Effects of pre-blossom temperatures on flower development and fruit set in apricot J. Rodrigo* and M. Herrero Unidad de Fruticultura, SIA - DGA, Campus de Aula Dei Apdo. 727, 50080 ZARAGOZA, SPAIN Tel: 34-976 716320/716316 Fax: 34-976 716335 e-mail: [email protected] * Corresponding author 1 Abstract The influence of pre-blossom temperatures on flower development and fruit set is ascertained in apricot (Prunus armeniaca L.), a species without previous records on the effect of pre-blossom temperature on fruit set, but that is particularly prone to erratic fruit set. A polyethylene cage was used during pre-blossom development of flower buds to increase maximum temperatures by 6-7ºC and mean temperatures by 3ºC in orchard conditions. This increase in temperature accelerated flower bud development, caused a hastening in flowering time and, following hand-pollination, reduced fruit set. At anthesis, flowers that had developed in warmer conditions weighed less and showed less development of the pistil than control flowers. Pistil growth of flowers under warm conditions did not differ from that of control flowers when both populations were compared on a real time scale, in spite of the fact that warmed buds were at an advanced external phenological stage. Thus, hastening of external floral development by warm pre-blossom temperatures was not accompanied by advance in pistil development. This lack of synchrony resulted in premature flowering of flowers with underdeveloped pistils that had a reduced capability to set fruit. The results are discussed in terms of flower quality and its implications in fruit set and subsequent crop load. Keywords: Apricot, Prunus armeniaca, flower development, fruit set, pistil, anthesis, temperature. 2 1. Introduction Irregularity of yield is one of the main problems in fruit production (Tromp, 1986). Year-to-year variations in crop production have been traditionally related to weather conditions in spring. Thus, wind and low temperatures affect bee activity and therefore pollination (Dennis, 1979), and frosts can reduce the number of buds, flowers and fruits (Rodrigo, 2000). Likewise, rain or high relative humidity can cause pollination failures and promote floral disease during bloom (Gradziel and Weinbaum, 1999), and temperature in the days following anthesis affects pollen tube growth and the effective pollination period (Williams, 1970). An effort to integrate all these variables influencing crop production in a model system based on multiple regression analysis (Beattie and Folley, 1977) showed that while meteorological variables at flowering time had an effect on the subsequent apple fruit production, surprisingly pre-blossom temperatures had also a clear effect on yield. These results were later further confirmed showing a negative correlation between crop load and warm pre- blossom temperatures in apple (Beattie and Folley, 1978; Jackson and Hamer, 1980; Jackson et al., 1983). Conversely, cold pre-blossom temperatures have been correlated to high yield in pear (Browning and Miller, 1992). While these mathematical models based on natural orchard conditions establish a clear correlation between pre-blossom temperatures and yield, the mechanisms leading to this relationship remain obscure. Several attempts have been made to evaluate the effects of pre-blossom temperatures on fruit set. This has been achieved mainly through the use of potted trees, but results are not always clear. Experiments on potted apple trees subjected under controlled pre-blossom temperatures have shown that fruit set is enhanced at low temperatures and reduced 3 at high temperatures (Abbott 1971; Jackson et al., 1983; Miller et al., 1986). Likewise, in pear and apple, low temperatures appeared to promote fruit set on potted trees exposed to different temperature regimes from February to harvest (Tromp and Borsboom, 1994). However, other experiments in apple have shown no significant differences in fruit set (Abbott, 1962; Miller et al., 1987) or even that low pre-blossom temperature reduces final fruit set (Tromp, 1986). In Prunus species, information is also scarce and contradictory. While high pre-blossom temperatures reduced fruit set on sweet cherry potted trees (Beppu et al., 1997), increased temperature of bagged branches did not affect the percentage of fruit set in almond (Egea and Burgos, 1995). These apparently contradictory results may be related to the different treatment conditions in the different experiments. Thus, depending on the experiments, temperature has been increased during the day (Beppu et al., 1997), overnight (Jackson et al., 1983; Miller et al., 1986, 1987) or continuously, either under constant temperature (Abbott, 1971) or with a day/night regime (Tromp, 1986; Tromp and Borsboom, 1994). Likewise, the duration of the treatments have been different. Temperature has been applied during three months before bloom (Jackson et al., 1983; Miller et al., 1986, 1987), from dormancy to petal fall (Abbott, 1971), from