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Maintenance of high epidermal cell density and reduced calyx-end cracking in developing ‘Pink Lady’ apples treated with a combination of cytokinin 6-benzyladenine and gibberellins A4+A7
Scientia Horticulturae 165 (2014) 324–330
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Maintenance of high epidermal cell density and reduced calyx-end cracking in developing ‘Pink Lady’ apples treated with a combination of cytokinin 6-benzyladenine and gibberellins A4 +A7 Idit Ginzberg a,∗ , Edna Fogelman a , Lior Rosenthal a , Raphael A. Stern b,c a
Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO Box 6, Bet Dagan 50250, Israel MIGAL, Galilee Technology Center, PO Box 831, Kiryat Shmona 11016, Israel c Department of Biotechnology, Faculty of Life Sciences, Tel-Hai College, Upper Galilee 12210, Israel b
a r t i c l e
i n f o
Article history: Received 18 July 2013 Received in revised form 27 October 2013 Accepted 13 November 2013 Keywords: Fruit cracking Malus × domestica Peel anatomy Plant growth regulators
a b s t r a c t Calyx-end cracking in ‘Pink Lady’ apple develops as concentric cracks located around the stylar-end. The factors that induce the cracking are unknown, however we previously demonstrated that the intensity of the disorder can be controlled by application of a mixture of synthetic gibberellins (GA4 plus GA7 ) and cytokinin (6-benzyladenine; BA) at different phenological stages of fruit development, and suggested that the plant growth regulators (PGRs) affect the elasticity of the epidermal layer at the calyx-end by increasing cell density. The present study focused on histological analyses of the peel at the calyx-end following the application of BA + GA4+7 . The experiments were conducted in Northern Israel during 2011 and 2012. In 2011 the PGRs were applied as 0.2% (v/v) SuperlonTM (i.e., 40 mg l−1 BA plus 40 mg l−1 GA4+7 ) once, twice or three times every 14 days since 60 days after full bloom (DAFB). In 2012, to test the efficacy of a lower concentration of the PGRs, 0.025% (v/v) SuperlonTM was used. Spraying was applied at 60 to 90 DAFB (cell expansion phase) as before, in addition to earlier spraying at 7 to 35 DAFB (cell division phase). In all treatments given at cell expansion phase the BA + GA4+7 mixture increased the epidermal cell density at the calyx-end, compared to control. Multiple spraying maintained the high cell density during fruit expansion compared to one-time application, and the higher concentration of the PGRs was more potent than the lower. Assessment of calyx-end cracking suggested that low concentration of the PGRs (i.e., 5 mg l−1 BA plus 5 mg l−1 GA4+7 ) is most efficient when applied three times at cell division phase. Interestingly, in 2011 the incidence of calyx-end cracking was significantly lower relative to 2012 and 2010 that were characterized by extended daily hours of high temperatures (>34 ◦ C). This association can be used by growers as a criterion in applying the PGR treatment early in the season when temperatures are higher than the average and high incidence of calyx-end cracking might be expected. © 2013 Elsevier B.V. All rights reserved.
1. Introduction ‘Pink Lady’ apple (Malus × domestica Borkh.) (Cripps et al., 1993) fruits are susceptible to pre-harvest physiological disorder of calyxend cracking (Fig. 1) (Opara et al., 1997). In Israel, the incidence of cracking may reach up to 80% of the total yield. ‘Pink Lady’ is popular in many countries due to its pinkish color, crisp texture, and a high sugar:acid ratio (Mackay et al., 1994); however the high incidence of cracking affect its marketability, resulting in substantial economic losses.
∗ Corresponding author. Tel.: +972 3 9683787; fax: +972 3 9669583. E-mail address: [email protected] (I. Ginzberg). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.11.020
Calyx-end cracking in ‘Pink Lady’ develops as concentric cracks located around the stylar-end (Fig. 1). In contrast to the wellstudied skin russeting disorder that results from microscopic cracks in the cuticle and the subsequent formation of a periderm (Faust and Shear, 1972; Fogelman et al., 2009; Khanal et al., 2013), calyxend cracking disorder is usually characterized with cracks that traverse the cuticle, and although corky periderm is develops at their margins, it is not sufficient to prevent pathogen invasion into inner fruit tissues. In addition, apples susceptible for russeting early in fruit development, while calyx-end cracking appear toward the end of the growth. The factors that promote calyx-end cracking were not defined yet. Preliminary observations indicated that the severity of the disorder was associated with orchard location (especially in high latitudes and dry climates), row orientation that exposed fruit to
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as well-drained protogromosol (68% clay) on terra rossa soil at Baraam. The soil pH is 7.7 with CaCO3 contents of approx. 7% (w/w). 2.2. Application of plant growth regulators (PGRs) A commercial product containing synthetic cytokinins and gibberellins (GA) was used: SuperlonTM , a liquid formulation containing 1.9% (v/v) BA and 1.9% (v/v) GA4+7 (Fine Agrochemicals, Whittington, UK).
Fig. 1. Calyx-end cracking in ‘Pink Lady’ (left) compared to a normal fruit (right). Scale bar = 1 cm.
high level of solar radiation, and high temperatures in the second half of the growing season. Experiments that were conducted by us in the growth seasons of 2008 to 2011 indicated that combined applications of the cytokinin 6-benzyladenine (BA) and the gibberellins A4 + A7 (GA4+7 ) to ‘Pink Lady’ fruitlets between 60 and 90 days after full bloom (DAFB) reduced significantly the incidence of calyx-end cracking, while treatment of each of the plant growth regulator (PGR) alone was ineffective (Stern et al., 2013). The optimal treatment was three consecutive applications of 40 mg l−1 BA plus 40 mg l−1 GA4+7 mixture at 14 d interval beginning 60 DAFB, applied as the commercial formulation SuperlonTM (0.2%, Fine Agrochemicals, Whittington, UK). The epidermal layer of the fruit appeared to be the target of the BA + GA4+7 mixture. The PGRs probably increase peel flexibility due to enhanced cell divisions and increased cell density at the fruit surface, thus reduce its susceptibility to cracking at the developmental stage of dramatic fruit expansion (Stern et al., 2013). Our results were in accordance with previous reports showing that treatments with gibberellins reduced cuticular cracking in apple, probably by affecting the epidermal tissue. Eccher (1975) showed that treatment with GA4+7 resulted in less irregularity among epidermal cells and reduced cuticle cracking compared to untreated fruit. Knoche et al. (2011) showed that water-induced russeting and micro-cracking in ‘Golden Delicious’ apple could be reduced by multiple applications of GA4+7 . Treatment had no effect on the amount or rate of cutin or wax deposition, suggesting that reduced russeting and micro-cracking were due to the effects of GA4+7 on the epidermal and hypodermal layers of cell. Fogelman et al. (2009) reported a similar result of less russeting on ‘Smoothy Golden Delicious’ after multiple applications of the SuperlonTM during the first month of fruit development. The present work focused on anatomical changes induced at the calyx end of ‘Pink Lady’ fruit by multiple treatments of BA + GA4+7 mixture. It is shown that multiple applications of the PRGs maintained high epidermal cell density compared to untreated control, and reduced the incidence of the cracking disorder.
