Scientia Horticulturae 162 (2013) 188–203 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti Review Apple pollination: A review Fernando Ramírez a,∗ , Thomas Lee Davenport b a b Facultad de Ciencias Sociales, Universidad Colegio Mayor de Cundinamarca, Calle 28 No 5B-02, Bogotá, Colombia Director of Research and Development, Vivafresh Technologies, 1452 North Krome Avenue, Suite 101i, Florida City, FL 33034, United States a r t i c l e i n f o Article history: Received 16 May 2013 Received in revised form 31 July 2013 Accepted 8 August 2013 Keywords: Compatibility Rosaceae Pollen Self-pollination Cross-pollination a b s t r a c t Pollination is a key event for fruit set. Worldwide, there has been an increasing interest in apple pollination. Apple pollen grains are elliptical and tricolpate. Pollen germination is highly dependent on temperature. Most apple pollination occurs through cross-pollination; however, some cultivars have been reported to self-pollinate. Most apple cultivars have a gametophytic self incompatibility (GSI) system; however, others are semi compatible, or fully self compatible. The most common insect pollinator of apple is the honey bee. Other effective pollinator species include Hymenopterans, Dipterans and Coleopterans. Wind seems not to be an effective mechanism for pollination. Environmental conditions such as temperature, rain and high wind speed negatively affect pollination. This article reviews recent developments in our knowledge of apple pollination focusing on recently developed cultivars growing in the tropics. © 2013 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pollen morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pollen germination, fertilization and physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro pollen germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Irradiated pollen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Floral parts and ﬂowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross- and self-pollination, ﬂoral bloom and overlap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Self incompatibility, semi compatibility and compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outcrossing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insect pollination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Artiﬁcial pollen application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction The apple is the most ubiquitous of temperate fruits and has been cultivated in Europe and Asia from antiquity (Janick et al., 1996; Adachi et al., 2009). Orchards are now found from Siberia and northern China, where winter temperatures fall to −40 ◦ C, to high elevations in Colombia and Indonesia straddling the ∗ Corresponding author. Tel.: +57 13109409. E-mail addresses: [email protected], [email protected] (F. Ramírez), [email protected]ﬂ.edu (T.L. Davenport). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.08.007 188 189 189 191 191 191 195 196 196 197 199 199 199 200 200 equator, where two crops can be produced in a single year (Janick, 1974). The center of origin of apples is Asia (Forsline et al., 2003), particularly the Republic of Kazakhstan (Dzhangaliev, 2010). Most wild apple species are found in the mountains of central and inner Asia, western and southwestern China, the Far East, and Siberia (Ignatov and Bodishevskaya, 2011). Apple cultivation dates back to a few centuries B.C. to the Greeks and Romans. Greeks and Romans were growing apples at least 2500 years ago (Hancock et al., 2008). The Romans spread the apple across Europe during their invasions (Hancock et al., 2008). Its introduction to the Americas by European colonists began in the 16th and 17th centuries. Nowadays, apples are commercially produced in numerous countries and have great F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 economic importance. The accepted scientiﬁc name for apple is Malus × domestica Borkh. It is also named Malus domestica Borkh. The cultivated apple is likely the result of interspeciﬁc hybridization (Forsline et al., 2003). Its primary wild ancestor is Malus sieversii found from the Heavenly Mountains (Tien Shan) on the border between western China and the former Soviet Union to the edge of the Caspian sea (Forsline and Aldwinckle, 2004; Hancock et al., 2008). The number of species in the genus Malus is uncertain and still controversial (Pereira-Lorenzo et al., 2009). Harris et al. (2002) reported 55 species, Zhou (1999) reported 30–35 species, Robinson et al. (2001) 25–47 species, Janick et al. (1996) reported 37 and Forsline et al. (2003) reported 27 primary apple species. Apples have been introduced into temperate, subtropical and tropical environments worldwide. There are over 6000 regionally important cultivars and land races across the world, but a few major cultivars dominate worldwide. Pollination is a key event in plant reproduction, stimulating ovary growth and development. Pollination is the mechanical transfer of pollen from anthers to stigmas within a plant species and is a prerequisite to the fertilization of the ovules to initiate development of seeds and fruit. Successful pollination is an important event for apple diversiﬁcation among different countries for it is critical for dependable fruit production. The current article discusses apple pollination in a wide range of environments. It reviews available information on pollen morphology, germination, tube growth, compatibility related features, cross-pollination, self-pollination and insect pollination in tropical, subtropical and temperate climates. 2. Pollen morphology Anthesis is the opening of ﬂowers coupled with anther dehiscence and pollen grain release (Jackson, 2003). Pollen grains are dormant, resistant structures containing lipid reserves for germination and early growth but are quickly dehydrated after anther dehiscence and must absorb water to germinate when deposited on stigmas (Jackson, 2003). When dry, they are ellipsoidal and tricolpate (Fig. 1) with three germinal furrows extending almost the full length of the grain (Adams, 1916; Currie et al., 1997). They swell when wet and become more globular in shape (Adams, 1916). The exine, or outer, layer of the typical pollen grain has a striated pattern and sometimes bears small pores on its surface. Estimated average pollen length is about 40 m and width about 20 m (Currie et al., 1997). It is heavy and not readily carried by wind (Dennis, 2003). The quantity of pollen produced by a cultivar depends on its ﬂower production and the yield of pollen per ﬂower (Jackson, 2003). 3. Pollen germination, fertilization and physiology Pollen germination is the ﬁrst of a series of steps leading to subsequent ovule fertilization, fruit development and growth. It occurs soon after a pollen grain contacts the ﬂoral stigmatic surface (Fig. 2). The stigma has a wet surface composed of extracellular secretions from its papilla cells, which collapse after anthesis (Sedgley, 1990). The hydrated pollen grain germinates in the secretion pool on the stigmatic surface, and the emergent pollen tube begins to grow through the interstitial material of the transmitting tract (Jackson, 2003). ‘Recognition’ of incompatibility takes place here to select the most compatible pollen grain (Fig. 2) (Stösser et al., 1996; Jackson, 2003). Pollen germination on apple stigmas is mediated by a series of complex processes that involve proteins and other molecules. RNA, protein and polyamine concentrations within a pollen grain remain relatively unchanged before germination. After germination, they begin to decrease (Bagni et al., 1981). Mature pollen grains contain two generative nuclei and the tube cell nucleus. Once 189 compatible pollen grains are deposited on stigmas, germination proceeds with pollen tube elongation, each carrying a tube nucleus and two generative nuclei down each style into the ovaries (Dennis, 2003). Pollen tube growth is mediated by proteins, but many details remain to be fully elucidated. Some of these proteins interact with stylar glycoproteins to anchor the pollen tube to the pollen/stylar extracellular matrix (Di Sandro et al., 2010). An extracellular form of the calcium-dependent protein-crosslinking enzyme TGase (transglutaminase) is involved in the apical growth of Malus domestica pollen tube (Di Sandro et al., 2010). This protein possibly interacts with the pollen tube and style during fertilization (Di Sandro et al., 2010); yet, further research is required to fully elucidate the mechanisms of various proteins involved in pollen tube growth. Another group of molecules, polyamines, which are organic compounds having two or more amino or nitrogen containing groups are also necessary during pollen tube growth (Speranza and Calzoni, 1980; Bagni et al., 1981). Their role may be related to the structure and assembly of vegetative cell walls (Berta et al., 1997; Lenucci et al., 2005); however, the precise role of polyamines secreted from the germinating pollen tube and their interaction with the pollen/stylar extracellular matrix is also not completely understood (Di Sandro et al., 2010). After continuous tube cell elongation, they enter the micropyles (a small opening on the surface of each ovule) and penetrate where they rupture, releasing the two generative nuclei in each. One nucleus unites with the egg cell in each ovule to produce the diploid zygote and the other unites with the two polar nuclei in the embryo sac, producing a triploid nucleus. The resulting zygote passes through successive cell divisions that occur rapidly to produce the embryo. The triploid nuclei divide to form a nuclear-free, liquid endosperm (Dennis, 2003; Jackson, 2003). The rate of pollen germination is affected by temperature and varies with the source of pollen (Jackson, 2003). Percent germination of ‘Manchurian’ crabapple pollen and ‘Golden Delicious’ apples on the stigmatic surface of ‘Golden Delicious’ pistils increased with increasing temperature from 13 to 29 ◦ C in the ﬁrst 24 and 48 h after pollination, respectively (Yoder et al., 2009). Pollen germination is directly correlated with physiological temperatures in the 24 h following its deposition on stigmas (Williams and Maier, 1977), but higher temperatures are detrimental. Dry ‘Golden Delicious’ apple pollen subjected to a range of temperatures (40, 50, 60, 70, 80 or 90 ◦ C) at different time intervals (0, 1/6, 1/3, 2/3, 1, 2, 4, 8, 26, 24, or 48 h) displayed the lowest germination rates (18.7%) after 1/3 h at the highest temperature, 90 ◦ C (Marcucci et al., 1982). Pollen grains exposed to 50, 60, 70 and 80 ◦ C for 1 h resulted in 68.7; 70.3; 55. 