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Advanced analysis of developmental and ripening characteristics of pollinated common-type fig (Ficus carica L.)
Scientia Horticulturae 198 (2016) 98–106
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Advanced analysis of developmental and ripening characteristics of pollinated common-type fig (Ficus carica L.) Yogev Rosianski a,b , Zohar E. Freiman a , Shira Milo Cochavi a,b , Zeev Yablovitz a , Zohar Kerem b , Moshe A. Flaishman a,∗ a
Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan 50250, Israel The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel b
a r t i c l e
i n f o
Article history: Received 21 August 2015 Received in revised form 12 November 2015 Accepted 17 November 2015 Available online 14 December 2015 Keywords: Fig fruit Ficus carica Caprification Storage
a b s t r a c t Development and ripening processes differ in pollinated and parthenocarpic fruit. While the facultative parthenocarpic common-type fig fruit serves as a receptacle for flower development, it becomes fleshy by either pollination or through a parthenocarpic process. Here we studied the effect of pollination on common-type fig fruit development and ripening characteristics compared to the parthenocarpic fruit under otherwise identical conditions. The effects of pollination on fruit development were investigated on the tree and in storage. Pollinated fruit showed altered developmental processes. Ripened pollinated fruit were round, in contrast to the pear-like shape of the parthenocarpic fruit. The pollinated fruit also had a larger diameter and weight and improved firmness compared to the parthenocarpic fruit. At harvest, the pollinated fruit exhibited more commercially desirable physical and taste characteristics, with advanced fertile nutlets compared to the sterile undeveloped non-bearing nutlets of the parthenocarpic fruit. During storage, senescence and spoilage of the pollinated fruit were slower than in parthenocarpic fruit, as manifested by firmness, internal texture, weight, size, shriveling, and decay. Thus, pollination of the common-type fig cultivar Brown Turkey delayed senescence and extended the shelf life of its fruit. The external and internal morphological differences throughout post-pollination development make common-type fig an excellent research tool for studies of physiological and molecular aspects of pollination. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The fig tree, Ficus carica L. (Moraceae), is one of the world’s oldest horticultural crops. It is indigenous to wide areas, ranging from Asiatic Turkey to northern India, and local varieties are cultivated in most Mediterranean countries (Küden, 1996). The fig fruit is well known for its attractive taste and nutritive value due to its antioxidant properties, and it is consumed fresh or dried worldwide (Solomon et al., 2006; Solomon et al., 2010a,b; Kirthikar and Basu, 1986). The fig is an aggregate fruit composed of individual small drupes, each termed a drupelet. The drupelets develop from the ovaries in a closed inflorescence, known as the syconium (the edible fig fruit), which encloses many unisexual female flowers (Flaishman et al., 2008).
∗ Corresponding author. Fax: +972 3 9683973. E-mail address: [email protected] (M.A. Flaishman). http://dx.doi.org/10.1016/j.scienta.2015.11.027 0304-4238/© 2015 Elsevier B.V. All rights reserved.
Fig fruit development can be described by a double sigmoid growth curve comprising three phases (Marei and Crane, 1971). Phase I is characterized by rapid growth in size as a result of extensive cell division. During phase II, fruit growth is arrested. Rapid fruit growth in phase III is mainly a result of cell expansion; during this phase, the fig is ripening, changing its skin and inner inflorescence colors, accumulating sugars and acids, and producing volatiles similar to fleshy fruits. The rate of the ripening process in fig is extremely rapid, lasting several days. At the end of phase III, the fruit is two to three times larger than in phase II, with changes in its color as a result of anthocyanin accumulation in the skin (Solomon et al., 2006); its dry weight is 70% higher and total sugars increase by 90% relative to phase II fruit; it accumulates water and becomes soft, and the pulp texture undergoes modifications to an edible state (Flaishman et al., 2008; Chessa and Mitra, 1997; Crane, 1986). Although fig fruit is categorized as climacteric, showing a rise in respiration rate and ethylene production at the onset of the ripening phase, when
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harvested prior to complete ripening, it never reaches the commercially desirable parameters of size, color, flavor and texture. Alternatively, when harvested late, the figs tend to perish due to over-ripening and high susceptibility to pathogens (Flaishman et al., 2008; Chessa and Mitra, 1997). Normally, fungal and yeast infection occurs in the orchard, and is fully demonstrated in ripening harvested fruits (Michailides, 2003). Rapid cooling together with controlled or modified atmosphere during storage enables sustaining fruit quality (Turk, 1989; Celikel and Karacali, 1998; Turk et al., 1993; Colelli et al., 1991). The fruit developmental stage for commercial harvest is cultivar-specific. For dark-skinned fig cultivars, this is generally at 50–80% color coverage of the skin area. At this stage, the fig is edible with a short storage capacity of only 7 days under cooling conditions (Chessa and Mitra, 1997). Fig cultivars are divided into four types according to their pollination requirement (called caprification in figs) and cropping season (Flaishman et al., 2008). The first type, the caprifig, commonly termed ‘male fig’, is not edible, and is the commercial planting source for pollen. Two edible fig types, Smyrna and SanPedro, are requiring caprification to bear their main crops. The unique San-Pedro types yield an early parthenocarpic crop known as ‘breba’, in addition to the pollinated main crop. The fourth type is the common female type, also called ‘persistent’ fig, which yields one or two crops per year, with or without caprification (Flaishman et al., 2008; Galil and Eisikowitch, 1968; Galil and Neeman, 1977). Pollination of the different fig types is a consequence of a complex symbiotic relationship with the fig wasp. The pollen-carrying wasp Blastophaga psenes accesses the inner fig cavity through the fruit ostiole and pollinates the female flowers (Galil and Neeman, 1977). The caprified figs are usually larger and greener than the noncaprified ones (Condit, 1947; Oukabli et al., 2001; Michailides et al., 2008). Studies on the effect of caprification on fruit quality in the common fig type cv. Kadota reported higher fruit weight together with a tendency toward splitting, and changes in several fruit characteristics, including darker skin and red-colored inflorescences in addition to the sweeter and richer flavor of the pollinated fruits (Gaaliche et al., 2011a; Condit, 1920). Caprified San-Pedro-type fruit showed modifications in their cell-wall components which moderately affected several textural parameters, as well as stimulation of anthocyanin and total polyphenol biosynthesis (Trad et al., 2013). In flowering plants, after pollination of the stigma, seed set and fruit progression proceed in a well-coordinated manner (Gillaspy et al., 1993). Plants can also set fruit in the absence of fertilization, a phenomenon called parthenocarpy. Thus, parthenocarpic cultivars overcome the environmental impact of adverse hot or cold temperatures on fruit production. A distinction is often made between obligatory parthenocarpy, which always results in seedless fruit, and facultative parthenocarpy, which results in seedless fruit only when pollination is prevented. Parthenocarpic, seedless horticultural fruit are preferred over their seeded counterparts in many crops. Parthenocarpic tomatos, as well as parthenocarpic banana, pineapple and some grape and citrus cultivars, are good examples of facultative parthenocarpy because they only produce seedless fruit in the absence of fertilization. However, detailed studies of the quality of parthenocarpic and pollinated fruit is lacking in many fruit. To the best of our knowledge, a comprehensive study of fig fruit development based on comparing pollinated and parthenocarpic fruit of the same cultivar and crop has never been performed. In this research, we examined the influence of pollination on the development of the common-type facultatively parthenocarpic fig fruit of cv. Brown Turkey, and on its performance under commercial fruit-storage conditions.