2. Materials and methods 2.1. Experimental orchards and conditions Experiments were conducted for two seasons 2011 and 2012 on 10-to-12 year-old ‘Pink Lady’ apple trees grafted on MM 106 rootstock in Baraam orchard, located 700 m a.s.l. in Upper Galilee, where all trees were 3.5 m high, spaced at 4.5 m × 5.5 m (900 trees ha−1 ). The orchard is located in a semi-arid region with high temperatures (ca. 35◦ C max.) and low humidity (<40% RH) during the Summer (May to October). Annual precipitation (November–April) was approx. 700 mm. The soil is 0.8 m to 1.2 m deep, and classed
2.2.1. Experiment 1 (2011) Multiple spraying of 0.2% (v/v) SuperlonTM (40 mg l−1 BA + 40 mg l−1 GA4+7 ) were applied at different times during the growing season; indicated as days after full bloom (DAFB). Four different treatments were tested (Table 1): one spray at 60 DAFB; two sprays applied at 60 and 75 DAFB; and three sprays applied at 60, 75 and 90 DAFB. Spraying of 0.2% (v/v) SuperlonTM are the accepted practice in the orchard. To test the efficiency of reduced concentration 0.025% SuperlonTM (5 mg l−1 BA + 5 mg l−1 GA4+7 ) was applied; however, at six time points, every two to three weeks since 7 DAFB (7, 21, 35, 60, 75 and 90 DAFB; Table 1). An additional set of trees served as non-treated controls. The PGRs were applied as a foliar spray at 3 up to 5 l per tree, using a high pressure-handgun (Kibbutz Degania, Israel) until runoff. Triton X-100, at 0.025% (v/v) was included in all sprays as a non-ionic surfactant. The experiments were conducted on whole trees bearing a similar crop load (ca. 240 fruits tree−1 , ca. 35 kg tree−1 ). Control trees, with the same crop load, were not sprayed. The experimental design was as randomised complete blocks, with four blocks of four trees per treatment. One buffer tree was always used between two treatment trees to avoid drift. 2.2.2. Experiment 2 (2012) To assess the optimum time and number of applications of the reduced PGR solution, the treatments were applied in two phenological stages of fruit development. Cell division phase of fruit growth (phase I), 7 to 60 DAFB, and the cell expansion phase of fruit growth (phase II), 60 to 90 DAFB. SuperlonTM , 0.025% (v/v) (5 mg l−1 BA + 5 mg l−1 GA4+7 ) was applied once, twice or three times during phase I or phase II (7, 21, 35 DAFB or 60, 75, 90 DAFB, respectively), or throughout both growth phases (Table 1). The effect of auxin on the cracking disorder was tested by a combination of two spraying of 0.025% SuperlonTM (60 and 75 DAFB) with one spray of 25 mg l−1 2,4-Dichlorprop-P (2,4-DPP; Prigan, Agan Chemicals, Israel) at 90 DAFB. Spraying of 0.2% SuperlonTM at the second phase, 60 to 90 DAFB, was applied similarly to the described above for the experimental year of 2011. An additional set of trees served as non-treated controls. The sprayings system and the experimental design were as described for Experiment 1. 2.3. Fruit size and assessment of fruit cracking Average fruit size and the incidence of the cracking disorder were assessed at harvest. Fifty randomized fruits from the trees’ outskirt were collected from each tree. Fruit diameter in mm was measured at the equatorial plan of the fruit, and fruit affected by calyx-end cracking were counted. Values were expressed as a percentage. 2.4. Tissue embedding and measurements of epidermal cell number and cuticle thickness Histological studies were conducted on fruit harvested seven days following the spraying at the second phase of growth (i.e.,
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Table 1 Schematic presentation of the dates (given as DAFB) of SuperlonTM application at the various treatments at two experimental years, 2011 and 2012. DAFBb
BA plus GA4+7 treatment 40 mg l−1
5 mg l−1
Sprayinga
Phase Ic 7
Phase IId 21
35
60
75
90
x x x x
x x x
x x
x x x x x x
x x x x x
A x x x
Experimental year 2011 x x x x Experimental year 2012 x x x x x x x x x a b c d e
Control x1 x2 x3 x6 Control x1 x2 x3 x1 x2 x2+Auxe x3 x6 x3
x
x
x
x x x
x x
x
x
x
x
Number of PGR spraying. DAFB, days after full bloom. Cell division phase of fruit growth, 7 to 35 DAFB. Cell expansion phase of fruit growth, 60 to 90 DAFB. Additional spray of 25 mg l−1 auxin (2,4-DPP) was applied at 90 DAFB.
67, 82 and 97 DAFB). Four fruit were collected from each repeat of SuperlonTM and control treatments (16 fruits per treatment). Apple surface tissue samples (blocks of 5 mm × 3 mm × 3 mm) were collected from the calyx-end of each fruit within 24 h since harvest. Samples were fixed in FAA [50% (v/v) ethanol, 5% (v/v) acetic acid, 3.7% (v/v) formaldehyde, and 41.3% (v/v) H2 O], dehydrated in an ethanol/Histoclear series (Finkelman Chemicals, Petach-Tikva, Israel) and embedded in ParaplastTM (Paraplast Plus; McCormick Scientific, St. Louis, MO, USA) according to standard methods (Ruzin, 1999). Tissue sections (15–20 m thick) were stained with Toluidine blue (Sigma Chemicals, Rehovot, Israel) for examinations of tissue morphology (Johansen, 1940). Sections were observed under a light microscope (Leica DMLB; Wetzlar, Germany) and images were displayed on a monitor through a CCD camera (Leica DC2000) using the Leica IM1000 programme. To measure epidermal cell numbers and cuticle thickness in each treatment, one cross-section was selected from each fruit. The density of epidermal cells was determined by counting cells along an arbitrary 500 m line, at three locations in the section. The average cell number 500 m−1 was calculated for each section. Cuticle thickness was determined by measuring the lengths (m) of arbitrary lines from the base of the epidermal cell layer to the outer edge of the cuticle at five locations along the section (Fig. 2). An average value was calculated for the section.
3. Results 3.1. BA + GA4+7 spraying during fruit development maintain high epidermis cell density 3.1.1. Experimental year 2011 Cross sections of apples collected at 67, 82 and 97 DAFB indicated significant reduction (32%) in density of epidermal cells during fruit development (control, Table 2). However, multiple application of BA + GA4+7 (40 mg l−1 each), at 60 and 75 DAFB or Table 2 Effect of multiple applications of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) during fruit development on the average number of epidermal cells (EC) and cuticle thickness (CT) of apples collected one week following the spraying (Experimental year 2011). BA + GA4+7 treatmenta
DAFBb
ECc
EC change (%)d
CTe
Control
67 82 97
51.83 A 43.83 AB 35.0 B
100 85 68
29.18 A 25.74 A 27.92 A
40 mg l−1 x1
67 82 97
61.22 A 44.67 B 39.11 B
100 73 64
32.98 A 28.89 A 31.36 A
40 mg l−1 x2
67 82 97
54.58 A 60.25 A 51.67 A
100 110 95
26.66 A 30.90 A 25.86 A
40 mg l−1 x3
67 82 97
55.58 A 49.17 A 44.08 A
100 89 79
26.69 A 28.34 A 28.85 A
5 mg l−1 x6
67 82 97
70.67 A 66.25 A 55.75 A
100 94 79
32.62 A 35.55 A 29.76 A
2.5. Statistical analysis Percentage data of fruit size and cracking were subjected to arcsine transformation before analysis in order to provide a normal distribution of variance. Data were analysed for statistical significance using the general linear model (GLM) procedure. Duncan’s new multiple range test was used to compare treatments when ANOVA showed significant differences between means. Data for cell density and cuticle thickness were analysed statistically by one-way ANOVA using JMP software (SAS Institute Inc., Cary, NC, USA).
Means values within a column, at each treatment, followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. b Dates of fruit collection given as days after full bloom. c Number of epidermal cells along an artificial line of 500 m tangential to fruit surface. d Change in EC value relative to value at 67 DAFB of each treatment. e Cuticle thickness in m as indicated in Fig. 2.
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Fig. 2. Anatomical study of the calyx end demonstrating the effect of PGR treatment on epidermal cell density and organization at two phenological stages of fruit development. Samples were collected at 67 and 97 DAFB from untreated control fruits (left) and fruits treated twice, at 60 and 75 DAFB, with 0.025% (v/v) SuperlonTM (5 mg l−1 BA plus 5 mg l−1 GA4+7 ) and at 90 DAFB with ‘Prigan’ (25 mg l−1 2,4-DPP) (right). In untreated control fruits, as fruit expands, epidermal cell density is reduced and the gap between neighboring cells increase. PGR induce epidermal cell accumulation that maintain high cell density and induce cell aggregations. Pictures width present 500 m. Arrow line demonstrates measurements of cuticle thickness by measuring the length (m) of an arbitrary line from the base of the epidermal cell layer to the outer edge of the cuticle.
three times at 60, 75 and 90 DAFB, prevented this reduction and maintained the high epidermal cell density, although the double spraying seems more efficient (Table 2). In an attempt to reduce the concentration of the PGR spraying, BA + GA4+7 were applied at 5 mg l−1 every 14 days since seven DAFB, and were found to similarly prevent the reduction in epidermal cell density during fruit expansion; notably, as this treatment begun prior to 60 DAFB, the initial EC value is higher than that of control and other treatments. Interestingly, cuticle thickness did not vary during fruit expansion in control and treated fruits (Table 2), and only weak correlation (R2 = 0.19) was found between cell number and cuticle thickness. The effect of the treatments on epidermal cell density was compared for each sampling date to determine the best timing, concentration and number of PGR applications. Data indicated that spraying with 5 mg l−1 BA + GA4+7 solution six times since 7 DAFB resulted with the highest increase in cell density in all sampling dates, compared to untreated control and to spraying with 40 mg l−1 solution (Table 3). Of the 40 mg l−1 treatments—one spray at 60 DAFB did not change significantly epidermal cell density at 67 DAFB (note, all 40 mg l−1 treatments got one spraying at that time point); two sprays at 60 and 75 DAFB (40 mg l−1 x2 and 40 mg l−1
Table 3 The effect of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) applications on the average number of epidermal cells (EC) was compared for each sampling date (Experimental year 2011). DAFBa
BA + GA4+7 treatmentb
ECc
EC change (%)d
67
Control 40 mg l−1 x1 40 mg l−1 x2 40 mg l−1 x3 5 mg l−1 x6
51.83 B 61.22 AB 54.58 B 55.58 B 70.67 A
100 118 105 107 136
Control 40 mg l−1 x1 40 mg l−1 x2 40 mg l−1 x3 5 mg l−1 x6
43.83 B 44.66 B 60.25 AB 49.17 AB 66.25 A
100 102 137 112 151
Control 40 mg l−1 x1 40 mg l−1 x2 40 mg l−1 x3 5 mg l−1 x6
35.00 B 39.11 AB 51.67 AB 44.08 AB 55.75 A
100 112 148 126 159
82
97
Means values within a column at each treatment, followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Dates of fruit collection given as days after full bloom. b Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. c Number of epidermal cells along an artificial line of 500 m tangential to fruit surface. d Change in EC values relative to Control of each date.
