Scientia Horticulturae, 35 (1988) 15-26 15 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Effects of Fruit Age, Fermentat ion and Storage on Germinat ion of Cucurbit Seeds
HAIM NERSON and HARRY S. PARIS
Department of Vegetable Crops, Agricultural Research Organization, Newe Ya'ar Experiment Station, P.O. Haifa (Israel)
Contribution No. 1705-E, 1986 series, from the Agricultural Research Organization, Bet Dagan, Israel
( Accepted for publication 9 September 1987 )
ABSTRACT
Nerson, H. and Paris, H.S., 1988. Effects of fruit age, fermentation and storage on germination of cucurbit seeds. Scientia Hortic., 35: 15-26.
The effects of fruit age, fermentation and storage on germinability and leakage from seeds of one cultivar of each of four cucurbit crops, cucumber (Cucumis sativus L. cultivar 'Bet Alfa'), melon (Cucumis melo L. 'Noy Yizre'el'), watermelon (CitruUus lanatus (Thunb.) Matsum. & Nakai 'Sugar Baby') and squash (Cucurbita pepo L. 'Vegetable Spaghetti') were studied under controlled conditions. Generally, germinability was best from fruits which were 49-54 days past anthesis (dpa), the most mature fruits studied. Fermentation of seeds from immature and mature fruits of the cucumber, melon and watermelon was either beneficial, or at least not harmful. How- ever, fermentation of seeds from immature (28 dpa) fruits of the squash cultivar resulted in no germination. Storage for up to 48 months generally did not affect seeds from the most mature fruits. Storage adversely affected germination of seeds from immature (26-28 dpa) fruits of the cucumber, melon and squash, but favorably affected germination of seeds from immature (26 dpa) fruits of the watermelon. Excessive seed leakage and a relatively low ratio of embryo weight to seed coat weight were associated with poor germination.
Production of high-quality seeds of cucurbit crops depends on harvesting the fruits at the proper stage of maturity as well as on proper extraction and storage procedures. Generally, cucurbit fruits must be mature in order for the seeds to be most vigorous (Odland, 1937; Young, 1949; Sakr and Mahmoud,
1952; Holmes, 1953; Harrington, 1959; Goldbach, 1978; Wallerstein et al., 1981; Alvarenga et al., 1984), seeds from immature and over-mature fruits being inferior. Although fermentat ion of cucurbit seeds in order more easily to ex- tract them from the surrounding pulp is a generally accepted practice (Whi- taker and Davis, 1962), little information is available concerning the effect of fermentation on seed quality. Fermented melon seeds germinated better than non-fermented seeds (Harrington, 1959), but in cucumber no advantage was achieved by fermentat ion and an extended fermentat ion period proved harm- ful (Nienhuis and Lower, 1981). Storage of cucurbit seeds for several weeks or months has been found to be beneficial for germination ( Odland, 1937; Watts, 1938).
Evaluation of seed viability, germination and vigor has a long history, and new methods have been developed for more accurate prediction of seed-lot behavior under optimal or sub-optimal field conditions (McDonald, 1980; Matthews, 1981; Halmer and Bewley, 1984).
The purposes of the present study were to determine the effect of fruit age, fermentation and storage duration on germination percentage and rate of seeds from four cucurbit crops. In order to obtain a better understanding of how fruit age, fermentation and storage affect germinability, the size of seed compo- nents, amount of water imbibed, effect of the leachate exuded by soaked seeds on germination, and composition of the leachate were also examined.
