Impaginato 61 1. Introduction Oil pumpkins play a significant role in human nutrition and health. The nutritional value of oil pumpkin seeds is based on high protein and antioxi- dant content, and high energy potential due to the high percentage of oil (Fruhwirth and Hermetter, 2007; Sari et al., 2008; Fokou et al., 2009; Lelley et al., 2009; Urbanek Krajnc et al., 2016). During the last decade, oil pumpkin cultivation declined regarding productivity and quality due to the outbreaks of the Zucchini yellow mosaic virus (ZYMV), extremely high temperatures, radiation stress and prolonged periods of drought (Lelley et al., 2009; Seebold et al., 2009; Gong et al., 2013). Years 2013 and 2015 have been excessively hot and we have seen serious problems in fruiting pumpkins related to weather conditions especially high day/night temperatures and drought stress (Yavuz et al., 2015). Heat stress depends on intensity, duration, and rate of increase in temperature. The extent to which it occurs, in specific climatic zones, depends on dura- tion and level of high temperatures occurring during the day and/or the night. In general, a transient ele- vation in temperature, 10-15°C above ambient, is considered as heat stress (Wahid et al., 2007). It cau- ses an array of morpho-anatomical, physiological and biochemical changes in plants, which affect plant growth and development and may lead to a drastic reduction in economic yield. On the morphological level, high temperature can cause considerable pre- and post-harvest damages such as scorching of lea- ves and stems, sunburns on leaves, stems and fruits, leaf senescence and wilting, shoot and root growth inhibition, fruit discoloration and damage, and redu- ced yield (Wahid et al., 2007; Ara et al., 2015; Johnson, 2015). The physiological changes caused by high temperatures include the negative effect on photosynthesis, respiration, water relations, and modulated levels of hormones and primary and secondary metabolites. On the biochemical and sub- cellular level the direct injuries due to high tempera- tures include protein denaturation and aggregation, disorganization of cytoskeleton and increased fluidity of membrane lipids. Indirect or slower heat injuries include inactivation of enzymes in chloroplast and mitochondria, inhibition of protein synthesis, protein degradation and loss of membrane integrity. These injuries eventually lead to starvation, inhibition of growth, reduced ion flux, production of toxic com- Adv. Hort. Sci., 2017 31(1): 61-73 DOI: 10.13128/ahs-20727 The impact of fruit temperature dynamics on heat stress tolerance of selected oil pumpkin genotypes A. Urbanek Krajnc *, J. Rakun, P. Berk, A. Ivančič Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, Hoče, Slovenia. Key words: breeding, Cucurbita argyrosperma var. argyrosperma, Cucurbita moschata, Cucurbita okeechobeensis, Cucurbita pepo, fruit temperatures. Abstract: Fruit temperature is a key parameter for fruit growth and quality which is affected by climate, plant vigorousi- ty, solar exposure and fruit thermal properties. In the present study, the variability in temperature dynamics of Styrian oil pumpkin fruits and selected interspecific hybrids involving Cucurbita argyrosperma, C. moschata, C. pepo was analy- sed in two different periods of hot weather. The temperatures were measured with thermistors on (a) attached fruits, (b) detached fruits exposed to the sun and (c) artificially black coloured fruits. The highest average temperatures were determined in the Styrian oil pumpkin, whereas the lowest temperatures were determined in genotypes with lighter fruit exteriors suggesting that those are less sensitive to heat stress conditions and may represent a good option for the improvements of adaptability to climatic changes. In order to combine lighter and harder pericarp, the most promising genotypes were crossed with wild Cucurbita okeechobeensis. The histological analysis showed that C. okeechobeensis was a good source of genes for obtaining a thicker sclerenchymatic layer within pericarp. (*) Corresponding author: andreja.urbanek@um.si Received for publication 8 September 2016 Accepted for publication 4 March 2017 Copyright: © 2017 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2017 31(1): 61-73 62 pounds and reactive oxygen species (ROS), which coincide with increased synthesis of antioxidants and activity of antioxidant enzymes. Furthermore, enhan- ced expression of a variety of heat shock proteins and other stress-related proteins constitute major plant responses to heat stress (Iba, 2002; Howarth, 2005; Wahid et al., 2007; Ara et al., 2013 a, b, 2015). During the ontogenetic development of plants, flowering and fruit set are the most sensitive stages; fruit set of pumpkins is affected at day/night tempe- ratures above 26/20°C and is severeliy affected above 35/26°C (Schrader et al., 2004, 2011; Saudreau et al., 2011; Lei et al., 2014; Johnson, 2015). The dec- rease in yield is due to the rapid decrease of photo- synthesis, which reduces the amounts of sugars and other storage products that can go into fruits and developing seeds. On the other hand, hot night tem- peratures lead to greater cell respiration. High tem- peratures can also cause increased developmental disorders in fruiting vegetables due to the reduced pollen production leading to a reduction in fruit and seed sets, smaller pods, and split sets. Another effect of heat stress in many plant species is induced sterili- ty when heat is imposed immediately before or during anthesis (Siddique et al., 1999; Wahid et al., 2007). Theoretical and experimental evidence shows that fruit temperature can be 10°C higher than the air temperature under sunny conditions (Schrader et al., 2004; Racskó et al., 2005; Schrader, 2011; Lei et al., 2014). Sunburn of fruits is a surface injury caused by solar radiation which, during the initial phase, results in a light corky layer, golden or bronze discoloura- tion. The damage occurs mainly in the surface and subsurface layers. There are two types of sunburn damage which may have effects on fruits and fruiting vegetables. The first, sunburn necrosis, appears due to the thermal death of cells on the sun exposed side of the fruit; cell membrane integrity is lost and cells start leaking their contents (Schrader, 2011; Johnson, 2015). The critical fruit tissues’ temperatures for sun- burn necrosis vary with the type of fruit. The fruit surface temperature (FST) threshold for sunburn necrosis for cucumbers and pumpkins is between 37 and 42°C (Rabinowitch et al., 1983, 1986; Ara et al., 2013 a, b; 2015). The second type of sunburn injury is sunburn browning, which is caused by the combina- tion of high FST and high solar radiation. It causes degradation of photosynthetic pigments resulting in yellow spots on the sun-exposed side of the fruit and occurs at a temperature about 5°C lower than sun- burn necrosis (Schrader, 2011; Johnson, 2015). Plants have three major ways in which they dissi- pate excess heat: (1) long-wave radiation, (2) heat convection into the air and (3) transpiration. If tran- spiration is interrupted by stomatal closure due to water stress, inadequate water uptake or other fac- tors, a major cooling mechanism is not functioning. This will cause internal leaf/fruit temperatures to rise. Without transpiration, the only way that plants can reduce heat is by heat radiation back into the air or wind cooling. Under high temperatures, radiated heat builds up in the atmosphere around plants, lim- iting further heat dissipation (Wahid et al., 2007; Schrader, 2011; Johnson, 2015). The adverse effects of high air and soil tempera- tures, and the high levels of solar radiation can be mitigated by developing plant genotypes with impro- ved thermotolerance. Some attempts to develop heat-tolerant genotypes via conventional breeding protocols have been successful (Ehlers and Hall, 1998; Camejo et al., 2005). Breeding of cultivated cucurbits was mainly focused in combinig good attri- butes of C. moschata and C. maxima. (Balkaya and Karaagac, 2005; Balkaya et al., 2009, 2010 a, b, 2011; Balkaya and Kandemir, 2015). It is well known that C. moschata is best adapted to hot climate and is suc- cessfully cultivated in tropical and subtropical regions (Balkaya et al., 2010 a, b; Balkaya and Kandemir, 2015). Recently, a study was conducted to determine the extent of heat tolerance of newly developed interspecific squash hybrid named as ‘Maxchata’ compared to its parents C. maxima and C. moschata (Ara et al., 2013 a, b, 2015) under three different temperature regimes. Results showed that various gas exchange and photosynthetic attributes dropped significantly with increasing temperature, while inter- cellular CO2 concentration increased showing the nonstomatal limitations. These trends were more abrupt in C. maxima, reflecting that C. maxima was the most susceptible, while ‘Maxchata’ showed inter- mediate response. C. moschata had the best photo- synthetic attributes to sustain the heat regimes (Ara et al., 2013 a, b). The ultramorphological, biochemi- cal, and transcriptional analyses gave similar results. The electron microscopy highlighted the maximum degradation of the leaf ultrastructure of C. maxima. C. moschata and ‘Maxchata’ exhibited lower degree of subcellular injury upon heat exposure. The antioxi- dant enzyme activities and their expression were found to be highest in C. moschata, moderate in ‘Maxchata’, and lowest in C. maxima (Ara et al., 2013 a, b; 2015). The authors concluded that the interspe- cific hybridization with C. moschata might significant- Urbanek Krajnc et al. - Fruit temperature dynamics of selected oil pumpkin genotypes 63 ly contribute to