Can “ Caricia ” and “ Princesa ” apples be considered low-chilling cultivars ?

The purpose of this work was to study the response of two apple cultivars bred for low chilling environments to artificial chilling accumulation. Two trials were carried out; in experiment one, excised shoots were randomly taken from “Caricia” and “Princesa”, and in experiment two, intact and excised shoots of “Caricia”, “Princesa” and “Gala” (control) were collected. After collection, both shoot types were exposed to artificial chilling accumulation (4.0 ± 0.5°C) from 0 to 1200 chill units (CU). Bud break of mixed buds of “Caricia” and “Princesa” was higher than 50% between 0 CU to 1200 CU, irrespective of shoot type. Bud break of “Gala” mixed buds exceeded 50% only in intact shoots after accumulating 900 CU. The mean time to bud break of “Caricia” and “Princesa” diminished with increasing chilling accumulation and stabilized after ~600 CU, depending on the type of shoot and the year of experimentation. The low-chill apple cultivars tested in this work showed shallow dormancy, but they required moderate cold accumulation (800 – 1150 CU) to fully satisfy their chilling requirements. Thus, although their shallow dormancy makes them suitable for cultivation in chill-deficient environments, they cannot be considered low-chill cultivars.

One of the major challenges of temperate-zone fruit production in warm-winter areas is to overcome the dormancy period (Erez, 2001).Dormancy has been defined as the inability to initiate growth from meristems or other organs and cells, with the capacity to resume growth under favorable conditions (Rohde, & Bhalerao, 2007).In natural conditions, dormancy release and growth resumption in apple and other temperate deciduous fruit trees is mediated by a quantitative accumulation of chilling (Alburquerque, García-Montiel, Carrillo, & Burgos, 2008;Gariglio, Weber, Castro, & Micheloud, 2012;Hauagge & Cummins, 1991c;Oukabli & Mahhou, 2007;Rahemi & Pakkish, 2009).However, the chilling requirement is highly variable, depending on the genotype (Hauagge & Cummins, 2001), the environmental conditions in autumn (Heide, 2003) and the type of bud (apical versus lateral) (Erez, 2001).
Low-chill apple cultivars cultivated in mildwinter areas frequently show symptoms of a lack of chilling.Such symptoms comprise low bud break, erratic and delayed flowering, low seed set under open pollination and a shift to self-fertility (Castro et al., 2016;Erez, 2001;Mohamed, 2008).Consequently, chemical dormancy-breakers are usually applied in these mild-winter areas to promote the growth and flowering of low-chill apple cultivars (Botelho, & Müller, 2007;Erez, 2001;Mohamed, 2008;Njuguna et al., 2004).
Knowledge of the behavior of the apical and lateral buds under a wide range of chilling accumulation conditions is necessary to predict their performance in warm-winter regions.This knowledge is essential in the global warming context that will affect the chilling availability in warm regions (Luedeling, Girvetz, Semenov, & Brown, 2011).
Hence, the aim of this work was to determine the chilling requirements of two low-chill apple cultivars ("Caricia" and "Princesa").We hypothesized that the chilling requirements of these low-chill apple buds (cv."Caricia" and "Princesa") reside within the range of 0 to 600 CU.We expected that within this range of cold accumulation, a stabilization of the mean time to bud break (MTB) and bud break over 50% after adequate forcing would be observed.