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2. Materials and methods 2.1. Plant material and treatment Mature fig trees (Ficus carica L., cv. Brown Turkey) growing in a commercial orchard near Beer Tuvia, Israel (31◦ 44’13.66 N, 34◦ 43’32.05 E) were used in two field experiments performed on May 2012 and 2013. ‘Brown Turkey’ belongs to the common-type figs and produces parthenocarpic and pollinated fruit. All of the trees were maintained according to commercial production practices. Fruit corresponding to growth phase I (15 mm in diameter, n = 2,000), sixth or seventh born along the shoot, were tagged on the trees. Of these, 1000 fruit were covered with transparent, 15 × 10 cm in size, 100-mesh bags to prevent natural pollination by wasps, and served as the non-pollinated control group, while the other 1000 were hand-pollinated. Pollen was extracted from five caprifig (male-type) fruit of selected varieties; at the mature ripening phase. The split fruit was gently tapped, and the pollen was shaken into a small vial and immediately stored at −20 ◦ C. A pollen mix of equal amounts from five selected caprifig trees (0.5 g) were dissolved in 100 mL of a 2% sucrose solution (2 g sucrose, 100 mL ddH2 O) and injected into the ‘Brown Turkey’ fruit through the ostiole, with a plastic syringe. To ensure that the whole inflorescence comes into contact with the pollination solution, fruit were filled until a drop of the pollination solution came out of the ostiole, resulting in 320 ± 25 pollinated flowers per fruit. Following hand-pollination, all fruit were covered with mesh bags (100 mesh) to avoid infection and to ensure uniform fruit development, the bags were removed at harvest. 50 pollinated and 50 parthenocarpic fruits were removed from the trees at each developmental or ripening stage to be measured or stored (ripening fruit only). Fruits from each treatment were collected every 7 days for 90 days post-pollination to create a development profile. At 90 days post-pollination, all ripened fruit were harvested and classified according to their ripening stage, which was determined by the percentages of color coverage on the outer fruit skin (30% of the fruit exterior area, 60% etc.). The non-ripened fruit were left on the tree and harvested a week later. At each developmental stage, 15 fruits were subjected to weight, diameter, height and deformation measurements and samples were kept at −20 ◦ C for later measurements of soluble solids content (SSC) and acidity. Five fruits from each treatment were fixed in freshly prepared FAA fixative (50% ethanol, 5% acetic acid, 10% formaldehyde and 35% ddH2 O) for further histological examination. 2.2. Storage trial For the storage trials, eight fruit per fruit-ripening stage—30%, 60% and 100% ripened and over-ripe were harvested and arranged in cartons with plastic-cavity insert trays, each fruit in a separate cavity. The cartons were stored at 1–2 ◦ C and 90–95% relative humidity (RH) for 7 days (storage period I) followed by 2 days at 20 ◦ C and 85% RH (shelf simulation), or for 14 days at 1–2 ◦ C and 90–95% RH (storage period II). 2.3. Ethylene-production measurements Eight fruits per stage or treatment were collected for ethyleneproduction analysis. The analysis was performed by enclosing one fruit in a 1-L airtight glass jar for 2 h at 20 ◦ C, then withdrawing the headspace gas using a syringe and injecting its content into a gas chromatograph (GC). GC analysis of ethylene was performed in a Varian 3300 GC instrument with flame-ionization detector (FID) (Varian Inc., Palo Alto, CA, USA), using a stainless-steel column (length 1.5 m, outside diameter 3.17 mm, internal diameter
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2.16 mm) packed with HayeSep T, particle size 0.125–0.149 mm (Alltech Associates Inc., Deerfield, IL, USA), and helium as the carrier gas (5 mL/min) (Freiman et al., 2012). 2.4. Histological analysis Each fruit was fixed in FAA (formalin, acetic acid, 96% ethanol and water 1:0.5:5:3.5) at room temperature for at least 3 weeks. Fixed tissue was cut into small 4- to 6-mm thick slices, dehydrated through a graded ethanol series (up to 100%) and embedded in Paraplast plus (Oxford Labware). Cross sections (15 m) were cut using a rotary model RM2245 microtome (Leica Biosystems) and collected on saline-coated glass slides. Sections were stained using Fast-Green FCF dye prior to light microscopy (Ruzin, 1999).
distilled water with 2 drops of 1% phenolphthalein as the indicator were mixed in a conical flask and titrated with aqueous 0.1 N NaOH until a slight but stable pink color appeared. The volume of NaOH used to neutralize the acidity of the juice was recorded and used to calculate TA. Each developmental fruit stage and storage batch measurements were performed on 10 fruits, two repeats per fruit. 2.5.4. Statistical analyses All experiments were designed in random distributions. Data are presented in tables and bar graphs as the mean ± standard error between 10 and 15 samples (SE). 3. Results 3.1. Effect of pollination on flower and seed development
2.5. Fruit quality 2.5.1. Fruit weight, width and firmness Fruit weight was measured using a SNOWREX LB-300 weightscale. Width was measured by a standard caliber, as the maximal horizontal fruit diameter. Fruit firmness was measured using an Inspekt Table Blue universal testing machine with 5 kN capacity (Hegewald & Peschke MTP, Nossen, Germany). Fruit firmness was expressed in Newtons as F(N) = the energy used to deform the fruit to 5% of its diameter.
Pollinated fruits showed altered developmental processes. Progression of the pollinated inflorescence pistils was more conspicuous than that of the parthenocarpic fruit, with style deterioration and color alterations beginning 1 week after pollination (WAP). Pollinated styles became brown (Fig. 1A, 1 WAP), whereas withering was absent in the parthenocarpic fig. At this stage, the embryo was already developing in the pollinated ovary (Fig. 1D). Pollinated pistils changed their color to red 5 WAP, 2 weeks earlier than the parthenocarpic pistils (Fig. 1A, G, F). Nevertheless, as the fruit reached the final green stage before ripening onset, both parthenocarpic and pollinated fruit displayed red inflorescences, even though the seeds of the pollinated fruit harbored a mature embryo while the styles of the parthenocarpic fruit pistils remained underdeveloped (Fig. 1H and I).
2.5.2. Soluble solids content (SSC) Fruit were frozen and stored until SSC (%) analysis. For this analysis, the fruit were thawed, squeezed through a double layer of gauze and SSC of the juice was measured using a digital refractometer (Atago, Tokyo, Japan). Eight different fruits were sampled for each measurement.
3.2. Effect of pollination on fig fruit development
2.5.3. Fruit acidity Titrable acidity (TA) was determined using an acid–base titration method (Sadka et al., 2000). Briefly, 2 mL fruit juice and 10 mL
Ripening pollinated fruit differed from parthenocarpic ones in shape: round for the former, pear-shaped for the latter (Fig. 2A). The differences could already be detected during the transition from
Fig. 1. Inflorescence progression in pollinated and parthenocarpic fig. (A) Inflorescence development of pollinated (top) and parthenocarpic (bottom) fruits. (B) Pollinated and (C) parthenocarpic fruit at pollination. (D) Pollinated and (E) parthenocarpic fruit 1 week after pollination (WAP). (F) Pollinated and (G) parthenocarpic fruit 5 WAP. (H) Pollinated and (I) parthenocarpic fruit 9 WAP.
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Fig. 2. Fruit development of parthenocarpic and pollinated fig. (A) Ripe pollinated (poll) and parthenocarpic (part) fruit. Fruit diameter in (B) phase I-II and (C) phase III of fruit development. Fruit weight during (D) phase I-II and (E) phase III. (F) Percentage of fruit in different ripening stages 9 weeks after pollination (WAP). (G) Percentage of fruit in different ripening stages at 10 weeks + 2 days after pollination Red—parthenocarpic, blue—pollinated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
phase I to phase II, 3 WAP, when the diameter of the pollinated fruit was slightly larger and their weight almost double that of the parthenocarpic fruit (Fig. 2B and D). The weight of parthenocarpic and pollinated fruits increased by 5.35 and 9.07 g, respectively, between 3 and 9 WAP (Fig. 2D). It should be noted that there were no differences in fruit length between parthenocarpic and pollinated fruit at any time during fruit development (data not shown).
Phase II of fruit development is conventionally known as the ‘quiescent’ period in which the fig fruit experiences major growth arrest. However, neither parthenocarpic nor pollinated fruit showed complete arrest during this phase as their diameter increased by 2 and 5 mm, respectively (Fig. 2B). During phase III, the differences in diameter and weight between parthenocarpic and pollinated fruit continued to expand (Fig. 2C and E). The strikingly rapid growth in
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phase III reached its maximum at the 100% ripened stage, in which the diameter of parthenocarpic and pollinated fruit was 4.5 and 5 cm, respectively (Fig. 2C). At this stage, both parthenocarpic and pollinated fruit reached their maximal weight, 80 and 100 g, respectively. The difference in fruit weight was noticeable from the 60% ripened stage onward (Fig. 2E). At 10 WAP, 67% of the parthenocarpic fruit were at least 60% ripened while 68% of the pollinated fruit were still green, as only 28% of them had reached the 60% ripening stage (Fig. 2F). Almost all of the pollinated fruit were fully ripe by the 11th WAP (Fig. 2G).
3.3. Effect of pollination on fig fruit ripening 3.3.1. Fruit color and ethylene production Fruit ripening stage was determined according to the fruit skin’s anthocyanin fluorescence. The skin anthocyanin increased during fruit ripening with no significant differences between parthenocarpic and pollinated fruit, indicating identical ripening stages (data not shown). On the other hand, the inner inflorescence showed significant and visible color differences throughout fruit development (Fig. 1A). The inner inflorescence of parthenocarpic fruit started to gain its red color at 6 WAP while in pollinated fruit it was already completely red at this time point (Fig. 1A). Anthocyanin level in the parthenocarpic inflorescence reached the same level as the pollinated fruit during later developmental stages (Fig. 1A). Ethylene production started to rise with ripening at 10% colorand peaked when fruits were at 60% color. There were no significant differences in ethylene production between parthenocarpic and pollinated fruit (Fig. 3A).