4 and 47.9% germination, respectively and were reduced to 57.6; 11.5; and 0% (for both 70 ◦ C and 80 ◦ C) after 16 h. In cross pollination of ‘M.9’ with ‘Marubakaido’ in Brazil, pollen germination began on the stigma 12 h after pollination, and 83% germination of deposited pollen was observed after 24 h at 25 ◦ C (Dantas et al., 2002). Pollen tube growth rate also increases linearly with increasing temperatures from 0 to 40 ◦ C (Jefferies and Brain, 1984). The time necessary for pollen to reach the ovary is a measure of the effectiveness of pollination. Pollen tube growth typically takes two days (48 h) to reach the ovary under typical temperature conditions (Namikawa, 1923; Yoder et al., 2009). de Albuquerque et al. (2010a) evaluated pollen tube growth in 34 crosses between Brazilian apple cultivars. Tube growth was observed 120 h after pollen deposition on the stigma; however, these authors failed to provide information about the temperature at which tube growth occurred; Moreover, 50–100% of the pollen tubes reached the ovaries (in most of the studied cultivars), but low compatibility was found between ‘Imperatriz’ × ‘Daiane’ (16%). Pollen germination ranged from 59 to 73% in cultivars such as Princesa, Condessa, Eva, Baronesa, Fred Hough, Imperatriz, Daiane, Duquesa, Gala and Suprema (de Albuquerque et al., 2010a). The effective pollination period (EPP) is determined by the longevity of the egg apparatus 190 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Fig. 1. Air-dried apple pollen grains for apple sports of ‘Red Delicious’. (A) Aversang and (B) Ultrared. Reproduced with permission after Currie et al. (1997). (Williams, 1966). The duration of EPP is highly variable in apples with values ranging from two to nine days depending on cultivar (Sanzol and Herrero, 2001). For example, ‘Cox’, ‘Jonathan’ and ‘Laxton’s Superb’ apples have EEPs of 2.5, 5.5 and 6.5 days, respectively (Williams, 1966). Flowers on young apple trees tend to have shorter EPPs and higher proportions of immature and degenerate ovules than those on older wood and trees (Robbie and Atkinson, 1994). EPP was evaluated in ‘Golden Delicious’, ‘Redchief Delicious’ and ‘Golden Delicious Tardío,’ a regional mutant of Golden Delicious in Cuauhtémoc, Mexico located in the mountains at 28◦ 24 N; 106◦ 52 W, 2060 m above sea level and with a semi-arid, temperate climate consisting of 400–600 mm rainfall and a mean annual temperature of 12–18 ◦ C (Guerrero-Prieto et al., 2009). The duration of EPP was six days for ‘Redchief Delicious’, four days for ‘Golden Delicious’, and 10 days for ‘Golden Delicious Tardío’. The average ovule viability seemed to be a limiting factor for ‘Golden Delicious’, leading to a reduced initial fruit set (Guerrero-Prieto et al., 2009). The fertility of pollen varies greatly among apple cultivars. Early apple pollen germination studies demonstrated the great variability within many of the apple varieties investigated (Knight, 1917; Florin, 1927; Branscreidt, 1930). Apple pollen germination rates are high in diploid (2n) cultivars such as: Cox’s Pomona, Oranie, P. J. Bergius, Signe Tillisch, Savstaholm, Vitgylling, and Yellow Richard, which also have high viabilities (98–99%) (Florin, 1927; Kvaale, 1927). Whereas, pollen of the diploid ‘Cox’s Pomona’ had germination rates of 71–100%, other diploid cultivars, such as Björkvik and Cellini specimens I, II, and III had lower viabilities ranging from 59 to 72% (Florin, 1927; Kvaale, 1927). Overall, triploid cultivars Fig. 2. Pollen interactions and compatibility. (A) Pollen near the stigma, (B) pollen lands on stigma but is unable to germinate, (C and D) Pollen is able to germinate and reaches the ovary. F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 are inferior to diploids with respect to pollen germination rates and the low number of pollen grains per anther (Larsen and Tung, 1950). Early studies demonstrated that diploid and triploid cultivars consist of 34 and 51 chromosomes, respectively (Rybin, 1926; Darlington and Moffett, 1930; Howlett, 1931). Diploid pollen averages over 70% germination compared to <10% germination in pollen from 40 triploid apple cultivars (Stott, 1972). Pollen germination was more than 50% in diploids such as ‘Golden Delicious’, ‘Mantet’ and ‘Summerred’ (Visser and Verhaegh, 1980). These observations contrasts with those of the diploid ‘Priscilla’, the only cultivar to have less than 10% pollen germination in a study conducted in the Netherlands (Visser and Verhaegh, 1980). 4. In vitro pollen germination In vitro studies have been conducted to investigate the effectiveness of various media and temperature on pollen germination. For example, ‘Golden Delicious’ and ‘Starkrimson’ pollen germination occurred after 120 min incubation in Petri dishes at 30 ◦ C in a medium containing 0.2 M sucrose, 20 g/ml H3 BO3 , and 300 g/ml Ca(NO3 )2 ·4H2 O. Optimum pH was 6.0 for ‘Starkrimson’ and 7.0 for ‘Golden Delicious’ (Calzoni et al., 1979). Apple pollen can be stored for in vitro germination on media containing boric acid, magnesium sulphate, potassium nitrate, calcium nitrate, sucrose and agar (at different concentrations) due to its effective viability after storage (Imani et al., 2011). Sucrose, in concentrations ranging from 15% to 25% is typically used as a carbon source for pollen germination. Boric acid alone was not effective in promoting in vitro germination. The largest germination rates in Fuji (51.1%), Imperatriz (31.7%), M.9 (20.8%), Catarina (19.2%), Gala (13.7%), and Marubakaido (6.1%) pollen were observed in 15% sucrose and an absence of boric acid (Dantas et al., 2002). Considerable advantage can be obtained from stored pollen when applied by hand cross pollinations since it remains viable for several days at room temperature (Hancock et al., 2008). It can remain viable for several weeks if refrigerated under low relative humidity. Loss of viability could be partly overcome by slow dehydration of the pollen (Hopping and Jerram, 1980). Apple pollen can also be satisfactorily stored over dry ice at −60◦ to −55◦ C (Griggs et al., 1953). It can be held for at least a year at −15 ◦ C in loosely stoppered vials in desiccators with calcium chloride (Hancock et al., 2008). Pollen grains of apple stored for nine months in small, closed, glass vessels at -15 ◦ C showed 95% germination, which was as good as fresh samples (Tupý, 1959). Others have reported that pollen germination ranged from 50 to 75% in 18 apple cultivars after storage at −1 ◦ C for about 70 days (Campo Dall’Orto et al., 1985). Imani et al. (2011) studied the viability of pollen in four apple cultivars (Primgold, Golab, M9 and Northern Spy) three and seven months after storage at three temperatures (4 ◦ C, −20 ◦ C and −80 ◦ C). After three months storage at −80 ◦ C, ‘Primgold’ pollen had the greatest germination rate of 96.21% and ‘Northern Spy’ stored at 4 ◦ C had the lowest germination rate of 58.33%. ‘Primgold’ pollen showed a germination rate of 90.66% after seven months storage at −80 ◦ C, and ‘Northern Spy’ showed the lowest germination rate of 36.67% when stored at 4 ◦ C (Imani et al., 2011). 5. Irradiated pollen Pollen irradiation studies began at the end of the 19th century with the discovery of X-rays (Sestili and Ficcadenti, 1996). The earliest investigations were aimed at evaluating the effects of radiation on pollen germination and tube growth (Lopriore, 1897). Pollen irradiation with gamma rays has a number of different effects on apple pollen viability and fruit development (Table 1). The primary impact is reduction of pollen germination (Marcucci 191 et al., 1984; Montalti and Filiti, 1984). Visser and Oost (1981) found that irradiated apple pollen stored at 4 ◦ C and 0–10% RH was much more sensitive to dry storage conditions and had less germination than untreated fresh pollen. Adverse effects on reproductive organ development in response to ionizing irradiation of pollen include abnormal seed formation (De Witte and Keulemans, 1994). Other affected features include fruit set, seed number per fruit, embryo set and embryo development. They were lower when ﬂowers were pollinated with irradiated pollen compared to non-treated pollen (Nicoll et al., 1987; De Witte and Keulemans, 1994). Positive effects of pollen irradiation are the formation of parthenocarpic fruits, which are devoid of embryo and endosperm and the development of parthenogenetic embryos (Marcucci et al., 1984; Zhang and Lespinasse, 1991). Other positive effects are stimulation of amylase, cellulase, ribonuclease and particularly acid phosphatase activities in the pollen of ‘Golden Delicious’ (Calzoni and Speranza, 1982). 