one month before anthesis to petal fall (Beppu et al., 1997), or from dormancy to harvest (Tromp, 1986; Tromp and Borsboom, 1994). Furthermore, the use of young potted trees further complicates the situation since it cannot entirely reflect the behaviour of adult trees in orchard conditions (Sedgley and Griffin, 1989). As an alternative, temperature conditions can be modified in whole trees by means of plastic-covered trees in the orchard. This 4 approach has been used to evaluate the effects of temperature and irrigation on fruit growth (Atkinson et al., 1998), but has not been used widely for pre-blossom temperature modification in orchard conditions. We have used a mobile greenhouse adapted to an adult tree in the orchard, similar to those used to protect valuable hybridizations from freeze injury and insect contamination to test whether elevated pre-blossom temperatures reduce fruit set. This approach increases both mean and maximum temperature, the two parameters shown to affect subsequent fruit set in previous analyses (Beattie and Folley, 1978; Jackson and Hamer, 1980). This work has been done in apricot (Prunus armeniaca L.), a species without previous records on the effect of pre-blossom temperature on fruit set, but which is particularly prone to erratic fruit set (Mehlenbacher et al., 1990). The aim of this work is to evaluate the effect of pre-blossom warm temperatures on flower bud development, flower morphology and subsequent fruit set. 5 2. Material and methods 2.1. Plant material and temperature monitoring The experiments were performed in two consecutive years, 1999 and 2000, on different trees from an eight-year-old orchard of the apricot cultivar 'Moniqui', which is particularly prone to erratic fruit set (Rodrigo and Herrero, 1996). When most flower buds presented separation of bud scales and an initial protrusion of sepals, a tree was enclosed within a metallic structure covered by a 0.178 mm thick polyethylene film in order to increase the daytime temperature. Another tree at the same phenological stage was left uncaged as control. Since ventilation was needed to prevent excess heat accumulation and moisture condensation, the cage was partially opened daily during daylight. Temperature measurements inside and outside the cage were logged at 5 min intervals using shaded data loggers (Testostor 175-3, Testo, Germany) placed at 60 cm high from soil level and orientated to the north. At full bloom, when 50% of the flower buds were opened, the cage was dismantled and flowering and subsequent fruit set proceeded under natural orchard conditions. 2.2. Flower development To follow flower bud development, several branches completing over 1500 flower buds per tree were monitored. To characterize the progression of bud phenophases, counts of flower buds at each phenological stage were made every two days until full bloom. Assessments were made using a previously adjusted scale, in which stage values are linearly related to apricot flower bud development: 1.6 (separation of bud scales); 3.0 (protrusion of sepals); 4.2 (broadening of exposed 6 sepals); 4.9 (expansion and rounding of sepals); 5.5 (initial protrusion of petals); 5.9 (expansion and rounding of petals); 6.1 (anthesis); 7.4 (abscission of petals) (Austin et al., 1998). Linear regressions were performed in both treatments to fit functions of chronological time through the adjusted phenophase data. Slopes were compared and tested to determine if they were significantly different. In both years, 30 flowers were collected every two days from the outset of the experiment up to anthesis (240 flower buds in all) and preserved in FAA [formalin - acetic acid - ethanol 70%, (1:1:18, v/v/v)] for further analysis. Subsequently, from anthesis onwards, a total of 275 pistils, 47 of them at anthesis and 12 per day thereafter over 19 days, were collected in each treatment and preserved in FAA. To follow pistil growth from anthesis onwards fresh pistils were individually weighed prior to fixation. Since differences were apparent between treatments, pistil size was also measured in the pistils previously preserved in FAA from the outset of the experiment, when bud scales started to separate. For this purpose, pistils were separated from other bud or flower structures and observed under a Wild Heerbrugg M8 binocular microscope. The images were collected using a Cohu 8310 RGB Colour Camera attached to the binocular microscope and processed using a Quantiment 570 Image Analysis System (Leica Cambridge, England). Pistil size values were obtained by measuring the surface of the image corresponding to the ovary and the length of the style. On the day of anthesis, individual weights of several flower structures were also obtained separately, these were petals, sepals and stamens, anthers, pistil and peduncles. This was done on 47 flowers per treatment and year. Since no significant differences were found between both years, data from both years were pooled 7 together. Data were subjected to analysis of variance. 