3.3.2. Fruit taste parameters 3.3.2.1. Acidity. Both parthenocarpic and pollinated fruits at different ripening stages were evaluated for their acidity level. In general, pollinated fruit had higher TA levels than parthenocarpic fruit (Fig. 3B). During the fruit-ripening process, TA level dropped sharply in both the parthenocarpic and pollinated groups. However, even at the fully ripened developmental stage, pollinated fruit consistently showed higher levels of acidity than parthenocarpic fruit (Fig. 3B).
3.3.2.2. SSC. SSC increased throughout the fruit-ripening process from 4% in the 30% ripened fruit to 9% in the 100% ripened fruit. No significant differences were found between parthenocarpic and pollinated fruit (Fig. 3C).
3.3.2.3. Fruit firmness. One of the most important commercial characteristics for figs is their firmness, which affects consumption quality and shipment ability. Pollinated fruit were significantly firmer than parthenocarpic fruit. The differences were already noticeable between 8 and 9 WAP in premature green fruit (Fig. 3D). During the ripening process, fruit firmness declined. However, the pollinated fruit remained firmer than the parthenocarpic fruit throughout the entire ripening process, showing the most significant difference at the 10% ripened stage (Fig. 3D). These results corresponded to a thicker cell wall in the pollinated fruit, as revealed by Ruthenium red staining (Fig. 3E). While both fruit groups showed a decline in cell-wall staining at the 100% ripened stage compared to the green fruit at 9 WAP, it was much more prominent in the pollinated vs. parthenocarpic fruit at the 9 WAP (Fig. 3E, 1 and 2). Cell-wall staining in the pollinated fruit remained at a higher intensity than in the parthenocarpic fruit at the 100% ripened stage (Fig. 3E, 3 and 4).
3.4. Effect of pollination on storage of fig fruits 3.4.1. Fruit diameter and weight under storage and shelf conditions The influence of storage and shelf conditions on parthenocarpic and pollinated fig fruit was studied by storing the fruits for 7 and 14 days under storage conditions, followed by 2 days of shelf conditions. Fruit diameter and weight significantly declined under storage and shelf conditions (Fig. 4A and B). After 7 and 14 days of storage, 60% ripened pollinated fruit showed significant superiority in fruit diameter over their parthenocarpic counterparts. However, after an additional 2 days of shelf conditions, both parthenocarpic and pollinated fruit displayed very similar diameters with no significant differences (data not shown). Weight loss after 7 and 14 days of storage and shelf conditions, respectively, was similar for both pollinated and parthenocarpic fruits (Fig. 4B). 3.4.2. Acidity and SSC in stored pollinated and parthenocarpic fruit There were no significant differences in TA or SSC level of parthenocarpic vs. pollinated 60% ripened fruit under the different storage and shelf conditions. At the end of the storage and shelf period, pollinated fruit had higher acidity levels with no significant difference in SSC compared to the parthenocarpic fruit (data not shown). 3.4.3. Firmness and spoilage of stored pollinated and parthenocarpic fruit A decrease in firmness was detected in both parthenocarpic and pollinated fruit, being more rapid in the former than the latter. After 7 of days in storage, no significant decline in firmness was detected in the pollinated fruit, whereas it declined by 40% in the parthenocarpic fruit. After 14 days, no additional decline of parthenocarpic fruit firmness was detected, while the pollinated fruit firmness declined by 25% (Fig. 4C). The pollinated fruit maintained their higher firmness at the end of both periods compared to the parthenocarpic fruit (Fig. 4C). Moreover, the high firmness of the pollinated fruit, prior to and after storage, was also noticeable in fully colored ripened fruit (data not shown). In addition to the storage experiments with commercially mature figs, we attempted to store fruit that had been harvested at an advanced stage of ripening. The pollinated fruit showed much better keeping quality, even when harvested fully ripe (data not shown). Parthenocarpic fruit showed higher susceptibility to mold and skin tearing than pollinated fruit. While being held at 1 ◦ C for 7 or 14 days, there was no mold formation or skin tears on any of the 60% ripened pollinated or parthenocarpic fruit. However, at the end of the 7- and 14-day storage periods, the outer skin and interior of the pollinated fruit was intact (Fig. 5A), whereas the parthenocarpic outer skin became more brown and the interior began to deteriorate (Fig. 5B). When kept for an additional 2 days of shelf life at 20 ◦ C, the marketable appearance of the pollinated fruit, which showed no susceptibility to mold, was maintained compared to the parthenocarpic fruit, which showed 55% susceptibility to mold (Fig. 5B). 4. Discussion The fig bears a unique closed inflorescence structure—the syconium—that is not present in any other fruit in the human diet. The multiple fig fruit is composed of individual drupelets that develop from the ovaries enclosed in the succulent receptacle to form a single accessory fruit (Flaishman et al., 2008; Storey, 1977). Fig is one of the earliest domesticated plants and human-assisted caprification of its Smyrna- and San-Pedro-type cultivars is one of the oldest known horticultural procedures
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Fig. 3. Fig-ripening parameters for pollinated and parthenocarpic fruit. (A) Ethylene production during fruit development and ripening. (B) Acidity level in ripening fruit. (C) Soluble solids content (SSC) of ripening fruit. (D) Firmness evaluation of fruit during development and ripening. (E) Cell-wall staining in green fruit 9 weeks after pollination (WAP) (1-pollinated, 2-parthenocarpic) and 100% ripened fruit (3-pollinated, 4-parthenocarpic). Red—parthenocarpic, blue—pollinated; Y AXIS: ethylene (L/kg h); soluble solids (SSC); PART E: cell-wall stain; pollinated; parthenocarpic. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Zohary and Spiegel-Roy, 1975; Kislev et al., 2006). These fig types have pollination-obligatory summer crops, but the San-Pedro-type cultivars produce a parthenocarpic spring crop. The third edible type, the common-type fig, has a facultative parthenocarpic fruit, producing both pollinated and parthenocarpic fruit during the summer. This feature makes the common-type fig a good platform to study the effect of fig caprification on fruit development, quality and post-harvest performance. In this study, we showed that fig caprification leads to substantial changes in fruit development as well as high quality of the mature fruit with better post-harvest performance. Normal fruit development is an outcome of successful pollination and fertilization events, where fertilization of the ovule usually leads to ovary expansion and fruit development (Gillaspy et al., 1993). Alternative pathways to normal fertilization involve exogenous hormone treatments or endogenous genetic stimuli that induce parthenocarpic fruit development. In the last few decades, parthenocarpic fruit development and ripening processes have been studied in many plants, such as tomato, watermelon, strawberry and citrus (Hayata et al., 1995; Gorguet et al., 2005; Serrani et al., 2007; Thompson, 1969; Vardi et al., 2008). Several studies have compared pollinated to parthenocarpic figs. In some, parthenocapy was induced by external application or osti-
ole injection of hormones (Crane and Blondeau, 1949; Crane and Van Overbeek, 1965). In others, pollination effects were examined in relation to development and fruit characteristics. Those studies applied varying degrees of caprification (Gaaliche et al., 2011a,b) or tested pollinated fruit from human-assisted caprification vs. fruit grown far from a pollen source as the non-pollinated group (Trad et al., 2013; Trad et al., 2012). One study was performed on two separate crops of a San-Pedro-type cultivar (Lodhi et al., 1969). The first (spring) crop constituted a naturally occurring parthenocarpic group, while the second (summer) crop was the pollination-obligatory group. Our study was performed on hand-pollinated fig fruit and naturally facultatively parthenocarpic common-type fig. The fruits were sampled weekly and evaluated throughout development until the 100% ripened stage. The effect of pollination on fruits under storage conditions was also monitored. Soon after fig pollination (1 WAP), the pollinated pistil showed style deterioration while the pistil of the parthenocarpic fruit remained intact throughout fruit development (Fig. 1). This phenomenon has been recorded in partially developed parthenocarpic fruit in Arabidopsis, where the unfertilized pistil does not degenerate, probably due to high gibberellin responsiveness (Carbonell-Bejerano et al., 2010). Crane and Van Overbeek (1965) noted that seed development in the pollinated fig inflorescence
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Fig. 4. Characteristics of 60% ripened fruit after storage. (A) Diameter, (B) weight, (C) firmness. Red—parthenocarpic fruit, blue—pollinated fruit. Storage I = 7 days, storage II = 14 days. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Fruit spoilage in 60% ripened fruit after storage for 14 days at 1 ◦ C. (A) Pollinated fruit. (B) Parthenocarpic fruit.
includes the formation of a rigid seed coat, whereas the parthenocarpic seed was smaller and lacked rigidity. This phenomenon can be detected in Fig. 1H and I, were few empty seeds are found in parthenocarpic fruit. This was also shown by Freiman et al. (2015). The color of the pollinated inflorescence turned red, while in the parthenocarpic fruit, inflorescence coloration was delayed. Previous studies have also mentioned color differences between the caprified fig inflorescence and hormone-induced parthenocarpic figs (Crane and Blondeau, 1949; Crane and Van Overbeek, 1965).