6. Floral parts and ﬂowering Morphology of the apple ﬂower is generally typical of the rose subfamily, Maloideae (Shefﬁeld et al., 2005). Flowers of different cultivars and seedlings vary considerably in size, petal shape, and color from white to deep pink (Janick et al., 1996). The apple tree is a monoecious species with hermaphroditic ﬂowers (Pratt, 1988; Pereira-Lorenzo et al., 2009). Mixed buds are composed of three to six ﬂowers in cymes (the apical ﬂower being the most advanced) (Dennis, 1986, 2003). Apple ﬂowers are deteriminate; however, it is variously described as a corymb, a corymbose raceme, a cyme and a false cyme (Foster et al., 2003; Jackson, 2003). Apple ﬂowers are borne on two types of shoots, spurs and long shoots (Wilkie et al., 2008). Spurs are short, lateral shoots in which extension growth is limited to the production of a rosette with few leaves (Abbott, 1970; Wilkie et al., 2008). Flower numbers in an inﬂorescence can vary from 3 to 20, but ﬁve is the most common for commercially grown cultivars (Racskó and Miller, 2010). The typical ﬂower consists of ﬁve petals, a calyx of ﬁve sepals, about 20 stamens and the pistil which divides into ﬁve styles (Janick et al., 1996) (Figs. 3 and 4). Domestic apples usually bear four to seven ﬂowers on an inﬂorescence from which the central initiates ﬁrst followed by the laterals. ‘Gala’, ‘Elstar’, ‘Golden Delicious’, ‘Granny Smith’ and ‘Fuji’ apple ﬂoral opening is greatly inﬂuenced by their position on the inﬂorescence (Racskó and Miller, 2010). The order in which the ﬂowers open corresponds to the order in which they develop, thus starting with the apical (terminal) ﬂower and proceeding downwards through laterals (Racskó and Miller, 2010). Within each individual ﬂower lays an ovary. The ovary has ﬁve carpels, each usually containing two ovules, so that in most cases, the maximum seed content is 10 but some cultivars may have more (Janick et al., 1996; Jackson, 2003). Apples are considered perfectly or imperfectly syncarpic depending on cultivar. Carpels are congenitally fused in the syncarpic condition (Endress, 1994). Some use the term syncarpic to describe taxa such as apple in which the pollen tube transmitting tissues of each carpel remain separate throughout their entire length despite the carpels being congenitally fused externally (Shefﬁeld et al., 2005). The ﬁve stigmas, which unite into a common style that leads to the ovary, are surrounded by 20–25 erect pollen-bearing stamens. The ﬂower is epigynous with the ovary being enclosed by non-ovarian tissue (fused base of sepals, petals and stamens or cortex of stem, depending on morphological interpretation) that remains attached to the ovary at harvest, giving rise to a ‘false’ fruit, or pome (Dennis, 2003) (Fig. 5). Nectar is secreted from nectaries located between the stamen and the ovary between the bases of the stamens and the style (McGregor, 1976). Within the apple genus, Malus, each of the ﬁve styles bears a single 192 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Table 1 Pollen irradiation effects. Cultivar(s) Effects Source Erovan, Golden Delicious R1-49 and X6677 Baskatong Idared Golden Delicious, Alkmene, Jonathan, James Grieve Hybrid TNR31.35 Reduced fruit set and seed number Formation of parthenocarpic fruits Fruit and seed set were reduced Reduced germination capacity and poor quality seeds Irradiation caused pollen cell membrane ﬂexibility loss Pollen grains germinated slowly relative to the control; chromosomic abnormalities – presence of chromatin bridges – uneven distribution of chromosomic material in the 2 daughter nuclei A low dose-rate (6.86 krad/h) reduced germination more than a higher rate (345 krad/h) Irradiation strongly impaired pollen vitality: only 1% of the tubes had reached the base of the style after 48 h Low irradiation dosage (0.1 krad) reduced seed production and pollination Higher irradiation doses no fruits formed Zhang and Lespinasse (1991) --------------------------------------------------Golden Delicious --------------------------------------------------- Nicoll et al. (1987) De Witte and Keulemans (1994) Visser and Oost (1981) Lecuyer et al. (1991) Speranza et al. (1982) Montalti and Filiti (1984) Marcucci et al. (1984) Fig. 3. ‘Criollo’ apple cultivar fom Bogotá, Cundinamarca State, Colombia. (A) Floral bud, (B) Young ﬂowers note the pink color of petals, (C) Floral opening, (D–F) Fully opened ﬂowers. Note the White-pink color pattern of the petals and pink petals of young ﬂowers. Photos by Fernando Ramírez. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of the article.) F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 193 Fig. 4. Apple Varieties at Nuevo Colón, Boyacá State, Colombia. (A) ‘Emilia’ apple. (A–B) Note the light pink-white petals, (C) young ﬂoral buds and (D) ﬂowering buds with fruit on the same shoot. (E) ‘Wilter’s ﬂowers. (F–H) ‘Criollo’s ﬂowers at Tibasosa, Boyacá State, Colombia. Photos by Fernando Ramírez. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of the article.) stigma (Shefﬁeld et al., 2005). Anthers are the male organs where microsporogenesis takes place. This event marks the beginning of pollen formation and subsequent maturation until anthesis takes place and pollen is shed from anthers. The number of pollen grains per anther ranged from 1170 to 3800 in 18 apple cultivars grown under subtropical conditions in São Paulo, Brazil (Campo Dall’Orto et al., 1985); however, these authors did not include temperature or other climatic variables to explain the source of variation in pollen grain number but mentioned only ‘mild climate conditions prevailing in the State of São Paulo, Brazil’. On average, Brazilian cultivars, such as Baronesa, Suprema, Imperatriz, Lisgala, Joaquina, Princesa, Fred Hough, Daiane, Catarina, Primícia, Duquesa and Condessa produce 16–20 anthers (de Albuquerque et al., 2010b). Pollen grains per ﬂower ranged from 23,000 to 74,000 (Campo Dall’Orto et al., 194 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Fig. 5. Pommes from Boyacá State, Colombia. (A) ‘Pennsylvania’ at Nuevo Colón. (B) ‘Anna’ at Sotaquirá. (C) An unknown dwarf cultivar from Nobsa. Photos by Fernando Ramírez. 1985). de Albuquerque et al. (2010b) found a variable number of pollen grains per ﬂower ranging between 53,000 and 103,700 in Brazilian apple cultivars. The variation in pollen grains per ﬂower is mainly due to cultivar differences. Flowering biology of various woody angiosperms grown in the tropics and sub-tropics, such as mango, avocado and citrus, and in temperate conditions, such as apple, plums and pears is well documented (Jackson, 2003; Kozma et al., 2003; Wilkie et al., 2008; Ramírez and Davenport, 2010, 2012). Floral induction of apple refers to the events causing the shift in buds from forming vegetative to forming reproductive structures (Dennis, 2003). Induction in apple also refers to the process during which previously repressed information is being transformed to form a new structure, namely the ﬂower bud (Koutinas et al., 2010). It occurs during the spring bloom in temperate conditions (Abbott, 1970) and has also been reported to occur during early summer, but it can extend into early autumn under some conditions (Dennis, 2003). Floral initiation follows when the meristem ﬂattens and becomes macroscopically visible. Floral bud development continues as primordial sepals, petals, stamens and pistils form centripetally on the apex and grow into fully formed appendages (Dennis, 2003). Apple ﬂowers can develop in both terminal and axillary buds of both spurs and shoots (Dennis, 2003). Floral development in apple is not continuous, but broken by a period of protective dormancy during the winter months (Wilkie et al., 2008). Buds of most temperatezone deciduous trees have a dormancy period in the winter (Naor et al., 2003). Low, chilling temperatures are the most signiﬁcant factor affecting dormancy completion, although, other factors such as light intensity, heat and mist during endodormancy affect dormancy completion to a certain extent (Naor et al., 2003). Chilling requirements differ among apple cultivars and even within a cultivar. There are great differences in chilling requirements between bud types (Dennis, 2003; Naor et al., 2003). This is the case of lateral vegetative buds which have a relatively high chilling hours requirement whereas terminal vegetative and ﬂoral buds have lower chilling temperature or hours requirements (Samish and Lavee, 1962). If accumulated chilling hours are insufﬁcient, both vegetative and ﬂower