The timing of oral development events is an

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1 Journal of Horticultural Science & Biotechnology (2002) 77 (4) 474±478 Flower differentiation and spur leaf area in almond By V. S. POLITO *, K. PINNEY, R. HEEREMA and S. A. WEINBAUM Department of Pomology, 1 Shields Avenue, University of California, Davis CA USA ( vspolito@ucdavis.edu) (Accepted 14 April 2002) SUMMARY In mature almond trees, owers are borne laterally and predominantly on spurs. The ower buds contain a single terminal ower and no leaves. We investigated variation in the timing of oral initiation and organogenesis among and within spurs on `Nonpareil' almond (Prunus dulcis [Mill.] D. A. Webb). Spurs were sampled twice during the latesummer period of ower initiation and differentiation. All lateral buds on individual spurs were dissected and the developmental status of the meristem was determined. Extensive variation in the progress of oral initiation and differentiation was evident among spurs. Spurs with high total leaf areas had more oral buds, and these buds underwent transition to owering and oral organogenesis earlier than buds on spurs with lower total leaf areas. Within spurs, there was no gradient in oral development relative to bud position on the spur. The results indicate that spurs function independently with regard to oral development events, and suggest that within spurs buds may function somewhat independently as well. The timing of oral development events is an important element in understanding the biology underlying crop-production practices in fruit- and nutcrop species (Sedgley and Grif n, 1989). Although ower development has been studied more or less widely in many important tree-crop species, the literature available for most of these is generally limited and does not address potential sources of variability in oral development in tree canopies. In research on phenology of oral differentiation, authors typically describe the onset of oral transition or oral organogenesis. Researchers frequently acknowledge the variation in timing of oral development within tree canopies, however, that variation is rarely described or documented. Comparisons of the phenology of oral development between and among cultivars within a species have been reported by Luza and Polito (1988) and Polito and Pinney (1997) for walnut, Juglans regia L., and Wetzstein and Sparks (1983) for pecan, Carya illinoinensis (Wangenh.) K. Koch. Differences in the timing of oral development among geographic locations may sometimes be inferred from cases in which researchers have examined the same species in different areas, such as the work on peach, Prunus persica L., conducted by Warriner et al. (1985), Bustamente-Garcia (1980) and Raseira and Moore (1986), although each of these investigations was conducted on different peach cultivars. Other work, such as that by Guimond et al. (1998) for sweet cherry (Prunus avium L. `Bing'), has focused on the effect of cultural manipulation on the timing of oral development. Almond, Prunus dulcis (Mill.) D. A. Webb, is particularly well suited to a study of variation in oral developmental phenology. Flower buds form on spurs that bear up to six lateral oral buds (Figure 1A, B). The spurs remain viable for 3±5 years (Weinbaum and Speigel-Roy, 1985). The ower buds contain no leaves *Author for correspondence. and only a single terminal ower. Thus, the stage of development within a bud can be determined precisely. Lamp et al. (2001) studied the timing of oral initiation and the onset of oral developmental stages in three almond cultivars growing in four locations. Their results documented the extensive variation that exists, not only among cultivars, locations and years, but within a single cultivar in a single location. They described eight stages of oral differentiation and found that during late summer to early autumn, when oral initiation and differentiation were underway, buds in all of the eight stages occurred within a single tree. In light of these results, we initiated the present work to identify and assess parameters that may contribute to the developmental variation within tree canopies that exists among potential ower buds. Speci cally, we studied the relationship between leaf area per spur (possibly indicative of photosynthate availability per spur) and bud position along the spur axis on the timing of oral initiation and the progress of oral organ differentiation. Studies of several other woody species have correlated the amount of owering per branch with leaf area per branch (Doust and Doust, 1988; Lovelock et al., 1999) but did not address temporal patterns of oral differentiation. MATERIALS AND METHODS Nonfruiting spurs were collected from `Nonpareil' almond trees growing in a well-managed, commercial orchard in Arbuckle, California (lat N, long W). Spurs were sampled on 23 August 2001 and 8 September On 23 August, 60 nonfruiting spurs representing the apparent range of leaf area per spur were collected. On 8 September, another 60 nonfruiting spurs exhibiting between one and six lateral buds (the range observed) were collected. At each sampling date we were careful to select only spurs in which no leaf abscission had occurred. Leaf area per 474

