Temperature mediated physiological dormancy in dry bitter gourd (Momordica charantia) seeds

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1 Bopper and Kruse (2018). Seed Science and Technology, 46, 1, Temperature mediated physiological dormancy in dry bitter gourd (Momordica charantia) seeds Sebastian Bopper and Michael Kruse Division of Seed Science and Technology, University of Hohenheim, Stuttgart, Germany ( (Submitted January 2017; Accepted August 2017; Published online December 2017) Abstract Bitter gourd (Momordica charantia) is a vegetable cultivated predominantly in tropical and subtropical Asia for its immature fruits and tender shoot tips. The Royal Botanic Gardens Kew Seed Information Database indicates that bitter gourd seeds are orthodox, thus tolerating desiccation and sub-zero temperature. However, genebanks have reported low germination after storage at sub-zero temperatures whereas storage at above 5 C resulted in acceptable germination. The objectives of this study were to evaluate the effect of storage temperature on the germination behaviour of the seeds and, as a practical outcome, to develop a successful dormancy breaking protocol for bitter gourd seeds after cold storage. Results of standard viability and germination tests of two bitter gourd seed lots confirmed that storage of dry seeds at sub-zero temperatures for more than five minutes induced a secondary dormancy in the embryo. A heat treatment of dry seeds at 50 C for at least two minutes could break this dormancy. Temperature cycling showed that dormancy induction and breakage were reversible without any measurable decline in viability. As temperature treatments did not affect seed imbibition behaviour, the mechanism is not hardseededness, but a type of physiological dormancy. Keywords: bitter gourd, dormancy, Momordica charantia, seed storage Introduction Momordica charantia L., bitter gourd, is a member of the cucurbit family. The vegetable is cultivated for its immature fruits and tender shoot tips (Vogel, 1996) and is also used in traditional African and Chinese medicine (Grover and Yadav, 2004), as well as in modern medicine where the focus is on its presumed beneficial effect in the treatment of diabetes (Ahmed et al., 2004). In the past, bitter gourd breeding mainly comprised the selection of suitable landraces on a semi-professional level. During the last decades, professional breeding programmes have been established because of the increasing attractiveness of the species for nutrition and drugs (Behera et al., 2010). Consequently, breeders increasingly turn to genebanks to get access to greater diversity to attain their breeding goals Bopper et al. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: 19

2 SEBASTIAN BOPPER AND MICHAEL KRUSE The seeds of bitter gourd are classified as orthodox (Doijode, 2001; Royal Botanic Gardens Kew, 2016), thus, seeds are expected to survive long-term dry storage at subzero temperatures (Hong and Ellis, 1996). For bitter gourd, rather short longevities have been reported of either a few years (Ullmann, 1949; Priestley, 1986) or not more than 12 months (Yeh et al., 2005; Fonseka and Fonseka, 2011). Surprisingly, some genebanks reported no germination of bitter gourd seed samples stored at sub-zero temperatures for six months, whereas unchanged germination percentages could be obtained after storage at 5 C (Ebert and Huang, 2015). The authors of this latter study assumed that freezing killed the dry seeds of this tropical species. This would not be in line with an orthodox storage type. However, in principle, secondary dormancy might have been induced by cool temperatures, although this is not observed for dry seeds in many species (Bewley and Black, 1982). Bharathi and John (2013) reported primary dormancy in some Momordica species, but not in M. charantia. Baskin and Baskin (2014) inferred a physiological dormancy (PD) for bitter gourd. Pandey et al. (2013) reported physical dormancy (PY) in the related species, M. cochinchinensis L. In the case of M. charantia, ISTA (2017) gives no recommendation for breaking dormancy but prescribes alternating temperatures of 20/30 C or constant temperatures of 30 C as the optimum temperature for germination. Bharathi and John (2013) recommend a constant temperature between 25 and 30 C. They also reported diverse methods for breaking dormancy from simple soaking to gut enzyme scarification. The objective of this study was to clarify the status of bitter gourd seeds after storage at sub-zero temperatures and, if seeds are dormant, to identify the dormancy mechanism and a suitable dormancy breaking treatment. Materials and methods Seed material Seeds of two accessions of bitter gourd from India (VI049940) and Thailand (VI049009) were supplied in 2015 by the AVRDC The World Vegetable Center in Taiwan (GENESYS Global