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Early flower initiation allows ample manipulation of flowering time in cherimoya (Annona cherimola Mill.)
Scientia Horticulturae 121 (2009) 327–332
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Early flower initiation allows ample manipulation of flowering time in cherimoya (Annona cherimola Mill.) L. Soler, J. Cuevas * Dpt. Crop Production, University of Almerı´a, La Can˜ada de San Urbano s/n, 04120 Almerı´a, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2008 Received in revised form 3 February 2009 Accepted 4 February 2009
Flower initiation date and readiness to flowering in buds of different age were studied in ‘Fino de Jete’ cherimoya (Annona cherimola) cultivar in order to establish the limits for the manipulation of its flowering date. Flower initiation was analyzed by light and scanning electron microscopy (SEM) collecting axillary buds from May to the following February, whereas the bud readiness to produce perfect flowers was determined by forcing buds of different age to sprout by means of leaf removal and tipping the new growth. SEM images confirm that cherimoya buds are differentiated into flowers almost a year before blooming. In this regard, axillary buds have already formed the sepals when the subtending leaf has just begun unfolding (week 0), while the petals are clearly visible in 1-week-old buds. Sectioning of paraffin-embedded buds illustrate that cherimoya buds are in fact a bud complex that 1 week after its inception comprises 4–5 buds of different size of which the two largest ones are reproductive, while the 2–3 smallest buds often remain undifferentiated at that time. The high capacity of flowering expressed by young buds that have been forced to grow proves that cherimoya meristems are early competent for flowering. No differences in fertility or in the time needed to reach anthesis after leaf removal were found among buds of different ages. Node position had no effect on bud break and flowering potential. The early flower initiation in cherimoya deduced from this work opens a wide temporal window for the experimental manipulation of flowering and harvest dates in this crop. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Annona cherimola Flower initiation Bud complex Defoliation Out of season crop
1. Introduction Spain is the world’s leading producer of cherimoya with a production of 22,451 t in 3218 ha (M.A.P.A., 2006). The commonest cherimoya cultivar, ‘Fino de Jete’, occupies 90% of the acreage and most of its crop is harvested in just a 2-month period (October– November). This fact and the high perishability of cherimoyas limit the period of marketing and threaten the future of its cultivation in Spain. Extended marketing by off season fruit production alleviates market oversupply and increases profits. In a few tropical fruit trees, this target has been reached by the modification of flowering dates (Menzel, 1983; Davenport, 2003; Manochai et al., 2005). This is the case of cherimoya and other annonas in which out of season flowering can be promoted by manipulating vegetative flushes (George and Nissen, 1987; Kavati and Piza, 1997; Soler and Cuevas, 2008). Cherimoya flowers develop laterally at leaf scars of 1-year-old shoots and in basal nodes of the current season’s growth (Hopping, 1982; Gardiazabal and Rosenberg, 1994; Gonza´lez and Cuevas,
* Corresponding author. Tel.: +34 950 015 559; fax: +34 950 015 939. E-mail address: [email protected] (J. Cuevas). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.02.005
2007). Late flowers may also be formed to a lesser extent in distal nodes of the current season’s growth (George and Nissen, 1988; Higuchi and Utsunomiya, 1999). Cherimoyas are remarkable in the sense that their axillary buds become enclosed below the leaf petiole soon after their inception (Schroeder, 1970). Hence, the presence of the leaf inhibits bud break. Natural leaf drop occurs after winter, bringing spring bud bursting and flowering. However, hand and chemical leaf removal can be used to relieve subpetiolar buds from this paradormancy (i.e. dormancy imposed on the bud by a different plant organ; in this case, the leaf) allowing out of season flowering and harvesting (Magdahl, 1989; Soler et al., 2002; Soler and Cuevas, 2008). The success of this technique depends, however, on achieving defoliation when the meristems of the subpetiolar buds have evolved into flowers, since a premature execution of defoliation may lead to the immediate promotion of vegetative growth (Cautı´n and Razeto, 1999). On atemoya, a hybrid between A. cherimola A. squamosa, successful leaf removal is performed in summer on lignified or semi-lignified shoots (George and Nissen, 1987; Lemos et al., 2003). Unfortunately, the information available about the date of flower initiation (i.e. the first morphological change which can be detected in a meristem evolving into a flower) in cherimoya is very scarce. Fouad et al. (1999) affirm that flower initiation occurs after
328
L. Soler, J. Cuevas / Scientia Horticulturae 121 (2009) 327–332
˜ a (1992) suggests, winter, only 3 months before bud bursting. Acun on the contrary, that flower initiation in cherimoya occurs the previous season to bloom since the flowers are already differentiated under the leaf, when the trees enter winter dormancy. Higuchi and Utsunomiya (1999) are more precise when they affirm that the flowers formed in the basal nodes of the new growth are differentiated a year prior to anthesis, whereas the flowers formed in the distal nodes of the current season’s growth differentiate in synchronization with shoot growth, about 5 weeks before their anthesis. Regardless of the conditions leading to flower formation, the anatomy of cherimoya axillary bud has not yet been studied in detail. The present investigation has two major aims: to document when flower initiation occurs in cherimoya and to determine bud readiness to produce perfect flowers after eliminating the paradormancy imposed by the presence of the subtending leaf. These aims have been achieved by a combination of microscopic observations of young buds and by field analyses of flowering response of buds forced to grow by hand defoliation and tipping. 2. Materials and methods
Fig. 1. Leaf removal followed by tipping of new growth leaves the subpetiolar bud exposed (arrow).