2.3. Pollination and fruit set Since differences in flower development were apparent quite early in the experiment and the two treatments flowered at different times, to avoid the influence of different pollination conditions in the orchard at full bloom, controlled pollinations were carried out by hand in both treatments. For this purpose, pollen from 'Canino', a cultivar compatible with 'Moniqui' (Rodrigo and Herrero, 1996), was obtained from flowers at the balloon stage by removing the anthers and placing them on paper at room temperature. Pollen was sieved 24 h later through a 0.26-mm mesh and frozen at -20ºC until required. Seven branches were tagged to complete over 1000 flower buds per tree. Receptive flowers were hand pollinated every other day until all flowers were opened. Since the cages were dismantled at full bloom, the first flowers opened were hand-pollinated already in the cage. To ascertain the final fruit set, in tagged branches weekly counts of flowers and developing fruits were made at anthesis and harvest. Fruit set was expressed as the percentage of fruits per total flowers (Williams, 1970). Finally, fruits of both treatments were weighed at harvest to determine the crop of the tagged branches. 8 3. Results The polyethylene cage induced a mean increase in the maximum temperature of 7.6ºC in 1999 and 6ºC in 2000. Minimum temperature was slightly altered and reduced 1.5ºC under the cage in 1999 and 0.4ºC in 2000. This resulted in an increase in the mean temperature of 3ºC in 1999 and 2.7ºC in 2000 (Table 1). This increase in temperature was achieved while the pattern of diurnal variation was maintained through the treatment period, increasing from 8:00 to 15:00 hours and decreasing gradually to 19:00 hours (Figure 1). This pattern was consistent over the two years. The influence of pre-blossom temperature on flower bud development was significantly different in both treatments. Regression slopes, and therefore rates of growth, differed significantly between warm treatment and the control (P<0.01) (Figure 2). Thus, warm temperature regime accelerated blooming time in both years. In spite to the fact that flowering time differed between the warm treatment and the control, no big differences in temperature were recorded in the 10 d following anthesis in each treatment. Thus, both treatments had similar mean temperature (9.6ºC in 1999 and 9.7ºC in 2000) during this time. A comparison of pistil growth at four phenological stages showed that although pistils in both treatments followed the same pattern of growth in the first days of the experiment (from separation of bud scales to expansion and rounding of sepals), differences in ovary size (Figure 3a) and style length (Figure 3b) were found in the subsequent flower bud stages in which both parameters were smaller under warm treatment. Moreover, at anthesis, a proportion of the flowers, in the warm treatment, showed small pistils. While on the control treatment most of the flowers (92%) had a morphologically well developed pistil, in the warm treatment 33% of the flowers 9 presented pistils not completely developed and 13% of the flowers showed short styles and unswelled ovaries (Figure 4). Thus, number of normal pistils in the warm treatment (54%) were significantly lower than in the control (χ2 = 136, 2 df, P<0.001). However, a daily observation and weighing of these pistils revealed that this proportion of underdeveloped pistils were not arrested in development but, on the contrary, were gradually completing their development, since the proportion of underdeveloped pistils gradually disappeared during the 5 days following anthesis, while no flower drop could be recorded during this time. These differences in pistil development were reflected in clear differences in flower weight at anthesis. While control flowers averaged 169 ± 3 mg, flowers from the warm treatment averaged approximately one third less (114 ± 3 mg). To evaluate whether these differences were entirely due to differences in pistil weight or other structures were involved, several flower parts (peduncle and receptacle, petals, sepals and filaments, anthers and pistil) from individual flowers were separately weighed. All the structures were significantly heavier in control than in warmed flowers (Table 2). The differences in pistil weight were maintained during the days following anthesis in which control pistils experimented a larger growth than warmed pistils (Figure 5). However, when pistil development from the outset of the experiment was plotted in real dates instead of related to anthesis, the pattern of growth was coincident in both treatments and there were not significant differences in ovary size (Figure 6a). Style length at anthesis was shorter in the warm treatment 10.9 ± 0.6 mm than in the control 16.8 ± 0.5 mm. These differences were maintained when style development was plotted in real dates, resulting in that warm treatment flowers attained a final shorted style (Figure 6b). 10