Portal, 2016). The coding in this study is lot A (VI049940) and lot B (VI049009). The two seed lots were produced by the AVRDC in the growing seasons between 2012 and 2014 and stored after receipt at the University of Hohenheim at between 18 and 20 C in aluminium foil bags prior to starting the experiments. Thousand seed mass (TSM), seed moisture content (SMC) and mass of embryo and seed coat The TSM and the SMC were determined as described by the ISTA Rules (ISTA, 2017). For determining embryo and seed coat masses and their proportions, 25 seeds were randomly taken per lot and separated into both components. The mass percentages per components were determined. Furthermore, the moisture content of both components was determined according to ISTA Rules (ISTA, 2017). 20

3 PHYSIOLOGICAL DORMANCY IN BITTER GOURD SEEDS Determination of fat content and fatty acid composition The fat content of the embryo was determined as well as the fatty acid composition. Random samples of both lots were taken, the seed coats were removed and about 9 g of naked embryos were used for the analyses. The analyses were done by the Landesanstalt für Landwirtschaftliche Chemie of the University of Hohenheim, Germany. The method described by the European Commission Regulation (EC) No 152/2009 III H was used to determine fat content and the in-house method LA Chemie (P ) based on a fatty acid methyl ester (FAME) standard with 37 components was used to determine the fatty acid composition. Tetrazolium test For testing the viability of the seeds, a tetrazolium (TTC) test was done following the principles of the ISTA Rules (ISTA, 2017). Seeds were imbibed in water at 20 C for 18 hours, then the seed coat and the endosperm layer were removed and the upper third of the cotyledons were cut off. Prepared embryos were incubated in a ph-buffered 1% TTC solution at 30 C for 18 hours. Embryos were classified as viable if the radicle was completely stained and not more than 50% of the distal end of the cotyledons remained unstained. Germination tests For all germination tests, seeds were disinfected by submerging them for 10 minutes in a 0.4% (w/v) a.i. thiram solution. The solution was prepared by a dilution of the commercial product Aatiram 65 (Cheminova Deutschland, Germany). After this treatment, the seeds were rinsed and transferred to the germination test without drying. Germination tests were done in rolled paper towels at constant 25 C and permanent light. Seeds were counted as germinated when their root was longer than 2 mm. The total number of seeds per test is indicated in the results. If the number of seeds was 50, the number was divided into at least two replicates with an equal number of seeds. Seed storage Sub-samples of dry seeds were hermetically sealed in aluminium foil bags from which as much air as possible was removed before closing. The foil bags were then stored at between 5 and -20 C in laboratory bath cryostats or in a freezer for between 15 seconds and seven days. If several bags were handled at the same time, care was taken that they did not attach to each other to ensure that the bags cooled evenly and quickly. Heat treatment Sub-samples of dry seeds in hermetically-sealed aluminium foil bags were kept in a laboratory water bath at between 32 and 50 C. A water bath was used because the high heat capacity of the water allows quick heating of the seeds without a measurable deviation from the temperature setting. Again, if several bags were handled at the same time, care was taken that they were not in contact with each other. When samples were already stored at sub-zero temperatures, samples were kept for at least 24 hours at room temperature before transferring them to the heat treatment without opening the foil bags. 21

4 SEBASTIAN BOPPER AND MICHAEL KRUSE Duration of the heat treatment started when the bags entered the water and ended when bags were removed from the water. Combinations of cold storage and heat treatment In a first experiment, seed samples were stored at -20 C for four days, followed by two days storage at room temperature. Then, samples were heat treated at 50 C for periods between 0 (no heat treatment) and 60 minutes. Afterwards, a germination test together with control seeds was performed. In a second experiment, seeds were stored at -20 C for four days, followed by two days storage at room temperature. Then, samples were heat-treated at 50 C for eight minutes followed by one day storage at room temperature. This procedure was repeated five times to seed samples in sequence and during each cycle, two sub-samples were taken for germination tests, one after the cold storage and one after the heat treatment. During all cycles, seeds were dry and sealed hermetically in foil bags from the beginning up to the start of the germination test. In a third experiment, one sample