A comparison between parthenocarpic and pollinated fruit development clearly revealed the superiority of the pollinated ripe fig fruit over their parthenocarpic counterparts in growth, width, weight, firmness and taste qualities (Figs. 2, 3). It was clear from our results that the pollinated ripened fruit are larger in both diameter and weight than the parthenocarpic fruit. Similarly, several authors have shown that strongly human-assisted caprified fruit are larger and heavier than non-caprified ones (Gaaliche et al., 2011a,b; Trad et al., 2012). It is likely that in all of the above cases, the larger and heavier fruit are a consequence of inflorescence swelling, seed development and closure of the inner cavity. However in Crane and Blondeau (Crane and Blondeau, 1949), hormonal induction produced parthenocarpic fruit that were similar in size to the caprified ones, probably because the hormone induction resembled caprified fertilization of the flowers. Inflorescence swelling, seed development and closure of the inner cavity together with cell-wall thickening, as shown in our work, could cause the pollinated fruit to be firmer than the parthenocarpic one. The softer caprified fruit described by Trad et al. (2013) could have been from a different variety or the fruit might have been measured at a different stage than ours. The taste of the ripened pollinated fruit was more sour than that of the parthenocarpic fruit, with higher TA, whereas the caprified fruit described by Gaaliche et al. (2011b) and Trad et al. (2012) showed no acidity differences with noncaprified fruit. The SSC was lower in our pollinated fruit, oppose to the caprified fruit in Gaaliche et al. (Gaaliche et al., 2011b) that showed no significant difference in total soluble solids compared to non-caprified fruit. Storage and shelf life of the pollinated common-type figs were improved compared to the parthenocarpic fruit. Stored pollinated fruit retained their commercially favored physiological characteristics over parthenocarpic fruit, while maintaining their superior size, higher firmness and better taste parameters. Pollination by pollens from different trees may carry some disease, none were found in our
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study and in addition, the stored parthenocarpic fruit were highly sensitive to mold growth and skin injury, dramatically reducing their commercial value (Fig. 5). We suggest that the thicker cell wall of the pollinated fruit delayed loss of cellular water, resulting in firmer stored fruits. Thicker cell wall may also be the cause of the high storability of pre-harvest 1-MCP treated figs. While cell wall degradation during ripening is ethylene induced, blocking its perception surely effects this process (Freiman et al., 2012). The assumption that fertilization creates a stimulus that strengthens the ovules and ovary as sinks and diverts the flow of metabolites from the vegetative organs, was mentioned by Crane and Van Overbeek in relation to fig fruit (Crane and Van Overbeek, 1965). Parthenocarpic fig induction by use of external applications of gibberellins, auxins and cytokinins has been proven in several studies, stating a number of variable parameters between the two systems—pollinated fruit and induced parthenocarpy (Crane and Blondeau, 1949; Crane and Van Overbeek, 1965). Other studies published in recent years have shown that fertilization efficiency and pollen source affect several fig fruit quality characteristics (Gaaliche et al., 2011a,b). Cultivars belonging to the Smyrna and San-Pedro types, in which the summer crop is caprification-obligatory, also presented distinctive parameters when comparing fully caprified and partially caprified fruits (Trad et al., 2013, 2012). Lodhi et al. (Lodhi et al., 1969), working with a San-Pedro-type cultivar, found higher auxin concentrations in the first parthenocarpic crop than in the second pollinated crop, whereas gibberellin concentrations were higher in the second pollinated crop. It was concluded that figs possessing functional seeds and those with non-bearing seeds present different developmental and ripening features. Similarly in our study, the differential development of pollinated and parthenocarpic figs resulted from the presence or absence of functional seeds. This research establishes an optimal experimental system to identify the signals originating from the seed that initiate fruit development, and to separate the specific pathways that also occur naturally in the parthenocarpic but otherwise identical fig. Identification of parthenocarpy inducers in the fig will contribute to our general understanding of this phenomenon in other plants.
Acknowledgments We thank Omer Ben-Yehoshua for use of his excellent fig orchard. The financial support for this research was provided by a grant from the Chief Scientist of the Israeli Ministry of Agriculture.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2015. 11.027. References Carbonell-Bejerano, P., Urbez, C., Carbonell, J., Granell, A., Perez-Amador, M.A., 2010. A fertilization-independent developmental program triggers partial fruit development and senescence processes in pistils of Arabidopsis. Plant Physiol. 154 (1), 163–172. Celikel, F.G., Karacali, I., 1998. Effects of harvest maturity and precooling on fruit quality and longevity of Bursa Siyahi figs (Ficus carica L.). Acta Hortic. 480, 283–288. Chessa, I., Mitra, S.K., 1997. Postharvest Physiology and Storage of Tropical and Subtropical Fruits. CAB International, Wallingford, UK, pp. 245–268. Colelli, G., Mitchell, F.G., Kader, A.A., 1991. Extension of postharvest life of ‘Mission’ figs by CO2 -enriched atmospheres. HortScience 26 (9), 1193–1195. Condit, I.J., 1920. Caprifigs and caprification. Calif. Agric. Expt. Stat. Bull. 319, 341–375. Condit, I.J., 1947. The Fig Chronica Botanica. Walthams, Massachusetts, 222p.
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Crane, J.C., Blondeau, R., 1949. The use of growth-regulating chemicals to induce parthenocarpic fruit in the calimyrna fig. Plant Physiology. 24 (1), 44–54. Crane, J.C., Van Overbeek, J., 1965. Kinin-induced parthenocarpy in the fig, Ficus carica L. Science 147 (3664), 1468–1469. Crane, J.C., 1986. CRC Handbook of Fruit Set and Development. CRC Press, Boca Raton, FL, pp. 153–165. Flaishman, M.A., Rodov, V., Stover, E., 2008. The fig: botany, horticulture, and breeding. Hortic. Rev. 34, 132–196. Freiman, Z.E., Rodov, V., Yablovitz, Z., Horev, B., Flaishman, M.A., 2012. Preharvest application of 1-methylcyclopropene inhibits ripening and improves keeping quality of ‘Brown Turkey’ figs (Ficus carica L.). Sci. Hortic. 138, 266–272. Freiman, Zohar E., Rosianskey, Yogev, Dasmohapatra, Rajeswari, Kamara, Itzhak, Flaishman, Moshe A., 2015. The ambiguous ripening nature of the fig (Ficus carica L.) fruit: a gene-expression study of potential ripening regulators and ethylene-related genes. J. Exp. Bot. 66 (11), 3309–3324, erv140. Gaaliche, B., Trad, M., Mars, M., 2011a. Effect of pollination intensity, frequency and pollen source on fig (Ficus carica L.) productivity and fruit quality. Sci. Hortic. 30 (4), 737–742. Gaaliche, B., Hfaiedh, L., Trad, M., Mars, M., 2011b. Caprification efficiency of some Tunisian local fig (Ficus carica L.) cultivars. Pakistan J. Agric. Sci. 48 (4), 295–298. Galil, J., Eisikowitch, D., 1968. Flowering cycles and fruit types of Ficus sycomorus in Israel. New Phytol. 