buds are retarded in development and cropping is reduced (Dennis, 2003). Exceptions to chilling requirement occur in some regions of the tropics, as in Indonesia and Colombia where defoliation soon after harvest induces bud break in low chilling cultivars, resulting in two crops per year (Edwards and Notodimedjo, 1987; Dennis, 2003). Flowering is inﬂuenced by a number of factors, such as accumulation of chilling temperature hours that affects ﬂoral initiation after winter dormancy (Tromp, 1980). Low irradiance inhibits ﬂoral initiation on spurs in spring (Cain, 1971). Water stress has been used to induce apple ﬂowering (Jones, 1987). Flowering can be attained by defoliating trees. Photoperiod plays little or no role in ﬂowering of apple (Dennis, 2003). Hormones, such as gibberellic acid (GA3 ), inhibit apple ﬂowering (Wilkie et al., 2008); however, C3 Epi-GA4 promotes ﬂowering (Looney et al., 1985; Pharis et al., 1992). Cytokinins also promote ﬂowering (McLaughlin and Greene, 1984; Wilkie et al., 2008). For a concise description of compounds involved in apple ﬂowering see Ramírez and Hoad (1981) Rohozinski et al. (1986) and Dennis (2003). Interest has recently focused on the ﬂowering genes of apple. The ﬂowering locus T (FT) gene is responsible for inducing ﬂowering in apple (Tränkner et al., 2010). Two FT-like genes have been identiﬁed, MdFT1 and MdFT2 (Kotoda et al., 2010). MdFT1 is expressed mainly in apical buds of fruit-bearing shoots, ﬂower buds (at the balloon stage), ﬂoral organs, such as stamens, and whole young fruits. Little expression is found in tissues, such as roots, stems, mature leaves and apical buds of vegetative shoots, with little detection in seeds and cultured shoots, which included apical buds, F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 stems and leaves. On the other hand, the transcript of MdFT2 was detected mainly in reproductive organs, such as ﬂower buds, sepals, petals, stamens, carpels, receptacles, peduncles and whole young fruits, with some expression also detected in mature fruit. The temporal and spatial expression of ﬂoral pattern genes, such as MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFTF were investigated in apple shoot apexes (Mimida et al., 2011a). Expression levels of AFL2 and MdAP1 were up regulated in young ﬂoral primordia (Mimida et al., 2011a). AFL2, MdFT, and MdAP1 affect the conversion from vegetative meristems to inﬂorescence meristems after the decline of MdTFL1 expression, and at a later stage, AFL2 and MdAP1 promote formation of the ﬂoral primordia and ﬂoral organs (Mimida et al., 2011a). Mimida et al. (2011b) suggested that MdFT1 and/or MdFT2 might be involved in regulation of cell proliferation and formation of new tissues and may affect leaf and fruit development. Flowering is the seminal event for pollen production in apple and its understanding can lead to increased pollination in temperate, sub-tropical and tropical environments. 7. Cross- and self-pollination, ﬂoral bloom and overlap Since Waite (1865) ﬁrst provided consistent evidence that apples beneﬁt from inter-planting and that cross-pollination occurs between cultivars, there has been ongoing research in this area. Cross-pollination has been documented as an important mechanism to establish genetic connection among and between populations of wild apples (Dzhangaliev, 2010). Cross-pollination between compatible cultivars depends on insects as pollen vectors during ﬂowering, and their activity is impaired by inclement weather (Broothaerts et al., 2004a,b). Most apple cultivars require cross-pollination with a compatible pollinizer to set commercial crops of fruit even in partially self-fruitful cultivars (Dennis, 2003). It is, thus, considered that cross-pollination increases apple tree productivity (Schneider et al., 2005). Some exceptions to this are the varieties ‘Newtown’ and, to a lesser extent, ‘Golden Delicious’ and ‘Rome Beauty’ (Delaplane and Mayer, 2000). Although, ‘Golden Delicious’ is partially self-fruitful, it will produce better crops with cross-pollination (Lerner and Hurst, 2002). Generally, closely related varieties, such as McIntosh, Early McIntosh, Cortland, and Macoun do not cross-pollinate each other well (Delaplane and Mayer, 2000). ‘Anna’ is commonly cross pollinated with ‘Dorset’ an important pollen donor cultivar in Colombia (Schwarz, 1994). In contrast, without ‘Dorset’ as a pollinizer, ‘Anna’ produces low quantities of small fruits (Schwarz, 1994). Cultivars, such as Anna, Dorsett Golden, Castaño, Uzcátegui, Winter Banana, Rome Beauty, Red Delicious, Golden Delicious, and Granny Smith are commonly pollinized by Dorsett Golden, Castaño, Anna, Reineta de Reinetas, Red Delicious, Rome Beauty, Granny Smith, and Golden Delicious respectively in Venezuela (Monteverde, 1989). Self-pollination occurs less than cross-pollination. ‘Fuji’ and ‘Golden Delicious’ produce only 1% and 1.8% fruit set after selfpollination (De Witte et al., 1996). Higher levels of self-pollination were reported in ‘Elstar’ (7%) and ‘Idared’ (12.3%) (De Witte et al., 1996). ‘Cox’s Orange Pippin’ exhibits low fruit set when selffertilized (0.7–17%) (Modlibowska, 1945; De Witte et al., 1996). Pollination studies conducted in tropical Asia revealed successful self-pollination. ‘Rome Beauty’ can be grown without pollinizer in high regions of East Java (Yuda et al., 1991). Self-compatibility is considered to be high in this tropical region. This result provides supportive evidence that seed and fruit can be formed owing to selfpollination in tropical highlands. Saito et al. (2007) reported that self-pollination of ‘Fuji’ showed percentages of fruit sets ranging from 0 to 4.5% over 4 years in Japan. In contrast, fruit set resulting from self-pollination of ‘Megumi’ and ‘Orin’ showed percentages ranging from 40 to 48% in the 2nd year and from 16.3 to 38% in 195 the 3rd year of a four year study. From self-pollination of ‘Fuji’, they obtained many progenies from fruits containing seeds by the application of embryo culture; however, the percentages of seed set in ‘Fuji’ were less than those in ‘Megumi’. PCR ampliﬁcation using S-allele-speciﬁc primers showed the possibility that some progeny were derived from self compatible fertilization (Saito et al., 2007). There is strong interest in the self-fertile character in many fruit and nut tree crops because self-pollination could ensure more consistently high production yields compared to cross-pollination (Broothaerts et al., 2004b). Floral overlap occurs when ﬂowers in an apple tree open synchronously with those of another one, hence promoting effective pollen transfer between them. Floral overlap is common among apple cultivars and is the main mechanism facilitating cross pollination. Cultivars with a long ﬂowering season, e.g. those that ﬂower profusely both on one-year-old, long shoots and on spurs, may be especially useful as cross pollinizers (Jackson, 2003). Pollinizers must bloom at the same time as the cultivar being pollinated and should be annual, rather than biennial, to ensure a supply of pollen every year (Dennis, 2003). To optimize pollination, it is necessary to plant both early- and late-blooming pollinizers so that the main variety blooms in between (Delaplane and Mayer, 2000). In that way, ample pollen will be available for early-blooming on the main variety, and if frost kills the blooms, the late-blooming pollinizers will provide pollen for those ﬂowers that remain (Delaplane and Mayer, 2000). The best pollinizer for apple and the effect of different pollinizers on fruit quality, were considered in sixteen cultivars (Bashir et al., 2010). The mean performance of ‘Spartan’ as pollinizer proved to be the best in terms of fruit set, followed by ‘Gala’ (Bashir et al., 2010). Some apple cultivars such as Sir Prize, Turley, Mutsu, Stayman, the Winesap group and others are poor pollinizers and should not be used as a pollen source (Lerner and Hurst, 2002). Modern apple orchards frequently use crabapple (Malus ﬂoribunda) pollinizers to provide pollination of solid blocks of diploid or triploid cultivars (Ko et al., 2010). In the Himalayan region, ‘Manchurian’ crabapple was found to be an efﬁcient apple pollinizer, followed by the spur types, ‘Stark Spur’ and ‘Oregon Spur’ on the basis of higher bloom density and fruit set variables (Das et al., 2011). Floral overlap has been reported to occur regularly over years between Gala and Fuji cultivars in southern Brazil (Petri et al., 2008). ‘Prof. Spengler’, ‘Profusion’, ‘Winter gold’ and ‘John Downil’ are apple cultivars with the greatest potential for use as pollinizers to supplement pollination of Gala and Fuji cultivars (Petri et al., 2008). ‘Rainha’ is used as the pollinizer for ‘Gala’ in Paraná state, Brazil (Hauagge and Bruckner, 2002). When ‘Marubakaido’ was used as the pollen donor for ‘M.9’, fruit set was 26% and 32% in 1999 and 2000 respectively. Alternatively, effective fruit set was 5% and 25%, when ‘M.9’ was used as pollen donor on ‘Marubakaido’ in the same two seasons (Dantas et al., 2001). Others have reported that ‘Rome Beauty’ overlapped with ‘Golden Delicious Tardío’ with a 20% fruit set in the latter when open pollinated, whereas fruit set increased to 90% when interplanted with ‘Golden Delicious’ in Mexico (Guerrero Prieto et al., 2006). ‘Golden Delicious’ initial fruit set was 98% when hand pollinated with ‘Snow Drift’ compared with 61% fruit set in open pollinated trees interplanted with the same pollinizer (Guerrero Prieto et al., 2006). ‘Manchurian’, ‘Snow Drift’ and ‘Winter Banana’ full bloom stages overlapped with those of ‘RedChief Delicious’. The full bloom stage of ‘Rome Beauty’ overlapped with ‘Golden Delicious Tardío’ in Chihuahua state, Mexico (Guerrero Prieto et al., 2006); however, high pollination was due to the occurrence of honeybees (three hives per hectare) (Guerrero Prieto et al., 2006). There is an extensive literature and internet resources on ﬂoral overlap that readers can consult for further details of this biological process. For further references see Berkett (1994), Delaplane and Mayer (2000), Phillips (2005) and Sanders (2010). 