was cold-stored for four days and another sample was cold-stored and heat-treated as described in experiment 2. After three days in the germination test, seed coats were taken off and the moisture content of the embryos was determined. In addition, the moisture content of embryos in dry control seeds was determined. In a fourth experiment, the seed coat was removed from two sub-samples of 25 seeds per lot and only the naked embryos were used. Another four sub-samples of 25 seeds were kept intact. All sub-samples were stored at -20 C for four days. Thereafter, from the naked embryos, one sub-sample was directly transferred to a germination test, the other sub-sample was heat-treated at 50 C for eight minutes before starting the germination test. From the four samples containing intact stored seeds, the first sample was directly transferred to the germination test after the four days at -20 C, the second sample was heat-treated at 50 C for eight minutes before starting the germination test. From the seeds of the third and fourth sub-samples, the seed coats were removed after cold storage; the third sample was directly transferred to the germination test and the fourth sub-sample was heat-treated at 50 C for eight minutes before starting the germination test. In a fifth experiment, seeds were scarified with a lancet opposite the radicle at the seam so that the two parts of the coat did not fall apart. Scarified and non-scarified seeds were then stored at -20 C for four days and thereafter transferred to a germination test. Statistics Where indicated, the differences between two binomial data points where tested by a chisquared test. This test was generated using SAS software (SAS Institute Inc., Cary, NC, USA). 22

5 PHYSIOLOGICAL DORMANCY IN BITTER GOURD SEEDS Results The initial germination of seed lots A and B was 99 and 45%, respectively, and the TSM was 241 and 53 g, respectively. Both seed lots contain ripe and fully developed seeds and had the same initial SMC of 6.4%. In both seed lots, about 40% of the seed mass is seed coat and 60% is embryonic tissue. For lots A and B, the moisture content of the coat was 8.2 and 9.7%, respectively, and the moisture content of the embryo was 5.6 and 5.8%, respectively. The raw fat content in the embryonic tissue of seed lots A and B was and 49.80%, respectively. Fatty acids with a concentration higher than 1% were stearic acid (C 18:0 ) at and 39.82% for lots A and B, respectively; palmitic acid (C 16:0 ) at 1.90 and 2.00%; oleic acid (C 18:1n9c ) at 1.3 and 1.70%; and linoleic acid (C 18:2n6c ) at 3.10 and 4.30%. The raw fat fraction also contained a 38 th unknown component, not part of the FAME standard, with a very high proportion within the raw fat fraction of and 50.90% for seed lots A and B, respectively. Impact of cold storage on germination Samples of 50 seeds from both seed lots were stored for seven days at temperatures between -20 and 5 C and in addition at room temperature. The proportion of germinated seeds after storage decreased with decreasing storage temperature (table 1). In particular, from 5 to -5 C, the germination percentage dropped drastically: from 98 to 8% in lot A and from 50 to 0% in lot B. The non-germinated seeds at the end of the germination tests were tested for viability in a tetrazolium test. The sum of germinated seeds plus nongerminated but viable seeds was always within the range of 92 96% in seed lot A and 40 56% in seed lot B. Table 1. Impact of different temperatures of storage of dry bitter gourd seeds for seven days on the proportion of germinated seeds (n = 50 seeds per germination test). Storage temperature ( C) Lot A Germinated seeds (%) Lot B Room temperature Samples of 100 or 50 seeds were stored at -20 C for periods between 15 seconds and 10 minutes. Periods longer than five minutes resulted in a decline in germination, from 99 to 0% in seed lot A and from 63 to 0% in seed lot B (figure 1A). Both declines are significant (chi-squared test with one degree of freedom, P ). The start of reduction of germination was already apparent after two minutes in the case of lot A and after 45 seconds in the case of lot B. 23