2.1. Plant material The experiments were carried out in 2004 using 8-year-old ‘Fino de Jete’ trees grown in a private orchard located in Motril, Southern Spain (lat. 368450 N, long. 38270 W). The trees were openvase-trained and spaced at a distance of 7 m 4 m. The orchard was irrigated (6000 m3 per ha) and fertilized (NK 150–150 UF) following standard practices. 2.2. Flower initiation and development To determine flower initiation date in cherimoya, 40 buds from the fifth node of the current season’s growth were collected from six ‘Fino de Jete’ trees at different time intervals. The first collection was carried out the day on which the subtending leaf had just unfolded. This date is hereafter referred to as week 0. Successive samples were taken after 1, 2, 4, 8, 16, 32 and 48 weeks. These buds were used for light microscopy and scanning electron microscopy (SEM) observations. For SEM studies, the buds were fixed in 2.5% glutaraldehyde in 0.04 M phosphate buffer (pH 7.2) and stored at 4 8C. When the bud was young and tender, the leaf stalk was not removed before fixation to avoid damage to the meristems. After fixation, the buds were dissected under a binocular microscope, dehydrated through a graded ethanol series, and then critical point dried, mounted on stubs and coated with gold in a Sputter Coater Bal-Tec SCD 005. The samples were observed in a Hitachi S-3500N SEM model at an acceleration voltage of 10 kV. For light microscopy, 1- and 4-week-old buds were fixed in FAA (formalin:glacial acetic acid:70% ethanol at 1:1:18 ratio), dehydrated through a graded ethanol series and embedded in Paraplast. The samples were then cut into 20 mm thick sections, mounted on slides and stained with Gerlach solution (Gerlach, 1969) before observation. 2.3. Bud readiness to complete flower development after leaf removal and shoot tipping Two field experiments were carried out to confirm the capacity of the young buds to produce perfect flowers. The first experiment followed a completely randomized block design with two treatments. Treatments were leaf removal plus tipping and control without intervention. In the first treatment, hand leaf removal was made on June 18th in 20 shoots per tree when the young leaf of the fifth node was just unfolding (week 0). Leaf removal was
immediately followed by the tipping of the new growth above the fifth node leaving the subpetiolar bud exposed (Fig. 1). Bud break and flowering percentages were recorded three and 6 weeks after and compared with control shoots. Four trees acted as blocks and replications. The number of flowers per node in response to the intervention was also counted and the time to reach anthesis recorded. In a second test, we explored the influence of bud age on flower production. To do so, we tagged the nodes in which the leaf was just unfolding in 100 randomly selected shoots per tree in four trees (replications). Subsequently, hand defoliation and tipping was achieved as above on June 28th (week 0) and after 1, 2, 4 and 8 weeks, in a set of 20 shoots per date. Bud bursting and flowering levels were recorded after three and 6 weeks and the response to the different dates of intervention compared by variance analysis using Statgraphics Plus 5.1 software (Manugistics, Rockville, MD). The experiment followed a completely randomized block design with five treatments (dates) in which each tree acted as block and replication. 3. Results 3.1. Flower initiation and development Flower initiation in cherimoya (Annona cherimola Mill.) cv. ‘Fino de Jete’ occurred even before the subtending leaf had completely expanded. SEM observations clearly show that axillary buds have already evolved into flowers when the subtending leaves have begun unfolding (week 0). At this time, the subpetiolar buds were partially covered by two bracts below which the enlargement of the floral dome had occurred and the incipient formation of the three sepals had started (Fig. 2A and B). Flower development progressed rapidly and sepals and petals could be clearly distinguished in 1-week-old buds (Fig. 2C). Several independent buds were recognized below the leaf petiole in 1-week-old axils and afterwards, although only the largest bud could be dissected in most samples processed by SEM due to technical hitches. During the following weeks, the most apparent change was an increase in the size of the main bud, whose sepals and outer petals were greatly enlarged (Fig. 2D and E). The inner petals were hardly visible due to their reduced size (Fig. 2F). After this time, a pause in floral organ development seemed to be established and not until week 16, an incipient ring of stamens started to be distinguished at
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Fig. 2. Cherimoya flower initiation under scanning electron microscopy. Axillary bud 1 week before the subtending leaves unfold (A); at leaf unfolding (week 0) (B); buds 1 (C), 2 (D), 4 (E), 32 (F) and 48 weeks (G–H) after leaf unfolding. Vm: vegetative meristem; B: bract; S sepal; P: outer petal; St: stamen; IP: inner petal; C: carpel. Bar A, B and F = 100 mm; C, D, E, G and H = 500 mm.
the base of the floral dome (Fig. 2F). Progress slowed down and no visible changes in the floral organs could be perceived afterwards. The development of individual carpel could still not be recognized on buds sampled 32 weeks after their inception. The formation of the carpels was, nonetheless, evident 48 weeks after bud formation (Fig. 2G and H). At this time, most of the leaves had abscised and bud bursting was taking place.