x3), increased dramatically although not significantly cell density at 82 DAFB compared to control and one time treatment (40 mg l−1 x1); third spray at 90 DAFB (40 mg l−1 x3) resulted with higher value than control at 97 DAFB but did not differ than the other 40 mg l−1 treatments (Table 3). Overall, calculating the percentage of change of EC values for each treatment compared to the control of the same sampling date suggested that one spray at 60 DAFB is not sufficient to get increase in epidermal cell density and that the third spray of 40 mg l−1 BA + GA4+7 at 90 DAFB may not be effective as the two applications at 60 and 75 DAFB (Table 3). 3.1.2. Experimental year 2012 As 5 mg l−1 of BA + GA4+7 was effective in maintaining high epidermal cell density during apple development, the best timing and number of its applications was tested in the experimental year of 2012. Anatomical analysis was performed to treatments that were applied between 60 and 90 DAFB, in accordance to the previous year Table 4 Effect of multiple applications of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) during fruit development at growth phase II on the average number of epidermal cells (EC) and cuticle thickness (CT) of apples collected one week following the spraying (Experimental year 2012). BA + GA4+7 treatmenta
DAFBb
ECc
EC change (%)d
CTe
Control
67 82 97
46.66 A 42.81 A 38.27 B
100 92 82
36.06 A 35.82 A 37.95 A
5 mg l−1 x2
67 82 97
49.42 A 46.02 AB 40.89 B
100 93 83
35.42 A 36.66 A 39.93 A
5 mg l−1 x3
67 82 97
48.08 A 43.46 AB 37.11 B
100 90 77
36.77 A 36.32 A 37.63 A
5 mg l−1 x2 +25 mg l−1 Aux
67 82 97
44.1 A 43.15 A 48.54 A
100 98 110
34.23B 34.62 B 41.71 A
5 mg l−1 x6
67 82 97
43.42 A 47.64 A 30.53 A
100 110 70
32.69 A 37.73 A 32.68 A
40 mg l−1 x3
67 82 97
44.33 A 52.38 A 48.45 A
100 118 109
36.11 A 39.10 A 42.78 A
Means values within a column at each treatment, followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. b Dates of fruit collection given as days after full bloom. c Number of epidermal cells along an artificial line of 500 m tangential to fruit surface. d Change in EC value relative to value at 67 DAFB of each treatment. e Cuticle thickness in m as indicated in Fig. 2.
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Fig. 3. Effect of PGR treatments at two phenological stages of fruit development – 7 to 35 DAFB (Phase I) and 60 to 90 DAFB (Phase II) – on the incidence of ‘Pink Lady’ fruits with calyx-end cracking. Fruits were sprayed with 0.025% (v/v) SuperlonTM (BA plus GA4+7 , 5 mg l−1 each), or 0.2% (v/v) SuperlonTM (40 mg l−1 each), once (x1), twice (x2) or three times (x3). One treatment included spraying of synthetic auxin using ‘Prigan’ (25 mg l−1 2,4-DPP). All treatments are detailed in Table 1. Treatments were applied by a handgun sprayer. Each data point is the mean of four trees per treatment (n = 50 fruit per tree). Different lower-case letters denote significant differences between means according to Duncan’s new multiple range test (P ≤ 0.05). The Experiment was conducted in 2012 (Experiment 2).
experiments. As expected, the density of the epidermis cells was reduced (28%) during fruit expansion in untreated fruits (Table 4). Two (60 and 75 DAFB) or three (60, 75 and 90 DAFB) spraying of 5 mg l−1 BA + GA4+7 did not maintain high cell density during fruit expansion, while three spraying with higher concentration of 40 mg l−1 (60, 75 and 90 DAFB) maintained the high cell density (Table 4), similar to the observation the year before. Results for treatment with six sprays of 5 mg l−1 BA + GA4+7 from 7 to 90 DAFB indicated of no statistical significance between the sampling dates, however EC values implied of a trend for reduced cell density following the 90 DAFB spraying (Table 4). Overall data implies that 5 mg l−1 of the PGR solution is not efficient in affecting epidermal cell density when applied at the second phase of fruit development. As auxin was shown to affect cell enlargement in apples (Wismer et al., 1995) a combined treatment was applied where two sprays of 5 mg l−1 BA + GA4+7 at 60 and 75 DAFB were followed by a spray of 25 mg l−1 synthetic auxin (2,4-DPP) at 90 DAFB. The combined treatment resulted with maintenance of cell density during fruit development, and increased cuticle thickness (Table 4). When treatments were compared at 97 DAFB, the BA + GA4+7 + 2,4DPP (5 mg l−1 x2 + 25 mg l−1 Aux) spraying resulted with significant higher cell density (+20% and +27%) compared to BA + GA4+7 alone (5 mg l−1 x2) or Control, respectively; values were similar to those obtained for the triple application of 40 mg l−1 BA + GA4+7 (Table 5). Nevertheless, similar to 2011 data only weak correlation (R2 = 0.15) was found between cuticle thickness and the EC number for all treatments and Control. 3.2. The effect of BA + GA4+7 spraying on fruit size and calyx-end cracking (Experimental year 2012) As BA + GA4+7 activities were to increase cell density probably by stimulating cell divisions, the effect on fruit size and yield was
monitored at the end of the growth season. Total yield per tree was similar for all treatments; however fruit size distribution differed between treatments (Table 6). Treatments of 5 mg l−1 BA + GA4+7 +25 mg l−1 Aux and 5 mg l−1 x6, resulted with significantly bigger fruits (≥75 mm) and higher fraction of big to medium size fruits (88.2% and 83.5%, respectively) compared to control (67.4%) or the 5 mg l−1 BA + GA4+7 x2 and x3 (44% and 41.9%, respectively) treatments. In addition, at the end of the growth season fruits from all treatments (Table 1, year 2012) were collected to evaluate the effect of BA + GA4+7 on calyx-end cracking. Results indicated highest rate of cracking in control and one application of 5 mg l−1 BA + GA4+7 at 60 DAFB, but lowest rate of cracking when 5 mg l−1 BA + GA4+7 was applied three times during phase-I or six times during both growth phases of fruit development; other treatments resulted with intermediate values (Fig. 3).
Table 5 The effect of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) that were applied at the second phase of growth on the average number of epidermal cells (EC) at 97 DAFB (Experimental year 2012). Treatmenta
ECb
EC change (%)c
5 mg l−1 x2 +25 mg l−1 Aux 40 mg l−1 x3 5 mg l−1 x2 Control 5 mg l−1 x3 5 mg l−1 x6
48.54 A 48.45A 40.89 AB 38.27 AB 37.11 AB 30.53 B
127 127 107 100 97 80
Means values within a column followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. b Number of epidermal cells along an artificial line of 500 m tangential to fruit surface c Change in EC values relative to Control.
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Table 6 The effect of BA + GA4+7 treatments at the second phase of growth on yield and fruit size. Fruits of each treatment were divided to three groups based on marketable size. Rate of each group was calculated out of the respective total yield. Treatment of BA + GA4+7 a
Control 5 mg l−1 x2 5 mg l−1 x3 5 mg l−1 x2 +25 mg l−1 Aux 5 mg l−1 x6 40 mg l−1 x3 a b
Total yield (kg/tree)
104 A 116 A 95 A 114 A 105 A 94 A
Rate of fruits at each group size (%) Big, ≥75 mm
Medium, 70 mmb
Small, ≤65 mm
14.5 B 2.7 C 5.1 C 38.9 A 30.6 AB 15.1 B
53.0 A 41.3 A 36.8 A 49.3 A 53.0 A 58.5 A
32.4 B 56.0 A 57.9 A 11.7 C 15.4 C 25.0 C
Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. Optimal marketable size.