MATERIALS AND METHODS
Material preparation. - Open-pollinated cultivars of four cucurbit crops, cuc- umber ( Cucumis sativus L. 'Bet Alfa' ), melon ( Cucumis melo L. 'Noy Yizre'el'), watermelon ( Citrullus lanatus ( T h u n b . ) Matsum & Nakai 'Sugar Baby' ) and squash (Cucurbita pepo L. 'Vegetable Spaghetti ') were grown in an isolated field at Newe Ya'ar (nor thern Israel) during the spring season of 1983. Ap- proximately 120 pistillate flowers of each species were tagged at anthesis, and 20 fruits which developed from them were harvested at 21, 28, 35 and 49 (cuc- umber and squash), or 26, 33, 40 and 54 (melon and watermelon) dpa. The seeds and the surrounding pulp and juice of each crop were collected and mixed in a container. About half of each seed lot was allowed to ferment in a closed plastic bag for 72 h at 25 ° C in the dark, while the other half was subjected to immediate cleaning, which consisted of separation of seeds from the pulp and juice and washing for 15 rain in running tap water. Clean seeds were dried for 72 h at room temperature ( 25 _+ 5 ° C ) and then stored at 10 ° C and 45% relative humidity. Fermented seeds underwent the same cleaning, drying and storage procedure.
Germination tests. - - The 32 seed lots ( 4 cucurbits, 4 fruit ages, fermented or not fermented) were subjected to germination tests immediately after drying
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and after 6, 12, 24, 36 and 48 months of storage. Germination was examined at a favorable temperature ( 25 ° C ) in the dark and the percentage of germinated seeds (radicle protrusion) was recorded after 2, 4 and 7 days of incubation. Germination rate was expressed as an index for mean days to germination and computed using the formula.
Index = $1 T1 -{- $2 T2 + ..... Sn Tn $1 + $2 + ..... Sn
where S is the number of germinated seeds and T is the incubation time in days (Loy and Evensen, 1979). In each germination test, four replicates of 25 seeds were germinated in a 9-cm-diameter petri-dish on Whatman No. 3 blot- ting paper moistened with 5-6 ml distilled water.
Seed size during fruit maturation. - - Seeds extracted, with or without fermen- tation, from fruits harvested at four different stages of maturity for each crop and stored for 24 months were sampled for seed coat and embryo size and water partitioning after imbibition. Dry seeds were dissected and their embryos and seed coats weighed separately. Other samples were weighed, imbibed for 24 h (25 ° C, dark) and then dissected to determine water absorption by embryos, seed coats and seed cavities (Nerson et al., 1985 ). Each sample consisted of 5 replicates of 10 seeds.
Effect of seed leachate on germination. - - After 24 months of storage, fermented and non-fermented melon and watermelon seeds extracted at 33 and 26 dpa, respectively, were soaked (5 g seeds per 100 ml distilled water) for 2-2 8 or 24 3, h at 25 °C in the dark. Four replicates of 25 seeds of commercial lots of the four cucurbit cultivars mentioned above were then incubated on blotting paper moistened with one of the 12 leachate solutions or water. In addition, the soaked (leachate producing) and non-soaked (control) seeds of the same four lots were germinated on moist blotting paper. Germination tests for all treatments were conducted at 25°C in the dark, and data were recorded daily during a week of incubation.
Seed leachate composition. ~ Four replicates of each of 31 seed lots (data miss- ing for 21-day-old fermented squash) were soaked in distilled water, 25 ml per g seeds, for 24 h at 25 ° C in the dark. Electrical conductivities of the leach- ates were measured using a type CDM-2e conductivity meter with a DCD-104 conductivity cell (Radiometer A.S. Danemark). K + and Na + in the leachates were determined by flame emission photometry (Perkin Elmer model 370 Atomic absorption spectrophotometer). Nucleic acids and protein concentra- tions were estimated from the ratio of A2so/A26o in the leachates ( Givelberg et al., 1984). Total soluble sugars in the leachates were determined using an- throne reagent (Johnson et al., 1964).
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Stat is t ical analysis. - - Analysis of variance was conducted for all the results and separation within cucurbits among treatments was by Duncan's multiple range test ( P = 0.01). Separations of percentage data were determined after arc sin transformation (Little, 1985).
