Received for publication: 17 August, 2011. Accepted for publication: 1 March, 2012. 1 Department of Ecophysiology, Palmira Research Center, Corporación Colombiana de Investigación Agropecuaria (Corpoica). Palmira (Colombia). 2 Department of Plant Biology, Universidad Politécnica de Valencia. Valencia (Spain). 3 Corresponding author. arebolledor@corpoica.org.co Agronomía Colombiana 30(1), 34-40, 2012 Effect of 2.4-D exogenous application on the abscission and fruit growth in Sweet orange. var. Salustiana Efecto de la aplicación exógena de 2,4-D en la abscisión y crecimiento del fruto de naranjo dulce var. Salustiana Alexander Rebolledo R.1, 3, Amparo García-Luis2, and José Luis Guardiola B.2 ABSTRACT RESUMEN The effect of 2.4-D applications in full bloom on the abscission and fruit growth process was studied on sweet orange fruit in 20-year-old trees of Citrus sinensis (L.) Osbeck cv. Salustiana with a high flowering level. Abscission was determined on the whole tree and on the leafy inflorescences. Growth variables of the fruit were characterized (diameter, fresh and dry weight). 2.4-D application (20 mg L-1, 3.6 L per tree) increased the growth rate of the fruits and fruits size at maturity, however reduced the number of fruits which kept constant the yield at harvest. Differences between the diameter of the control fruits and the fruits treated with 2.4-D were observed during the early fruitlet development and until day 43 after anthesis. These differences increased with time following a linear relationship. For all the studied variables the diary increase level reaches the maximum by day 53, when the cell expansion of the vesicles starts. Se evaluó el efecto de la aplicación de 2,4-D en floración com- pleta sobre los procesos de abscisión y crecimiento del fruto del naranjo dulce Citrus sinensis (L.) Osbeck de la variedad Salustiana, en árboles de 20 años de edad con un alto nivel de floración. Se determinó la abscisión en todo el árbol, al igual que en inflorescencias con hojas, caracterizando las variables de crecimiento del fruto (diámetro, peso fresco y peso seco). La aplicación de 2,4-D (20 mg L-1, 3.6 L por árbol) incremento la velocidad de crecimiento del fruto y su tamaño final al mo- mento de la cosecha, reduciendo su número pero sin modifi- carla. Se presentaron diferencias en diámetro en relación con el testigo sin tratar, durante el desarrollo temprano del fruto y hasta el día 43 después de antesis. A partir de este momento estas diferencias se incrementaron con el tiempo. La tasa de aumento diaria para todas las variables estudiadas alcanzó un máximo el día 53, momento en que dio inicio el crecimiento en expansión de las vesículas. Key words: Citrus sinensis, flowering, set fruit, fruit growth and development. Palabras clave: Citrus sinensis, floración, cuajado del fruto, crecimiento y desarrollo del fruto. Introduction The sweet orange Citrus sinensis (L.) Osbeck variety Salustiana is a parthenocarpic cultivar (González-Sicilia, 1968). Parthenocarpic cultivars usually show lower fruit set and fruit size than seeded cultivars. However, many citrus cultivars produce a high number of flowers that are enough to obtain a high yield. Flower number is inversely correlated to the percentage of the final fruit set and fruit size (Goldschmidt and Monselise, 1977; Agustí et al., 1981; Becerra and Guardiola, 1984). Citrus trees show two successive abscission waves that affect flowering and fruit development. In some cases the abscission is a continuous process with the highest peak between 6 and 8 weeks after full flowering (Duarte and Guardiola, 1996; Laskowski, 2006). The first wave induces a massive abscission of flowers and ovaries while the second reduces the fruit number, reaching a significant growth during June drop (Goldschmidt, 1999; Guardiola, 2000; Goren et al., 2000; Iglesias et al., 2006; Pérez and Jiménez, 2009). The abscission can appear in the flowers and ovaries around the pedicel zone (AZ-A) while in the developing fruit dur- ing June drop can appear around the calyx (AZ-C) (Schnei- der, 1968; Spiegel-Roy and Goldschmidt, 1996; Guardiola and García-Luis, 2000; Iglesias et al., 2006; Laskowski et al., 2008; Pérez and Jiménez, 2009). In both cases the abscission is mediated by ethylene synthesis (Ortolá et al., 1991; Burns et al., 1992; Goren, 1993; Iglesias et al., 2006). Some authors had suggested in oranges an increase in fruit abscission by a direct or indirect stimulation in the ethylene production (Goren, 1993; Kazokas and Burns, 1998). 