196 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 8. Self incompatibility, semi compatibility and compatibility There are two main causes of unfruitfulness in apple: sterility and sexual incompatibility (Janick et al., 1996). Incompatibility that is due to the failure of pollen to grow down the style and bring about fertilization is widespread in apple. Self incompatibility is particularly common, although cases of cross incompatibility are also known (Janick et al., 1996). The essential feature of the incompatibility system is that pollen tube growth is inhibited in a style or ovary containing the same incompatibility alleles (Figure 2) (Dennis, 2003). Depending on their S loci, pairs of apple cultivars can be incompatible when both loci are identical, semi compatible when they carry one different and one similar S locus or fully compatible when they differ in their S loci (Schneider et al., 2005). Self incompatibility has developed as one of the mechanisms that prevent successive self fertilizations and deleterious inbreeding. The S-RNase-based gametophytic self-incompatibility (GSI) system has been found among three plant families Solanaceae, Rosaceae, and Plantaginaceae (Minamikawa et al., 2010; McClure et al., 2011). Rosaceae family has the gametophytic self incompatibility (GSI) system wherein pollen tube growth inhibition is controlled by the S locus (Schneider et al., 2005; Kubo et al., 2010). Self-incompatibility (SI) is an intraspeciﬁc reproductive barrier adopted by angiosperms that allows the pistil to distinguish between self (genetically related) and non-self (genetically unrelated) pollen (Kubo et al., 2010). The pistil and pollen determinants of S-speciﬁcity in Rosaceae are a ribonuclease and an F-box protein, respectively (Yamane and Tao, 2009). The S haplotype contains two closely linked S-speciﬁcity genes, pistil S and pollen S. (Li et al., 2007; Sassa et al., 2007). The single, multiallelic gene encodes ribonucleases (S-RNases), present in the pistil of mature ﬂowers that recognizes and inhibits pollen development (Jackson, 2003). The presence of S-RNases in the pistil constitutes a selective barrier through which the pollen tubes have to pass: selfincompatible pollen tubes are recognized and retained, whereas compatible tubes are allowed to grow further down the style to fertilize the egg cells (Broothaerts et al., 1995; Kitahara et al., 2000; Li et al., 2007). S-RNases are found in the intercellular space and distributed evenly in the cytoplasm of pollen tubes in vivo and in vitro (Li et al., 2007). Most apple varieties are self-incompatible (Table 2). Recently, a number of S alleles have been identiﬁed in apple ranging from S1 to S32 (Yamane and Tao, 2009); however due to inconsistent labeling of S-alleles and some erroneous data in the literature, there has been confusion in the S-allele genotypes in apple (Table 2) (Broothaerts et al., 2004a). Using pollination tests, Matsumoto et al. (2006) found that S19 and S28 behaved as different alleles, whereas S17 and S19 appeared to be the same allele. Matsumoto et al. (2003) found that S6 and S12 were identical, as were S17 and S19 . S11 was assigned in place of S13 and S14 . Kim et al. (2008) determined the S-genotypes of ‘Charden’ (S2 S3 S4 ), ‘Winesap’ (S1 S28 ), ‘York Imperial’ (S2 S31 ), ‘Stark Earliblazel’ (S1 S28 ), and ‘Burgundy’ (S20 S32 ), by S-RNase sequencing and S-allele-speciﬁc PCR analysis. Two new S-RNases, S-31 and S-32, were also identiﬁed from ‘York imperial’ and ‘Burgundy’, respectively (Kim et al., 2008). It has long been recognized that most apple cultivars are effectively self incompatible, or very largely so, and that fruit set usually depends on cross pollination between genetically different cultivars (Jackson, 2003). This is particularly the case of ‘Red Delicious’ apple that exhibits full self incompatibility (Stern et al., 2001). All cultivars in the Delicious group such as: ‘Red Delicious’, ‘Oregon Spur’, ‘Starkrimson’ ‘Red Chief’ and ‘Well Spur’, among others, share common S-genotypes. The multigene complex comprises a S-RNase gene in the pistil and S-haplotype speciﬁc F-box gene in the pollen tube (Hegedus, 2006). ‘Starkrimson’, too, has been identiﬁed as totally self incompatible (Calzoni et al., 1979). Schneider et al. (2005) determined that the cross pollination rate of semi-compatible cultivars was signiﬁcantly lower than that of a fully compatible pollinizer, based on PCR analysis of S-RNAase. Pseudo compatibility (semi-compatible pollen tubes produced by self pollination) (Fig. 2) was maximized by pollinating old ﬂowers with large quantities of pollen (provided self pollination) and maintaining a temperature of about 20 ◦ C during the period of pollen tube growth (Williams and Maier, 1977). In contrast, selfcompatible apple cultivars have also been identiﬁed (Fig. 2 and Table 2) (Matsumoto et al., 1999). Others found that the autotetraploid cultivars were self-compatible (Table 2) (Adachi et al., 2009). Goldschmidt-Reischel (1993) found no indications of pollen incompatibility in ‘Cox’s Orange’ between ornamental cultivars and dessert cultivars of Malus in controlled conditions. Experiments with ﬂowers of ‘Cox’s Orange Pippin’ apples have shown that semicompatible pollen tubes produced by self-pollination may affect fertilization and therefore fruit set (Williams and Maier, 1977). Apples require at least two genetically different cultivars for fruit production (Matsumoto et al., 2008). The identiﬁcation of S-locus F-box brother (SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus (Li et al., 2011). Li et al. (2011) recently identiﬁed ﬁve MdSLFB (SRNase linked F-box) genes from four different apple S-genotypes. These genes showed pollen- and S-allele speciﬁc expression with a high polymorphism among S-alleles. Transgenic trees with the self-fertile phenotype were associated with the complete absence of pistil S-RNase proteins, which are the products of the targeted Sgene; therefore, self fertility was due to inhibition of expression of the S-RNase gene in the pistil, resulting in un-arrested self-pollen tube growth, and subsequent egg fertilization (Broothaerts et al., 2004b). Heterogeneity of the S-RNase allelic distribution is much higher in cultivated apples than in wild types, which shows that breeding leads to strong departure from the expected homogeneity of genes under negative frequency-dependent selection (Dreesen et al., 2010). Domestication of apple has led to higher levels of genetic uniformity (Dreesen et al., 2010); however, S-RNase allelic richness of modern cultivars is poor compared to old cultivars (Dreesen et al., 2010). 9. Outcrossing Pollen dispersal is essential for cross pollination to occur. (Kron and Husband, 2006). Several studies have estimated pollen dispersal distances by measuring the rate of fruit or seed set decline with distance from a pollinizing cultivar (Milutinovic et al., 1996); however, the most reliable estimates of pollen dispersal may come from following pollen directly or tracking their alleles represented in the DNA (Kron et al., 2001a). Molecular markers have been used to reconstruct and understand pollen dispersal and pollination in apple orchards (Kron et al., 2001a,b). Kron et al. (2001a) conducted experiments on principal cultivars, such as Red Delicious, Empire, McIntoch, Northern Spy, Mutsu, Gala, Cortland, Paulared and Idared, and a distribution of pollinizer trees across (Idared, Vista Bella and Granny Smith) and along (Fuji, Paulared and Golden Russet) rows of 62.4 m versus 13.7 m, respectively. They found that pollen dispersal across rows averaged 17.4 m and along rows 5.8 m from the nearest pollinizer tree. Those ﬁndings provided quantitative insight from many guidelines of orchard design and management to enhance pollination efﬁciency in high density orchards (Kron et al., 2001a). Kron et al. (2001b) examined the patterns of pollen dispersal from the single pollenizer cultivar Idared throughout an 18-row area consisting of several pollen recipient cultivars. They found that pollen dispersed at least 15 rows (73.5 m) at one study location and 18 rows (86 m) at another F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 197 Table 2 Self incompatible, pseudo compatible and compatible alleles. Data represents most up to date corrected information. Cultivar(s) Alleles Country Source Self incompatible alleles 20 cultivars 137 diploid and 14 