Paraffin bud sectioning confirms SEM observations and enlightens the compound nature of cherimoya subpetiolar buds. Sectioning revealed that cherimoya buds are in fact a bud complex containing 4–5 buds of decreasing size, each one enclosed by two brown hairy bracts. The inner bud, closer to the stalk, was of larger size and showed a clear advancement in development with respect to the others; therefore it can be
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labelled as the primary bud. The second one in size is referred to as the secondary bud. Confirming previous SEM observations, 1-week-old primary buds showed the initiation of sepal and petals, while most secondary buds have only initiated the sepals (Fig. 3A). Two or three undifferentiated buds (accessory buds), of decreasing size along the axis, usually followed (Fig. 3B). The first change indicative of flower initiation observed in the sectioning of 1week-old buds was the elevation of a narrow pedicel above which a floral receptacle is being formed. Above the bracts, a slender pedicel with a single bracteole continued (see arrow in Fig. 3C). In 4-week-old samples, five independent buds were often recognized in the bud complex. The primary and secondary buds have formed 1 or 2 flowers, while the others had no trace of flower formation. The third bud commonly adopted a narrow shape indicative of a vegetative condition; while the smallest buds showed an undifferentiated stage that did not allow them to be labelled as either vegetative or reproductive (Fig. 3D). The mixed nature of reproductive cherimoya buds is confirmed by the common
observation of a second structure (floral or vegetative) in primary and secondary buds (Fig. 3E and F). 3.2. Bud readiness to complete flower development after leaf removal and shoot tipping Leaf removal plus shoot tipping on June 18th, 9–10 months before natural leaf drop, was highly successful in releasing young subpetiolar buds (week 0) from paradormancy. Bud bursting occurred rapidly and no significant differences appeared between the results evaluated three and 6 weeks after leaf removal. Therefore, from now on the results here presented will correspond to the final observations carried out 6 weeks after intervention. The axillary buds of the fifth node sprouted in a high proportion (69 12%) after the intervention, and bloomed profusely (0.87 0.19 flowers per node; 1.20 0.07 flowers per node taking into consideration only the buds that sprouted). Many reproductive buds also formed leaves, confirming the mixed nature of the primary bud. On average floral meristems took 42 days to reach anthesis after
Fig. 3. Bud complex under light microscopy 1 (A–C and E–F) and 4 (D) weeks after leaf unfolding: (A) primary and secondary buds with sepals and petals initiated. (B) Bud complex with two reproductive and two undifferentiated buds. (C) Bud complex showing the elevation of the floral dome over a narrow pedicle. The arrow marks a single bracteole. (D) Bud complex 4 weeks after leaf unfolding with five independent buds of decreasing size. (E–F) One-week-old bud complex showing two flowers (arrows) in the primary bud (18).
L. Soler, J. Cuevas / Scientia Horticulturae 121 (2009) 327–332
Fig. 4. Bud bursting and flowering percentages and number of flowers per axillary bud complex after leaf removal and tipping at different dates after leaf unfolding.
Fig. 5. Bud bursting and flowering percentages and number of flowers per node in forced buds according to node position (nodes are numbered from the base to the apex).
leaf detachment. No axillary bud sprouting or blooming came about in control shoots. In the second trial, most buds burst and developed flowers in response to hand defoliation and shoot tipping regardless of their age. The percentage of sprouting buds was between 87 and 97% (p = 0.82), while the percentage of them forming flowers was between 82 and 92%, depending on their age. The average number of flowers formed was between 0.86 and 1.08 per node with slight variations among buds of different age (Fig. 4). No differences occurred as a function of bud age neither in the percentage of buds developing flowers (p = 0.70) nor in the amount of flowers per node (p = 0.71). No trend can be deduced from the values obtained by buds of different age in the range of 0–8-week-old buds (Fig. 4). This confirms that all buds are able to produce flowers regardless their age. Bud position within the range studied (buds from sixth to tenth node) had no effect on bud bursting (p = 0.11) and flowering (p = 0.55) (Fig. 5). 4. Discussion The first discernible change indicative of flower initiation could be detected in the buds of ‘Fino de Jete’ cherimoya as soon as the subtending leaf started to unfold. At this moment (week 0), the floral dome has enlarged and three sepals could be distinguished in the primary bud of the axillary bud complex. Actually, flower initiation could have occurred even earlier, since non-systematic observations of younger buds showed floral dome swelling in the primary bud. This result coincides with Higuchi and Utsunomiya (1999), who observed floral morphogenesis below 1-week-old leafstalks in ‘Big Sister’ cherimoya. The development of the floral
331