6 SEBASTIAN BOPPER AND MICHAEL KRUSE (A) (B) Germinated seeds (%) Germinated seeds (%) seed lot A seed lot B 0 Initial Duration (minutes) Duration (minutes) Figure 1. Impact of duration of (A) cold storage of dry bitter gourd seeds at -20 C and (B) heat treatment of dry seeds at 50 C after four days cold storage on the proportion of germinated seeds (n = 100 seeds per germination test, except in (B) for duration 10 minutes where n = 50). In addition, the initial germination is indicated by open symbols in (A). Impact of heat treatment after cold storage In the first experiment, seeds were stored for four days at -20 C and then heat-treated at 50 C for different durations. Heat treatment of dry seeds in the foil bags for only two minutes raised the percentage of germinated seeds in seed lot A from 0 to nearly 100% and in seed lot B from 0 to nearly 65% (figure 1B). Both increases are significant (chisquared test with one degree of freedom, P ). In seed lot A, a heat treatment for up to 60 minutes had no negative impact on germination, whereas in lot B, a heat treatment for longer than 10 minutes decreased the germination down to nearly 0%, when seeds were treated for 60 minutes. Here, all non-germinated seeds showed decayed tissue and were obviously dead. The second experiment comprised five replications of cold storage and heat treatment in a sequence. In all five subsequent cycles, the germination percentages after cold storage ranged from 0 to 8% in both seed lots (table 2). After the heat treatments, the germination percentages were within the range 90 to 100% in seed lot A and 26 to 36% in seed lot B. In seed lot A, the range includes the initial germination percentage of 98%, in seed lot B the range is below the initial germination percentage of 45%. The third experiment showed that the moisture content increased during the 3-day imbibition period, irrespective of whether a heat treatment was applied or not. In lot A, the initial moisture content of the dry embryos, the moisture content of the embryos stored at -20 C and the moisture content of embryos stored at -20 C and heat-treated were 5.6, 30.1 and 38.8%, respectively. In lot B the moisture contents were 5.8, 25.2 and 25.8%, respectively. 24

7 PHYSIOLOGICAL DORMANCY IN BITTER GOURD SEEDS Table 2. Impact of five temperature cycles of cold storage and heat treatment on the proportion of germinated bitter gourd seeds (number of seeds per germination test, n = 50). Germinated seeds (%) Cycle no. Seed lot Control Temperature step ( C) of a cycle A B The fourth experiment with different points in time of decoating showed (table 3) that the presence or non-presence of the seed coat during the cold storage, the heat treatment or the germination test did not affect the germination test result. The fifth experiment revealed in both seed lots that the scarification had no impact on germination after storage at -20 C, since all seeds, scarified and non-scarified, did not germinate. Table 3. Impact of seed coat removal (decoating), cold storage (four days storage at -20 C) and heat treatment (50 C for eight minutes) in different sequences on germination capacity and viability of bitter gourd seeds (number of seeds per test, n = 25). Seed treatment(s) before starting germination test Germination A Germination + viability Seed lot Proportion (%) Germination B Germination + viability Cold storage Decoating + cold storage Cold storage + decoating Cold storage + heat treatment Decoating + cold storage + heat treatment Cold storage + decoating + heat treatment Discussion The increasing interest in bitter gourd genetic resources has resulted in an increase in the number of accessions stored in genebanks. Results of germination tests after comparatively short storage periods of less than one year under sub-zero temperatures have shown disappointing results, even though the seeds were stored as other orthodox seeds, under sub-zero temperature at low SMC (Anonymous, 2017). Authors therefore 25

8 SEBASTIAN BOPPER AND MICHAEL KRUSE questioned the orthodox classification of the species (Ebert and Huang, 2015). Our results show that bitter gourd seeds are not cold sensitive, as they were not killed by freezing but acquired secondary dormancy. Keeping seeds at -20 C for only five minutes already induced this secondary dormancy completely in all seeds tested (figure 1A). A period of only 45 seconds at -20 C caused germination to decrease from 63 to 16% for lot B. The results shown in table 1 were obtained by transferring seeds directly to the indicated temperatures. So, freezing and thawing were very fast. In a separate experiment, slow freezing and thawing with rates of -0.1 and 0.1 C minute -1, respectively, were applied to reach the intended storage temperatures. The results suggest that there is no influence of the freezing and thawing rate on the percentage of germinated seeds after storage (data not shown). The heat treatment at 50 C was overall very efficient in recovering germination after cold storage. Nevertheless, the results for the two lots differed. In seed lot A, the germination after a heat treatment for two minutes or longer was as high as before the cold storage. There was no deterioration effect detected with increasing heat treatment duration up to 60 minutes. In seed lot B, the heat treatment after cold storage was able to raise germination above the initial value when the duration was 10 minutes or shorter (figure 1B). However, above that range, germination decreased with increasing duration and got