RESULTS
I m m e d i a t e germina t ion . - - Seeds extracted from the most mature fruits gen- erally had the highest percentage germination and fastest germination rate (Tables I and II). Cucumber and squash seeds from the youngest fruits (21 dpa) did not germinate. In cucumber, 35-day-old fruits were sufficiently ma- ture for maximal germination percentage and rate. More time was required to obtain complete germination of the other 3 crops. Fermentation did not affect the germination percentage of seeds from the most mature fruits of cucumber, squash and melon, but did increase germination in watermelon. In seeds from immature (28 dpa) fruits, fermentation increased the germination of cucum- ber, but decreased germination of squash and 33 dpa melon seeds (Table I). Germination was accelerated by increased fruit maturity and by fermentation (Table II).
G e r m i n a t i o n al ter storage. - - Seeds from immature fruits of cucumber, melon and squash ( 28, 26 and 28-35 dpa, respectively) tended to lose their ability to germinate after storage (Table I). Seeds from fruits that were more mature either maintained or had increased germination after storage. Fermentation and storage of immature watermelon seeds resulted in much improved germination.
Germination rates of immature fermented cucumber and immature non- fermented squash seeds were improved after storage (Table II). Storage gen- erally increased the germination rate of melon and watermelon.
S e e d d e v e l o p m e n t a n d wa ter absorpt ion d u r i n g imbibi t ion. - - The whole seed and the embryo of cucumber and squash were still growing and accumulating dry matter at 28 dpa (Fig. 1 ). On the other hand, seeds and embryos of melon and watermelon had almost reached their maximal dry weight by 26 dpa. Over- all, fermentation tended to decrease seed and embryo weight of each species except squash.
Embryo weight/coat weight ratios in dry seeds increased with fruit age in cucumber and squash, but remained unchanged in melon and watermelon (Fig. 1 ). Water absorption by immature growing seeds and their components in each species were higher than in mature seeds (Table III ). The 4 cucurbits differed markedly in water partitioning among embryo, coat and cavity. Water uptake by the watermelon embryo was less than that of the other 3 crops.
Fermentation did not significantly affect either the embryo weight/coat
F i g . 1. Growth of whole cucurbit seeds, their embryos and their coats during fruit maturation. Each of the numbers on the right is the standard error of 8 treatments ( 4 fruit ages, fermented or non-fermented).
weight ratio or the water absorption by the seed components (data not pre- sented). However, in most cases more water was accumulated in seed cavities of non-fermented seeds as compared with fermented seeds (Table III).
G e r m i n a t i o n in s e e d leachate . - - Non-fermented watermelon seed leachate de- creased germination rate of squash and watermelon seeds and germination percentage of melon seeds (data not presented). Germination percentage of pre-soaked non-fermented watermelon seeds was improved significantly over the non-soaked control (pre-soaked 33%, non-soaked 11%). Melon non-fer- mented seed leachate did not affect the germination of any cucurbit seeds, and the germination of pre-soaked non-fermented melon seeds was not improved over the non-soaked control (data not presented).
S e e d l eacha te c o m p o s i t i o n . - - Electrical conductivity (Table IV) and leakage of K +, Na + (data not presented) and organic compounds (Table IV) de-
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TABLE III
Effects of fruit age on water absorption (24 h of imbibition) by the embryo and seed coat (mg H20/mg dry weight) and by the seed cavity (mg H20/seed) in 2-year-old seeds of 4 cucurbits. Water absorption for embryos and seed coats was calculated as the means of fermented and non- fermented seeds. - F = w i t h o u t fermentation; + F = w i t h fermentation
Cucurbit Fruit Water absorption maturity (dpa) Embryo Coat Cavity
- F + F
Cucumber 21 1.07 a 1.49 a 21.1 a 17.7 b 28 0.44 c 1.01 b 14.3 c 6.1 de 35 0.59 bc 0.65 c 7.2 d 5.2 e 49 0.63 b 0.58 c 5.9 de 5.0 e
Squash 21 1.02 a 2.35 a 37.3 a 29.4 b 28 0.52 b 2.46 a 21.5 cd 19.0 de 35 0.44 b 1.78 b 23.0 c 16.6 ef 49 0.49 b 1.65 b 14.9 f 10.7 g
Melon 26 0.73 a 1.04 a 8.3 a 5.9 b 33 0.54 b 0.94 a 5.6 bc 5.5 bc 40 0.49 b 0.89 ab 5.6 bc 4.9 bcd 54 0.57 b 0.72 b 3.5 d 4.4 cd
Watermelon 26 0.35 a 0.86 a 10.8 a 8.5 b 33 0.37 a 0.82 a 8.2 b 5.8 c 40 0.33 a 0.84 a 7.6 b 7.4 b 54 0.33 a 0.89 a 7.0 bc 7.5 b
creased with increasing fruit age. Fermentation of seeds during extraction sig- nificantly decreased the electrical conductivity in leachates from 21-28 dpa seeds of cucumber, squash and melon and in leachates from all seeds of water- melon. Potassium content of leachates corresponded closely with electrical conductivity values, but no relationship was evident between sodium content and electrical conductivity (data not presented). Fermentation decreased leakage of proteins, nucleic acids and soluble sugars from immature seeds of cucumber, melon and watermelon. In squash, fermentation also decreased leakage of proteins and nucleic acids, but increased leakage of soluble sugars.