heat tolerance (Ara et al., 2013 a, b, 2015). The presented study is associated with creating heat tolerant pumpkins characterised by lighter exo- carp. The work began in 1996 and is based on a mod- ified recurrent selection approach. Its aim is to create cultivars characterised by bushy growth, resistance to all major pests and diseases, tolerance to drought and high temperatures, and large and thick seeds having thin seed coats and high concentrations of high quality oil. The basic population (i.e., population of the cycle- 0) was established by inter-crossing all available sources of genes (i.e., numerous local and commer- cial cultivars, populations and hybrids of C. pepo, fol- lowing the semi-diallel scheme). In 1997, the most valuable progenies were planted in New Caledonia, at the CIRAD centre near Pouembout (South Pacific). Due to the favourable semi-tropical climate, it was possible to execute three cycles per year. The prob- lems, however, were seed germination within fruits and rotting fruits due to overheating. As the genetic resources within C. pepo were found to be insuffi- cient for overcoming these problems, it was decided to incorporate interspecific hybridisation and change the exterior fruit colour. The main sources of genes for lighter fruit exterior were C. argyrosperma var. argyrosperma and C. moschata. Cucurbita argyros- perma, which was the main source of genes for whitish exocarp and was also used as a genetic bridge between C. pepo and C. moschata. Another interesting trait obtained by interspecific crosses was dark yellow fruit exterior which was associated with the same colour of mesocarp. Some years later, a wild species C. okeechobeensis (Small) L. H. Bailey was added to the hybridisation programme, in order to improve the resistance to viruses and harder peri- carp. The interspecific hybrids included in this study were indirect progenies developed within the recur- rent selection programme which involved intra- and inter-population crosses, self-pollinations, and back- crosses. The progress in breeding for heat stress tolerance strongly depends upon understanding the genetic and physiological mechanisms associated with stress tolerance of the whole plants well as at the molecu- lar and cellular levels. Our study involved thin-coated seed pumpkins with lighter exocarp because they were considered to have the highest level of toler- ance to high air temperatures and high levels of solar radiation during summer. In order to investigate their tolerance to heat stress, pericarp tissue temperature profiles were monitored on the exocarp surface, as well as in the meso- and endocarp of fully developed attached and detached fruits during two different periods of hot weather. 2. Materials and Methods Plant material Four different plant materials with thin coated seed were used in the study: (1) Styrian oil pumpkin Cucurbita pepo subsp. pepo var. styriaca (O), (2) progeny with whitish fruit derived from the cross C. pepo (non-lignified seed coat, oil type) × C. argyros- p er m a va r. a r g y ro s p er m a ( O / A) , ( 3 ) a p ro gen y derived from crosses involving C. pepo (non-lignified seed type, oil type), C. argyrosperma and tropical C. moschata characterised by yellow fruits (A/Mo x O/A) and (4) a three-species hybrid involving C. pepo (non- lignified seed coat, oil type), C. argyrosperma var. argyrosperma and C. okeechobeensis (Oke × O/A) (Table 1, Fig. 1). The temperatures were measured on (a) attached fruits (1st period, 27th July to 5th August 2012), (b) detached fruits exposed to the sun (2nd period, 1st to 11th September 2012) and (c) artificially black coloured fruits (on both periods). The fruits were exposed to sunlight due to loss of foliage caused by drought stress and diseases. For each experiment, three fruits of each studied material were used. Fruit temperature measurements The temperatures were measured during two dif- ferent periods of hot weather, between the 27th July Table 1 - List of key plant materials with short explanations of abbreviations Plant material Abbreviations Species Cucurbita argyrosperma. var. argyrosperma A Cucurbita moschata Mo Cucuribita pepo subsp. pepo var. styriaca O Cucurbita okeechobeensis Oke Interspecific hybrids Material obtained from the cross C. pepo (non-lignified seed coat, oil type) × C. argyrosperma. var. argyrosperma, followed by several seasons of intrapopulation recombina- tions and selection O/A Three-species hybrid involving C. argyrosperma (used as a genetic bridge), C. moschata and C. pepo (non-lignified seed coat, oil type), after several seasons of intrapopula- tion crosses and selection A/Mo x O/A A three-species hybrid involving C. pepo (non-lignified seed coat, oil type), C. argyrosperma var. argyrosperma and C. okeechobeensis Oke x O/A Adv. Hort. Sci., 2017 31(1): 61-73 64 to 5th August 2012 and 1st