Material and methods
The experiments were carried out in the experimental field of the Facultad de Ciencias Agrarias of the Universidad Nacional del Litoral (CECIF), Santa Fé, Argentina (31° 26' S; 60° 56' W.; 40 m above sea level) during two years, 2011 and 2014.
In May (end of autumn in the southern hemisphere) of 2011, one-year-old shoots without apical mixed buds were randomly collected from 20 plants of each cultivar.The chilling that had accumulated before the collection date was 10 chilling hours below 7ºC.The remaining leaves were removed, and the collected shoots were cut into pieces 15 cm long.Their basal and nonlignified apical portions were discarded.In the resulting excised shoots, the uppermost buds (near the wound) were eliminated.Thus, only the three central mixed buds were left on every excised shoot.Then, the excised shoots were treated with carbendazim [methylbenzimidazol-2-ylcarbamate] (2 mL L -1 ) for 10 minutes and allowed to dry naturally on absorbent paper.
Five groups of 40 excised shoots per cultivar were placed in plastic bags and exposed to artificial low temperature (4.0 ± 0.5°C) in a cold chamber to simulate five chilling accumulation treatments: 0, 300, 600, 900 and 1200 chilling hours (CH).According to the Utah Model, one hour at 4ºC is equivalent to 1 Chilling Unit (Richardson, Seeley, & Walker, 1974).Thus, the chilling accumulation treatments were expressed as chilling units.All shoots were placed in darkness and horizontally within the cold chamber until cold treatment was finished.
After cold treatment, the shoots from each treatment were divided into 8 groups of 5 shoots each.Each of these groups was placed in a 250-cm 3 plastic container, with their basal tip in a sodium hypochlorite solution (1:1000 v/v), and kept for 30 days in a growth chamber at 25 ± 0.5°C, with a 16hour photoperiod, and 50 μmol m -2 s -1 light intensity to force bud break.The basal tips of the shoots were cut weekly, and water was replaced daily.
The bud break times of the mixed buds were recorded every other day.Bud break occurrence was defined as the time when the mixed buds reached stage 53 on the pome fruit BBCH scale (Meier, 2001).The bud break percentage (BP) and the mean time to bud break of the mixed buds (MTB) were obtained using the following equations: where j is the number of excised shoots per experimental unit (j = 5), B i is the i-th bud in the j-th excised shoot broke within the forcing period, and n is the number of buds per excised shoot (n = 3).
where j is the number of excised shoots per experimental unit (j = 5) and Ti is the time from the beginning of the forcing period at 25 ± 0.5ºC to the occurrence of budbreak of the i-th bud in the j-th excised shoot and n is the number of buds per excised shoot (n = 3).We considered the MTB to have stabilized when an inflection point was observed above which the reduction in its value was less than one day (e.g.; the MTB value tended to a limiting value).
The experimental model was a 2×5 factorial and completely randomized design (CRD) with two cultivars and five chilling accumulation treatments.The experimental unit was a container with 5 excised shoots.Each experimental unit was repeated eight times (n = 40 containers and 200 excised shoots per cultivar).
The data were analysed with general linear models (GLM) adjusted with the lme function of the nlme package (Pinheiro, Bates, DebRoy, & Sarkar, 2011) of the R statistical language (R Development Core Team, 2011) using the InfoStat interface (Di Rienzo et al., 2012).The results from the GLM indicated the significance of a cultivar × chilling treatment effect for MTB; hence, regression analysis was performed.To test the effect of each cultivar within a chilling treatment interval, data for the cultivars within the chilling interval were pooled, and cultivar was included as a dummy variable in the regression model.The linear, quadratic and cubic components of chilling treatment were included in this analysis.The selection of variables was made, and the best model, chosen by the backward elimination procedure of InfoStat (Di Rienzo et al., 2012).
Normality and homoscedasticity were tested graphically with a Q-Q plot, and a plot of residuals vs. predictors, respectively).
In May (end of autumn in the southern hemisphere) of 2014, one-year-old shoots with (intact shoots) and without (excised shoots) apical buds were randomly collected from 20 plants of each variety.No chilling accumulation had been recorded before the collection date.The remaining leaves were removed.All shoots were cut into pieces 15 cm long; the basal and non-lignified apical portions of excised shoots (without apical buds) were discarded.Furthermore, in the resulting excised shoots, the uppermost buds (near the wound) were eliminated.Thus, only the three central mixed buds were left on each excised shoot.
However, in the intact shoots (with apical bud), only the basal portion was removed.Intact shoots were used in addition to excised shoots to compare the effect of chilling accumulation on lateral versus apical mixed buds.Thus, in the intact shoots, only the apical and three central mixed buds were left.
The experimental conditions, sample handling procedures and variables analysed were the same as in Experiment 1.
The experimental model was a 3×2×5 factorial CRD with three cultivars, two types of shoots and five levels of chilling accumulation.The experimental unit was a container with 5 excised shoots.Each experimental unit was repeated eight times (n = 80 containers and 400 shoots per cultivar).The data were analysed using GLMs as in experiment 1.As interactions on MTB and BP were detected, data for each cultivar and shoot type within the chilling interval were pooled.Thus, "cultivar" and "shoot type" effects were included as dummy variables in the regression model.In this analysis, the linear, quadratic and cubic components of chilling treatment and dummy variables were included.Variables were selected, and the best model, chosen by a backward elimination procedure using InfoStat (Di Rienzo et al., 2012).
To compare the season effect on the chilling response of "Caricia" and "Princesa" mixed buds, data of excised shoots of both experiments (2011 and 2014) were analyzed together in a 2×2×5 factorial in a CRD (two seasons, two cultivars and five levels of cold accumulation).The data were analysed using GLM, as previously described.The MTB and BP response was modelled by regression analysis using "cultivar" and "season" as dummy variables.As in the previous regression analysis, variables were selected, and the best model, chosen by a backward elimination procedure using InfoStat (Di Rienzo et al., 2012).