organs before bud break is, however, limited. We have observed sepals, petals and stamen initiation before sprouting, although the individual carpels could not be recognized in 32-week-old ‘Fino de Jete’ buds. Fouad et al. (1999) identified the sepals and the petals in dormant buds of ‘Hindy’, ‘Balady’ and ‘Abd-El-Razik’ cherimoyas, but the development of stamens and carpels were not detected before bud bursting. SEM observation and paraffin sectioning have shown the complex nature of the cherimoya bud. In agreement with Higuchi and Utsunomiya (1999), the cherimoya bud complex contains up to five single independents buds in 4-week-old samples. Cautı´n and Razeto (1999) interpret, on the contrary, that 8-week-old axillary buds of cherimoya contain only one meristem, because most often only one shoot emerges from the leaf scar. This assumption is wrong because most nodes enclose below the leaf petiole no less than two mixed buds with developing flowers, plus two–three undifferentiated meristems (Fig. 3). Certainly, however, on most occasions only one flower per node was initially formed after defoliation (Figs. 4 and 5). This circumstance suggests a secondary role for the accompanying meristems. Up to six meristems arranged in a vertical series at each node have also been found in atemoya where the same secondary role is assigned to the small meristems of the bud complex (Marler and Crane, 1994). In this hybrid, only the selective removal of the primary shoot allows the accessory meristems to sprout (Marler and Crane, 1994). Higuchi and Utsunomiya (1999) conclude that flowers formed at the basal nodes of the new growth differentiated almost a year before anthesis, while the late flowers formed at the apical nodes of the new growth differentiated in the new season just a few weeks before anthesis. We fully agree with these conclusions. In fact, according to our observations, the solitary flowers that develop at the leaf scar and the basal flowers of the new growth share the same origin, the primary mixed bud and they have the same flower initiation date (almost a year before bloom). Apical flowers studied by Higuchi and Utsunomiya (1999) could not be observed in our dormant buds because they were not yet formed at the time of sampling. However, it should be stressed that although basal and apical flowers are both formed in the new shoot growth, they are initiated at different times, exposed to different stimuli, and they respond differently to environmental constraints (Higuchi and Utsunomiya, 1999). Finally, our results prove that the paradormancy exerted on axillary buds can be successfully removed by defoliation as soon as the new leaves emerge. Shoot tipping is, nonetheless, needed for the accomplishment of this intervention (Cautı´n and Razeto, 1999; Soler and Cuevas, 2008). Contrary to our expectations, no differences in fertility exist among buds of different age, demonstrating an early competence of young buds for flowering. Likewise, no differences seems to occur among buds located at different nodes of the shoot, providing therefore the opportunity to tip shoots of different length and nonetheless produce a second set of flowers in the new growth. In summary, early flower initiation in cherimoya allows a second bloom as soon as on July, 10–11 months before ordinary blooming dates. Year round cherimoya production is, therefore, only limited by environmental factors affecting pollination and fruit development. Previous results have demonstrated the possibility of producing cherimoya during winter and spring in addition to the common autumn crop (Soler et al., 2002; Soler and Cuevas, 2008). The success of pollination during cool winter months is under study to further extend to summer the period of cherimoya commercialisation. Acknowledgement We are indebted to Esmeralda Urea who was a great help with SEM images.
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References ˜ a, P.E., 1992. Aproximacio´n al periodo de diferenciacio´n floral y descripcio´n de Acun yemas de chirimoyo (Annona cherimola Mill.) cv. Concha Lisa para la zona de Quillota. Universidad Cato´lica de Valparaı´so, Chile, 76 pp. Cautı´n, R., Razeto, B., 1999. Evaluacio´n del comportamiento de yemas de chirimoyo sometidos a tratamientos de floracio´n forzada. In: II Congreso Internacional de Anona´ceas, Chiapas, pp. 127–133. Davenport, T.L., 2003. Management of flowering in three tropical and subtropical fruit tree species. HortScience 38, 1331–1335. Gardiazabal, F., Rosenberg, G., 1994. El cultivo del chirimoyo. Universidad de Valparaı´so, Chile, 145 pp. Fouad, M.M., Mohamed, S.M., Ghonaim, I.E., 1999. Effect of chemical defoliants on leaf defoliation and flower bud differentiation of some Annona cherimola cultivars. Bull. Fac. Agric. Cairo Univ. 50, 245–264. George, A.P., Nissen, R.J., 1987. Effects of cincturing, defoliation and summer pruning on vegetative growth and flowering of custard apple (Annona cherimola x Annona squamosa) in subtropical Queensland. Aust. J. Exp. Agric. 27, 915–918. George, A.P., Nissen, R.J., 1988. The effects of temperature, vapour pressure deficit and soil moisture stress on growth, flowering and fruit set of custard apple (Annona cherimola X Annona squamosa) ‘African Pride’. Sci. Hortic. 34, 183–191. Gerlach, D., 1969. A rapid safranin.crystal violet-light green staining sequence for paraffin sections of plant material. Stain Technol. 44, 210–211. Gonza´lez, M., Cuevas, J., 2007. Optimal crop load and positioning of fruit in cherimoya (Annona cherimola Mill.) trees. Sci. Hortic. 115, 129–134. Higuchi, H., Utsunomiya, N., 1999. Floral differentiation and development in cherimoya Annona cherimola Mill.) under warm (30/25 8C) and cool (20/ 15 8C) day/night temperatures. J. Jpn. Soc. Hortic. Sci. 68, 707–716.