to 11th September 2012. During both periods, three pumpkins of each progeny (O, O/A, A/Mo x O/A) were chosen and the thermis- tors were placed on exocarp the sun-exposed side, as well as inserted in meso- (2 cm deep) and endocarp (10 cm deep) (Fig. 1A, C, E). The temperatures were recorded every 15 seconds and stored as 10-min averages. The data represented in figures 3-6 show a diurnal and maximum day temperature as average of three fruits out of each progeny. In order to measure temperatures, 32 BETA- THERM 10K3A542I thermistors connected to a Campbell’s CR1000 datalogger (Campbell Scientific Inc., Logan, UT, USA) were used. The data logger recorded the current times for each iteration supply voltage and analogue input voltage. The thermistors’ readings were performed sequentially as they were all connected to the same ADC converter through a multiplexor; so only one of the thermistors was con- nected to the converter at a time. The selected thermistors had accuracies of 0.2°C and were of negative-temperature-coefficient (NTK) type, which meant that their resistances decreased with the increases of temperature. As the data logger was unable to read the resistance of the thermistor directly, the thermistors were connected in a form of voltage divider, with an additional resistor with 1 KΩ±0.1% fixed resistance. As the resistive responses of the thermistors were nonlinear, the measured temperatures were calculated according to the tem- perature table from the datasheet (BETHATERM). The results are represented by mean values (N=3), and were statistically evaluated by one-way analysis of variance (ANOVA), using the SPSS 21 software (SPSS 21 software, SPSS Inc., Chicago, IL, USA). Significant differences between mean values were d e t e r m i n e d u s i n g t h e p o s t - h o c D u n c a n t e s t . Significant differences (α<0.05) between means were indicated by different letters. Air/soil measurements Furthermore, air/soil temperatures and relative humidity were measured on the sun-exposed side and within the canopy (5 and 20 cm below soil sur- face, on soil surface as well as 10 and 20 cm above soil surface using 215 and 107 Temperature Probe sensors (Campbell Scientific Inc., Logan, UT, USA). The temperatures were recorded every 15 seconds and stored as 10-min averages. Histochemical evaluation of pericarp From each studied material, 3-6 fruits were taken for histological evaluation. Out of each fruit six pieces (diameter 8 mm) of pericarp were sampled on equa- tor of the fruit positioned around the pumpkin in reg- ular intervals. The pieces were cut with a cryotom in order to evaluate the size of the lignified cell layer. An Olympus microscope (Provis AX 70) with a 100 W mercury arc lamp was used to take analogue images with a 3-chip-colour video camera (Sony DXL 950 P, 3 CCD). Fluorescence images were obtained through an UplanFI 40x dry objective (n.a., 0.75), a PlanApo 60x oil immersion objective (n.a., 1.40), and an UplanApo 100 x oil immersion objective (n.a., 1.35). Lignified cells were visualised using an Olympus filter set (U- MWU) with 330-385 excitation and 420 nm emission. Fig. 1 - Fruits of selected genotypes with attached sensors, which were selected for lighter and harder fruit exterior and thin coated seeds: A, B) Styrian oil pumpkin (O). C, D) Three-species hybrid involving C. argyrosperma var. argyrosperma (used as a genetic bridge), C. moschata and C. pepo (non-lignified seed coat, oil type), after sev- eral seasons of intrapopulation crosses and selection (A/Mo × O/A). E, F) Genotype with lighter (i.e., whitish, white-green) fruits material obtained from the cross C. pepo (non-lignified seed coat, oil type) × C. argyrosper- ma, followed by several seasons of intrapopulation recombinations and selection (O/A). G) C. okeechobeen- sis. H) A three-species hybrid involving C. pepo (non-lig- nified seed coat, oil type), C. argyrosperma and C. okee- chobeensis (Oke × O/A). Urbanek Krajnc et al. - Fruit temperature dynamics of selected oil pumpkin genotypes 65 3. Results and Discussion Our research was based on the hypothesis that interspecific hybridization aimed in creating lighter exocarp, thicker hypodermal sklerenchyma layer and cuticle would reduce the heat load on the fruit. Air/soil measurements During the first period of measurements (Jul.