67, 745–758. Galil, J., Neeman, G., 1977. Pollen transfer and pollination in the common fig (Ficus carica L.). New Phytol. 79 (1), 163–171. Gillaspy, G., Ben-David, H., Gruissem, W., 1993. Fruits: a developmental perspective. Plant Cell 5 (10), 1439–1451. Gorguet, B., Heusden, V.A.W., Lindhout, P., 2005. Parthenocarpic fruit development in tomato. Plant Biol. 7 (2), 131–139. Hayata, Y., Niimi, Y., Iwasaki, N., 1995. Synthetic cytokinin-1-(2 = chloro = 4 = pyridyl)-3-phenylurea (CPPU)-promotes fruit set and induces parthenocarpy in watermelon. J. Am. Soc. Hortic. Sci. 120 (6), 997–1000. Küden, A.B., 1996. Mediterranean Selected Fruits Intercountry Network (MESFIN) under the aegis of FAO. Plant Resources of Fig. Kirthikar, K.R., Basu, B.D., 1986. In: Blatter, E., Caius, I.F. (Eds.), Indian Medicinal Plants. , second ed. International Book Distributors, Dehradun, India, pp. 2329–2331. Kislev, M.E., Hartmann, A., Bar-Yosef, O., 2006. Early domesticated fig in the Jordan Valley. Science 312 (5778), 1372–1374. Lodhi, F., Bradley, M.V., Crane, J.C., 1969. Auxins and gibberellin-like substances in parthenocarpic and non-parthenocarpic syconia of Ficus carica L., cv. King. Plant Physiol. 44 (4), 555–561. Marei, N., Crane, J.C., 1971. Growth and respiratory response of fig (Ficus carica L. cv. Mission) fruits to ethylene. Plant Physiol. 48 (3), 249–254. Michailides, T.J., Morgan, D.P., Felts, D., Doster, M.A., 2008. Control of decay in caprifigs and calimyrna figs with fungicides. Acta Hortic. 798, 269–275. Michailides, T.J., 2003. In: Ploetz, R.C. (Ed.), Diseases of Fig. CAB International, Wallingford, UK, pp. 253–273. Oukabli, A., Mamouni, A., Laghezali, M., Ater, M., Roger, J.P., Khadari, B., 2001. Local caprifig tree characterization and analysis of interest for pollination. In: II International Symposium on Fig. 605. pp 61–64. Ruzin, S.E., 1999. Plant Microtechnique and Microscopy. Oxford University Press, Cambridge, UK. Sadka, Avi, et al., 2000. Arsenite reduces acid content in citrus fruit, inhibits activity of citrate synthase but induces its gene expression. J. Am. Soc. Hortic. Sci. 125 (3), 288–293. Serrani, J.C., Fos, M., Atare´ıs, A., Garcı´ıa-martı´ınez, J.L., 2007. Effect of gibberellin and auxin on parthenocarpic fruit growth induction in the cv Micro-Tom of tomato. J. Plant Growth Regul. 26 (3), 211–221. Solomon, A., Golubowicz, S., Yablowicz, Z., Grossman, S., Bergman, M., Gottlieb, H.E., Altman, A., Kerem, Z., Flaishman, M.A., 2006. Antioxidant activities and anthocyanin content of fresh fruits of common fig (Ficus carica L.). J. Agric. Food Chem. 54 (20), 7717–7723. Solomon, A., Golubowicz, S., Yablowicz, Z., Bergman, M., Grossman, S., Altman, A., Kerem, Z., Flaishman, M.A., 2010a. EPR studies of O2- , OH, and 1 O2 scavenging and prevention of glutathione depletion in fibroblast cells by cyanidin-3rhamnoglucoside isolated from fig (Ficus carica L.) fruits. J. Agric. Food Chem. 58, 7158–7165. Solomon, A., Golubowicz, S., Yablowicz, Z., Bergman, M., Grossman, S., Altman, A., Kerem, Z., Flaishman, M.A., 2010b. Protection of fibroblasts (NIH-3T3) against oxidative damage by cyanidin-3-rhamnoglucoside isolated from fig fruits (Ficus carica L.). J. Agric. Food Chem. 58, 6660–6665. Storey, W.B., 1977. The Fig: Its Biology, History, Culture, and Utilization. Jurupa Mountains Cultural Center, California USA. Thompson, P.A., 1969. The effect of applied growth substances on development of the strawberry fruit: II: interactions of auxins and gibberellins. J. Exp. Bot. 20 (3), 629–647. Trad, M., Ginies, C., Gaaliche, B., Renard, C.M.G.C., Mars, M., 2012. Does pollination affect aroma development in ripened fig (Ficus carica L.) fruit? Sci. Hortic. 134, 93–99. Trad, M., Le Bourvellec, C., Gaaliche, B., Ginies, C., Renard, C.M.G.C., Mars, M., 2013. Caprification modifies polyphenols but not cell wall concentrations in ripe figs. Sci. Hortic. 160, 115–122.
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Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Advanced analysis of developmental and ripening characteristics of pollinated common-type fig (Ficus carica L.) Yogev Rosianski a,b , Zohar E. Freiman a , Shira Milo Cochavi a,b , Zeev Yablovitz a , Zohar Kerem b , Moshe A. Flaishman a,∗ a
Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan 50250, Israel The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel b
a r t i c l e
i n f o
Article history: Received 21 August 2015 Received in revised form 12 November 2015 Accepted 17 November 2015 Available online 14 December 2015 Keywords: Fig fruit Ficus carica Caprification Storage
a b s t r a c t Development and ripening processes differ in pollinated and parthenocarpic fruit. While the facultative parthenocarpic common-type fig fruit serves as a receptacle for flower development, it becomes fleshy by either pollination or through a parthenocarpic process. Here we studied the effect of pollination on common-type fig fruit development and ripening characteristics compared to the parthenocarpic fruit under otherwise identical conditions. The effects of pollination on fruit development were investigated on the tree and in storage. Pollinated fruit showed altered developmental processes. Ripened pollinated fruit were round, in contrast to the pear-like shape of the parthenocarpic fruit. The pollinated fruit also had a larger diameter and weight and improved firmness compared to the parthenocarpic fruit. At harvest, the pollinated fruit exhibited more commercially desirable physical and taste characteristics, with advanced fertile nutlets compared to the sterile undeveloped non-bearing nutlets of the parthenocarpic fruit. During storage, senescence and spoilage of the pollinated fruit were slower than in parthenocarpic fruit, as manifested by firmness, internal texture, weight, size, shriveling, and decay. Thus, pollination of the common-type fig cultivar Brown Turkey delayed senescence and extended the shelf life of its fruit. The external and internal morphological differences throughout post-pollination development make common-type fig an excellent research tool for studies of physiological and molecular aspects of pollination. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The fig tree, Ficus carica L. (Moraceae), is one of the world’s oldest horticultural crops. It is indigenous to wide areas, ranging from Asiatic Turkey to northern India, and local varieties are cultivated in most Mediterranean countries (Küden, 1996). The fig fruit is well known for its attractive taste and nutritive value due to its antioxidant properties, and it is consumed fresh or dried worldwide (Solomon et al., 2006; Solomon et al., 2010a,b; Kirthikar and Basu, 1986). The fig is an aggregate fruit composed of individual small drupes, each termed a drupelet. The drupelets develop from the ovaries in a closed inflorescence, known as the syconium (the edible fig fruit), which encloses many unisexual female flowers (Flaishman et al., 2008).
∗ Corresponding author. Fax: +972 3 9683973. E-mail address: [email protected] (M.A. Flaishman). http://dx.doi.org/10.1016/j.scienta.2015.11.027 0304-4238/© 2015 Elsevier B.V. All rights reserved.