Hopping, M.E., 1982. Pollination and fruit set of cherimoya. The Orchadist of New Zealand, March, 56–58. Kavati, R., Piza Jr., C. de T., 1997. Formac¸ao e manejo do pomar de fruta-do-conde, atemoya e cherimoia. In: Sao Jose´, A.R.,Souza, I.V.B.,Morais, O.M.,Rebouc¸as, T.N.H. (Eds.), Anona´ceas: produc¸ao e mercado. Vito´ria da Conquista-BA, pp. 75–83. Lemos, E.E.P., Marhino, G.A., Almeida, M.C., 2003. Efeito da desfolha de ramos sobre a induc¸ao de brotos e flores em atemoia (Annona cherimola Mill. x Annona squamosa L.). Rev. Bras. Frutic. Jaboticabal 25, 170–171. Magdahl, C., 1989. Efecto de la defoliacio´n anticipada sobre la brotacio´n, floracio´n y desarrollo de frutos en chirimoyo (Annona cherimola Mill.) cv. Concha Lisa y efectividad de algunos productos como defoliantes. Tesis. Universidad Cato´lica de Valparaı´so. Chile, 103 pp. M.A.P.A., 2006. Anuario de estadı´stica agroalimentaria. Ministerio de Agricultura y Pesca, Madrid, Spain, 973 pp. Marler, T.E., Crane, J.H., 1994. Increasing scaffold branch angle of atemoya by manipulation of the primary complex. HortScience 29, 818–820. Manochai, P., Sruamsiri, P., Wiriya-alongkorn, W., Naphrom, D., Hegele, M., Bangerth, F., 2005. Year around off season flower induction in longan (Dimocarpus longan, Lour.) trees by KCLO3 applications: potentials and problems. Sci. Hortic. 104, 379–390. Menzel, C.M., 1983. The control of floral initiation in lychee: a review. Sci. Hortic. 21, 201–215. Schroeder, C.A., 1970. Fruits of the annonas. Calif. Avocado Soc. Yearbook 54, 117–118. Soler, L., Cuevas, J., 2008. Development of a new technique to produce winter cherimoyas. HortTechnology 18, 24–28. Soler, L., Mateos, J., Ruiz, J., 2002. Estrategias para la produccio´n de chirimoyos en primavera. In: III Congreso Internacional de Anona´ceas, Quillota, Chile, p. 15.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Early flower initiation allows ample manipulation of flowering time in cherimoya (Annona cherimola Mill.) L. Soler, J. Cuevas * Dpt. Crop Production, University of Almerı´a, La Can˜ada de San Urbano s/n, 04120 Almerı´a, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2008 Received in revised form 3 February 2009 Accepted 4 February 2009
Flower initiation date and readiness to flowering in buds of different age were studied in ‘Fino de Jete’ cherimoya (Annona cherimola) cultivar in order to establish the limits for the manipulation of its flowering date. Flower initiation was analyzed by light and scanning electron microscopy (SEM) collecting axillary buds from May to the following February, whereas the bud readiness to produce perfect flowers was determined by forcing buds of different age to sprout by means of leaf removal and tipping the new growth. SEM images confirm that cherimoya buds are differentiated into flowers almost a year before blooming. In this regard, axillary buds have already formed the sepals when the subtending leaf has just begun unfolding (week 0), while the petals are clearly visible in 1-week-old buds. Sectioning of paraffin-embedded buds illustrate that cherimoya buds are in fact a bud complex that 1 week after its inception comprises 4–5 buds of different size of which the two largest ones are reproductive, while the 2–3 smallest buds often remain undifferentiated at that time. The high capacity of flowering expressed by young buds that have been forced to grow proves that cherimoya meristems are early competent for flowering. No differences in fertility or in the time needed to reach anthesis after leaf removal were found among buds of different ages. Node position had no effect on bud break and flowering potential. The early flower initiation in cherimoya deduced from this work opens a wide temporal window for the experimental manipulation of flowering and harvest dates in this crop. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Annona cherimola Flower initiation Bud complex Defoliation Out of season crop
1. Introduction Spain is the world’s leading producer of cherimoya with a production of 22,451 t in 3218 ha (M.A.P.A., 2006). The commonest cherimoya cultivar, ‘Fino de Jete’, occupies 90% of the acreage and most of its crop is harvested in just a 2-month period (October– November). This fact and the high perishability of cherimoyas limit the period of marketing and threaten the future of its cultivation in Spain. Extended marketing by off season fruit production alleviates market oversupply and increases profits. In a few tropical fruit trees, this target has been reached by the modification of flowering dates (Menzel, 1983; Davenport, 2003; Manochai et al., 2005). This is the case of cherimoya and other annonas in which out of season flowering can be promoted by manipulating vegetative flushes (George and Nissen, 1987; Kavati and Piza, 1997; Soler and Cuevas, 2008). Cherimoya flowers develop laterally at leaf scars of 1-year-old shoots and in basal nodes of the current season’s growth (Hopping, 1982; Gardiazabal and Rosenberg, 1994; Gonza´lez and Cuevas,
* Corresponding author. Tel.: +34 950 015 559; fax: +34 950 015 939. E-mail address: [email protected] (J. Cuevas). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.02.005