According to the described above a negative correlation was found between the rate of fruits with calyx-end cracking and fruit size, taking into account all treatments and control (data from Table 6 and Fig. 3). The rate of cracking reduced as the rate of fruit size increased (R2 = 0.49); noticeably, treatments that increased epidermal cell density probably affected fruit expansion as well, possibly by an independent manner. 3.3. The effect of temperature on calyx-end cracking As can be seen in Fig. 3, the rate of calyx-end cracking in the experimental plots of 2012 was very high, up to 75% of the untreated control fruits, similar to the high rate monitored in 2010 (72%). However in 2011 the rate of calyx-end cracking was only 14.3%. The years 2010 and 2012 were characterized with high temperatures – 107 and 59 h with temperatures above 34 ◦ C, respectively – while only 23 h in 2011, implying correlation between prolonged high temperatures and incidence of calyx-end cracking. Accordingly data on local climate and rate of calyx-end cracking was collected from several orchards since 2008 up to 2012 and indicated of high correlation (R2 = 0.78) between number of hours with temperatures higher than 34 ◦ C and rate of calyx-end cracking (Fig. 4). 4. Discussion Fruit development can be divided to two main developmental stages. At the first phase, following fruit-set, the fruitlet grow by
Fig. 4. Association study between rate of fruits with calyx-end cracking and the duration of high temperatures in the growth seasons. Graph includes data of the years 2008 up to 2012 from 14 orchards at the north of Israel that did not get any treatment. A strong correlation was found, R2 = 0.78; P = 0.05.
intensive cell divisions, while at the second phase fruit expand due to cell enlargement and increase in intercellular spaces (Dennis, 2003; Westwood, 1993; Tromp and Wertheim, 2005). Observations conducted in Israel over several years indicated that cell-division phase is terminated at approx. 60 DAFB; the phase of major fruit expansion occurs from June to July (60 to 100 DAFB) with an increase of fruit volume of 16–18 ml week−1 . After 100 DAFB, fruit expansion dropped to 4–6 ml week−1 . Accordingly, BA + GA4+7 treatments aimed at increasing the flexibility of the peel were conducted at that developmental stage. Our previous experiments showed that application of BA + GA4+7 , during the critical stage of fruit expansion (60 to 90 DAFB) reduced calyx-end cracking (Stern et al., 2013). This PGR combination was potent at reducing the incidence of russeting in ‘Golden Delicious’ as well (Fogelman et al., 2009). BA probably increases the rate of cell division, as was previously shown for apple and pear, by Stern et al. (2003a,b, 2006) and for other species by Davis (2010). The gibberellins A4+7 increase cell volume (Stern et al., 2007; Zhang et al., 2007; Davis, 2010) and contribute as well to increased epidermal cell density (Curry, 2012). Additional treatment that was shown to increase epidermal cell density included application of a synthetic auxin following two applications of low concentration of BA + GA4+7 (5 mg l−1 ) (Table 5). Auxin was shown to contribute to cell enlargement (Wismer et al., 1995) and although epidermal cell size was not monitored at the present study, this treatment resulted with increased rate of big size fruits, indicating cell enlargement activity in the fruit (Table 6). Interestingly, values for cuticle thickness, as measured from the base of the epidermal cell layer to the outer edge of the cuticle, remained the same at the tested period of 60 to 90 DAFB – although fruit expanded significantly – with no significant difference between control and PGR treated fruits. Knoche et al. (2011) suggest that GA4+7 had no effect on the amounts or rates of cutin or wax deposition, and that the decrease in russeting and formation of microcracks in the cuticle of GA4+7 -treated fruit must be accounted for effects on underlying epi- and hypodermal tissues. If dermal cell layers contributed to the apple skin mechanics (Khanal et al., 2013), increased cell division and cell size can maintain cell-to-cell adhesion under tensional stresses providing a stronger structural support of fruit surface. Whether PGRs treatments altered cuticular composition and modified the mechanical properties of the apple skin (Domínguez et al., 2011), remains to be clarified. In 2011, multiple spraying of BA + GA4+7 – 40 mg l−1 each applied as 0.2% SuperlonTM – increased epidermal cell density (Table 3). Actually, comparing the effect of each treatment during the experimental period indicated that the PGR treatment maintained high cell density along fruit development while in control cell density decreased (Table 2). It is notable that one spray at 60 DAFB was not sufficient to maintain the high cell density. In 2012, application of BA + GA4+7 at a lower concentration – 5 mg l−1 each, applied as 0.025% SuperlonTM – at the same fruit developmental stage (60, 75 and 90 DAFB) did not maintain the
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high epidermal cell density during fruit expansion as observed for the application of the higher concentration (40 mg l−1 ) that year and the year before (Tables 2 and 4), implying that BA + GA4+7 at a concentration of 5 mg l−1 was not as potent at that late stage. To compensate for the weak activity of the low concentration of BA + GA4+7 (5 mg l−1 ) the effect of its spraying was tested in earlier stages of fruit development, from 7 to 35 DAFB (growth phase I). These fruits were not used for the anatomical study, but were included in the evaluation of calyx-end cracking severity. The severity of the cracking disorder was monitored at the end of the growing season of 2012 and included all treatments as detailed in Table 1. Results suggested that all treatments (except 5 mg l−1 x1 at phase II) reduced the cracking incidence compared to control (Fig. 3). The best treatments were those that included three sequential treatments at 7, 21 and 35 DAFB—i.e., 5 mg l−1 x3 phase I, and 5 mg l−1 x6 phases I + II (Fig. 3). It is notable that the 5 mg l−1 x6 at phases I + II also resulted with increased fruit size (Table 6). Comparing fruit size and rate of cracking showed a correlation with increased fruit size due to the PGRs treatment, and reduced calyx-end cracking. An association of climate conditions and the incidence of calyxend cracking was suggested by the growers. Calyx-end cracking incidence in 2011 and 2012 was 14.3% and 61.7%, respectively. The former year was cooler than the latter—in 2012 there were 59 h with temperatures above 34 ◦ C (in June to August), compared to only 23 h in 2011. Accordingly, data that was collected from several orchards during 2008 to 2012 indicated of positive and strong association between the duration of high temperatures and the incidence of the disorder (R2 = 0.78; Fig. 4). 5. Conclusions Application of BA + GA4+7 to the developing fruit increase epidermal cell density at the calyx-end, probably enhancing the elasticity of the skin and reinforcing the cuticular membrane, consequentially reduce the susceptibility of the fruit to calyx-end cracking. Results suggest that when temperatures higher than average are expected to increase the incidence of calyx-end cracking, BA + GA4+7 can be applied to minimize the damage. If indications for hot climate will be detected at the beginning of the growth season, multiple treatments of 5 mg l−1 will be effective at the first phase of fruit developments, however if decision will be made at a later stage, a higher concentration of 40 mg l−1 should be applied. Acknowledgments This work was supported by the Fruit Grower Association, Israel, and is a contribution of ARO, the Volcani Center No. 107/2013).
References Cripps, J.E.L., Richards, L.A., Mairata, A.M., 1993. ‘Pink Lady’ apple. HortScience 28, 1057. Curry, E., 2012. Increase in epidermal planar cell density accompanies decreased russeting of ‘Golden Delicious’ apples treated with gibberellins A4 + 7. HortScience 47, 232–237. Davis, P.J., 2010. In: Davis, P.J. (Ed.), The Plant Hormones: Their Nature Occurrence and Functions Plant Hormones. Springer, Dordrecht, The Netherlands, pp. 1–15. Dennis, J.R.F., 2003. Flowering Pollination and Fruit set and Development. In: Ferree, D.C., Warrington, I.J. (Eds.), Apples: Botany, Production and Uses. CABI Publishing, Wallingford, Oxon, UK, pp. 153–166. Domínguez, E., Heredia-Guerrero, J.A., Heredia, A., 2011. The biophysical design of plant cuticles: an overview. New Phytol. 189, 938–949. Eccher, T., 1975. Influenza di alcuni fitormoni sulla rugginosita della ‘Golden Delicious’. Riv. dell’ Ortoflorofrutticoltura Ital. 59, 246–261. Faust, M., Shear, C.B., 1972. Russeting of apples, an interpretive review. HortScience 11, 233–235. Fogelman, E., Redel, G., Doron, I., Naor, A., Ben-Yashar, E., Ginzberg, I., 2009. Control of apple russeting in warm and dry climate. J. Hortic. Sci. Biotechnol. 84, 279–284. Johansen, D.A., 1940. Plant Microtechniques. McGraw-Hill Book Company Inc, New York NY, USA, pp. 523. Khanal, B.P., Grimm, E., Knoche, M., 2013. Russeting in apple and pear: a plastic periderm replaces a stiff cuticle. AoB Plants 5, pls048, http://dx.doi.org/ 10.1093/aobpla/pls048. Knoche, M., Khanal, B.P., Stopar, M., 2011. Russeting and microcracking of ‘Golden Delicious’ apple fruit concomitantly decline due to gibberellins A4 + 7 application. J. Am. Soc. Hortic. Sci. 136, 159–164. Mackay, A.G., Godley, G., Cripps, J.E.L., Melvin-Carter, E., Paterson, J., Wood, P., 1994. Pink Lady Sundowner Apples: Bulletin No. 4285. Department of Agriculture, South Perth, WA, Australia, pp. 32. Opara, L.U., Studan, C.J., Banks, N.H., 1997. Fruit skin splitting and cracking. Hortic. Rev. 19, 217–262. Ruzin, S.E., 1999. Plant Microtechniques and Microscopy. Oxford University Press, New York NY, USA, pp. 322. Stern, R.A., Ben-Arie, R., Neria, O., Flaishman, M., 2003a. CPPU and BA increase fruit size of ‘Royal Gala’ (Malus domestica) apple in a warm climate. J. Hortic. Sci. Biotechnol. 78, 297–302. Stern, R.A., Ben-Arie, R., Ginzberg, I., 2013. Reducing the incidence of calyx cracking in ‘Pink Lady’ apple using a combination of cytokinin 6-benzyladenine and gibberellins (GA4 + 7). J. Hortic. Sci. Biotechnol. 88, 147–153. Stern, R.A., Shargal, A., Flaishman, M., 2003b. Thidiazuron increases fruit size of ‘Spadona’ and ‘Coscia’ pear (Pyrus communis L. J. Hortic. Sci. Biotechnol. 78, 51–55. Stern, R.A., Ben-Arie, R., Appleboum, S., Flaishman, M., 2006. Cytokinins increase fruit size of ‘Delicious’ and ‘Golden Delicious’ (Malus domestica) apple in a warm climate. J. Hortic. Sci. Biotechnol. 81, 51–56. Stern, R.A., Doron, I., Ben-Arie, R., 2007. Plant growth regulators increase the fruit size of ‘Spadona’ and ‘Coscia’ pear (Pyrus communis L.) in a warm climate. J. Hortic. Sci. Biotechnol. 82, 803–807. Tromp, J., Wertheim, S.J., 2005. Fruit growth and development. In: Tromp, J., Webster, A.D., Wertheim, S.J. (Eds.), Fundamentals of Temperate Zone Tree Fruit Production. Backhuys Publishers, Leiden, The Netherlands, pp. 240–266. Westwood, M.N., 1993. Temperate-Zone Pomology: Physiology and Culture, third ed. Timber Press, Portland, OR, USA, pp. 523. Zhang, C., Tanabe, K., Tani, H., Nakajima, H., Mori, M., Sakuno, E., 2007. Biologically active gibberellins and ABA in fruit of two late-maturing Japanese pear (Pyrus pyrifolia ‘Nakai’) cultivars with contrasting fruit size. J. Am. Soc. Hortic. Sci. 132, 452–458. Wismer, P.T., Proctor, J.T.A., Elfving, D.C., 1995. Benzyladenine affects cell division and cell size during apple fruit thinning. J. Am. Soc. Hortic. Sci. 120, 802–807.