35Rebolledo R., García-Luis, and Guardiola B.: Effect of 2.4-D exogenous application on the abscission and fruit growth in Sweet orange. var. Salustiana The first abscission wave does not seem to be correlated with the supply of carbohydrates. The reserves of carbohy- drates in the tree stay high during the first abscission wave (García-Luis et al., 1988; Erner, 1989; Ruiz and Guardiola, 1994; Ruiz et al., 2001). Fruit drop in the early stages of fruit development in the first abscission wave is preceded by a reduction in the growth rate. The reduction in the trans- port of carbohydrates is related with lower sink strength and not with the supply of metabolites (limitation at the source) (Ruiz and Guardiola, 1994; García-Luis et al., 2002). In citrus, the sink strength of the developing fruit as well as yield of commercially valuable large-size fruit increases with exogenous auxin applications (Guardiola and García- Luis, 2000; Chao and Lovatt, 2010). The result is a faster fruit growth until ripening (Guardiola and Lázaro, 1987; Ortolá et al., 1988; Agustí et al., 1994). The in vivo responses to exogenous auxin applications have been correlated with in vitro behaviour of fruit tissue. Guardiola et al. (1993) provided evidence that the sensitivity of the fruit tissues to growth regulators changes markedly during early fruit development. Studies made by Laskowski et al. (2008) dem- onstrated a strong correlation of higher endogenous indole acetic acid content with the growth rate in the Salustiana variety during early fruit development. The greatest response had been obtained when auxin was applied during the flowering period or short after (Cog- gins and Hield, 1968; Duarte and Guardiola, 1996). In Clementine Esbal mandarin, application of 2.4-D during flowering increased the fruit growing rate and delayed the abscission more intensely several weeks after application (Duarte and Guardiola, 1996). Reduction in fruit number was compensated with an in- creased in fruit size. A similar response was found in Nova mandarin (Guardiola, 1996). In several orange cultivars 2.4-D application was effective in increasing fruit size. This response was obtained when the application was made 6-8 weeks after flowering (Erner et al., 1993). Applications of 2.4-D during the flowering period were not effective. We have found evidence that applications of 2.4-D dur- ing flowering increased fruit size in sweet oranges of the Salustiana cultivar without affecting the yield. The object of this study was to evaluate the effect of 2.4-D on the abscis- sion and fruit development in Sweet orange. var. Salustiana. Materials and methods Plant material The study was carried out in Valencia (Spain) and conduc- ted on adult tress (20 years old) of Salustina sweet orange (Citrus sinensis (L.) Osbeck) grafted on rootstock citrange Troyer (Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.). The trees displayed alternate bearing and its flowering intensity was determined by the previous flowering. In the orchard in a same year, trees with different flowering intensities were often present. Hormone treatment The application of 2.4-D was made once the trees showed 50% of flowers in anthesis on single and multi flowered leafy inflorescences. The 2.4-D application (Viriman, 10% (p/v) solution of isopropyl ester) was made on 11 trees. The concentration was 20 mg L-1 and 3.6 L/tree. The application was made to the entire tree foliar surface. A non-ionic etting agent, alkyl polyglycol ether, at a final concentration of 20% (p/v) was added to all solutions Abscission and fruit growth Abscission and fruit growth studies were conducted on single and multiple-flowered leafy inflorescences. Hundred inflorescences of each type were tagged. The multiple- flowered leafy inflorescences had five leaves and flowers respectively. It was taken into consideration that the final flower bud of each inflorescence was next to open. Anthesis took place 7 d later. Fruit number of each inflorescence was recorded every 10 d. These records were used to calculate the relative and absolute abscission rate. Before the flower buds appeared, a canvas was installed underneath each tree. The flowers and developing fruit that felt on the canvas were collected periodically to determine the tree total abscission. The fruit variables were fresh and dry weight and diam- eter. Measurements were made every 10 d when data were recorded. Fruit diameter was determined measuring the equatorial region of the apical fruit in each inflorescence. Twenty fruits were collected in order to determine fresh and dry weight and taking into reference the fruit diameter of the tagged inflorescences. Statistical analyses The characteristics of the inflorescences, as related to fruit abortion, were compared using an ANOVA. The statistical package IBM®-SPSS (IBM Corporation, New York, NY) was utilised throughout. Statistical analyses include DMS and DUNCAN. Results Effect of 2.4-D on abscission pattern The 2.4-D applications produced a reduction in the fruit number with an increase in the average weight without 36 Agron. Colomb. 