2007). Late flowers may also be formed to a lesser extent in distal nodes of the current season’s growth (George and Nissen, 1988; Higuchi and Utsunomiya, 1999). Cherimoyas are remarkable in the sense that their axillary buds become enclosed below the leaf petiole soon after their inception (Schroeder, 1970). Hence, the presence of the leaf inhibits bud break. Natural leaf drop occurs after winter, bringing spring bud bursting and flowering. However, hand and chemical leaf removal can be used to relieve subpetiolar buds from this paradormancy (i.e. dormancy imposed on the bud by a different plant organ; in this case, the leaf) allowing out of season flowering and harvesting (Magdahl, 1989; Soler et al., 2002; Soler and Cuevas, 2008). The success of this technique depends, however, on achieving defoliation when the meristems of the subpetiolar buds have evolved into flowers, since a premature execution of defoliation may lead to the immediate promotion of vegetative growth (Cautı´n and Razeto, 1999). On atemoya, a hybrid between A. cherimola A. squamosa, successful leaf removal is performed in summer on lignified or semi-lignified shoots (George and Nissen, 1987; Lemos et al., 2003). Unfortunately, the information available about the date of flower initiation (i.e. the first morphological change which can be detected in a meristem evolving into a flower) in cherimoya is very scarce. Fouad et al. (1999) affirm that flower initiation occurs after
328
L. Soler, J. Cuevas / Scientia Horticulturae 121 (2009) 327–332
˜ a (1992) suggests, winter, only 3 months before bud bursting. Acun on the contrary, that flower initiation in cherimoya occurs the previous season to bloom since the flowers are already differentiated under the leaf, when the trees enter winter dormancy. Higuchi and Utsunomiya (1999) are more precise when they affirm that the flowers formed in the basal nodes of the new growth are differentiated a year prior to anthesis, whereas the flowers formed in the distal nodes of the current season’s growth differentiate in synchronization with shoot growth, about 5 weeks before their anthesis. Regardless of the conditions leading to flower formation, the anatomy of cherimoya axillary bud has not yet been studied in detail. The present investigation has two major aims: to document when flower initiation occurs in cherimoya and to determine bud readiness to produce perfect flowers after eliminating the paradormancy imposed by the presence of the subtending leaf. These aims have been achieved by a combination of microscopic observations of young buds and by field analyses of flowering response of buds forced to grow by hand defoliation and tipping. 2. Materials and methods
Fig. 1. Leaf removal followed by tipping of new growth leaves the subpetiolar bud exposed (arrow).
2.1. Plant material The experiments were carried out in 2004 using 8-year-old ‘Fino de Jete’ trees grown in a private orchard located in Motril, Southern Spain (lat. 368450 N, long. 38270 W). The trees were openvase-trained and spaced at a distance of 7 m 4 m. The orchard was irrigated (6000 m3 per ha) and fertilized (NK 150–150 UF) following standard practices. 2.2. Flower initiation and development To determine flower initiation date in cherimoya, 40 buds from the fifth node of the current season’s growth were collected from six ‘Fino de Jete’ trees at different time intervals. The first collection was carried out the day on which the subtending leaf had just unfolded. This date is hereafter referred to as week 0. Successive samples were taken after 1, 2, 4, 8, 16, 32 and 48 weeks. These buds were used for light microscopy and scanning electron microscopy (SEM) observations. For SEM studies, the buds were fixed in 2.5% glutaraldehyde in 0.04 M phosphate buffer (pH 7.2) and stored at 4 8C. When the bud was young and tender, the leaf stalk was not removed before fixation to avoid damage to the meristems. After fixation, the buds were dissected under a binocular microscope, dehydrated through a graded ethanol series, and then critical point dried, mounted on stubs and coated with gold in a Sputter Coater Bal-Tec SCD 005. The samples were observed in a Hitachi S-3500N SEM model at an acceleration voltage of 10 kV. For light microscopy, 1- and 4-week-old buds were fixed in FAA (formalin:glacial acetic acid:70% ethanol at 1:1:18 ratio), dehydrated through a graded ethanol series and embedded in Paraplast. The samples were then cut into 20 mm thick sections, mounted on slides and stained with Gerlach solution (Gerlach, 1969) before observation. 2.3. Bud readiness to complete flower development after leaf removal and shoot tipping Two field experiments were carried out to confirm the capacity of the young buds to produce perfect flowers. The first experiment followed a completely randomized block design with two treatments. Treatments were leaf removal plus tipping and control without intervention. In the first treatment, hand leaf removal was made on June 18th in 20 shoots per tree when the young leaf of the fifth node was just unfolding (week 0). Leaf removal was
immediately followed by the tipping of the new growth above the fifth node leaving the subpetiolar bud exposed (Fig. 1). Bud break and flowering percentages were recorded three and 6 weeks after and compared with control shoots. Four trees acted as blocks and replications. The number of flowers per node in response to the intervention was also counted and the time to reach anthesis recorded. In a second test, we explored the influence of bud age on flower production. To do so, we tagged the nodes in which the leaf was just unfolding in 100 randomly selected shoots per tree in four trees (replications). Subsequently, hand defoliation and tipping was achieved as above on June 28th (week 0) and after 1, 2, 4 and 8 weeks, in a set of 20 shoots per date. Bud bursting and flowering levels were recorded after three and 6 weeks and the response to the different dates of intervention compared by variance analysis using Statgraphics Plus 5.1 software (Manugistics, Rockville, MD). The experiment followed a completely randomized block design with five treatments (dates) in which each tree acted as block and replication. 