2 V. S. Polito, K. Pinney, R. Heerema and S. A. Weinbaum 475 Fig. 1 A, B. Almond spur shoots showing terminal vegetative bud and lateral ower buds. A. Spur with one lateral ower bud. B. Spur with four lateral ower buds. L, lateral ower bud; T, terminal vegetative bud. C, D. Shoot apices of lateral buds on almond spur shoots. C. Apex at transition to owering stage. D. Apex after oral transition with sepal and petal primordia initiated. Ap, apex; Br, bract; BS, bud scale; Pe, petal primordia; Se, sepal primordium. spur was determined for each of the 120 spurs collected using a Delta T Area Meter (Decagon, Pullman, Washington). Every lateral bud on each spur was dissected under a Wild 5A stereomicroscope with a 503 objective lens and 163 wide- eld ocular lenses. Bud position on the spur was recorded at the time of oral dissection, and the apical meristem of each bud was scored according to six developmental stages: vegetative (bud-scale producing), transition to oral condition (apparent as a broadening and elongation of the meristem, see Figure 1C), sepal primordia initiated, petal primordia initiated (Figure 1D), stamen primordia initiated and pistil primordium initiated. Working at the resolution of the stereo dissection microscope did not permit the ne distinction of transitional stages noted by Lamp et al. (2001), who used scanning electron microscopy, but these six stages were readily apparent at this level of resolution. A developmental index, based on the extent of bud apical meristem development, was used to score individual lateral buds. Buds containing only a vegetative terminal meristem had an index value of zero. Buds that had progressed beyond the vegetative state to or past the oral transition stage received one index point and one additional index point was assigned for each round of oral organogenesis (sepal, petal, stamen and pistil initiation) that occurred subsequent to oral initiation. Buds with necrotic apices were scored as ±1 index point. Spur developmental index (SDI) was calculated as the sum of the developmental indices of all lateral buds on the spur divided by the total number of lateral buds on the spur. SDI provides a measure of developmental progress for the buds on a given spur.

3 476 Almond spur development Table I Mean leaf areas for almond spurs having 0 to 6 lateral ower buds. Mean values with different lower case letters differ signi cantly (P<0.05, Student-Newman-Keuls method) No. of ower buds per spur Mean leaf area per spur (mm 2 ) a ab ab b c c c. 3 N RESULTS Among nonfruiting spurs, the number of oral buds per spur (Table I) and the relative development of the most advanced of those oral buds (Figure 2) exhibited a strong positive trend with spur leaf area. On 23 August, 30.4% of spurs that had one or more lateral buds had no lateral buds showing development beyond the vegetative condition (Figure 2A). By 8 September, the percentage of vegetative spurs had decreased by more than 50%, and only 11.7% of spurs had all buds in the vegetative state (Figure 2B). For both sampling dates, mean leaf areas for spurs in which buds had advanced beyond the vegetative stage were greater than that for those spurs Fig. 3 Relationship between spur development index (SDI) and corrected leaf area per bud (CLA) for almond spurs on 8 September SDI = CLA (r = 0.70, F = 56.7, P<0.0001). that had not yet undergone transition to owering. Among spurs collected on either sampling date, no viable lateral buds ± whether vegetative or oral ± were evident on spurs with mean leaf areas less than 1000.mm 2 (Figure 2), and those spurs appeared dead. Spurs with mean leaf areas between 1000.mm 2 and 1273.mm 2 appeared viable as of 8 September but supported only vegetative or transitional buds (Figure 2B). To establish the correlation between leaf area per spur and owering intensity (e.g. ower number per spur), we calculated a ``corrected'' leaf area by subtracting 1273.mm 2 from the mean leaf area