triploid cultivars Northern Spy Akane Ralls Janet Idared Fiesta Elstar Gala Golden Delicious Senshu Spijon Tohoku Toyo Nebuta Ambitious Seven cultivars Charden Winesap York Imperial Stark Earliblazel Burgundy 20 cultivars Various Daiane, Imperatriz and Princesa Lisgala Suprema Catarina Joaquina and Fred Hough Baronesa Duquesa Primícia Condessa S1 –S11 Sixty diploid compatibiliy groups S1 S3 S7 S24 S1 S2 S3 S7 S3 S5 S3 S5 S2 S5 S2 S3 S1 S7 S3 S7 S9 S24 S5 S28 S3 S9 S2 S9 Found S2,S9 identical; S17,S19 identical S2 S3 S4 S1 S28 S2 S31 S1 S28 S20 S32 S1 S3 , S1 S5 , S1 S9 , S3 S5 , S3 S7 , S3 S9 and S7 S9 S44 S45 S46 S3 S5 S2 S5 S1 S9 S1 S19 S5 S19 S3 S9 S2 S3 S24 S2 Germany Various USA USA USA USA USA USA USA USA Japan Japan Japan Japan Japan Japan Japan Korea Korea Korea Korea Korea Korea China Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Kobel et al. (1939) Broothaerts (2003) and Broothaerts et al. (2004a) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al., 2000 Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Sakurai et al. (2000) Matsumoto et al. (2003) Kim et al. (2008) Kim et al. (2008) Kim et al. (2008) Kim et al. (2008) Kim et al. (2008) Heo et al. (2011) Long et al. (2010) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) de Albuquerque et al. (2011) USA Williams and Maier (1977) Japan Japan Japan Japan Japan Japan Matsumoto et al. (1999) Matsumoto et al. (1999) Matsumoto et al. (1999) Matsumoto et al. (1999) Matsumoto et al. (1999) Matsumoto et al. (1999) Pseudo compatibility Cox’s Orange Pippin Self compatible alleles Megumi Doud Golden Delicious Sweden Spartan Sweden Alpha 68A Tensei Welday Jonathan S2 S9 S2 S2 S3 S3 S9 S9 S10 S10 Unknown S1 S1 S9 S9 S7 S7 S9 S9 orchard. Moreover, 44% to 80% of all dispersal occurred within three rows (≈14.5 m) of the pollen donor. Pollen dispersal generally declined with distance. Others, found that pollen dispersal inferred from fruit set had occurred up to 35–80 m from the nearest pollen donor (Milutinovic et al., 1996). Matsumoto et al. (2008) selected ‘Maypole’ and ‘Dolgo’ as pollinizers for the cultivar ‘Fuji’, and investigated the rate of fruit and seeds in ‘Fuji’ fruits produced by pollen of the pollinizers. These investigators developed a method for tracing pollen ﬂow based on the leaf color of progeny and SRNase allele of ‘Maypole’, and on Simple Sequence Repeat (SSR) analyses of ‘Maypole’ and ‘Dolgo’. Fruit production decreased with increasing distance from the pollinizer. The rate of fruit produced when ‘Fuji’ was 2.5 m from pollinizers was 84% in ‘Maypole’ and 77% in ‘Dolgo’. When ‘Fuji’ was at 5 m from the pollinizers, fruit set was 71% in ‘Maypole’ and 64% in ‘Dolgo’. When ‘Fuji’ was 10 m from the pollinizers, fruit set was 47% and 39% in ‘Maypole’ and ‘Dolgo’, respectively. Pollination of Malus sylvestris occurred mostly between nearby trees with a median of observed pollination distances of approximately 23 m; however, longer distance pollination occurred at a lower extent at 60 m (Larsen and Kjær, 2009). Therefore, the closer the distance between trees, the higher likelihood for mating (Larsen and Kjær, 2009). The effective distance between the main apple cultivar and pollinizers should be approximately at 6–15 m depending upon tree vigor to ensure best pollen transfer across the orchard (Warmund, 2002). Maggs et al. (1971) found that 12 m was the limit distance for cross pollination to occur in ‘Granny Smith’ in Australia. 10. Insect pollination Honeybees (Apis mellifera) appear to be the most numerically important apple pollinators (Kendall and Smith, 1975; Boyle and Philogène, 1983; Jackson, 2003; Dag et al., 2005). Honeybees are the most important pollinator of apple in North America (Delaplane and Mayer, 2000) and elsewhere worldwide. This is evidenced by fruit quality and increases in yield to varying degrees as a result of pollination by domesticated honeybees in countries such as Australia (Keogh et al., 2010a,b). For example, more than 97% of the insects that visit apple blossoms are wild bee or honeybees in New South Wales, Australia (Somerville and White, 2005). Bee exclusion from apple trees causes signiﬁcant reductions in fruit set, yield/tree, seed and fruit number (Langridge and Jenkins, 1970). Apple is an important crop in New Zealand. Honeybee pollination among apple cultivars in New Zealand has been covered by Palmer-Jones and Clinch (1966, 1967, 1968). They found that the only pollinating insects were honeybees and negligible numbers of Bombus terrestris Linn. A density of about 40 honeybees per 30,000 ﬂowers were estimated per minute, and this appeared adequate for all apple cultivars studied (Palmer-Jones and Clinch, 1967). Insect visitation of apple varieties: Granny Smith, Kid’s Orange Red, and 198 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Golden Delicious were studied in the Henderson and Warkworth area near Auckland, New Zealand (Palmer-Jones and Clinch, 1968). Visiting insects were solely honeybees and negligible numbers of Bombus terrestris Linn. and unidentiﬁed long-tongued bumble bees (Palmer-Jones and Clinch, 1968). The number of honeybees per 30,000 ﬂowers averaged 53 for the three varieties (Palmer-Jones and Clinch, 1968). Insect visitation of ‘Cox’s Orange Pippin’ were studied near Richmond, Nelson, New Zealand (Palmer-Jones and Clinch, 1966). This study reported that the only insects found as pollinators were honeybees and negligible numbers of Bombus terrestris Linn. that were mainly nectar collectors. Honeybees were 18 times as numerous as bumble bees (Palmer-Jones and Clinch, 1966). These authors concluded that apple fruit set depended almost exclusively upon insect visitation to ﬂowers and honeybees were the most important pollinator of the ﬂowers. Honeybees had low pollination percentages in ‘Granny Smith’ (6–16%), ‘Kid’s Orange Red’ (7–22%) and ‘Golden Delicious’ (10–20%) in various orchards near Auckland in New Zealand (Palmer-Jones and Clinch, 1968); however, these authors did not record temperature or other climatic conditions in the region. It is not possible to determine if the low pollination rates were due to low pollinator availability or low temperature conditions affecting insect behavior and pollination. The attractiveness of an apple cultivar could be correlated with the abundance of its ﬂowers (Kendall and Smith, 1975). Bee foraging behavior on apple ﬂowers is key to understanding cross pollination in apple trees. The stamens of apple ﬂowers allow nectar-gathering bees to obtain nectar by pushing their tongues between the ﬁlaments without touching the anthers or stigma (Free, 1960). Pollen gathering bees prefer to approach the nectary from the top of stamens, collecting pollen in the process, or by scrabbling over the anthers (Free, 1960). The proportion of nectar to pollen gatherers depends on the structure of the stamens and if the stamens are ﬂexible enough (Free, 1960). The ratio of nectar gatherers to pollen gatherers, and probably the behavior of individuals, varied greatly on different days and at different times on the same day. Bees visited some ﬂowers for pollen only and others for nectar during the time they were observed; however, some apple cultivars such as the Delicious apples have a ﬂoral structure that reduces pollination efﬁciency (Roberts, 1945). Gaps at the base of stamens on these ﬂowers enable side-working honeybees to gather pollen without contacting the anthers and stigmas of the blossoms. Other conditions can also alter the effectiveness of bee pollination. Characteristics of nectar reward and ﬂoral morphology revealed that ‘Jonathan’ and ‘Topred’ ﬂowers had similar nectar contents; however, the morphology of the ﬂowers forced different honeybee behaviors in the two cultivars (Schneider et al., 2002). ‘Jonathan’ ﬂowers attracted fewer honeybees, but due to their anther arrangement, more of the ﬂowers were approached from the top by honeybees collecting nectar than those ﬂowers on ‘Topred’. Although honeybees pollinate apple well, they are not the most efﬁcient apple pollinator (Delaplane and Mayer, 2000). They sometimes rob an apple ﬂower of its nectar without pollinating it, such as previously described for the Delicious apple variety. Honeybees make fewer contacts with the sexual column of the apple ﬂower, compared to certain solitary bees (Delaplane and Mayer, 2000). Caged tree experiments provided evidence that preventing bee contact with ﬂowers had a negative effect on pollination. Enclosed trees of ‘Yates’ apples, each grafted with a limb of ‘Jonathan’, in bee-proof cages caused signiﬁcant reduction in the number of fruit set, weight of fruit harvested, and the number of seeds per fruit as compared with un-caged trees, although airborne apple pollen concentrations were 4.07 times higher inside the cages than outside (Langridge and Jenkins, 1970). The ﬂowers of Malus attract a wide range of pollinators. In addition to Apis, other bee pollinators of apples include the genera, Andrena, (McGregor, 1976; Gardner and Ascher, 2006), Bombus, (Palmer-Jones and Clinch, 1966, 1967; McGregor, 1976), Halictus (McGregor, 1976), and Osmia (McGregor, 1976; Kuhn and Ambrose, 1984; Torchio and Asensio, 1985; Torchio et al., 1987; Maeta et al., 1992; Sekita, 2001; Wei et al., 2002; Shefﬁeld et al., 