30(1) 2012 0 20 40 60 80 100 -30 0 30 60 90 120 Fl ow er a nd fr ui t a bs ci ss io n (% ) Days after anthesis (daa) 0 2 4 6 -30 0 30 60 90 120 A bs ci ss io n ra te ( fr ui tle ts /d ) Days after anthesis (daa) 20 40 60 80 -30 0 30 60 90 120 Fl ow er a nd fr ui t a bs ci ss io n (% ) Days after anthesis (daa) 0 0 20 40 60 80 100 120 -30 0 30 60 90 120 Fl ow er a nd fr ui t a bs ci ss io n (% ) Days after anthesis (daa) affecting the yield (P≤0.03; Tab. 1). Also the 2.4-D applica- tions produced a delay in the abscission. Until 42 daa (days after anthesis), the abscission was lower in the treated trees. From 42 daa, an increase in the accumulated abscission with a final fruit set of 3.1% in comparison with 3.7% in the control trees was observed (Fig. 1). Abscission occurred in two periods. The first period is re- lated to flowers and fruit that fall only at the pedicel. This was presented from anthesis to 42 daa. The second period occurred between 42-74 daa. This period is related to fruit that fall from both the pedicel and calyx base (Fig. 2). The change in the abscission pattern with 2.4-D applications was reflected as a differential effect in the abscission of both the pedicel and the calyx base. Until 42 daa the abscission by the pedicel was higher in control trees. From 42 to 67 daa the abscission was presented by the calyx base being higher in the treated trees. Between 42 and 52 daa a tran- sition period was presented with fruits that fall by both abscission zones. In absolute terms (number of flowers/fruitlets shed per day), two main peaks of abscission occurred. The first one at 22 daa with 2.8 and 2.1% in untreated and 2.4-D treated trees respectively. The second main peak occurred at 42 daa with 3.8 and 4.7% in untreated and 2.4-D treated trees respectively (Fig. 3). In multiple flowered leafy inflorescences, the abscission was slow until 22 daa. After 22 daa a considerable increase in the abscission was observed (Fig. 4). Differences in the time in which abscission stopped taking place were observed with 63 daa and 53 daa in untreated and 2.4-D treated trees respectively (Fig. 4). FIGURE 1. Accumulated abscission in 2.4-D treated ( ) and untreated ( ) ‘Salustiana’ trees. TABLE 1. Behaviour of the yield on 2.4-D treated and untreated ‘Salustiana’ trees. Fruit weight (g) Yield (kg) Fruit number/tree Control 2.4-D Control 2.4-D Control 2.4-D 115±7 b 133±5 a 180±11 a 192±14 a 2,088±232 a 1,551±159 b Mean values followed by distinct letters are significantly different according to Duncan’s test (P≤0.05). FIGURE 2. Accumulated abscission of flowers and fruitlets in 2.4-D treat- ed ( ) and untreated ( ) ‘Salustiana’ trees according to the abscis- sion zone. Pedicel abscission zone ( ) and calyx abscission zone ( ). FIGURE 3. Daily absolute (fruitlets / d) abscission rates in 2.4-D treated ( ) and untreated ( ) ‘Salustiana’ trees. FIGURE 4. Accumulated abscission in multiple-flowered leafy inflores- cences on 2.4-D treated ( ) and untreated ( ) ‘Salustiana’ trees. 37Rebolledo R., García-Luis, and Guardiola B.: Effect of 2.4-D exogenous application on the abscission and fruit growth in Sweet orange. var. Salustiana 0 2 4 6 -30 0 30 60 90 120 Fl ow er a nd fr ui t n um be r Days after anthesis (daa) FIGURE 5. Reduction in the fruit number per inflorescence in multiple – flowered leafy inflorescence on 2.4-D treated ( ) and untreated ( ) ‘Salustiana’ trees. FIGURE. 6. Percentage of inflorescences with one fruit of multiple (A) and single (B) flowered leafy inflorescence on 2.4-D treated ( ) and untreated ( ) ‘Salustiana’ trees. 0 20 40 60 80 100 120 -30 0 30 60 90 120 Fl ow er a nd fr ui t p er in flo re sc en ce ( % ) Days after anthesis (daa) 0 20 40 60 80 100 120 -30 0 30 60 90 120 Fl ow er a nd fr ui t p er in flo re sc en ce ( % ) Days after anthesis (daa) A B The abscission pattern was correlated with the reduction in the fruit number per inflorescence. Until 33 daa a reduction from 5.5 fruit per inflorescence to 4.4 in untreated trees, and 4.7 in 2.4-D treated trees was observed. From this day the reduction increased to stabilized at 63 daa with an fruit average of 1.2 in untreated trees and 1 in 2.4-D treated trees (Fig. 5). There was a different behaviour in the percentage of in- florescences that retain at least one fruit. The reduction in the number of multiple-flowered leafy inflorescences that retain at least one fruit occurred from 26 daa while in single flowered leafy inflorescences this occurred im- mediately after anthesis. In both