3. Results 3.1. Flower initiation and development Flower initiation in cherimoya (Annona cherimola Mill.) cv. ‘Fino de Jete’ occurred even before the subtending leaf had completely expanded. SEM observations clearly show that axillary buds have already evolved into flowers when the subtending leaves have begun unfolding (week 0). At this time, the subpetiolar buds were partially covered by two bracts below which the enlargement of the floral dome had occurred and the incipient formation of the three sepals had started (Fig. 2A and B). Flower development progressed rapidly and sepals and petals could be clearly distinguished in 1-week-old buds (Fig. 2C). Several independent buds were recognized below the leaf petiole in 1-week-old axils and afterwards, although only the largest bud could be dissected in most samples processed by SEM due to technical hitches. During the following weeks, the most apparent change was an increase in the size of the main bud, whose sepals and outer petals were greatly enlarged (Fig. 2D and E). The inner petals were hardly visible due to their reduced size (Fig. 2F). After this time, a pause in floral organ development seemed to be established and not until week 16, an incipient ring of stamens started to be distinguished at
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Fig. 2. Cherimoya flower initiation under scanning electron microscopy. Axillary bud 1 week before the subtending leaves unfold (A); at leaf unfolding (week 0) (B); buds 1 (C), 2 (D), 4 (E), 32 (F) and 48 weeks (G–H) after leaf unfolding. Vm: vegetative meristem; B: bract; S sepal; P: outer petal; St: stamen; IP: inner petal; C: carpel. Bar A, B and F = 100 mm; C, D, E, G and H = 500 mm.
the base of the floral dome (Fig. 2F). Progress slowed down and no visible changes in the floral organs could be perceived afterwards. The development of individual carpel could still not be recognized on buds sampled 32 weeks after their inception. The formation of the carpels was, nonetheless, evident 48 weeks after bud formation (Fig. 2G and H). At this time, most of the leaves had abscised and bud bursting was taking place.
Paraffin bud sectioning confirms SEM observations and enlightens the compound nature of cherimoya subpetiolar buds. Sectioning revealed that cherimoya buds are in fact a bud complex containing 4–5 buds of decreasing size, each one enclosed by two brown hairy bracts. The inner bud, closer to the stalk, was of larger size and showed a clear advancement in development with respect to the others; therefore it can be
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labelled as the primary bud. The second one in size is referred to as the secondary bud. Confirming previous SEM observations, 1-week-old primary buds showed the initiation of sepal and petals, while most secondary buds have only initiated the sepals (Fig. 3A). Two or three undifferentiated buds (accessory buds), of decreasing size along the axis, usually followed (Fig. 3B). The first change indicative of flower initiation observed in the sectioning of 1week-old buds was the elevation of a narrow pedicel above which a floral receptacle is being formed. Above the bracts, a slender pedicel with a single bracteole continued (see arrow in Fig. 3C). In 4-week-old samples, five independent buds were often recognized in the bud complex. The primary and secondary buds have formed 1 or 2 flowers, while the others had no trace of flower formation. The third bud commonly adopted a narrow shape indicative of a vegetative condition; while the smallest buds showed an undifferentiated stage that did not allow them to be labelled as either vegetative or reproductive (Fig. 3D). The mixed nature of reproductive cherimoya buds is confirmed by the common
observation of a second structure (floral or vegetative) in primary and secondary buds (Fig. 3E and F). 3.2. Bud readiness to complete flower development after leaf removal and shoot tipping Leaf removal plus shoot tipping on June 18th, 9–10 months before natural leaf drop, was highly successful in releasing young subpetiolar buds (week 0) from paradormancy. Bud bursting occurred rapidly and no significant differences appeared between the results evaluated three and 6 weeks after leaf removal. Therefore, from now on the results here presented will correspond to the final observations carried out 6 weeks after intervention. The axillary buds of the fifth node sprouted in a high proportion (69 12%) after the intervention, and bloomed profusely (0.87 0.19 flowers per node; 1.20 0.07 flowers per node taking into consideration only the buds that sprouted). Many reproductive buds also formed leaves, confirming the mixed nature of the primary bud. On average floral meristems took 42 days to reach anthesis after
Fig. 3. Bud complex under light microscopy 1 (A–C and E–F) and 4 (D) weeks after leaf unfolding: (A) primary and secondary buds with sepals and petals initiated. (B) Bud complex with two reproductive and two undifferentiated buds. (C) Bud complex showing the elevation of the floral dome over a narrow pedicle. The arrow marks a single bracteole. (D) Bud complex 4 weeks after leaf unfolding with five independent buds of decreasing size. (E–F) One-week-old bud complex showing two flowers (arrows) in the primary bud (18).