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Maintenance of high epidermal cell density and reduced calyx-end cracking in developing ‘Pink Lady’ apples treated with a combination of cytokinin 6-benzyladenine and gibberellins A4 +A7 Idit Ginzberg a,∗ , Edna Fogelman a , Lior Rosenthal a , Raphael A. Stern b,c a
Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO Box 6, Bet Dagan 50250, Israel MIGAL, Galilee Technology Center, PO Box 831, Kiryat Shmona 11016, Israel c Department of Biotechnology, Faculty of Life Sciences, Tel-Hai College, Upper Galilee 12210, Israel b
a r t i c l e
i n f o
Article history: Received 18 July 2013 Received in revised form 27 October 2013 Accepted 13 November 2013 Keywords: Fruit cracking Malus × domestica Peel anatomy Plant growth regulators
a b s t r a c t Calyx-end cracking in ‘Pink Lady’ apple develops as concentric cracks located around the stylar-end. The factors that induce the cracking are unknown, however we previously demonstrated that the intensity of the disorder can be controlled by application of a mixture of synthetic gibberellins (GA4 plus GA7 ) and cytokinin (6-benzyladenine; BA) at different phenological stages of fruit development, and suggested that the plant growth regulators (PGRs) affect the elasticity of the epidermal layer at the calyx-end by increasing cell density. The present study focused on histological analyses of the peel at the calyx-end following the application of BA + GA4+7 . The experiments were conducted in Northern Israel during 2011 and 2012. In 2011 the PGRs were applied as 0.2% (v/v) SuperlonTM (i.e., 40 mg l−1 BA plus 40 mg l−1 GA4+7 ) once, twice or three times every 14 days since 60 days after full bloom (DAFB). In 2012, to test the efficacy of a lower concentration of the PGRs, 0.025% (v/v) SuperlonTM was used. Spraying was applied at 60 to 90 DAFB (cell expansion phase) as before, in addition to earlier spraying at 7 to 35 DAFB (cell division phase). In all treatments given at cell expansion phase the BA + GA4+7 mixture increased the epidermal cell density at the calyx-end, compared to control. Multiple spraying maintained the high cell density during fruit expansion compared to one-time application, and the higher concentration of the PGRs was more potent than the lower. Assessment of calyx-end cracking suggested that low concentration of the PGRs (i.e., 5 mg l−1 BA plus 5 mg l−1 GA4+7 ) is most efficient when applied three times at cell division phase. Interestingly, in 2011 the incidence of calyx-end cracking was significantly lower relative to 2012 and 2010 that were characterized by extended daily hours of high temperatures (>34 ◦ C). This association can be used by growers as a criterion in applying the PGR treatment early in the season when temperatures are higher than the average and high incidence of calyx-end cracking might be expected. © 2013 Elsevier B.V. All rights reserved.
1. Introduction ‘Pink Lady’ apple (Malus × domestica Borkh.) (Cripps et al., 1993) fruits are susceptible to pre-harvest physiological disorder of calyxend cracking (Fig. 1) (Opara et al., 1997). In Israel, the incidence of cracking may reach up to 80% of the total yield. ‘Pink Lady’ is popular in many countries due to its pinkish color, crisp texture, and a high sugar:acid ratio (Mackay et al., 1994); however the high incidence of cracking affect its marketability, resulting in substantial economic losses.
∗ Corresponding author. Tel.: +972 3 9683787; fax: +972 3 9669583. E-mail address: [email protected] (I. Ginzberg). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.11.020
Calyx-end cracking in ‘Pink Lady’ develops as concentric cracks located around the stylar-end (Fig. 1). In contrast to the wellstudied skin russeting disorder that results from microscopic cracks in the cuticle and the subsequent formation of a periderm (Faust and Shear, 1972; Fogelman et al., 2009; Khanal et al., 2013), calyxend cracking disorder is usually characterized with cracks that traverse the cuticle, and although corky periderm is develops at their margins, it is not sufficient to prevent pathogen invasion into inner fruit tissues. In addition, apples susceptible for russeting early in fruit development, while calyx-end cracking appear toward the end of the growth. The factors that promote calyx-end cracking were not defined yet. Preliminary observations indicated that the severity of the disorder was associated with orchard location (especially in high latitudes and dry climates), row orientation that exposed fruit to
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as well-drained protogromosol (68% clay) on terra rossa soil at Baraam. The soil pH is 7.7 with CaCO3 contents of approx. 7% (w/w). 2.2. Application of plant growth regulators (PGRs) A commercial product containing synthetic cytokinins and gibberellins (GA) was used: SuperlonTM , a liquid formulation containing 1.9% (v/v) BA and 1.9% (v/v) GA4+7 (Fine Agrochemicals, Whittington, UK).
Fig. 1. Calyx-end cracking in ‘Pink Lady’ (left) compared to a normal fruit (right). Scale bar = 1 cm.
high level of solar radiation, and high temperatures in the second half of the growing season. Experiments that were conducted by us in the growth seasons of 2008 to 2011 indicated that combined applications of the cytokinin 6-benzyladenine (BA) and the gibberellins A4 + A7 (GA4+7 ) to ‘Pink Lady’ fruitlets between 60 and 90 days after full bloom (DAFB) reduced significantly the incidence of calyx-end cracking, while treatment of each of the plant growth regulator (PGR) alone was ineffective (Stern et al., 2013). The optimal treatment was three consecutive applications of 40 mg l−1 BA plus 40 mg l−1 GA4+7 mixture at 14 d interval beginning 60 DAFB, applied as the commercial formulation SuperlonTM (0.2%, Fine Agrochemicals, Whittington, UK). The epidermal layer of the fruit appeared to be the target of the BA + GA4+7 mixture. The PGRs probably increase peel flexibility due to enhanced cell divisions and increased cell density at the fruit surface, thus reduce its susceptibility to cracking at the developmental stage of dramatic fruit expansion (Stern et al., 2013). Our results were in accordance with previous reports showing that treatments with gibberellins reduced cuticular cracking in apple, probably by affecting the epidermal tissue. Eccher (1975) showed that treatment with GA4+7 resulted in less irregularity among epidermal cells and reduced cuticle cracking compared to untreated fruit. Knoche et al. (2011) showed that water-induced russeting and micro-cracking in ‘Golden Delicious’ apple could be reduced by multiple applications of GA4+7 . Treatment had no effect on the amount or rate of cutin or wax deposition, suggesting that reduced russeting and micro-cracking were due to the effects of GA4+7 on the epidermal and hypodermal layers of cell. Fogelman et al. (2009) reported a similar result of less russeting on ‘Smoothy Golden Delicious’ after multiple applications of the SuperlonTM during the first month of fruit development. The present work focused on anatomical changes induced at the calyx end of ‘Pink Lady’ fruit by multiple treatments of BA + GA4+7 mixture. It is shown that multiple applications of the PRGs maintained high epidermal cell density compared to untreated control, and reduced the incidence of the cracking disorder.