Fig fruit development can be described by a double sigmoid growth curve comprising three phases (Marei and Crane, 1971). Phase I is characterized by rapid growth in size as a result of extensive cell division. During phase II, fruit growth is arrested. Rapid fruit growth in phase III is mainly a result of cell expansion; during this phase, the fig is ripening, changing its skin and inner inflorescence colors, accumulating sugars and acids, and producing volatiles similar to fleshy fruits. The rate of the ripening process in fig is extremely rapid, lasting several days. At the end of phase III, the fruit is two to three times larger than in phase II, with changes in its color as a result of anthocyanin accumulation in the skin (Solomon et al., 2006); its dry weight is 70% higher and total sugars increase by 90% relative to phase II fruit; it accumulates water and becomes soft, and the pulp texture undergoes modifications to an edible state (Flaishman et al., 2008; Chessa and Mitra, 1997; Crane, 1986). Although fig fruit is categorized as climacteric, showing a rise in respiration rate and ethylene production at the onset of the ripening phase, when
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harvested prior to complete ripening, it never reaches the commercially desirable parameters of size, color, flavor and texture. Alternatively, when harvested late, the figs tend to perish due to over-ripening and high susceptibility to pathogens (Flaishman et al., 2008; Chessa and Mitra, 1997). Normally, fungal and yeast infection occurs in the orchard, and is fully demonstrated in ripening harvested fruits (Michailides, 2003). Rapid cooling together with controlled or modified atmosphere during storage enables sustaining fruit quality (Turk, 1989; Celikel and Karacali, 1998; Turk et al., 1993; Colelli et al., 1991). The fruit developmental stage for commercial harvest is cultivar-specific. For dark-skinned fig cultivars, this is generally at 50–80% color coverage of the skin area. At this stage, the fig is edible with a short storage capacity of only 7 days under cooling conditions (Chessa and Mitra, 1997). Fig cultivars are divided into four types according to their pollination requirement (called caprification in figs) and cropping season (Flaishman et al., 2008). The first type, the caprifig, commonly termed ‘male fig’, is not edible, and is the commercial planting source for pollen. Two edible fig types, Smyrna and SanPedro, are requiring caprification to bear their main crops. The unique San-Pedro types yield an early parthenocarpic crop known as ‘breba’, in addition to the pollinated main crop. The fourth type is the common female type, also called ‘persistent’ fig, which yields one or two crops per year, with or without caprification (Flaishman et al., 2008; Galil and Eisikowitch, 1968; Galil and Neeman, 1977). Pollination of the different fig types is a consequence of a complex symbiotic relationship with the fig wasp. The pollen-carrying wasp Blastophaga psenes accesses the inner fig cavity through the fruit ostiole and pollinates the female flowers (Galil and Neeman, 1977). The caprified figs are usually larger and greener than the noncaprified ones (Condit, 1947; Oukabli et al., 2001; Michailides et al., 2008). Studies on the effect of caprification on fruit quality in the common fig type cv. Kadota reported higher fruit weight together with a tendency toward splitting, and changes in several fruit characteristics, including darker skin and red-colored inflorescences in addition to the sweeter and richer flavor of the pollinated fruits (Gaaliche et al., 2011a; Condit, 1920). Caprified San-Pedro-type fruit showed modifications in their cell-wall components which moderately affected several textural parameters, as well as stimulation of anthocyanin and total polyphenol biosynthesis (Trad et al., 2013). In flowering plants, after pollination of the stigma, seed set and fruit progression proceed in a well-coordinated manner (Gillaspy et al., 1993). Plants can also set fruit in the absence of fertilization, a phenomenon called parthenocarpy. Thus, parthenocarpic cultivars overcome the environmental impact of adverse hot or cold temperatures on fruit production. A distinction is often made between obligatory parthenocarpy, which always results in seedless fruit, and facultative parthenocarpy, which results in seedless fruit only when pollination is prevented. Parthenocarpic, seedless horticultural fruit are preferred over their seeded counterparts in many crops. Parthenocarpic tomatos, as well as parthenocarpic banana, pineapple and some grape and citrus cultivars, are good examples of facultative parthenocarpy because they only produce seedless fruit in the absence of fertilization. However, detailed studies of the quality of parthenocarpic and pollinated fruit is lacking in many fruit. To the best of our knowledge, a comprehensive study of fig fruit development based on comparing pollinated and parthenocarpic fruit of the same cultivar and crop has never been performed. In this research, we examined the influence of pollination on the development of the common-type facultatively parthenocarpic fig fruit of cv. Brown Turkey, and on its performance under commercial fruit-storage conditions.
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2. Materials and methods 2.1. Plant material and treatment Mature fig trees (Ficus carica L., cv. Brown Turkey) growing in a commercial orchard near Beer Tuvia, Israel (31◦ 44’13.66 N, 34◦ 43’32.05 E) were used in two field experiments performed on May 2012 and 2013. ‘Brown Turkey’ belongs to the common-type figs and produces parthenocarpic and pollinated fruit. All of the trees were maintained according to commercial production practices. Fruit corresponding to growth phase I (15 mm in diameter, n = 2,000), sixth or seventh born along the shoot, were tagged on the trees. Of these, 1000 fruit were covered with transparent, 15 × 10 cm in size, 100-mesh bags to prevent natural pollination by wasps, and served as the non-pollinated control group, while the other 1000 were hand-pollinated. Pollen was extracted from five caprifig (male-type) fruit of selected varieties; at the mature ripening phase. The split fruit was gently tapped, and the pollen was shaken into a small vial and immediately stored at −20 ◦ C. A pollen mix of equal amounts from five selected caprifig trees (0.5 g) were dissolved in 100 mL of a 2% sucrose solution (2 g sucrose, 100 mL ddH2 O) and injected into the ‘Brown Turkey’ fruit through the ostiole, with a plastic syringe. To ensure that the whole inflorescence comes into contact with the pollination solution, fruit were filled until a drop of the pollination solution came out of the ostiole, resulting in 320 ± 25 pollinated flowers per fruit. Following hand-pollination, all fruit were covered with mesh bags (100 mesh) to avoid infection and to ensure uniform fruit development, the bags were removed at harvest. 50 pollinated and 50 parthenocarpic fruits were removed from the trees at each developmental or ripening stage to be measured or stored (ripening fruit only). Fruits from each treatment were collected every 7 days for 90 days post-pollination to create a development profile. At 90 days post-pollination, all ripened fruit were harvested and classified according to their ripening stage, which was determined by the percentages of color coverage on the outer fruit skin (30% of the fruit exterior area, 60% etc.). The non-ripened fruit were left on the tree and harvested a week later. At each developmental stage, 15 fruits were subjected to weight, diameter, height and deformation measurements and samples were kept at −20 ◦ C for later measurements of soluble solids content (SSC) and acidity. Five fruits from each treatment were fixed in freshly prepared FAA fixative (50% ethanol, 5% acetic acid, 10% formaldehyde and 35% ddH2 O) for further histological examination. 2.2. Storage trial For the storage trials, eight fruit per fruit-ripening stage—30%, 60% and 100% ripened and over-ripe were harvested and arranged in cartons with plastic-cavity insert trays, each fruit in a separate cavity. The cartons were stored at 1–2 ◦ C and 90–95% relative humidity (RH) for 7 days (storage period I) followed by 2 days at 20 ◦ C and 85% RH (shelf simulation), or for 14 days at 1–2 ◦ C and 90–95% RH (storage period II). 2.3. Ethylene-production measurements Eight fruits per stage or treatment were collected for ethyleneproduction analysis. The analysis was performed by enclosing one fruit in a 1-L airtight glass jar for 2 h at 20 ◦ C, then withdrawing the headspace gas using a syringe and injecting its content into a gas chromatograph (GC). GC analysis of ethylene was performed in a Varian 3300 GC instrument with flame-ionization detector (FID) (Varian Inc., Palo Alto, CA, USA), using a stainless-steel column (length 1.5 m, outside diameter 3.17 mm, internal diameter
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2.16 mm) packed with HayeSep T, particle size 0.125–0.149 mm (Alltech Associates Inc., Deerfield, IL, USA), and helium as the carrier gas (5 mL/min) (Freiman et al., 2012). 2.4. Histological analysis Each fruit was fixed in FAA (formalin, acetic acid, 96% ethanol and water 1:0.5:5:3.5) at room temperature for at least 3 weeks. Fixed tissue was cut into small 4- to 6-mm thick slices, dehydrated through a graded ethanol series (up to 100%) and embedded in Paraplast plus (Oxford Labware). Cross sections (15 m) were cut using a rotary model RM2245 microtome (Leica Biosystems) and collected on saline-coated glass slides. Sections were stained using Fast-Green FCF dye prior to light microscopy (Ruzin, 1999).
distilled water with 2 drops of 1% phenolphthalein as the indicator were mixed in a conical flask and titrated with aqueous 0.1 N NaOH until a slight but stable pink color appeared. The volume of NaOH used to neutralize the acidity of the juice was recorded and used to calculate TA. Each developmental fruit stage and storage batch measurements were performed on 10 fruits, two repeats per fruit. 2.5.4. Statistical analyses All experiments were designed in random distributions. Data are presented in tables and bar graphs as the mean ± standard error between 10 and 15 samples (SE). 3. Results 3.1. Effect of pollination on flower and seed development
2.5. Fruit quality 2.5.1. Fruit weight, width and firmness Fruit weight was measured using a SNOWREX LB-300 weightscale. Width was measured by a standard caliber, as the maximal horizontal fruit diameter. Fruit firmness was measured using an Inspekt Table Blue universal testing machine with 5 kN capacity (Hegewald & Peschke MTP, Nossen, Germany). Fruit firmness was expressed in Newtons as F(N) = the energy used to deform the fruit to 5% of its diameter.
Pollinated fruits showed altered developmental processes. Progression of the pollinated inflorescence pistils was more conspicuous than that of the parthenocarpic fruit, with style deterioration and color alterations beginning 1 week after pollination (WAP). Pollinated styles became brown (Fig. 1A, 1 WAP), whereas withering was absent in the parthenocarpic fig. At this stage, the embryo was already developing in the pollinated ovary (Fig. 1D). Pollinated pistils changed their color to red 5 WAP, 2 weeks earlier than the parthenocarpic pistils (Fig. 1A, G, F). Nevertheless, as the fruit reached the final green stage before ripening onset, both parthenocarpic and pollinated fruit displayed red inflorescences, even though the seeds of the pollinated fruit harbored a mature embryo while the styles of the parthenocarpic fruit pistils remained underdeveloped (Fig. 1H and I).