- Aug.), on the sun-exposed sides, the highest temper- atures were registered on the ground surface reach- ing 45°C, whereas 20 cm above ground the maximum daily air temperatures were about 38°C. On the sides shaded by a canopy, the ground temperatures remained cooler and more stable, varying between 18 during the nights and 32°C on the hottest days. Twenty cm above ground, the air temperatures reached 43°C. The underground temperatures (20 cm deep) remained stable with 20-22°C day/night varia- tion (Fig. 2 A). Additionally, the ground temperatures were also measured within the proximities of the studied pumpkins. In the proximity of Styrian oil pumpkins, from the 30th of July onwards, they increased from 27°C (daily maximum) to more than 47°C on the 1st August, and remained above 40°C for the next five days (Fig. 3 A, B). The maximum day ground tempera- ture in the proximity of white pumpkins were similar, reaching 45°C during the first three days of August (Fig. 3 E, F). At the beginning of September, the minimum night temperature was 10°C. One day, on the 7th September, it was particularly hot and the air tem- perature 20 cm above ground, on the sun-exposed side, reached 45°C, whereas the temperature of the ground surface reached 58°C. A similar situation was observed within the canopy. At 20 cm height, the temperature reached 45°C (Fig. 2 B). On attached Styrian oil pumpkins (1st period), the maximum day temperatures measured on the exo- carp varied between 38 and 50°C. In mesocarp the t e m p e r a t u r e s r a n g e d b e t w e e n 3 2 a n d 5 1 ° C . Endocarp was more or less 2°C cooler than mesocarp (Fig. 3 A, B). On detached Styrian oil pumpkins (2nd period) the maximum day temperatures of exocarp varied between 20 and 54°C, whereas mesocarp heated up above the temperature of exocarp to 61°C on the hottest day (7th September), on later days, the maximum day temperatures were around 45°C (Fig. 4 A, B). In the ‘yellow fruit’ pumpkin material (A/Mo × O/A), the temperatures of exocarp ranged between 42°C and 50°C on attached fruits during the first peri- od and 17°C and 50°C on detached fruits during the second period of hot weather. The temperatures of endocarp varied between 33°C and 42°C during the first period, whereas during the second period the temperatures of mesocarp of detached pumpkins ranged between 16°C and 45°C (Fig. 3 C, D, Fig. 4 C, D). ‘White fruit’ pumpkin material (O/A) was charac- terised by the lowest exocarp temperatures, which ranged between 30°C and 41°C during the first peri- od, whereas during the second period the tempera- tures ranged between 17°C and 44°C. The mesocarp temperatures varied between 35 and 44°C, the endo- carp temperatures were more or less 2°C above the mesocarp temperatures. During the second period, where temperatures were measured on detached fruits, the average temperatures of mesocarp were Fig. 2 - Relative humidity and the average air/soil temperatures measured within canopy and on sun exposed side during the period between 27 July-6 August 2012 (A) and between 1-11 September 2012 (B). Adv. Hort. Sci., 2017 31(1): 61-73 66 generally 2°C lower when compared to exocarp but the fruit flesh heated up to 56°C on the extremely hot days of the 6th and 7th September, similarly as was observed in the Styrian oil pumpkin (Fig. 4 A, B, E , F , F i g . S 1 s e e s u p p l e m e n t a r y m a t e r i a l ) . Transpiration appeared to be vital for maintaining optimal growth temperatures in growing plants. Detached fruits, however, lacked the protective effects of transpiration, and direct sources of heat, such as sunlight, can rapidly elevate the internal fruit Fig. 3 - Average and maximum day temperatures measured in exocarp, mesocarp, endocarp of attached pumpkins and ground in the period between 27. July- 6. August 2012: A, B) Styrian oil pumpkin (O). C, D) Genotype with yellow fruits involving C. argyrosper- ma, C. moschata and C. pepo (A/Mo × O/A). E, F) Genotype with white fruits obtained from the cross C. pepo (non-lignified seed coat, oil type) × C. argyrosperma (O/A). 67 Urbanek Krajnc et al. - Fruit temperature dynamics of selected oil pumpkin genotypes temperatures to above that of exocarp and towards the thermal death points of their cells. In case of Styrian oil pumpkin these lead to localised bleaching a n d n e c r o s i s ( s u n b u r n o r s u n s c a l d ) . S i m i l a r l y , R a b i n o w i t c h e t a l . ( 1 9 8 3 , 1 9 8 6 ) r e p o r t e d t h a t detached cucumbers (Cucumis sativus L.) and pep- pers (Capsicum annuum L.) had significantly higher surface temperatures and more serious sunburn injuries than attached fruit. Comparing the measured results of the different Fig. 4 - Average and maximum day temperatures measured in exocarp and mesocarp of detached pumpkins in the period between 1.- 11.September 2012: A, B) Styrian oil pumpkin (O). C, D) Genotype with yellow fruits involving C. argyrosperma, C. moschata and C. pepo (A/Mo × O/A). E, F) Genotype with white fruits obtained from the cross C. pepo (non-lignified seed coat, oil type) × C. argyrosperma (O/A). 