to interact with Pfr (Smith 1975; Duke 1978) and that this interaction is very sensitive to water stress (Duke 1978).

In Citrullus lanatus seeds water stress apparently does not affect the formation or development of the hypothetical component X with which Pfr presumably interacts, as water stress remains inhibitory for germination after 'normal' development for 24 h in water (Table 3). It therefore appears more likely that the inhibitory effect of water stress on germination lies in the prevention of the interaction ofPfr with the hypothetical component X.

Acknowledgement

The authors wish to thank the University of Pretoria and the C.S.I.R. for financial support.

References

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