2.5.2. Soluble solids content (SSC) Fruit were frozen and stored until SSC (%) analysis. For this analysis, the fruit were thawed, squeezed through a double layer of gauze and SSC of the juice was measured using a digital refractometer (Atago, Tokyo, Japan). Eight different fruits were sampled for each measurement.
3.2. Effect of pollination on fig fruit development
2.5.3. Fruit acidity Titrable acidity (TA) was determined using an acid–base titration method (Sadka et al., 2000). Briefly, 2 mL fruit juice and 10 mL
Ripening pollinated fruit differed from parthenocarpic ones in shape: round for the former, pear-shaped for the latter (Fig. 2A). The differences could already be detected during the transition from
Fig. 1. Inflorescence progression in pollinated and parthenocarpic fig. (A) Inflorescence development of pollinated (top) and parthenocarpic (bottom) fruits. (B) Pollinated and (C) parthenocarpic fruit at pollination. (D) Pollinated and (E) parthenocarpic fruit 1 week after pollination (WAP). (F) Pollinated and (G) parthenocarpic fruit 5 WAP. (H) Pollinated and (I) parthenocarpic fruit 9 WAP.
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Fig. 2. Fruit development of parthenocarpic and pollinated fig. (A) Ripe pollinated (poll) and parthenocarpic (part) fruit. Fruit diameter in (B) phase I-II and (C) phase III of fruit development. Fruit weight during (D) phase I-II and (E) phase III. (F) Percentage of fruit in different ripening stages 9 weeks after pollination (WAP). (G) Percentage of fruit in different ripening stages at 10 weeks + 2 days after pollination Red—parthenocarpic, blue—pollinated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
phase I to phase II, 3 WAP, when the diameter of the pollinated fruit was slightly larger and their weight almost double that of the parthenocarpic fruit (Fig. 2B and D). The weight of parthenocarpic and pollinated fruits increased by 5.35 and 9.07 g, respectively, between 3 and 9 WAP (Fig. 2D). It should be noted that there were no differences in fruit length between parthenocarpic and pollinated fruit at any time during fruit development (data not shown).
Phase II of fruit development is conventionally known as the ‘quiescent’ period in which the fig fruit experiences major growth arrest. However, neither parthenocarpic nor pollinated fruit showed complete arrest during this phase as their diameter increased by 2 and 5 mm, respectively (Fig. 2B). During phase III, the differences in diameter and weight between parthenocarpic and pollinated fruit continued to expand (Fig. 2C and E). The strikingly rapid growth in
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phase III reached its maximum at the 100% ripened stage, in which the diameter of parthenocarpic and pollinated fruit was 4.5 and 5 cm, respectively (Fig. 2C). At this stage, both parthenocarpic and pollinated fruit reached their maximal weight, 80 and 100 g, respectively. The difference in fruit weight was noticeable from the 60% ripened stage onward (Fig. 2E). At 10 WAP, 67% of the parthenocarpic fruit were at least 60% ripened while 68% of the pollinated fruit were still green, as only 28% of them had reached the 60% ripening stage (Fig. 2F). Almost all of the pollinated fruit were fully ripe by the 11th WAP (Fig. 2G).
3.3. Effect of pollination on fig fruit ripening 3.3.1. Fruit color and ethylene production Fruit ripening stage was determined according to the fruit skin’s anthocyanin fluorescence. The skin anthocyanin increased during fruit ripening with no significant differences between parthenocarpic and pollinated fruit, indicating identical ripening stages (data not shown). On the other hand, the inner inflorescence showed significant and visible color differences throughout fruit development (Fig. 1A). The inner inflorescence of parthenocarpic fruit started to gain its red color at 6 WAP while in pollinated fruit it was already completely red at this time point (Fig. 1A). Anthocyanin level in the parthenocarpic inflorescence reached the same level as the pollinated fruit during later developmental stages (Fig. 1A). Ethylene production started to rise with ripening at 10% colorand peaked when fruits were at 60% color. There were no significant differences in ethylene production between parthenocarpic and pollinated fruit (Fig. 3A).
3.3.2. Fruit taste parameters 3.3.2.1. Acidity. Both parthenocarpic and pollinated fruits at different ripening stages were evaluated for their acidity level. In general, pollinated fruit had higher TA levels than parthenocarpic fruit (Fig. 3B). During the fruit-ripening process, TA level dropped sharply in both the parthenocarpic and pollinated groups. However, even at the fully ripened developmental stage, pollinated fruit consistently showed higher levels of acidity than parthenocarpic fruit (Fig. 3B).
3.3.2.2. SSC. SSC increased throughout the fruit-ripening process from 4% in the 30% ripened fruit to 9% in the 100% ripened fruit. No significant differences were found between parthenocarpic and pollinated fruit (Fig. 3C).
3.3.2.3. Fruit firmness. One of the most important commercial characteristics for figs is their firmness, which affects consumption quality and shipment ability. Pollinated fruit were significantly firmer than parthenocarpic fruit. The differences were already noticeable between 8 and 9 WAP in premature green fruit (Fig. 3D). During the ripening process, fruit firmness declined. However, the pollinated fruit remained firmer than the parthenocarpic fruit throughout the entire ripening process, showing the most significant difference at the 10% ripened stage (Fig. 3D). These results corresponded to a thicker cell wall in the pollinated fruit, as revealed by Ruthenium red staining (Fig. 3E). While both fruit groups showed a decline in cell-wall staining at the 100% ripened stage compared to the green fruit at 9 WAP, it was much more prominent in the pollinated vs. parthenocarpic fruit at the 9 WAP (Fig. 3E, 1 and 2). Cell-wall staining in the pollinated fruit remained at a higher intensity than in the parthenocarpic fruit at the 100% ripened stage (Fig. 3E, 3 and 4).
3.4. Effect of pollination on storage of fig fruits 3.4.1. Fruit diameter and weight under storage and shelf conditions The influence of storage and shelf conditions on parthenocarpic and pollinated fig fruit was studied by storing the fruits for 7 and 14 days under storage conditions, followed by 2 days of shelf conditions. Fruit diameter and weight significantly declined under storage and shelf conditions (Fig. 4A and B). After 7 and 14 days of storage, 60% ripened pollinated fruit showed significant superiority in fruit diameter over their parthenocarpic counterparts. However, after an additional 2 days of shelf conditions, both parthenocarpic and pollinated fruit displayed very similar diameters with no significant differences (data not shown). Weight loss after 7 and 14 days of storage and shelf conditions, respectively, was similar for both pollinated and parthenocarpic fruits (Fig. 4B). 3.4.2. Acidity and SSC in stored pollinated and parthenocarpic fruit There were no significant differences in TA or SSC level of parthenocarpic vs. pollinated 60% ripened fruit under the different storage and shelf conditions. At the end of the storage and shelf period, pollinated fruit had higher acidity levels with no significant difference in SSC compared to the parthenocarpic fruit (data not shown). 3.4.3. Firmness and spoilage of stored pollinated and parthenocarpic fruit A decrease in firmness was detected in both parthenocarpic and pollinated fruit, being more rapid in the former than the latter. After 7 of days in storage, no significant decline in firmness was detected in the pollinated fruit, whereas it declined by 40% in the parthenocarpic fruit. After 14 days, no additional decline of parthenocarpic fruit firmness was detected, while the pollinated fruit firmness declined by 25% (Fig. 4C). The pollinated fruit maintained their higher firmness at the end of both periods compared to the parthenocarpic fruit (Fig. 4C). Moreover, the high firmness of the pollinated fruit, prior to and after storage, was also noticeable in fully colored ripened fruit (data not shown). In addition to the storage experiments with commercially mature figs, we attempted to store fruit that had been harvested at an advanced stage of ripening. The pollinated fruit showed much better keeping quality, even when harvested fully ripe (data not shown). Parthenocarpic fruit showed higher susceptibility to mold and skin tearing than pollinated fruit. While being held at 1 ◦ C for 7 or 14 days, there was no mold formation or skin tears on any of the 60% ripened pollinated or parthenocarpic fruit. However, at the end of the 7- and 14-day storage periods, the outer skin and interior of the pollinated fruit was intact (Fig. 5A), whereas the parthenocarpic outer skin became more brown and the interior began to deteriorate (Fig. 5B). When kept for an additional 2 days of shelf life at 20 ◦ C, the marketable appearance of the pollinated fruit, which showed no susceptibility to mold, was maintained compared to the parthenocarpic fruit, which showed 55% susceptibility to mold (Fig. 5B). 4. Discussion The fig bears a unique closed inflorescence structure—the syconium—that is not present in any other fruit in the human diet. The multiple fig fruit is composed of individual drupelets that develop from the ovaries enclosed in the succulent receptacle to form a single accessory fruit (Flaishman et al., 2008; Storey, 1977). Fig is one of the earliest domesticated plants and human-assisted caprification of its Smyrna- and San-Pedro-type cultivars is one of the oldest known horticultural procedures