68 Adv. Hort. Sci., 2017 31(1): 61-73 varieties on attached fruits, we can conclude that Styrian oil pumpkin was characterised by the highest temperatures within all three tissues (Fig. S2), fol- lowed by yellow genotype, whereas white genotype had the lowest temperature, confirming the hypoth- esis that varieties with lighter fruit colour are less h e a t e d u p . O u r r e s u l t s o f t h e e x p e r i m e n t o n detached fruits, however, suggest that the most tol- erant is A/Mo × O/A which is a progeny derived from the interspecific cross involving the Styrian oil pump- kin, C. argyrosperma and C. moschata. It is well known that C. moschata is best adapted to hot cli- mate and is successfully cultivated in the tropical and subtropical regions (Balkaya et al., 2010 a, b; Balkaya and Kandemir, 2015). Heat tolerance of C. moschata can be very useful in breeding involving interspecific hybridisation. One of the successful examples, men- tioned earlier in the text, is interspecific hybrid of squash named as ‘Maxchata’. The authors concluded that the interspecific hybridization involving C. moschata might significantly contribute to heat tol- erance (Ara et al., 2013 a, b; 2015). In order to test further the resistance of selected genotypes to heat stress, the selected pumpkins were sprayed with black colour and exposed to the sun in order to induce bleaching by the excess heat. The exocarp of the black coloured Styrian oil pump- kin fruit heated up to 52°C whereas the mesocarp temperature rose up to 43°C in the afternoon hours between 3 and 4 p.m. during both periods. When analysing the daily temperature curves of attached pumpkins during the July-August period, a depres- sion in the curve was observed at around 12 a.m., reflecting a higher transpiration of fruits in response to heat stress; later, a rapid increase in temperature, may indicate stomata closure (Fig. 5A). It is well- Fig. 5 - Average and maximum day temperatures measured in exocarp and endocarp of black coloured attached pumpkins measured in the period between 27 July- 6 August 2012: A, B) Styrian oil pumpkin (O). C, D) Genotype with yellow fruits involving C. argyros- perma, C. moschata and C. pepo (A/Mo × O/A). 69 Urbanek Krajnc et al. - Fruit temperature dynamics of selected oil pumpkin genotypes known that if transpiration is interrupted by stomatal closure a major cooling mechanism is lost and the internal fruit temperatures raise (Wahid et al., 2007; Johnson et al., 2015). The temperatures of coloured fruits were measured for three days since a general collapse and tissue death of the inner pericarp layers was observed later on. The fruits were too much damaged by sunburn and measurements of tempera- tures no longer made sense. In the September peri- o d , w h e n t h e e x p e r i m e n t w a s p e r f o r m e d o n detached fruits, the exocarp of Styrian oil pumpkin heated up to 51°C, similarly as on attached fruits. Mesocarp temperature raised to a higher level on detached fruits (61°C) in comparison to attached fruits (43°C) (Fig. 6A, Fig. 6B, Fig. S2). I n t e r e s t i n g l y , d u r i n g t h e 1 s t e x p e r i m e n t o n Fig. 6 - Average and maximum day temperatures measured in exocarp and mesocarp of black coloured detached pumpkins measured in the period between 1.-11.September 2012: A, B) Styrian oil pumpkin (O). C, D) Genotype with yellow fruits involving C. argyros- perma, C. moschata and C. pepo (A/Mo × O/A). E, F) Genotype with white fruits obtained from the cross C. pepo (non-lignified seed coat, oil type) × C. argyrosperma (O/A). 70 Adv. Hort. Sci., 2017 31(1): 61-73 attached pumpkins the exocarp of the black coloured ‘yellow’ pumpkin heated up to 64°C, whereas the endocarp up to 48°C during the July-August period (Fig. 5C, D). Compared to the Styrian oil pumpkin the temperatures were higher. A rapid increase of tem- perature was determined in the late morning hours until noon. Later, an abrupt decrease of temperature followed and maintained at around 38°C between 3 and 6 p.m. After that, during the night hours, the temperature dropped below 20°C (Fig. 5C). Similarly, in September, during the early afternoon hours of the hottest day (7th September), the maximum day temperatures on exocarp reached 64°C, whereas in mesocarp the temperatures reached 57°C. On later dates the daily maximum temperatures of exocarp constantly reached 42°C, whereas mesocarp was heated up to 57°C (Fig. 6 C, D). The ‘white fruit’ genotype (O/A) was charac- terised by the highest mesocarp temperatures during the hottest days of the 2th period when compared to O and A/Mo × O/A progenies. During the first five days, the maximum exocarp temperatures were around 40°C, whereas the mesocarp reached maxi- mum day temperatures between 48°C and 55°C. During the following two days, the exocarp tempera- tures reached 60°C and 63°C, respectively, whereas the mesocarp 62°C and 71°C. Later, the exocarp max- imum temperature was comparable to that of the black coloured ‘yellow’ pumpkin, reaching 42°C in the early afternoon hours, whereas the mesocarp was heated up to approx. 