close to 0% with 60 minutes heat treatment. Viability tests showed that in seed lot B, seeds decayed with heat treatments at 50 C longer than 10 minutes. Thus, seed lot B is more sensitive to heat treatments than seed lot A, which might be due to the low initial quality of seed lot B. Induction of secondary dormancy by low temperatures has seldom been described (Popcov, 1935; Nikolaeva, 1969; Bewley and Black, 1982). Many authors report dormancy induced by supra-optimal temperatures and call this phaenomenon thermo-dormancy (Keys et al., 1975; Bewley et al., 2006, 2012; Baskin and Baskin, 2014). Thermodormancy induced by high temperature can be broken by chilling at low temperatures for many species. However, in bitter gourd just the opposite is observed: low temperatures induce dormancy and high temperatures break dormancy. Therefore, bitter gourd dormancy could be an example of a new dormancy type that could be called cold-induced dormancy, which can be broken by warm-stratification, which is already described as a method of artificial after-ripening for breaking a primary dormancy in grasses or rice (ISTA, 2017). Interestingly, the sub-zero temperatures can induce dormancy in dry seeds whereas chilling for breaking dormancy is only effective at above-zero temperatures and in imbibed seeds. Thus, the mechanisms of cold-induced dormancy and chilling for breaking dormancy are different. Physical dormancy, i.e. hardseededness, was reported for many species of the genus Momordica (Bharathi and John, 2013). However, our results clearly show that seeds of M. charantia show no hardseededness. Imbibition in bitter gourd seeds is slow in contrast to seeds of other crops because of the relative thick seed coat as we have shown by the seed coat mass. Thus, pre-treatments supporting water uptake are often recommended including imbibition in water for several days at room or elevated temperatures (Lin and Sung, 2001; Hsu et al., 2003). Those treatments will increase the speed of water uptake but this slow uptake can only happen if seeds are not hardseeded. 26

9 PHYSIOLOGICAL DORMANCY IN BITTER GOURD SEEDS The firm seed coat could also generate a mechanical dormancy, i.e. the high resistance force of the seed coat is hindering the embryo from emerging from the seed. However, we found that naked embryos could become dormant during a cold storage, so the presence and intactness of the seed coat is not the dormancy mechanism. Lin and Sung (2001) studied the influence of different treatments on the puncture resistance force in the zone of the hilum with the focus on germination. They concluded that the different observed mechanical restrictions exerted by the seed coat do not regulate the germination response of bitter gourd. This is further evidence that the seed coat is not involved in the dormancy mechanism in bitter gourd, but that there is a kind of endogenous dormancy. Since the mechanism of inducing dormancy is active in dry seeds and happens very quickly (in less than two minutes at -20 C), it must be a kind of chemical or physical switch. It could be that there is a conformational change of molecules or membranes when seed temperature gets below 0 C and changed back at 50 C. This assumption is supported by the observation that the induction and breakage of dormancy in dry seeds can be repeated several times without any imbibition in-between and without any major loss in the effect of either (table 3). In the context of a conformational change, there are two options: either an essential part of the germination process is switched off by a conformational change; or another molecule, which is not essential for germination, blocks the germination process due to a conformational change. Further investigations are needed to answer the question where this dormancy switch is physiologically located and how this switch works. Here, a study by Crane et al. (2003) showed noticeable parallels to our work and gave a basis for further investigations of the dormancy mechanism in bitter gourd. The authors showed that seeds of some Cuphea species did not germinate after storage at -18 C and that a brief heat pulse of 45 C could unblock the germination process. This phenomenon was attributed to the unique lipid composition of these seeds. The seeds had high concentrations of lauric acid (C 12 ) and/or myristic acid (C 14 ). During storage at sub-zero temperature, the lipids solidified and so, before germination could commence, it was necessary to melt these lipids in the dry seeds. In our samples, lauric acid was not determinable in either lot and the concentration of myristic acid was only 0.02%. It was assumed that the 38 th unknown component of the fatty acid pattern is a type of a triterpene glycoside, which seems likely because the bitterness of the fruit is buildup of the triterpene glycoside