Results and discussion
In experiment 1, the MTB of lateral mixed buds decreased significantly with increasing chilling accumulation, but the response differed between the varieties, with a highly significant interaction between   *CU 3 ).The excis nt 1. r 2 = 0.59).to chilling n both apple ely 800 CU re 1A).The = 0.10), but t differences t, "Caricia" to chilling the MTB of 0 to 1200 cised shoots same range The bud affected both (p = 0.001) The BP w (Figure 1B).slightly grea cultivars sho range betwee The fact t with respect trait of those to warm-win low-chill gen more related degree days (Hauagge, & 2003)    chilling acc 0.01; r 2 = "Caricia" M cubic comp well as the were statist (Figure 4A) "Princesa" 4E    Cummins, 1991b;Oukabli, & Mahhou, 2007).Following the above discussion, we consider the MTB to have stabilized when an inflection point was observed on the chilling accumulation curve, above which the reduction in its value was less than one day (e.g., the MTB value tended to a limiting value).Using this criterion, we found that the chilling requirement of apical and lateral buds of "Caricia" and "Princesa" was over 600 CU in both experiments, which refutes our research hypothesis.This value represents almost twice the requirement cited for these cultivars (Denardi et al., 1988;Hauagge, & Tsuneta, 1999).Using this same metric, the chilling requirement of apical and lateral buds of "Gala" was over 1200 CU.
It is important to mention that some methodological factors can affect the results.On excised shoots, the cut performed on the shoots can itself promote bud break (wound effect) (Naor, Flaishman, Stern, Moshe, & Erez, 2003).Additionally, the absence of the apical buds, which may dominate lateral buds (Cook, & Jacobs, 1999), can increase the percentage of bud break on the lateral buds of excised shoots.Neither Naor et al. (2003) or Cook, and Jacobs (1999) evaluated the wound effect or the absence of the apical bud on the MTB response or any other variable that expresses the time needed for buds to break (e.g., T50) in their experiments.Furthermore, according to Hauagge and Cummins (1991a), the low-chill apple cultivars never enter into deep endo-dormancy, so low temperatures may not be required to promote bud break in excised shoots.Nevertheless, our data demonstrate that up to 800-1150 CU of chilling accumulation reduces the heat requirement and accelerates the bud break on "Caricia" and "Princesa" apples, with additional chilling having minimal effects.
Despite showing some disadvantages, the methodology used in our work has been suitable for evaluating the chilling requirement or dormancy progression in many fruit species, both under artificial chilling accumulation (Mohamed, 2003;Putti et al., 2003;Rahemi, & Pakkish, 2009) and under natural chilling accumulation (Campoy et al., 2011;Dennis, 2003;Hauagge, & Cummins, 1991c;Mohamed, 2008;Oukabli, & Mahhou, 2007).Moreover, this methodology is one of the most suitable for evaluating chilling requirements because it is possible to control several key factors, such as light, thermal amplitude and temperature (Dennis, 2003).
Despite the above-discussed factors, lateral buds of excised shoots of the low-chill apple cultivars studied in our work showed a significant reduction in dormancy intensity (MTB) with increased chilling accumulation.Consequently, both apical and lateral buds of the tested cultivars showed shallow dormancy but required a moderate chilling accumulation (800-1150 CU) to stabilize their MTB value.The shallow dormancy of "Caricia" and "Princesa" makes them suitable for cultivation in areas with very low chilling availability (Denardi et al., 1988;Hauagge, & Cummins, 2001;Hauagge, & Tsuneta, 1999) despite the occurrence of symptoms of lack of chilling, such us a shift to selffertility and a wide flowering period (Castro et al., 2016).

Conclusion
The apple cultivars "Caricia" and "Princesa", bred for chill-deficient environments, showed shallow dormancy but required moderate cold accumulation (800-1150 CU) at 4ºC to fully satisfy their chilling requirements.These characteristics make these cultivars suitable for successful cultivation in mild-winter areas, but chill-deficiency symptoms may still be seen in some years without dormancy-breaking treatments."Caricia" and "Princesa" therefore cannot be considered low-chill cultivars.