55°C (Fig. 6 E, F). Summarising the results of temperature measure- ments within the black coloured pumpkins, we can conclude that for the attached pumpkins, the ‘yellow’ genotype was heated up more than the Styrian oil pumpkin. By analysing the results of detached pump- kins during the first days of measurements, the high- est temperatures of exo- and mesocarp were deter- mined in the Styrian oil pumpkin, whereas the maxi- mum day temperatures of both ‘yellow’ and ‘white’ genotypes did not differ significantly and were about 5°C lower than those of the Styrian oil pumpkin. H o w e v e r , o n t h e h o t t e s t d a y s ( 6 t h a n d 7 t h September), the temperatures of exocarp within ‘yel- low’ and ‘white’ genotypes were 8°C higher than those of the Styrian oil pumpkin reaching 64°C. Consequently, when compared to the Styrian oil pumpkin, higher temperatures were also determined in fruit tissues of both studied interspecific hybrids. During the last two centuries, pumpkins have been selected for their softer pericarp in order to ease the laborious and time-consuming hand-har- vesting of seeds. However, the resulting pumpkins’ genotypes became more susceptible not only to dis- eases but also to various types of abiotic stress such as heat stress and drought. In order to create a vari- ety with harder pericarp and also better abscission of the fruit peduncle, those pumpkins with lighter exo- carp were crossed with the wild species C. okee- chobeensis (Fig. 1 G, H), which is characterised by hard perikarp, good abscission of the pedincle and resistance to viruses. The selected hybrids were his- tologically evaluated and compared to the Styrian oil pumpkin and O/A hybrids with lighter pericarps. The Styrian oil pumpkin (O) was characterised by its thick cuticle, a 15-cell layer of chlorenchyma cells and a 2- 3 cell layer of more or less isodiametric lignified cells between the chlorenchyma and parenchyma of the mesocarp (Fig. 7 A, B). Both the ‘yellow’ (A/Mo × O/A) as well as the ‘white’ (O/A) genotypes were characterised by a similar 2-3 cell layer of thick ligni- fied cells although the cell walls of this layer as well as the cuticle were thicker (Fig. 7 C, F). Crosses of O/A genotypes with C. okeechobeensis (Oke × O/A) became characterised by a thicker scklerenchymatic layer of 4-5 oblong cells in a radial direction, which were approx. 100 µm long (Fig. 7 G, H). This progeny represents a good material for the future breeding for increased heat tolerance. Furthermore, C. okee- chobeensis has been recognised as promising for interspecific hybridization due to its central position in the genus Cucurbita (Gong et al., 2013). We may conclude that the genetic breeding of oil pumpkins for heat tolerance is still in its infancy stage and warrants more attention than it has been gi ven i n th e p a s t . C o n s i d era b l e i n f o rma ti o n i s presently available regarding the physiological and metabolic aspects of plant heat-stress tolerance (Ara et al., 2013 a, b, 2015). Furthermore, attempts have been made to include molecular marker technology for genetic characterization and/or development of plants with improved heat tolerance. Gong et al. (2013) analysed SSR polymorphisms on a large col- lection of Cucurbita materials in order to obtain an improved insight into the relationships amongst most of the species of the genus. Wild species C. foe- tidissima has been identified as resistant to numer- ous pathogens and pests but is most distant to other Cucurbita species. Cucurbita pepo and C. ficifolia were the most outlying of the mesophytic species. The clusters of the six remaining species form three pairs, C. maxima with C. ecuadorensis, C. okee- chobeensis with C. lundelliana, and C. moschata with C. argyrosperma. Due to the genetic distances 71 Urbanek Krajnc et al. - Fruit temperature dynamics of selected oil pumpkin genotypes amongst the Cucurbita species, various breeding strategies and biotechnological approaches have been employed (Merrick, 1995; Lebeda et al., 2007; Orti z-Al ami l l o et al. , 2007; L el l ey et al. , 2009; Karaağaç and Balkaya, 2013) but the success in intro- gressing desirable traits from one species to another have been limited. Cucurbita pepo excels in plant earliness and productivity but lacks genetic resources for disease resistance. Cucurbita moschata, on the other hand, carries resistance to various pathogens and is adapted to humid tropics but lacks earliness and productivity (Lebeda et al., 2007; Lelley et al., 2009; Karaağaç and Balkaya, 2013). Relatively high successes and fertilities have been observed for the c r o s s - c o m b i n a t i o n o f C . a r g y r o s p e r m a a n d C . moschata (Montes-Hernandez and Eguiarte, 2002; Ortiz-Alamillo et al., 2007) and C. maxima × C. moschata (Ara et al., 2013 a, 2015). However, despite all the complexity of heat toler- ance and difficulties encountered during the genetic transfer of tolerance, few heat-tolerant inbred lines and hybrid cultivars with commercial acceptability h a v e b e e n d e v e l o p e d a n d r e l e a s e d ( M o n t e s - Hernandez and Eguiarte, 2002; Ortiz-Alamillo et al., 2007; Ara et al., 2013 a, b, 2015). 