inflorescence types this parameter stopped at 67 daa. The 55% of multiple-flowered leafy inflorescences retain at least one fruit in comparison with the 39% in single flowered leafy inflorescences (Fig. 6). The 2.4-D applications increased the percentage of mul- tiple-flowered leafy inflorescences that retain at least one fruit. Between 33 and 53 daa this parameter increased to reach the maximum with 55% in untreated and 36% 2.4-D treated trees (Fig. 6). In single flowered leafy inflorescences a delay occurred until 33 daa, with a first peak in the rela- tive and absolute abscission rate of 2% in untreated and 1.1% in 2.4-D treated trees. From this day the abscission increased in treated trees to stop at 63 daa in untreated trees with 61% while in 2.4-D treated trees it stopped at 76 daa with 70% (Fig. 6). Effect of 2.4-D on fruit growth pattern The 2.4-D applications increased fruit size. The effect on the growth variables was observed immediately after anthesis. The differences between untreated and treated trees increased with the advance in the fruit developing stage. At time of harvest, the differences in diameter were of 5 mm in fruit of a single flowered leafy inflorescences and 4 mm in multiple-flowered leafy inflorescences (Fig. 7a). There was a higher effect on fruit from single flowe- red leafy inf lorescences than multiple-f lowered leafy inflorescences. The increase in fresh weight was low throughout early fruit growth until 43 daa. From this day a lineal increase was observed until 240 daa as well as in the diameter. At the time of harvest, the differences in fresh weight were of 35.5 g in fruit of single flowered leafy inflorescences and 24 g in multiple-flowered leafy inflorescences, being higher in fruit treated with 2.4-D (Fig. 7b). Like the fresh fruit weight, the dry matter accumulation was low in the early fruit growth period until 43 daa. The differences during this time were kept close to zero. In fruit of single flowered leafy inflorescences there was a first period of slow accumulation of dry matter between 53 and 133 daa. In fruit of multiple-flowered leafy inflorescences this pe- riod was extended to 148 daa. From this day there was an increased, to stabilize at 198 daa in the first inflorescence type (single flowered leafy) and 40 d later in the other inflorescence type (Fig. 7c).  38 Agron. Colomb. 30(1) 2012 Discussion The early drop of flower and ovaries after flowering time occurs by abscission of the pedicel zone (AZ-A). Once this zone becomes inactive, the calyx abscission zone (AZ-C) is activated during the June drop (Schneider, 1968; Spiegel- Roy and Goldschmidt, 1996; Guardiola and García-Luis, 2000; Iglesias et al., 2006). In both cases the abscission is mediated by the ethylene synthesis (Goren, 1993; Ortolá et al., 1997; Burns et al., 1998; Iglesias et al., 2006). The endogenous hormonal balance in the fruit controls the abscission process, especially auxins and ethylene. An ad- ditional contribution of auxins to the fruit can change the abscission process depending on the auxins concentration and type, as well as the fruit developing stage and type of cultivar (Guardiola and García-Luis, 2000). Applications of 2.4-D produced a delay in the abscission until 42 daa reducing the drop of flower and small developing fruit of the AZ-A and increasing fruit drop of the AZ-C. This pattern was found also on Clementine Esbal mandarin (Duarte and Guardiola, 1996) as well as on 'Owari Sat- suma' (Ortolá et al., 1997). After the application of the auxins, the abscission delay occurs by the inhibition of enzymes that degrade the cell wall in the abscission zone (Goren, 1993; Goren et al., 2000). Phenoxyacetic auxins were found to be more effective in delaying abscission (Ortolá et al., 1997). Although 2.4-D applications produced a reduction in the fruit set, the increase in fruit weight compensated this reduction without affecting the yield. This behaviour was previously reported in Clementine Esbal mandarin (Du- arte and Guardiola, 1996) and Nova mandarin (Guardiola, 1996). In Valencia and Shamouti orange cultivars, 2.4-D applications were not effective in increasing the fruit size (Erner et al., 1993). A higher response was obtained both in fruit set and fruit size when the applications were made 6-8 weeks after anthesis. The increase in the fruit sink strength produced by the auxins applications is reflected in a faster growth of the fruit until ripening (Guardiola and Lázaro, 1987; Ortolá et al., 1988; Agustí et al., 1994). In several cultivars, the maximum response had been obtained when the auxin application was made during or after flowering (Coggins and Hield, 1968; Duarte and Guardiola, 1996). Most stud- ies had