2. Materials and methods 2.1. Experimental orchards and conditions Experiments were conducted for two seasons 2011 and 2012 on 10-to-12 year-old ‘Pink Lady’ apple trees grafted on MM 106 rootstock in Baraam orchard, located 700 m a.s.l. in Upper Galilee, where all trees were 3.5 m high, spaced at 4.5 m × 5.5 m (900 trees ha−1 ). The orchard is located in a semi-arid region with high temperatures (ca. 35◦ C max.) and low humidity (<40% RH) during the Summer (May to October). Annual precipitation (November–April) was approx. 700 mm. The soil is 0.8 m to 1.2 m deep, and classed
2.2.1. Experiment 1 (2011) Multiple spraying of 0.2% (v/v) SuperlonTM (40 mg l−1 BA + 40 mg l−1 GA4+7 ) were applied at different times during the growing season; indicated as days after full bloom (DAFB). Four different treatments were tested (Table 1): one spray at 60 DAFB; two sprays applied at 60 and 75 DAFB; and three sprays applied at 60, 75 and 90 DAFB. Spraying of 0.2% (v/v) SuperlonTM are the accepted practice in the orchard. To test the efficiency of reduced concentration 0.025% SuperlonTM (5 mg l−1 BA + 5 mg l−1 GA4+7 ) was applied; however, at six time points, every two to three weeks since 7 DAFB (7, 21, 35, 60, 75 and 90 DAFB; Table 1). An additional set of trees served as non-treated controls. The PGRs were applied as a foliar spray at 3 up to 5 l per tree, using a high pressure-handgun (Kibbutz Degania, Israel) until runoff. Triton X-100, at 0.025% (v/v) was included in all sprays as a non-ionic surfactant. The experiments were conducted on whole trees bearing a similar crop load (ca. 240 fruits tree−1 , ca. 35 kg tree−1 ). Control trees, with the same crop load, were not sprayed. The experimental design was as randomised complete blocks, with four blocks of four trees per treatment. One buffer tree was always used between two treatment trees to avoid drift. 2.2.2. Experiment 2 (2012) To assess the optimum time and number of applications of the reduced PGR solution, the treatments were applied in two phenological stages of fruit development. Cell division phase of fruit growth (phase I), 7 to 60 DAFB, and the cell expansion phase of fruit growth (phase II), 60 to 90 DAFB. SuperlonTM , 0.025% (v/v) (5 mg l−1 BA + 5 mg l−1 GA4+7 ) was applied once, twice or three times during phase I or phase II (7, 21, 35 DAFB or 60, 75, 90 DAFB, respectively), or throughout both growth phases (Table 1). The effect of auxin on the cracking disorder was tested by a combination of two spraying of 0.025% SuperlonTM (60 and 75 DAFB) with one spray of 25 mg l−1 2,4-Dichlorprop-P (2,4-DPP; Prigan, Agan Chemicals, Israel) at 90 DAFB. Spraying of 0.2% SuperlonTM at the second phase, 60 to 90 DAFB, was applied similarly to the described above for the experimental year of 2011. An additional set of trees served as non-treated controls. The sprayings system and the experimental design were as described for Experiment 1. 2.3. Fruit size and assessment of fruit cracking Average fruit size and the incidence of the cracking disorder were assessed at harvest. Fifty randomized fruits from the trees’ outskirt were collected from each tree. Fruit diameter in mm was measured at the equatorial plan of the fruit, and fruit affected by calyx-end cracking were counted. Values were expressed as a percentage. 2.4. Tissue embedding and measurements of epidermal cell number and cuticle thickness Histological studies were conducted on fruit harvested seven days following the spraying at the second phase of growth (i.e.,
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Table 1 Schematic presentation of the dates (given as DAFB) of SuperlonTM application at the various treatments at two experimental years, 2011 and 2012. DAFBb
BA plus GA4+7 treatment 40 mg l−1
5 mg l−1
Sprayinga
Phase Ic 7
Phase IId 21
35
60
75
90
x x x x
x x x
x x
x x x x x x
x x x x x
A x x x
Experimental year 2011 x x x x Experimental year 2012 x x x x x x x x x a b c d e
Control x1 x2 x3 x6 Control x1 x2 x3 x1 x2 x2+Auxe x3 x6 x3
x
x
x
x x x
x x
x
x
x
x
Number of PGR spraying. DAFB, days after full bloom. Cell division phase of fruit growth, 7 to 35 DAFB. Cell expansion phase of fruit growth, 60 to 90 DAFB. Additional spray of 25 mg l−1 auxin (2,4-DPP) was applied at 90 DAFB.
67, 82 and 97 DAFB). Four fruit were collected from each repeat of SuperlonTM and control treatments (16 fruits per treatment). Apple surface tissue samples (blocks of 5 mm × 3 mm × 3 mm) were collected from the calyx-end of each fruit within 24 h since harvest. Samples were fixed in FAA [50% (v/v) ethanol, 5% (v/v) acetic acid, 3.7% (v/v) formaldehyde, and 41.3% (v/v) H2 O], dehydrated in an ethanol/Histoclear series (Finkelman Chemicals, Petach-Tikva, Israel) and embedded in ParaplastTM (Paraplast Plus; McCormick Scientific, St. Louis, MO, USA) according to standard methods (Ruzin, 1999). Tissue sections (15–20 m thick) were stained with Toluidine blue (Sigma Chemicals, Rehovot, Israel) for examinations of tissue morphology (Johansen, 1940). Sections were observed under a light microscope (Leica DMLB; Wetzlar, Germany) and images were displayed on a monitor through a CCD camera (Leica DC2000) using the Leica IM1000 programme. To measure epidermal cell numbers and cuticle thickness in each treatment, one cross-section was selected from each fruit. The density of epidermal cells was determined by counting cells along an arbitrary 500 m line, at three locations in the section. The average cell number 500 m−1 was calculated for each section. Cuticle thickness was determined by measuring the lengths (m) of arbitrary lines from the base of the epidermal cell layer to the outer edge of the cuticle at five locations along the section (Fig. 2). An average value was calculated for the section.
3. Results 3.1. BA + GA4+7 spraying during fruit development maintain high epidermis cell density 3.1.1. Experimental year 2011 Cross sections of apples collected at 67, 82 and 97 DAFB indicated significant reduction (32%) in density of epidermal cells during fruit development (control, Table 2). However, multiple application of BA + GA4+7 (40 mg l−1 each), at 60 and 75 DAFB or Table 2 Effect of multiple applications of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) during fruit development on the average number of epidermal cells (EC) and cuticle thickness (CT) of apples collected one week following the spraying (Experimental year 2011). BA + GA4+7 treatmenta
DAFBb
ECc
EC change (%)d
CTe
Control
67 82 97
51.83 A 43.83 AB 35.0 B
100 85 68
29.18 A 25.74 A 27.92 A
40 mg l−1 x1
67 82 97
61.22 A 44.67 B 39.11 B
100 73 64
32.98 A 28.89 A 31.36 A
40 mg l−1 x2
67 82 97
54.58 A 60.25 A 51.67 A
100 110 95
26.66 A 30.90 A 25.86 A
40 mg l−1 x3
67 82 97
55.58 A 49.17 A 44.08 A
100 89 79
26.69 A 28.34 A 28.85 A
5 mg l−1 x6
67 82 97
70.67 A 66.25 A 55.75 A
100 94 79
32.62 A 35.55 A 29.76 A
2.5. Statistical analysis Percentage data of fruit size and cracking were subjected to arcsine transformation before analysis in order to provide a normal distribution of variance. Data were analysed for statistical significance using the general linear model (GLM) procedure. Duncan’s new multiple range test was used to compare treatments when ANOVA showed significant differences between means. Data for cell density and cuticle thickness were analysed statistically by one-way ANOVA using JMP software (SAS Institute Inc., Cary, NC, USA).
Means values within a column, at each treatment, followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. b Dates of fruit collection given as days after full bloom. c Number of epidermal cells along an artificial line of 500 m tangential to fruit surface. d Change in EC value relative to value at 67 DAFB of each treatment. e Cuticle thickness in m as indicated in Fig. 2.