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Fig. 3. Fig-ripening parameters for pollinated and parthenocarpic fruit. (A) Ethylene production during fruit development and ripening. (B) Acidity level in ripening fruit. (C) Soluble solids content (SSC) of ripening fruit. (D) Firmness evaluation of fruit during development and ripening. (E) Cell-wall staining in green fruit 9 weeks after pollination (WAP) (1-pollinated, 2-parthenocarpic) and 100% ripened fruit (3-pollinated, 4-parthenocarpic). Red—parthenocarpic, blue—pollinated; Y AXIS: ethylene (L/kg h); soluble solids (SSC); PART E: cell-wall stain; pollinated; parthenocarpic. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Zohary and Spiegel-Roy, 1975; Kislev et al., 2006). These fig types have pollination-obligatory summer crops, but the San-Pedro-type cultivars produce a parthenocarpic spring crop. The third edible type, the common-type fig, has a facultative parthenocarpic fruit, producing both pollinated and parthenocarpic fruit during the summer. This feature makes the common-type fig a good platform to study the effect of fig caprification on fruit development, quality and post-harvest performance. In this study, we showed that fig caprification leads to substantial changes in fruit development as well as high quality of the mature fruit with better post-harvest performance. Normal fruit development is an outcome of successful pollination and fertilization events, where fertilization of the ovule usually leads to ovary expansion and fruit development (Gillaspy et al., 1993). Alternative pathways to normal fertilization involve exogenous hormone treatments or endogenous genetic stimuli that induce parthenocarpic fruit development. In the last few decades, parthenocarpic fruit development and ripening processes have been studied in many plants, such as tomato, watermelon, strawberry and citrus (Hayata et al., 1995; Gorguet et al., 2005; Serrani et al., 2007; Thompson, 1969; Vardi et al., 2008). Several studies have compared pollinated to parthenocarpic figs. In some, parthenocapy was induced by external application or osti-
ole injection of hormones (Crane and Blondeau, 1949; Crane and Van Overbeek, 1965). In others, pollination effects were examined in relation to development and fruit characteristics. Those studies applied varying degrees of caprification (Gaaliche et al., 2011a,b) or tested pollinated fruit from human-assisted caprification vs. fruit grown far from a pollen source as the non-pollinated group (Trad et al., 2013; Trad et al., 2012). One study was performed on two separate crops of a San-Pedro-type cultivar (Lodhi et al., 1969). The first (spring) crop constituted a naturally occurring parthenocarpic group, while the second (summer) crop was the pollination-obligatory group. Our study was performed on hand-pollinated fig fruit and naturally facultatively parthenocarpic common-type fig. The fruits were sampled weekly and evaluated throughout development until the 100% ripened stage. The effect of pollination on fruits under storage conditions was also monitored. Soon after fig pollination (1 WAP), the pollinated pistil showed style deterioration while the pistil of the parthenocarpic fruit remained intact throughout fruit development (Fig. 1). This phenomenon has been recorded in partially developed parthenocarpic fruit in Arabidopsis, where the unfertilized pistil does not degenerate, probably due to high gibberellin responsiveness (Carbonell-Bejerano et al., 2010). Crane and Van Overbeek (1965) noted that seed development in the pollinated fig inflorescence
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Fig. 4. Characteristics of 60% ripened fruit after storage. (A) Diameter, (B) weight, (C) firmness. Red—parthenocarpic fruit, blue—pollinated fruit. Storage I = 7 days, storage II = 14 days. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Fruit spoilage in 60% ripened fruit after storage for 14 days at 1 ◦ C. (A) Pollinated fruit. (B) Parthenocarpic fruit.
includes the formation of a rigid seed coat, whereas the parthenocarpic seed was smaller and lacked rigidity. This phenomenon can be detected in Fig. 1H and I, were few empty seeds are found in parthenocarpic fruit. This was also shown by Freiman et al. (2015). The color of the pollinated inflorescence turned red, while in the parthenocarpic fruit, inflorescence coloration was delayed. Previous studies have also mentioned color differences between the caprified fig inflorescence and hormone-induced parthenocarpic figs (Crane and Blondeau, 1949; Crane and Van Overbeek, 1965).
A comparison between parthenocarpic and pollinated fruit development clearly revealed the superiority of the pollinated ripe fig fruit over their parthenocarpic counterparts in growth, width, weight, firmness and taste qualities (Figs. 2, 3). It was clear from our results that the pollinated ripened fruit are larger in both diameter and weight than the parthenocarpic fruit. Similarly, several authors have shown that strongly human-assisted caprified fruit are larger and heavier than non-caprified ones (Gaaliche et al., 2011a,b; Trad et al., 2012). It is likely that in all of the above cases, the larger and heavier fruit are a consequence of inflorescence swelling, seed development and closure of the inner cavity. However in Crane and Blondeau (Crane and Blondeau, 1949), hormonal induction produced parthenocarpic fruit that were similar in size to the caprified ones, probably because the hormone induction resembled caprified fertilization of the flowers. Inflorescence swelling, seed development and closure of the inner cavity together with cell-wall thickening, as shown in our work, could cause the pollinated fruit to be firmer than the parthenocarpic one. The softer caprified fruit described by Trad et al. (2013) could have been from a different variety or the fruit might have been measured at a different stage than ours. The taste of the ripened pollinated fruit was more sour than that of the parthenocarpic fruit, with higher TA, whereas the caprified fruit described by Gaaliche et al. (2011b) and Trad et al. (2012) showed no acidity differences with noncaprified fruit. The SSC was lower in our pollinated fruit, oppose to the caprified fruit in Gaaliche et al. (Gaaliche et al., 2011b) that showed no significant difference in total soluble solids compared to non-caprified fruit. Storage and shelf life of the pollinated common-type figs were improved compared to the parthenocarpic fruit. Stored pollinated fruit retained their commercially favored physiological characteristics over parthenocarpic fruit, while maintaining their superior size, higher firmness and better taste parameters. Pollination by pollens from different trees may carry some disease, none were found in our
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study and in addition, the stored parthenocarpic fruit were highly sensitive to mold growth and skin injury, dramatically reducing their commercial value (Fig. 5). We suggest that the thicker cell wall of the pollinated fruit delayed loss of cellular water, resulting in firmer stored fruits. Thicker cell wall may also be the cause of the high storability of pre-harvest 1-MCP treated figs. While cell wall degradation during ripening is ethylene induced, blocking its perception surely effects this process (Freiman et al., 2012). The assumption that fertilization creates a stimulus that strengthens the ovules and ovary as sinks and diverts the flow of metabolites from the vegetative organs, was mentioned by Crane and Van Overbeek in relation to fig fruit (Crane and Van Overbeek, 1965). Parthenocarpic fig induction by use of external applications of gibberellins, auxins and cytokinins has been proven in several studies, stating a number of variable parameters between the two systems—pollinated fruit and induced parthenocarpy (Crane and Blondeau, 1949; Crane and Van Overbeek, 1965). Other studies published in recent years have shown that fertilization efficiency and pollen source affect several fig fruit quality characteristics (Gaaliche et al., 2011a,b). Cultivars belonging to the Smyrna and San-Pedro types, in which the summer crop is caprification-obligatory, also presented distinctive parameters when comparing fully caprified and partially caprified fruits (Trad et al., 2013, 2012). Lodhi et al. (Lodhi et al., 1969), working with a San-Pedro-type cultivar, found higher auxin concentrations in the first parthenocarpic crop than in the second pollinated crop, whereas gibberellin concentrations were higher in the second pollinated crop. It was concluded that figs possessing functional seeds and those with non-bearing seeds present different developmental and ripening features. Similarly in our study, the differential development of pollinated and parthenocarpic figs resulted from the presence or absence of functional seeds. This research establishes an optimal experimental system to identify the signals originating from the seed that initiate fruit development, and to separate the specific pathways that also occur naturally in the parthenocarpic but otherwise identical fig. Identification of parthenocarpy inducers in the fig will contribute to our general understanding of this phenomenon in other plants.
Acknowledgments We thank Omer Ben-Yehoshua for use of his excellent fig orchard. The financial support for this research was provided by a grant from the Chief Scientist of the Israeli Ministry of Agriculture.
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