4. Conclusions The presented study suggests that the colour of exocarp is probably one of the key parameters of tol- erance to high temperatures. Darker colours are gen- erally associated with higher fruit temperatures. In general, for attached pumpkin fruits higher tempera- tures were measured on exocarp, followed by meso- and endocarp. However, for the detached fruits on extremely hot days in September, the temperatures within mesocarp increased above those of exocarp. It is well known that if transpiration is interrupted by stomatal closure due to water stress and heat stress, a major cooling mechanism is not functioning. This was the reason of rising the internal fruit tempera- tures in case of detached fruits. One could expect that the genotypes with whitish exocarp (i.e., O/A progenies derived from the cross Styrian oil pumpkin × C. argyrosperma) would be the most tolerant to heat stress. The experiment on detached fruits, however, suggest that the most tol- erant is the A/Mo x O/A progeny derived from the cross involving the Styrian oil pumpkin, C. argyrosper- ma and C. moschata, which is characterized by yel- low exocarp. One of the reasons for this could be the introgression of desirable traits of C. moschata, which is more adapted to high temperatures. This parental species was brought from the Island of Espiritu Santo, Vanuatu (tropical Pacific) and was obviously more tolerant to heat stress than the other two involved pumpkin species. The second important parameter appears to be the histological structure of Fig. 7 - Histochemical analyses of perikarp. A, B) Styrian oil pumpkin (O) characterised by its thick cuticle, a 15-cell layer of chlorenchyma cells and a 1-2 cell layer of isodia- metric cells and one layer of oblong cells with thick cell walls. C, D) Genotype with yellow fruits (A/Mo × O/A), with two layers of isodiametric cells with lignified cell walls. E, F) Genotype with white fruits (O/A) and three layer of lignified cells below the chlorenchyma. G, H) A three-species hybrid involving C. pepo (non-lignified seed coat, oil type), C. argyrosperma and C. okee- chobeensis (Oke × O/A) exhibiting 4-5 cell layers of oblong sklerenchymatic cells below the chlorenchyma. 72 Adv. Hort. Sci., 2017 31(1): 61-73 the pericarp. The genetic improvement of this com- p l e x t r a i t s h o u l d c o n s i d e r b o t h p a r a m e t e r s . Nevertheless, to accelerate such progresses, major areas of emphasis in the future should be: (1) devel- opment of accurate screening procedures at each stage of plant development; (2) identification and characterization of additional genetic resources asso- ciated with heat tolerance; (3) discerning the genetic inheritance of heat tolerance; (4) development and efficient screening of large breeding populations to facilitate transfer of genes for heat tolerance to com- mercial cultivars. Acknowledgements This research was funded by the Slovenian Research Agency (ARRS, Z1-9602, P-0164). The aut- hors would like to express their gratitude to Prof. Dr. Borut Bohanec, Prof. Dr. Metka Sisko and Anja Ivanus for their genorous help and cooperation. 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YAVUZ D., SEYMEN M., YAVUZ N., TURKMEN O., 2015 - Effects of irrigation interval and quantity on the yield and quality of confectionary pumpkin grown under field conditions. - Agric. Water Manag., 159: 290-298. SM i Adv. Hort. Sci., 2017 31(1): 61-73, SM i-ii DOI: 10.13128/ahs-20727 The impact of fruit temperature dynamics on heat stress tolerance of selected oil pumpkin genotypes A. Urbanek Krajnc *, J. Rakun, P. Berk, A. Ivančič Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, Hoče, Slovenia. SUPPLEMENTARY MATERIAL Copyright: © 2017 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2017 31(1): 61-73, SM i-ii SM ii Fig. S1 - Comparison of the maximum day temperatures measured in the period between 27 July - 6 August 2012 in: A) Exocarp of Styrian oil pumpkin, yellow and white genotype; B) Endocarp of Styrian oil pumpkin, yellow and white genotype; C) Exocarp of black coloured Styrian oil pumpkin and yellow genotype; D) Endocarp of black coloured Styrian oil pumpkin and yellow genotype. Fig. S2 - Comparison of the maximum day temperatures measured in the period between 1-11 September 2012 in: A) Exocarp of Styrian oil pumpkin, dark yellow and white genotype; B) Mesocarp of Styrian oil pumpkin, yellow and white genotype; C) Exocarp of black coloured Styrian oil pumpkin, yellow and yellow genotype; D) Mesocarp of black coloured Styrian oil pumpkin, yellow and yellow genotype.