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Fig. 2. Anatomical study of the calyx end demonstrating the effect of PGR treatment on epidermal cell density and organization at two phenological stages of fruit development. Samples were collected at 67 and 97 DAFB from untreated control fruits (left) and fruits treated twice, at 60 and 75 DAFB, with 0.025% (v/v) SuperlonTM (5 mg l−1 BA plus 5 mg l−1 GA4+7 ) and at 90 DAFB with ‘Prigan’ (25 mg l−1 2,4-DPP) (right). In untreated control fruits, as fruit expands, epidermal cell density is reduced and the gap between neighboring cells increase. PGR induce epidermal cell accumulation that maintain high cell density and induce cell aggregations. Pictures width present 500 m. Arrow line demonstrates measurements of cuticle thickness by measuring the length (m) of an arbitrary line from the base of the epidermal cell layer to the outer edge of the cuticle.
three times at 60, 75 and 90 DAFB, prevented this reduction and maintained the high epidermal cell density, although the double spraying seems more efficient (Table 2). In an attempt to reduce the concentration of the PGR spraying, BA + GA4+7 were applied at 5 mg l−1 every 14 days since seven DAFB, and were found to similarly prevent the reduction in epidermal cell density during fruit expansion; notably, as this treatment begun prior to 60 DAFB, the initial EC value is higher than that of control and other treatments. Interestingly, cuticle thickness did not vary during fruit expansion in control and treated fruits (Table 2), and only weak correlation (R2 = 0.19) was found between cell number and cuticle thickness. The effect of the treatments on epidermal cell density was compared for each sampling date to determine the best timing, concentration and number of PGR applications. Data indicated that spraying with 5 mg l−1 BA + GA4+7 solution six times since 7 DAFB resulted with the highest increase in cell density in all sampling dates, compared to untreated control and to spraying with 40 mg l−1 solution (Table 3). Of the 40 mg l−1 treatments—one spray at 60 DAFB did not change significantly epidermal cell density at 67 DAFB (note, all 40 mg l−1 treatments got one spraying at that time point); two sprays at 60 and 75 DAFB (40 mg l−1 x2 and 40 mg l−1
Table 3 The effect of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) applications on the average number of epidermal cells (EC) was compared for each sampling date (Experimental year 2011). DAFBa
BA + GA4+7 treatmentb
ECc
EC change (%)d
67
Control 40 mg l−1 x1 40 mg l−1 x2 40 mg l−1 x3 5 mg l−1 x6
51.83 B 61.22 AB 54.58 B 55.58 B 70.67 A
100 118 105 107 136
Control 40 mg l−1 x1 40 mg l−1 x2 40 mg l−1 x3 5 mg l−1 x6
43.83 B 44.66 B 60.25 AB 49.17 AB 66.25 A
100 102 137 112 151
Control 40 mg l−1 x1 40 mg l−1 x2 40 mg l−1 x3 5 mg l−1 x6
35.00 B 39.11 AB 51.67 AB 44.08 AB 55.75 A
100 112 148 126 159
82
97
Means values within a column at each treatment, followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Dates of fruit collection given as days after full bloom. b Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. c Number of epidermal cells along an artificial line of 500 m tangential to fruit surface. d Change in EC values relative to Control of each date.
x3), increased dramatically although not significantly cell density at 82 DAFB compared to control and one time treatment (40 mg l−1 x1); third spray at 90 DAFB (40 mg l−1 x3) resulted with higher value than control at 97 DAFB but did not differ than the other 40 mg l−1 treatments (Table 3). Overall, calculating the percentage of change of EC values for each treatment compared to the control of the same sampling date suggested that one spray at 60 DAFB is not sufficient to get increase in epidermal cell density and that the third spray of 40 mg l−1 BA + GA4+7 at 90 DAFB may not be effective as the two applications at 60 and 75 DAFB (Table 3). 3.1.2. Experimental year 2012 As 5 mg l−1 of BA + GA4+7 was effective in maintaining high epidermal cell density during apple development, the best timing and number of its applications was tested in the experimental year of 2012. Anatomical analysis was performed to treatments that were applied between 60 and 90 DAFB, in accordance to the previous year Table 4 Effect of multiple applications of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) during fruit development at growth phase II on the average number of epidermal cells (EC) and cuticle thickness (CT) of apples collected one week following the spraying (Experimental year 2012). BA + GA4+7 treatmenta
DAFBb
ECc
EC change (%)d
CTe
Control
67 82 97
46.66 A 42.81 A 38.27 B
100 92 82
36.06 A 35.82 A 37.95 A
5 mg l−1 x2
67 82 97
49.42 A 46.02 AB 40.89 B
100 93 83
35.42 A 36.66 A 39.93 A
5 mg l−1 x3
67 82 97
48.08 A 43.46 AB 37.11 B
100 90 77
36.77 A 36.32 A 37.63 A
5 mg l−1 x2 +25 mg l−1 Aux
67 82 97
44.1 A 43.15 A 48.54 A
100 98 110
34.23B 34.62 B 41.71 A
5 mg l−1 x6
67 82 97
43.42 A 47.64 A 30.53 A
100 110 70
32.69 A 37.73 A 32.68 A
40 mg l−1 x3
67 82 97
44.33 A 52.38 A 48.45 A
100 118 109
36.11 A 39.10 A 42.78 A
Means values within a column at each treatment, followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. b Dates of fruit collection given as days after full bloom. c Number of epidermal cells along an artificial line of 500 m tangential to fruit surface. d Change in EC value relative to value at 67 DAFB of each treatment. e Cuticle thickness in m as indicated in Fig. 2.
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Fig. 3. Effect of PGR treatments at two phenological stages of fruit development – 7 to 35 DAFB (Phase I) and 60 to 90 DAFB (Phase II) – on the incidence of ‘Pink Lady’ fruits with calyx-end cracking. Fruits were sprayed with 0.025% (v/v) SuperlonTM (BA plus GA4+7 , 5 mg l−1 each), or 0.2% (v/v) SuperlonTM (40 mg l−1 each), once (x1), twice (x2) or three times (x3). One treatment included spraying of synthetic auxin using ‘Prigan’ (25 mg l−1 2,4-DPP). All treatments are detailed in Table 1. Treatments were applied by a handgun sprayer. Each data point is the mean of four trees per treatment (n = 50 fruit per tree). Different lower-case letters denote significant differences between means according to Duncan’s new multiple range test (P ≤ 0.05). The Experiment was conducted in 2012 (Experiment 2).
experiments. As expected, the density of the epidermis cells was reduced (28%) during fruit expansion in untreated fruits (Table 4). Two (60 and 75 DAFB) or three (60, 75 and 90 DAFB) spraying of 5 mg l−1 BA + GA4+7 did not maintain high cell density during fruit expansion, while three spraying with higher concentration of 40 mg l−1 (60, 75 and 90 DAFB) maintained the high cell density (Table 4), similar to the observation the year before. Results for treatment with six sprays of 5 mg l−1 BA + GA4+7 from 7 to 90 DAFB indicated of no statistical significance between the sampling dates, however EC values implied of a trend for reduced cell density following the 90 DAFB spraying (Table 4). Overall data implies that 5 mg l−1 of the PGR solution is not efficient in affecting epidermal cell density when applied at the second phase of fruit development. As auxin was shown to affect cell enlargement in apples (Wismer et al., 1995) a combined treatment was applied where two sprays of 5 mg l−1 BA + GA4+7 at 60 and 75 DAFB were followed by a spray of 25 mg l−1 synthetic auxin (2,4-DPP) at 90 DAFB. The combined treatment resulted with maintenance of cell density during fruit development, and increased cuticle thickness (Table 4). When treatments were compared at 97 DAFB, the BA + GA4+7 + 2,4DPP (5 mg l−1 x2 + 25 mg l−1 Aux) spraying resulted with significant higher cell density (+20% and +27%) compared to BA + GA4+7 alone (5 mg l−1 x2) or Control, respectively; values were similar to those obtained for the triple application of 40 mg l−1 BA + GA4+7 (Table 5). Nevertheless, similar to 2011 data only weak correlation (R2 = 0.15) was found between cuticle thickness and the EC number for all treatments and Control. 3.2. The effect of BA + GA4+7 spraying on fruit size and calyx-end cracking (Experimental year 2012) As BA + GA4+7 activities were to increase cell density probably by stimulating cell divisions, the effect on fruit size and yield was
monitored at the end of the growth season. Total yield per tree was similar for all treatments; however fruit size distribution differed between treatments (Table 6). Treatments of 5 mg l−1 BA + GA4+7 +25 mg l−1 Aux and 5 mg l−1 x6, resulted with significantly bigger fruits (≥75 mm) and higher fraction of big to medium size fruits (88.2% and 83.5%, respectively) compared to control (67.4%) or the 5 mg l−1 BA + GA4+7 x2 and x3 (44% and 41.9%, respectively) treatments. In addition, at the end of the growth season fruits from all treatments (Table 1, year 2012) were collected to evaluate the effect of BA + GA4+7 on calyx-end cracking. Results indicated highest rate of cracking in control and one application of 5 mg l−1 BA + GA4+7 at 60 DAFB, but lowest rate of cracking when 5 mg l−1 BA + GA4+7 was applied three times during phase-I or six times during both growth phases of fruit development; other treatments resulted with intermediate values (Fig. 3).