momordicine. Therefore, this triterpene glycoside in bitter gourd could be a candidate to cause the same characteristics as lauric and myristic acid in Cuphea. This is supported by the observation that the extracted crude fat of the embryos showed crystallisation during cooling down to room temperature, like the lipids investigated by Crane et al. (2003). Unfortunately, the melting point of the crude fat fraction was not determined. This could have giving an indication of the relevance of the fat fraction for the germination behaviour, in particular, if coinciding with the minimum temperature effective in the heat treatment. This leads to the general question, whether the phenomenon described here is a chemical or biophysical reaction to environmental conditions with the side effect on germination, or whether this behaviour has a biological, ecological meaning and is therefore a form of secondary dormancy. By looking at the climatic requirements for crop production of 27

10 SEBASTIAN BOPPER AND MICHAEL KRUSE Momordica species, it is reported that annual species of the genus Momordica are killed by frost and, in particular, bitter gourd requires a minimum temperature of 18 C during early growth (Bharathi and John, 2013). Therefore, we see the phenomenon described here for bitter gourd seeds as being in line with the growing conditions of that species and, therefore, not only as a biophysical phenomenon but also a type of secondary dormancy. The fact that the seed coat is not involved in the dormancy mechanism is very clear for lot A (table 3), but it seems that decoating leads to higher germination percentages in the case of lot B. The initial germination percentage of lot B was 45% tested with four samples of 100 seeds according to ISTA (2017), so this is representing the best estimate of the true value in this study. However, by means of the beta distribution it is possible to calculate a binomial proportion confidence interval for this scenario with a typical number of 50 seeds for a germination test used in this study. The 95% confidence interval is between the lower endpoint of 31% and the upper endpoint of 60%. Therefore, the medium quality of seed lot B and the small number of seeds used for germination experiments means there is a wide confidence interval and hence greater variance within the results of lot B, if germination tests are done with non-dormant seeds. Further, the results of the fourth experiment (table 3) suggests that decoating is beneficial in the case of lot B. Here, germination percentage values are above the prior discussed confidence interval. However, we also observed higher germination percentages up to 71% for lot B, if seeds were not decoated but heat-treated (figure 1B). So, it is not a general rule that decoating is of great advantage. In fact, we believe that in lot B, a certain level of dormancy was present when starting the experiment. Because dormancy induction by subzero temperatures was so effective and due to the shipment of both lots from Taiwan to Germany by airplane, it is possible that lot B be was exposed to cool temperatures during transport. Our assumption is similar to the findings of Lin and Sung (2001). They studied the effect of priming on germination under sub-optimal temperatures. Beside an observed general positive effect of priming, they assumed the reason for a higher proportion of germinated seeds after priming was that dormancy was present in their material from the start of their experiments. In addition, they postulated that the positive effect of warm water soaking on germination, often promoted by other authors, is based on the potential to break dormancy. We recommend that seeds are checked for the presence of dormancy before applying any priming pre-treatment and if dormancy is detected, to apply the heat treatment as described here first. This example of cold-induced dormancy during seed storage is an evidence that failure to germinate after cold storage is not necessarily due to loss of viability. In our study, we clarified that seeds of bitter gourd pass into a state of secondary dormancy during storage at sub-zero temperatures and low SMC, and such dormant seeds can be detected only by a viability test. We could not clarify the physiological basis of this dormancy, but we have shown that this dormancy is switched on and off at certain temperatures. Last, we identified a heat treatment as a simple and suitable dormancy breaking treatment. 28