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Fig. 4. Bud bursting and flowering percentages and number of flowers per axillary bud complex after leaf removal and tipping at different dates after leaf unfolding.
Fig. 5. Bud bursting and flowering percentages and number of flowers per node in forced buds according to node position (nodes are numbered from the base to the apex).
leaf detachment. No axillary bud sprouting or blooming came about in control shoots. In the second trial, most buds burst and developed flowers in response to hand defoliation and shoot tipping regardless of their age. The percentage of sprouting buds was between 87 and 97% (p = 0.82), while the percentage of them forming flowers was between 82 and 92%, depending on their age. The average number of flowers formed was between 0.86 and 1.08 per node with slight variations among buds of different age (Fig. 4). No differences occurred as a function of bud age neither in the percentage of buds developing flowers (p = 0.70) nor in the amount of flowers per node (p = 0.71). No trend can be deduced from the values obtained by buds of different age in the range of 0–8-week-old buds (Fig. 4). This confirms that all buds are able to produce flowers regardless their age. Bud position within the range studied (buds from sixth to tenth node) had no effect on bud bursting (p = 0.11) and flowering (p = 0.55) (Fig. 5). 4. Discussion The first discernible change indicative of flower initiation could be detected in the buds of ‘Fino de Jete’ cherimoya as soon as the subtending leaf started to unfold. At this moment (week 0), the floral dome has enlarged and three sepals could be distinguished in the primary bud of the axillary bud complex. Actually, flower initiation could have occurred even earlier, since non-systematic observations of younger buds showed floral dome swelling in the primary bud. This result coincides with Higuchi and Utsunomiya (1999), who observed floral morphogenesis below 1-week-old leafstalks in ‘Big Sister’ cherimoya. The development of the floral
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organs before bud break is, however, limited. We have observed sepals, petals and stamen initiation before sprouting, although the individual carpels could not be recognized in 32-week-old ‘Fino de Jete’ buds. Fouad et al. (1999) identified the sepals and the petals in dormant buds of ‘Hindy’, ‘Balady’ and ‘Abd-El-Razik’ cherimoyas, but the development of stamens and carpels were not detected before bud bursting. SEM observation and paraffin sectioning have shown the complex nature of the cherimoya bud. In agreement with Higuchi and Utsunomiya (1999), the cherimoya bud complex contains up to five single independents buds in 4-week-old samples. Cautı´n and Razeto (1999) interpret, on the contrary, that 8-week-old axillary buds of cherimoya contain only one meristem, because most often only one shoot emerges from the leaf scar. This assumption is wrong because most nodes enclose below the leaf petiole no less than two mixed buds with developing flowers, plus two–three undifferentiated meristems (Fig. 3). Certainly, however, on most occasions only one flower per node was initially formed after defoliation (Figs. 4 and 5). This circumstance suggests a secondary role for the accompanying meristems. Up to six meristems arranged in a vertical series at each node have also been found in atemoya where the same secondary role is assigned to the small meristems of the bud complex (Marler and Crane, 1994). In this hybrid, only the selective removal of the primary shoot allows the accessory meristems to sprout (Marler and Crane, 1994). Higuchi and Utsunomiya (1999) conclude that flowers formed at the basal nodes of the new growth differentiated almost a year before anthesis, while the late flowers formed at the apical nodes of the new growth differentiated in the new season just a few weeks before anthesis. We fully agree with these conclusions. In fact, according to our observations, the solitary flowers that develop at the leaf scar and the basal flowers of the new growth share the same origin, the primary mixed bud and they have the same flower initiation date (almost a year before bloom). Apical flowers studied by Higuchi and Utsunomiya (1999) could not be observed in our dormant buds because they were not yet formed at the time of sampling. However, it should be stressed that although basal and apical flowers are both formed in the new shoot growth, they are initiated at different times, exposed to different stimuli, and they respond differently to environmental constraints (Higuchi and Utsunomiya, 1999). Finally, our results prove that the paradormancy exerted on axillary buds can be successfully removed by defoliation as soon as the new leaves emerge. Shoot tipping is, nonetheless, needed for the accomplishment of this intervention (Cautı´n and Razeto, 1999; Soler and Cuevas, 2008). Contrary to our expectations, no differences in fertility exist among buds of different age, demonstrating an early competence of young buds for flowering. Likewise, no differences seems to occur among buds located at different nodes of the shoot, providing therefore the opportunity to tip shoots of different length and nonetheless produce a second set of flowers in the new growth. In summary, early flower initiation in cherimoya allows a second bloom as soon as on July, 10–11 months before ordinary blooming dates. Year round cherimoya production is, therefore, only limited by environmental factors affecting pollination and fruit development. Previous results have demonstrated the possibility of producing cherimoya during winter and spring in addition to the common autumn crop (Soler et al., 2002; Soler and Cuevas, 2008). The success of pollination during cool winter months is under study to further extend to summer the period of cherimoya commercialisation. Acknowledgement We are indebted to Esmeralda Urea who was a great help with SEM images.
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