per spur. Only leaf areas per spur in excess of 1273.mm 2 were associated with oral differentiation. Spurs carrying 4±6 oral buds had mean spur leaf areas greater than 3000.mm 2 which was signi cantly greater than mean leaf areas on spurs carrying three or fewer ower buds (Table I). When oral development (SDI) was plotted against corrected Fig. 2 Relationships between the stage of the most advanced bud on individual almond spurs and total leaf area per spur. A. Spurs sampled 23 August 2001 (n = 57). B. Spurs sampled 8 September 2001 (n = 60). Values in parentheses indicate number of cases. Error bars = 2 times the standard error of the mean. Different lower case letters indicate signi cant differences (P<0.05, Student-Newman-Keuls method). Cases without error bars are represented by only a single event. D, all buds on spur are dead; NB, no lateral buds present on spur; Pe, petal primordia; Pi, pistil primordia; Se, sepal primordia; St, stamen primordia; Tr, transitional apex; Ve, vegetative apex. Fig. 4 Mean spur leaf area, spur developmental index and number of oral buds for almond spur shoots on 8 September Error bars = 2 times the standard error of the mean.

4 V. S. Polito, K. Pinney, R. Heerema and S. A. Weinbaum 477 Table II Distribution of shoot apex differentiation for buds on almond spurs collected 23 August Bud position refers to relative position of the bud on the spur. N = 57 spurs Spurs with single bud All buds at same stage No. of spurs % of spurs % of spurs with differences Position of most advanced bud Distal Proximal Distal and proximal Mid-spur Position of Least Advanced Bud Distal Proximal Distal and proximal Mid-spur leaf area per bud, a strong positive correlation was apparent (Figure 3). Thus, spurs with relatively low leaf areas are more likely to remain completely vegetative, or, if they undergo transition to reproductive development, their development is delayed relative to buds on spurs with greater leaf areas (Figure 2B) and leaf areas per bud (Figure 3). Floral development, as indicated by SDI, tended to increase with leaf area per spur and ower buds per spur up to four buds per spur (Figure 4). Leaf area per spur did not differ signi cantly among spurs carrying 4±6 buds (Table I). To determine if there was a spatial gradient in oral differentiation along the spur axis, the stage of oral development and the relative position of each lateral oral bud on the spur were considered. There was no consistent relationship between stage of oral differentiation and bud position on the spur (Tables II, III). On 23 August, 36.8% of spurs had either no buds, a single bud or all buds at the same developmental stage. Thus, there were no developmental differences among buds on these spurs (Table II). On 8 September, buds of 38.3% of spurs did not differ (Table III). For spurs with multiple buds and bud apices at different developmental stages, the relative positions of the most and least advanced buds on the spur showed no consistent developmental gradient relative to bud position on the spur. On 23 August, the most avanced lateral bud was in the distal position for 17.5% of all spurs and in the proximal position for 14% of all spurs. This represents 40.0 and 32.0% of those spurs in which there were differences among the buds present. The least advanced lateral buds were in the distal position in 17.5% and in the proximal position in 21.1% of all spurs, or 40 and 48% of spurs with differences among buds. Similar results were evident for the 8 September sampling date when the most advanced lateral bud was in the distal position on 15% of all spurs (24.3% of spurs with differences) and in the proximal position of 25% of spurs (40.5% of spurs with differences), and the least developed lateral buds were in distal positions on 21.7% of all spurs (35.1% of those with differences), and in the proximal position in 18.3% of all spurs (29.7% of spurs with differences). For both samplng dates, there were several instances in which either the most developed or least developed buds were present at mid-spur bud positions, and several others where the distal and proximal bud position had attained the same stage of development (Tables II, III). DISCUSSION These results suggest a relatively high level of autonomy of almond spurs with regard to the ability of lateral buds to transition from the vegetative to the reproductive state and with the timing of that transition. Flower bud number has been correlated with leaf area per branch in several other woody species (Doust and Doust, 1988; Lovelock et al., 1999), but we are not aware of previous associations