focused in the characterization of the response of several mandarin cultivars to hormonal treatments before June drop (Guardiola and Lázaro, 1987; Ortolá et al., 1991; Agustí et al., 1991; Georgiu, 1998). The effect on fruit growth when 2.4-D is applied at flow- ering time coincides with peak values in the endogenous content of IAA (Laskowski et al., 2008). This effect was verified with the histological development pattern in dif- ferent developing stages of the fruit. The maximum cell division rate in the explants was found in 6 d-old-fruits that produced callus with higher IAA concentration (Re- bolledo et al., 2007). The cell types of the callus were a key characteristic of the in vitro development pattern. In the untreated explants the callus cells were large, elongated and vacuole shaped. In the IAA treated explants the cell callus was small. This behaviour was related with the IAA action on cell division (Krikorian, 1995; Khan, 1996; Laskowski et al., 2008; Rebolledo et al., 2007). FIGURE 7. Difference in diameter (A), fresh (B) and dry (C) weight bet- ween 2.4-D treated and untreated ‘Salustiana’ trees in single ( ) and multiple ( ) flowered leafy inflorescence. 0 2 4 6 D ry w ei gh t ( g) Days after anthesis (daa) -30 0 30 60 90 120 150 180 210 240 270 300 0 10 20 30 40 Fr es ch w ei gh t ( g) -30 0 30 60 90 120 150 180 210 240 270 300 0 2 4 6 8 -30 0 30 60 90 120 150 180 210 240 270 300 D ia m et er ( m m ) A B C 39Rebolledo R., García-Luis, and Guardiola B.: Effect of 2.4-D exogenous application on the abscission and fruit growth in Sweet orange. var. Salustiana There was a reduction in the capacity response when mesocarp explants were used. The mesocarp explants of 46 d-old-fruits showed a higher response with a minor concentration (10-6 M) than explants of 61 d-old-fruits that only were affected for the higher concentration of IAA (10-5 M). At 46 daa cell division activity was still observed in the external mesocarp (Amo-Marco and Picazo, 1994; Rebolledo et al., 2007). This activity can explain the effect of IAA on the callus growth. On 76 d-old-fruits the explants weren’t affected by the evaluated IAA concentrations. With the advance in the developing stage of the mesocarp there were only differentiated, vacuole-shaped and polygonal cells with large intercellular spaces. Applications of 2.4-D increased fruit size in the Sweet orange Salustiana cultivar. This effect was evident imme- diately after anthesis and until harvest time when there was a difference of 5 mm in fruit size. This behaviour was previously reported in Clementine Esbal mandarin with the same 2.4-D concentrations used in this study (17 and 20 mg L-1). The same effect was reported in Nova mandarin cultivar with a reduction in the final harvest (around 10%) (Guardiola, 1996). Applications of 2.4-D increased the fruit growth rate. The diameter differences between fruit treated and untreated with 2.4-D on single flowered leafy inflorescences and multiple-flowered leafy inflorescences were established from fruit early development. These differences increased linearly with the advance in the developing stage. Dur- ing the fruit early development (43 daa) the fresh weight increased and the dry matter accumulation showed the lower values. From this time the growth rate increased but with a different trend. The fresh weight difference increased almost linearly while dry matter accumulation slowly increase until 63 daa, being constant until 150 daa and then increasing to stabilize at 240 dda. The same has been reported by Chao and Lovatt (2010) who found an increase of commercially valuable large-size fruit of “Fina Sodea” clementine mandarin using triclopyr. The daily increase rate in all variables reached a maximum at 53 daa when the cell expansion growth of the juice sacs began, being higher in fruit of 2.4-D treated trees. From this time, both the fresh fruit weight as well as the dry fruit weight showed other peaks, but in all cases were higher in the 2.4-D treatment. Similar results concerning triclopyr treatment has been reported for mandarin (Agustí et al., 2002; Roussos and Tassis, 2011). Acknowledgements I am thankful to the memory of José Luis Guardiola Bár- cena for his scientific guidance and input on a personal level. I was fortunate to work alongside a brilliant scientist. Thankful to Department of Agriculture, Fisheries and Food of the Valencian Community for the grants awarded to Professor Dr. D. José Luis Guardiola Bárcena (R.I.P.), who have allowed the funding of this project and my time du- ring the course of this research (Project GV-CAPA00-11). Literature cited Agustí, M., V. Almela, and J.L. Guardiola. 1981. 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