2008; Matsumoto et al., 2009; Matsumoto and Maejima, 2010; Gruber et al., 2011). Moreover, Gardner and Ascher (2006) found that 31 native bee species pollinated apples in the Finger Lakes region of New York State. Of these, 14 species belong to eight subgenera of Andrena. Apis and Bombus removed similar amounts of pollen from apple ﬂowers, but Bombus deposited more pollen on the stigmas. Large numbers of bee foragers per tree directly increase the amount of pollination. High bee mobility between rows increase the amount of cross pollination, and a high proportion of ‘top workers’ increase pollination efﬁciency (Stern et al., 2001). However, excess pollination can result in over-cropping, leading to many small fruit of low quality (Schneider et al., 2002). Other bees, such as those in the genus, Osmia, visit ﬂowers at lower temperatures than do honeybees (McGregor, 1976). The megachilid bee, Osmia cornifrons, has been selected as an apple pollinator and used extensively in Aomori Prefecture, a leading apple-producing region in Japan (Sekita, 2001). O. cornifrons and Osmia lignaria propinqua are important apple tree pollinators in other parts of Japan as well (Maeta et al., 1992). Matsumoto et al. (2009) found that individual O. cornifrons bees showed strong ﬂower constancy for 4–8 min during one pollen-nectar foraging trip and foraged for different types of apple ﬂowers, e.g. from a red to a white petaled blossoms, during their 16–22 pollen-nectar foraging trips based on the S-RNase allele and simple sequence repeat (SSR) analyses. O. cornifrons bees are common visitors of ‘Maypole’, ‘Fuji’, ‘Pink Pearl’, ‘Prima’ apple trees in Japan (Matsumoto et al., 2009). Since 1996, over 80% of the total area of an apple orchard near Nagano Japan, has been pollinated using O. cornifrons. These bees were superior to honeybees in the number of ﬂowers foraged each day, the number of visitations to ﬂowers during low temperatures, strong winds, and reduced sunshine (Matsumoto and Maejima, 2010). O. cornifrons acts as a useful pollinator in apple orchards with pollinizers planted not more than 10 m from the primary commercial cultivars (Matsumoto and Maejima, 2010). In the Annapolis Valley, Nova Scotia, Canada, the wild species, Osmia tersula Cockerell (Megachilidae), accounted for almost 45% of all bees captured and was the most abundant species nesting in all habitats evaluated. It has potential as a commercial pollinator of spring-ﬂowering crops (Shefﬁeld et al., 2008). Two species of bees native to China, Osmia excavata Alfken and Osmia jacoti Cockerell, enhanced apple pollination in orchards in Shandong Province (Wei et al., 2002). Observations on the behavior of individuals showed that O. excavata averaged 49.6 foraging trips per day and was deemed responsible for set of an estimated 3108 individual fruit on ‘Ralls Janet’. O. jacoti averaged 31.2 foraging trips per day and was deemed responsible for set of an estimated 1831 individual fruit on ‘Ralls Janet’. Both species were more efﬁcient pollinators, than Apis mellifera (Wei et al., 2002). Other Osmia species such as O. bicornis have been used to pollinate apple orchards in Central Saxony, Germany (Gruber et al., 2011). O. cornuta has been evaluated as a potential apple pollinator in Japan, Spain, U.S.A. and Yugoslavia (Torchio and Asensio, 1985; Torchio et al., 1987). In North Carolina, O. lignaria lignaria, O. lignaria propinqua, and O. cornifrons improved fruit-set, seed number, and fruit shape in ‘Delicious’ apples, even in areas of orchards that already had honeybee hives (Kuhn and Ambrose, 1984). Other important apple pollinators are included in the bee families, Andrenidae and Halictidae (Boyle and Philogène, 1983). These hymenopterans had more pollen on their bodies than did Diptera (Boyle and Philogène, 1983); however pollen on bee’s bodies does not insure successful pollination. These authors did not use any molecular tool or methodology to effectively prove that pollen F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 deposition and actual pollination occurred. Bees such as Andrena carantonica Perez have been reported to gather important quantities of pollen from apple trees near Versailles, France. Wild bees from the families, Andrenidae and Halictidae and the honeybee, Apis mellifera L., are important pollinators in Wisconsin, USA, apple orchards (Watson et al., 2011). Other, bees and insects pollinate apple trees. Kendall (1973) examined the viability and compatibility of a variety of insects visiting apple ﬂowers. This study reported that Hymentopteras, Dipterans and Coleopterans (beetles) were among the most common ﬂoral visitors; however, relationships between the number of pollen grains deposited on ﬂowers by these insects and the percentage of fertilized ovules are highly variable. Pollen carried on the body hairs of all the examined insects had about the same viability as pollen from freshly dehisced anthers. Consistently less viable pollen was carried only by the syrphid, Rhingia campestris Mg,. and the males of a solitary bee, Andrena wilkella (Kirby) (Kendall, 1973). The only insects other than bees with pollen loads containing a high proportion of compatible fruit pollen were some syrphids (Eristalis spp.) and a conopid ﬂy (Myopa sp.) (Kendall, 1973; Kendall and Smith, 1975). Anthomyiidae (Diptera) was an important pollinator during two consecutive years in Nova Scotia (Shefﬁeld et al., 2003). Botero and Morales (2000) studied ‘Anna’ insect pollination at Carmen de Viboral Municipality, Western Antioquia State, Colombia. Temperature conditions were between 14 and 24 ◦ C, at an elevation of 2200 m above sea level with 1800 mm of annual rainfall. Caged and non-caged branches were used to evaluate the effect of wild insects on apple pollination. Daily observations were recorded between 7 am and 6 pm. Six insect orders were found visiting the non-caged ﬂowers: Hymenoptera, Diptera, Lepidoptera, Coleoptera, Hemiptera and Blattaria (Botero and Morales, 2000). The most abundant ﬂoral visitor was Apis mellifera with 76% (no hives provided) followed by Diptera (Syrphidae, Muscidae, Tachinidae, Calliphoridae) 8.7%, native bees (Trigonidae, Meliponas, Halíctidae) 4.5%, Bibionidae, Sciaridae, Tipulidae (Diptera) 3.7%, Beatles (Coleoptera) 3.1%, Lepidoptera 2.2%, Hemiptera 1.1% (Botero and Morales, 2000). Noncaged branches had greater pollination conducive to fruit set (41 fruits produced). In contrast, caged branches had lower pollination conducive to fruit set (10 fruits produced); however, these authors failed to explain if the number of fruits is per branch or for all branches examined. They did not measure pollination per se and only based their results on fruit set as a measure of pollination. ‘Anna’ grown in Colombia depends largely on insect pollination (Botero and Morales, 2000); however, it is self-compatible to some extent as evidenced by other studies (Petri, 1993). Moreover ‘Anna’ self-pollinates according to pollination studies at Aserri (1500 m above sea level) province of San Jose, Costa Rica (Guevara, 1992). Others report that ‘Anna’ has a high autogamy level (7%) (Díaz, 1993) and is well adapted to cross pollination (Petri, 1993). 11. Environmental conditions Apple breeding programs have developed cultivars that adapt well to a variety of climates. In the northern hemisphere, they are cultivated from northern Europe down to the tropics where two crops per year can be obtained at high altitudes. Apples have been introduced in South America, South-Africa, New Zealand, and Australia (Pereira-Lorenzo et al., 2009), and elsewere in the world. Environmental conditions, such as frost, precipitation, and temperature, negatively impact apple pollination (Williams and Maier, 1977; Dzhangaliev, 2010). Precipitation has been known to negatively impact ﬂight activity of wild bees that promote cross pollination (Dzhangaliev, 2010). Low winter temperatures can reduce both the number of pollen grains produced and their 199 viability as well as inhibit pollen tube growth (Jackson, 2003). High temperatures inhibit ﬂoral induction, pollen production and reduce its viability (Van Marrewijk, 1993). Keogh et al. (2010b) reported that bee activity is limited below temperatures of 13 ◦ C, with increasing activity as temperatures increase to around 19 ◦ C, above which activity tends to remain at a relatively high level; however there are many bee species that can be active at cool and/or high temperatures under topical conditions. Decreases in both the numbers of bees visiting blossoms and the distance traveled from the hive occur with low temperatures. Under rainy conditions, bees ﬂy between showers but only for short distances (Keogh et al., 2010b). Physiologically high temperatures also have adverse effects on insects and plants. As the temperature rises following pollination, pollen-tubes grow more rapidly, within limits, but the time during which the ovule is receptive is reduced. Unusually high temperatures are detrimental to fruit set (Dennis, 2003). Under a mean daily temperature of 15 ◦ C, pollen tubes take 2 days to reach the ovules compared with 4 days at 13 ◦ C and 8 days at 9 ◦ C (Williams, 1970). Wind is not considered an important factor for apple pollination (Jackson, 2003); however, wind speeds above 15 to 20 m.p.h. inhibit bee ﬂight (Jackson, 2003) and could have adverse effects on pollination. Particularly strong wind tends to reduce the ground speed of bees and hence reduces the number of ﬂights per day (Keogh et al., 2010b). A. mellifera activity was signiﬁcantly dependent on temperature, wind speed, and solar radiation, and O. cornuta activity depended on wind speed and solar radiation. Honeybees remain near hives during overcast and rainy days. Their ﬂight speed is 22 Km/h, thus, higher wind velocities affect their ﬂight (Mayer et al., 1985). 