between leaf area per branch and the earliness or rate of oral initiation. It is not known whether the earliness of oral initiation persists throughout differentiation and affects the date of bloom, oral quality at bloom, the likelihood of fruit set, or the growth potential of the resulting fruit. The association between leaf area per spur and the accumulation of nonstructural carbohydrates in spurs prior to oral Table III Distribution of shoot apex differentiation for buds on almond spurs collected 8 September Bud position refers to relative position of the bud on the spur. N = 60 spurs Spurs with single bud All buds at same stage No. of spurs % of spurs % of spurs with differences Position of most advanced bud Distal Proximal Distal and proximal Mid-spur Position of least advanced bud Distal Proximal Distal and proximal Mid-spur

5 478 Almond spur development differentiation was not tested in this study. Carbohydrate accumulation has long been associated with owering in temperate-zone, woody plants (Chandler, 1957; Robbie and Atkinson, 1994), and treatments such as girdling, root restriction, and limb bending, etc., which increase carbohydrate levels in shoots, also tend to promote oral initiation (Goldschmidt et al., 1985 and pers. comm.; Jackson and Sweet, 1972). Similarly, effects of shading, and observations of ower distribution within tree canopies are all consistent with the involvement of photosynthesis, the accumulation of nonstructural carbohydrates and the apparent carbon autonomy of individual branches (Jackson and Sweet, 1972; Sprugel et al., 1991; and Watson and Casper, 1984). The correlation between leaf number per spur and ower number per spur was much weaker than was the correlation between ower number and leaf area per spur (data not presented). Thus, ower number appeared to be limited less by the number of axillary bud positions than by the functional leaf area at those nodes. Floral differentiation of individual buds on spurs did not vary considerably along the spur axis. Positional patterns of development along an axis are typically constant or nearly constant for a given developmental event. Such a pattern may be basipetal, acropetal or bidirectional. Development that is random with respect to position on an axis, such as is the case for buds within almond spurs, is much less common. The lack of positional gradient in the pattern of bud differentiation within shoots suggests that within-spurs, buds may be functioning independently. This raises the possibility that localized differences in carbohydrate availability within spur shoots, perhaps due to variations in leaf size along the spur or the speci city of vascular connections between leaves and speci c spurs, may affect the rate of oral initiation as well. Lamp et al. (2001) examined the phenology of almond ower initiation and differentiation for three cultivars in four locations spanning 48 latitude. An objective of that study was to develop a predictive model based on time, measured as calendar days or accumulated heat units, from bloom. They found, however, that extensive within-tree variation in timing of oral differentiation events precluded the modelling effort. The present results con rm the within-tree variation in oral development phenology and provide insights into localized physiological factors that may contribute to that variation. Analyses of oral induction in herbaceous plants that respond to photoperiodic stimuli have emphasized the important role of localized carbohydrate uxes in oral induction (Bernier et al., 1998). Furthermore, Corbesier et al. (1998) showed that photosynthesis and the availability of current photosynthate, rather than previously assimilated, non-structural carbohydrates (storage), is the primary source of the carbohydrate involved in oral induction. The relationship between spur leaf area and timing of oral initiation and development suggests that localized variations in photosynthate supply may in uence oral initiation in almond buds. Bernier, G., Corbesier, L., PeÂrilleux, C., Havelange, A. and Lejeune, P. (1998). Physiological analysis of the oral transition. In: Genetic and environmental manipulation of horticultural crops. (Cockshull, K. E., Gray, D., Seymour, G. G. and Thomas, B., Eds). CAB International, Wallingford, UK, 103±9. Bustamente-Garcia, M. A. (1980). In uence of different irrigation regimes on ower bud formation and development in peach trees. Thesis, Univ. of California, Davis, USA. Chandler, W. H. (1957). Deciduous orchards, 3rd ed. Lea and Febiger, Philadelphia. 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