11 PHYSIOLOGICAL DORMANCY IN BITTER GOURD SEEDS Acknowledgements We are very thankful to Mr. A.W. Ebert from the AVRDC The World Vegetable Center for providing the seeds and Mrs. M. Wacker who did many initial experiments, which were the basis of this study. References Ahmed, I., Adeghate, E., Cummings, E., Sharma, A.K. and Singh, J. (2004). Beneficial effects and mechanism of action of Momordica charantia juice in the treatment of streptozotocin-induced diabetes mellitus in rat. Molecular and Cellular Biochemistry, 261, Anonymous (2017). Storage Solutions for Indigenous Vegetable Seeds. < php?a=3143> (accessed 12 January 2017). Baskin, C.C. and Baskin, J.M. (2014). Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination, Academic Press, Amsterdam, Heidelberg. Behera, T.K., Behera, S., Bharathi, L.K., John, K.J., Simon, P.W. and Staub, J.E. (2010). Bitter gourd: Botany, horticulture, breeding. In Horticultural Reviews, Volume 37, (ed. J. Janick), pp , John Wiley & Sons, Inc., Hoboken, NJ, USA. Bewley, J.D. and Black, M. (1982). Physiology and Biochemistry of Seeds in Relation to Germination: Volume 2: Viability, Dormancy, and Environmental Control, Springer-Verlag, Berlin. Bewley, J.D., Black, M. and Halmer, P. (2006). Thermodormancy. In The Encyclopedia of Seeds: Science, Technology and Uses, (eds. J.D. Bewley, M. Black and P. Halmer), CABI, Wallingford. Bewley, J.D., Bradford, K., Hilhorst, H. and Nonogaki, H. (2012). Seeds: Physiology of Development, Germination and Dormancy, 3 rd Edition, Springer, New York. Bharathi, L.K. and John, K.J. (2013). Momordica Genus in Asia: An Overview, Springer, New Delhi, New York. Crane, J., Miller, A.L., van Roekel, J.W. and Walters, C. (2003). Triacylglycerols determine the unusual storage physiology of Cuphea seed. Planta, 217, doi.org/ / s Doijode, S.D. (2001). Seed Storage of Horticultural Crops, Haworth Press, New York. Ebert, A.W. and Huang, Y.-K. (2015). Are Momordica charantia (bitter gourd) seeds truly orthodox? In Seeds for Future Generations Determinants of Longevity: International Society for Seed Science (ISSS) Seed Longevity Workshop; Book of Abstracts, (eds. U. Lohwasser and A. Börner), p. 58, Wernigerode. Fonseka, H.H. and Fonseka, R.M. (2011). Studies on deterioration and germination of bitter gourd seed (Momordica charantia L.) during storage. In Proceedings of the Vth International Symposium on Seed, Transplant and Stand Establishment of Horticultural Crops: Integrating Methods for Producing More with Less, (eds. J.A. Pascual and F. Pérez-Alfocea), Acta Horticulturae, Secretariat of the International Society for Horticultural Science, Leuven. GENESYS Global Portal (2016). Data accessed through GENESYS Global Portal on Plant Genetic Resources, Plant genetic resources accession level data provided by: The World Vegetable Center (AVRDC), Taiwan. Grover, J.K. and Yadav, S.P. (2004). Pharmacological actions and potential uses of Momordica charantia: a review. Journal of Ethnopharmacology, 93, dx.doi.org/ / j.jep Hong, T.D. and Ellis, R.H. (1996). A Protocol to Determine Seed Storage Behaviour, IPGRI technical bulletin, no. 1, IPGRI, Rome, Italy. Hsu, C.C., Chen, C.L., Chen, J.J. and Sung, J.M. (2003). Accelerated aging-enhanced lipid peroxidation in bitter gourd seeds and effects of priming and hot water soaking treatments. Scientia Horticulturae, 98, dx.doi.org/ / S (03) ISTA (2017). International Rules for Seed Testing, International Seed Testing Association, Bassersdorf, Switzerland. Keys, R.D., Smith, O.E., Kumamoto, J. and Lyon, J.L. (1975). Effect of gibberellic acid, kinetin, and ethylene plus carbon dioxide on the thermodormancy of lettuce seed (Lactuca sativa L. cv. Mesa 659). Plant Physiology, 56,

12 SEBASTIAN BOPPER AND MICHAEL KRUSE Lin, J.M. and Sung, J.M. (2001). Pre-sowing treatments for improving emergence of bitter gourd seedlings under optimal and sub-optimal temperatures. Seed Science and Technology, 29, Nikolaeva, M.G. (1969). Physiology of Deep Dormancy in Seeds, Israel Program for Scientific Translations Ltd., Jerusalem. Pandey, S., Devi, C., Kak, A., Khan, Y.J. and Gupta, V. (2013). Breaking seed dormancy in sweet gourd (Momordica cochinchinensis). Seed Science and Technology, 41, < doi.org/ / sst > Popcov, A.V. (1935). Vtorichnyi pokoi u semian krymsagyza. [Secondary dormancy in seeds of krym-saghyz]. Doklady Akademii Nauk SSSR, 2, Priestley, D.A. (1986). Seed Aging: Implications for Seed Storage and Persistence in the Soil, Comstock Publishing Associates, Ithaca, New York. Royal Botanic Gardens Kew (2016). Seed Information Database (SID): Version 7.1. < (accessed October 2016). Ullmann, W. (1949). Über die Keimfähigkeitsdauer (Lebensdauer) von landwirtschaftlichen und gartenbaulichen Samen. [About the duration of the ability to germinate (longevity) of agricultural and horticultural seeds]. Saatgut-Wirtschaft, 1, Vogel, G. (1996). Handbuch des Speziellen Gemüsebaues, [Handbook of Special Vegetable Production], Ulmer, Stuttgart (Hohenheim). Yeh, Y.M., Chiu, K.Y., Chen, C.L. and Sung, J.M. (2005). Partial vacuum extends the longevity of primed bitter gourd seeds by enhancing their anti-oxidative activities during storage. Scientia Horticulturae, 104, dx.doi.org/ / j.scienta