Table 5 The effect of 0.025% (v/v) or 0.2% (v/v) SuperlonTM (5 or 40 mg l−1 BA plus 5 or 40 mg l−1 GA4+7 ) that were applied at the second phase of growth on the average number of epidermal cells (EC) at 97 DAFB (Experimental year 2012). Treatmenta
ECb
EC change (%)c
5 mg l−1 x2 +25 mg l−1 Aux 40 mg l−1 x3 5 mg l−1 x2 Control 5 mg l−1 x3 5 mg l−1 x6
48.54 A 48.45A 40.89 AB 38.27 AB 37.11 AB 30.53 B
127 127 107 100 97 80
Means values within a column followed by different upper-case letters differ significantly at P ≤ 0.05 by one-way ANOVA using JMP software. a Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. b Number of epidermal cells along an artificial line of 500 m tangential to fruit surface c Change in EC values relative to Control.
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Table 6 The effect of BA + GA4+7 treatments at the second phase of growth on yield and fruit size. Fruits of each treatment were divided to three groups based on marketable size. Rate of each group was calculated out of the respective total yield. Treatment of BA + GA4+7 a
Control 5 mg l−1 x2 5 mg l−1 x3 5 mg l−1 x2 +25 mg l−1 Aux 5 mg l−1 x6 40 mg l−1 x3 a b
Total yield (kg/tree)
104 A 116 A 95 A 114 A 105 A 94 A
Rate of fruits at each group size (%) Big, ≥75 mm
Medium, 70 mmb
Small, ≤65 mm
14.5 B 2.7 C 5.1 C 38.9 A 30.6 AB 15.1 B
53.0 A 41.3 A 36.8 A 49.3 A 53.0 A 58.5 A
32.4 B 56.0 A 57.9 A 11.7 C 15.4 C 25.0 C
Concentration (mg l−1 ) and number of sprayings as detailed in Table 1. Optimal marketable size.
According to the described above a negative correlation was found between the rate of fruits with calyx-end cracking and fruit size, taking into account all treatments and control (data from Table 6 and Fig. 3). The rate of cracking reduced as the rate of fruit size increased (R2 = 0.49); noticeably, treatments that increased epidermal cell density probably affected fruit expansion as well, possibly by an independent manner. 3.3. The effect of temperature on calyx-end cracking As can be seen in Fig. 3, the rate of calyx-end cracking in the experimental plots of 2012 was very high, up to 75% of the untreated control fruits, similar to the high rate monitored in 2010 (72%). However in 2011 the rate of calyx-end cracking was only 14.3%. The years 2010 and 2012 were characterized with high temperatures – 107 and 59 h with temperatures above 34 ◦ C, respectively – while only 23 h in 2011, implying correlation between prolonged high temperatures and incidence of calyx-end cracking. Accordingly data on local climate and rate of calyx-end cracking was collected from several orchards since 2008 up to 2012 and indicated of high correlation (R2 = 0.78) between number of hours with temperatures higher than 34 ◦ C and rate of calyx-end cracking (Fig. 4). 4. Discussion Fruit development can be divided to two main developmental stages. At the first phase, following fruit-set, the fruitlet grow by
Fig. 4. Association study between rate of fruits with calyx-end cracking and the duration of high temperatures in the growth seasons. Graph includes data of the years 2008 up to 2012 from 14 orchards at the north of Israel that did not get any treatment. A strong correlation was found, R2 = 0.78; P = 0.05.
intensive cell divisions, while at the second phase fruit expand due to cell enlargement and increase in intercellular spaces (Dennis, 2003; Westwood, 1993; Tromp and Wertheim, 2005). Observations conducted in Israel over several years indicated that cell-division phase is terminated at approx. 60 DAFB; the phase of major fruit expansion occurs from June to July (60 to 100 DAFB) with an increase of fruit volume of 16–18 ml week−1 . After 100 DAFB, fruit expansion dropped to 4–6 ml week−1 . Accordingly, BA + GA4+7 treatments aimed at increasing the flexibility of the peel were conducted at that developmental stage. Our previous experiments showed that application of BA + GA4+7 , during the critical stage of fruit expansion (60 to 90 DAFB) reduced calyx-end cracking (Stern et al., 2013). This PGR combination was potent at reducing the incidence of russeting in ‘Golden Delicious’ as well (Fogelman et al., 2009). BA probably increases the rate of cell division, as was previously shown for apple and pear, by Stern et al. (2003a,b, 2006) and for other species by Davis (2010). The gibberellins A4+7 increase cell volume (Stern et al., 2007; Zhang et al., 2007; Davis, 2010) and contribute as well to increased epidermal cell density (Curry, 2012). Additional treatment that was shown to increase epidermal cell density included application of a synthetic auxin following two applications of low concentration of BA + GA4+7 (5 mg l−1 ) (Table 5). Auxin was shown to contribute to cell enlargement (Wismer et al., 1995) and although epidermal cell size was not monitored at the present study, this treatment resulted with increased rate of big size fruits, indicating cell enlargement activity in the fruit (Table 6). Interestingly, values for cuticle thickness, as measured from the base of the epidermal cell layer to the outer edge of the cuticle, remained the same at the tested period of 60 to 90 DAFB – although fruit expanded significantly – with no significant difference between control and PGR treated fruits. Knoche et al. (2011) suggest that GA4+7 had no effect on the amounts or rates of cutin or wax deposition, and that the decrease in russeting and formation of microcracks in the cuticle of GA4+7 -treated fruit must be accounted for effects on underlying epi- and hypodermal tissues. If dermal cell layers contributed to the apple skin mechanics (Khanal et al., 2013), increased cell division and cell size can maintain cell-to-cell adhesion under tensional stresses providing a stronger structural support of fruit surface. Whether PGRs treatments altered cuticular composition and modified the mechanical properties of the apple skin (Domínguez et al., 2011), remains to be clarified. In 2011, multiple spraying of BA + GA4+7 – 40 mg l−1 each applied as 0.2% SuperlonTM – increased epidermal cell density (Table 3). Actually, comparing the effect of each treatment during the experimental period indicated that the PGR treatment maintained high cell density along fruit development while in control cell density decreased (Table 2). It is notable that one spray at 60 DAFB was not sufficient to maintain the high cell density. In 2012, application of BA + GA4+7 at a lower concentration – 5 mg l−1 each, applied as 0.025% SuperlonTM – at the same fruit developmental stage (60, 75 and 90 DAFB) did not maintain the
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high epidermal cell density during fruit expansion as observed for the application of the higher concentration (40 mg l−1 ) that year and the year before (Tables 2 and 4), implying that BA + GA4+7 at a concentration of 5 mg l−1 was not as potent at that late stage. To compensate for the weak activity of the low concentration of BA + GA4+7 (5 mg l−1 ) the effect of its spraying was tested in earlier stages of fruit development, from 7 to 35 DAFB (growth phase I). These fruits were not used for the anatomical study, but were included in the evaluation of calyx-end cracking severity. The severity of the cracking disorder was monitored at the end of the growing season of 2012 and included all treatments as detailed in Table 1. Results suggested that all treatments (except 5 mg l−1 x1 at phase II) reduced the cracking incidence compared to control (Fig. 3). The best treatments were those that included three sequential treatments at 7, 21 and 35 DAFB—i.e., 5 mg l−1 x3 phase I, and 5 mg l−1 x6 phases I + II (Fig. 3). It is notable that the 5 mg l−1 x6 at phases I + II also resulted with increased fruit size (Table 6). Comparing fruit size and rate of cracking showed a correlation with increased fruit size due to the PGRs treatment, and reduced calyx-end cracking. An association of climate conditions and the incidence of calyxend cracking was suggested by the growers. Calyx-end cracking incidence in 2011 and 2012 was 14.3% and 61.7%, respectively. The former year was cooler than the latter—in 2012 there were 59 h with temperatures above 34 ◦ C (in June to August), compared to only 23 h in 2011. Accordingly, data that was collected from several orchards during 2008 to 2012 indicated of positive and strong association between the duration of high temperatures and the incidence of the disorder (R2 = 0.78; Fig. 4). 5. Conclusions Application of BA + GA4+7 to the developing fruit increase epidermal cell density at the calyx-end, probably enhancing the elasticity of the skin and reinforcing the cuticular membrane, consequentially reduce the susceptibility of the fruit to calyx-end cracking. Results suggest that when temperatures higher than average are expected to increase the incidence of calyx-end cracking, BA + GA4+7 can be applied to minimize the damage. If indications for hot climate will be detected at the beginning of the growth season, multiple treatments of 5 mg l−1 will be effective at the first phase of fruit developments, however if decision will be made at a later stage, a higher concentration of 40 mg l−1 should be applied. Acknowledgments This work was supported by the Fruit Grower Association, Israel, and is a contribution of ARO, the Volcani Center No. 107/2013).
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