DISCUSSION
Seeds from the most mature fruits (49-54 dpa) generally had a high ger- mination percentage and fast germination rate, which was retained for 4 years ( Tables I and II ). As seeds from over-mature fruits can have impaired germin- ability (Odland, 1937; Goldbach, 1978), a fruit age of approximately 50 dpa
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T A B L E IV
Effects of fruit maturity and seed fermentation on seed leachate composition of 4 cucurbits. - F = without fermentation; + F = with fermentation
Cucurbit Fru i t Electrical Proteins Nucleic acids Soluble sugars maturity conductivity (mgm1-1) (mgml 1) (mgl 1)
(dpa) (micro MHO c m - 1 )
- F + F - F + F - F + F - F + F
Cucumber 21 1940 a 905 b 10.8 a 8.0 b
28 445 c 146 d 5.9 c 3.2 d
35 l l 0 e 7 3 f 4 .1cd 2 .9d
49 4 9 f 5 6 f 2 .4d 3 .0d
Squash 21 2085 a - - 19.4 a - -
28 900 b 640 c 9.5 b 6.6 c
35 4 0 5 d 4 3 8 d 6.5c 4 .3cd
49 145 e 183 e 3.5 d 3.9 cd
Melon 26 624 a 424 b 5.8 a 4.4 ab
33 180d 343c 3 .7b 3 .5b
40 102e 196d 3 .5b 3 .4b
54 206d 298c 3 .9b 3 .1b
Watermelon 26 390 a 100 de 24.7 a 6.5 cd
33 231b 142cd 12.8b 8.2 c
40 178 bc 72 e 6.4 cd 3.5 e
54 148 cd 87 e 5.3 de 3.5 e
0.78 a 0.46 b 182 a 85 b
0.27 c 0.14 de 38 c 21 d
0.19 d 0.12 e 19 d 13 e
0.10 e 0.13 e 12 e 14 e
0.88 a - - 259 a - -
0.43 b 0.33 c 100 c 163 b
0.25 d 0.18 e 31 e 41 d
0.13 e 0.17 e 19 f 29 e
0 .28a 0.18b 2 7 a 16b
0 .16b 0.14b 14bc 8 b c d
0 .16b 0 .13b 6 c d 10bcd
0 .17b 0 .12b 3 d 13bc
0.16 abc 0.10 c 484 a 115 c
0 .19ab 0 .20ab 224b 114c
0 .19ab ~).12bc l l 8 c 15e
0.22 a 0.13 bc 44 d 21 e
1No seeds available.
can generally be considered ideal for seed harvest of field-grown cucurbits. The poor immediate germinability of cucumber and squash seeds from young fruits (28 and 28-35 dpa, respectively) and the rapid decline of germinability of these seed lots after several months of storage are probably related to the seeds being harvested while their embryos were still developing (Fig. 1 ). Similarly, soybean seeds 42 and 46 days after flowering had still-growing and final-size embryonic axes, and 20 and 95% germination, respectively, (Oberdorf et al., 1980 ). In contrast, melon and watermelon embryos almost reached their max- imal dry weight as early as 26 dpa and had markedly improved germination rates and percentages after storage. Embryo weight/coat weight ratios may be a useful indicator of seed maturation and germinability in each of the 4 cucur- bits (Fig. 1 ). Cucumber seeds with an embryo weight/coat weight ratio of less that 3.0 had poor germination. For seeds of squash, melon and watermelon, minimum ratios for good germination were 2.5, 1.7 and 1.0, respectively. The
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amount of water absorbed by the seed during imbibition is also a good indicator of seed maturation and germinability; immature seeds absorbed considerablay more water than mature seeds (Table III).
Fermentation had little or no effect on germination percentage, but in some cases it increased germination rate of seeds from mature (49-54 dpa) fruits (Tables I and II). In seeds from younger fruits, fermentation had different effects in the different cucurbits. The present data are based on only one cul- tivar per species and thus are not unequivocal, but from these and other results (H. Nerson, unpublished data) it appears that fermentation not only facili- tates seed extraction, but is also of benefit for improving germinability of seeds extracted from immature fruits of cucumber, melon and watermelon. As cu- curbits are usually harvested once-over when grown for seeds, and in the field there are fully mature as well as immature fruits, it appears that fermentation would improve overall germinability of seed lots of these three crops. On the other hand, fermentation as an aid to extraction of squash seeds should be employed only with fully mature fruits.
Storage generally decreased germinability of immature ( 28-35 dpa) squash and ( 28 dpa) cucumber seeds and the least mature (26 dpa) melon seeds ( Ta- ble I). More mature seeds of melon and seeds of watermelon generally had a better germination percentage and rate after several months of storage ( Tables I and II). Percentage germination of unstored, fully mature or nearly mature (40-54 dpa) seeds was lowest in the melon, suggesting that in the cultivar used, at least, seeds undergo dormancy. Dormancy has been noted for seeds of a cucumber plant introduced from India (Shifriss and George, 1965) and for other Cucumis species (Odland, 1937; Watts, 1938; Heit et al., 1978). In wa- termelon, another mode of germination inhibition may have occurred; in this crop fermentation enhanced germination in almost all circumstances (Tables I and II ). Moreover, water-soaking of non-fermented seeds, extracted from 26 dpa fruits and stored for 2 years, increased their germinability, while the leach- ates decreased the germination rate of commercial seed lots of watermelon and squash (data not shown). The results here, which are similar to an earlier report on oats ( Haggquist et al., 1984 ), suggest the existence of a water-soluble inhibitor in the immature watermelon seeds which can be neutralized by fer- mentation and/or long storage.
The results of the present study show that fermentation can have several effects on cucurbit seeds, beyond being a technical process for easier extraction and cleaning. Fermentation decreased embryo dry weight in seeds extracted from immature cucumber, melon and watermelon fruits (Fig. 1). Fermenta- tion also decreased the amount of water imbibed between the embryo and seed coat ( Table III ). Increased germination percentage and rate (Tables I and II) and reduced leakage ( Table IV ) following fermentation presumably reflect the removal of unidentified inhibitors and increased membrane integrity.
New methods have been adopted for more accurate prediction of seed via-
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bility under optimal and sub-optimal conditions. In particular, analysis of seed leachate composition has been useful in predicting seed-lot performance under field conditions (Matthews and Bradnock, 1968) as well as becoming increas- ingly common as a tool in seed physiology research (Givelberg et al., 1984). From a comparison of the data in Table IV with those in Tables I and II, leak- age during imbibition appears to be a reliable indicator of germinability. Fur- ther investigation is required to determine whether poor germination results from much leakage of important metabolites ( Givelberg et al., 1986), or whether high leakage and poor germination of cucurbit seeds are coincidental, without a cause-and-effect relationship.
ACKNOWLEDGEMENTS
The authors thank Dr. A. Givelberg of the Department of Weed Science at Newe Ya'ar for her helpful suggestions concerning the manuscript, and Y. Burger, M. Edelstein and A. Govers of the Department of Vegetable Crops for dedicated technical assistance.
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