12. Artiﬁcial pollen application Artiﬁcial pollen application is a technique that ensures that pollen will be effectively delivered to ﬂowers for fertilization to occur. Pollen can be ‘dusted’ on trees by dropping it into the updraft created by an air-blast sprayer. Some growers use helicopters to apply pollen from the air, after mixing it with a suitable diluent (Dennis, 2003); however pollen can be wasted as a result of broad dispersal with this artiﬁcial method. Other studies in countries such as Colombia, have used artiﬁcial pollen applications with a sprayer (pollen in aqueous solution) to improve fruit set. Pollen applications of 30, 60 and 90 g pollen/ha, were made on ‘Anna’ in Caldas State (Roldán et al., 1999); however a descriptive methodology on how pollen was applied with the sprayer was missing. The best pollination was obtained when applying 90 g/Ha (Roldán et al., 1999). Orozco Corral and Valverde Flores (2010) examined artiﬁcial pollen applications with a sprayer (25, 50 and 75 g/l). Pollen was placed in water inside the sprinkler and then applied to trees at one study orchard in Chihuahua State, Mexico. Effective artiﬁcial pollination was obtained by applying of 200 g/Ha and 100 g/Ha of pollen to ‘Gala’ and ‘Golden Delicious’ in another orchard location in Municipio de Guerrero, Mexico, (Orozco Corral and Valverde Flores, 2010). Two- or three-time repeated pollinations at 4-h intervals contributed to increased seed number per fruit and decreasing lopsided fruits, suggesting that multiple artiﬁcial pollinations within one day provided more complete pollination in Nagano, Japan (Matsumoto et al., 2012). 13. Conclusion Apple pollination studies have been conducted in many temperate, sub-tropical and tropical environments. Best documented are those in temperate environments. Investigations on pollen germination have provided insight about the great variability that 200 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 exists among germinating pollen grains among different cultivars. Temperature is a key factor for pollen germination. There has been increasing interest in pollination of commercial, economically important cultivars native to the United States, such as the Delicious group that currently has great demand throughout the world. Recently, there has been increasing interest in selfincompatibility gene systems of apple involving a great number of genes. Many genes involved within the GSI have been misidentiﬁed or re-named. This is evidence that the current molecular tools for allele identiﬁcation need more reﬁnement. Currently, there are more than 30 S alleles known to confer self-incompatibility; however, due to the great number of cultivars worldwide, this number is likely to increase. Particular interest in the S genotypes has led to recent discoveries in South American cultivars particularly in Brazil, but much more remains to be elucidated from other cultivars worldwide. Molecular markers, such as Random Ampliﬁed Polymorphic DNA (RAPDS) and Simple Sequence Repeats (SSR) provide insights into pollinizer distance and could also lead to determination of paternal inheritance among pollinizers. More remains to be elucidated on the role of speciﬁc genes during pollination, especially during germination and stigmatic contact. Gene-protein and other molecular interaction events are largely unclear and require further research. Pollen irradiation was an effective methodology for generating haploids in the 1980s; however, today irradiation with gamma rays has been used less in breeding programs. This is partly a consequence of radiation use regulations and the expanding use of molecular approaches. Among temperate environments, there are a great number of studies that have provided substantial evidence that crosspollination among trees is necessary for efﬁcient pollination and ample fruit set. Self-incompatibility from GSI systems prevents or inhibits pollen tube growth of pollen derived from same or related cultivars so self-pollination contribution to fruit set is almost negligible under temperate and sub-tropical environments. Wild apple pollination should be investigated more. Native wild-type cultivars and insect pollinators should be investigated in their places of origin. This could lead to a better understanding of pollinators that could be applied to commercial orchards. There is better understanding about insect pollinators and pollinizer cultivars in temperate conditions than in tropical or sub-tropical climates as evidenced by the numerous ﬁeld studies of pollinizer densities and interplanting distance for effective pollination in temperate climates. This knowledge contrasts with the relatively few tropical studies and the need for more information about the impact of warm, tropical conditions on pollination. Insect pollination in the tropics should focus more on ﬁnding new, effective native pollinators of apple as well as utilizing the already known honeybee. Bees, other than honeybees have been effectively used as pollinators in many parts of the world to provide better pollinating efﬁciencies than those of honeybees. Native bees in South America have not been investigated in depth to determine potentially effective pollinizers of apples under tropical conditions. Insects other than bees should also be considered. Many countries around the world and particularly apple producing counties in South America are lacking pollination studies. This may be due to little attention or interest by the private and governmental sectors. Pollination studies in the tropics should use modern methodologies that clearly prove successful pollination in order to determine that pollen grains are effectively transferred by pollinators. Many investigators assume that pollination is taking place just because insects are visiting the ﬂowers, but they have not determined if pollen deposition or egg fertilization actually occurred. Further research should include molecular markers, such as SSRs or new creative methodologies that can be used to determine the pollen parent. Successful pollination leads to apple fruit set. Understanding the interactions of environmental factors, such as temperature, relative humidity, wind speed and precipitation and their impacts on apple ﬂower and insect behavior are key to understanding pollination from the temperate to tropical growing areas. More investigations, particularly in the tropics, should focus on the effects of temperature on pollen germination. Although Brazil is one of the leading countries investigating pollen germination among the South American countries, there is much to be learned about pollen germination. Further investigations in tropical countries should focus on outcrossing, native insects as pollinators, self-, semi- and full-compatibility systems in local cultivars. Evaluation of pollination-enhancement methodologies, such as artiﬁcial pollination, and studies on the effects of temperature on local pollen viability, pollen-stigma interactions should also be among other research priorities. Without pollination, today’s apple industry would not be as efﬁcient. Our interest is to motivate young researchers, apple growers, horticulturalists and research scholars to take a closer look at apple pollination and deﬁne new, innovative ways that will lead to a better understanding and use of apples in diets throughout the world. Acknowledgement Special thanks to L. Marien for her valuable support. References Abbott, D.L., 1970. The role of budscales in the morphogenesis and dormancy of the apple fruit bud. In: Luckwill, L.C., Cutting, C.V. (Eds.), Physiology of Tree Crops. Academic Press, New York, pp. 64–82. Adachi, Y., Komori, S., Hoshikawa, Y., Tanaka, N., Abe, K., Bessho, H., Watanabe, M., Suzuki, A., 2009. Characteristics of fruiting and pollen tube growth of apple autotetraploid cultivars showing self-compatibility. J. Jpn. Soc. Hortic. Sci. 78, 402–409. Adams, J., 1916. On the germination of the pollen grains of apple and other fruit trees. Bot. Gaz. 61, 131–147. Bagni, N., Adamo, P., Seraﬁni-Fracassini, D., Villanueva, V.R., 1981. RNA, proteins and polyamines during tube growth in germinating apple pollen. Plant Physiol. 68, 727–730. Bashir, R., Sharma, G., Sharma, N., 2010. Studies on fruit set and fruit characteristics as affected by different pollinizers in apple (Malus × domestica Borkh.). Adv. Hort. Sci. 24, 137–144. Berta, G., Altamura, M.M., Fusconi, A., Cerruti, F., Capitani, F., Bagni, N., 1997. The plant cell wall is altered by inhibition of polyamine biosynthesis. New Phytol. 137, 569–577. Berkett, L.P., 1994. Management Guide for Low-input Sustainable Apple Production. USDA. Nothheast LISA Apple Production Project, USA. Botero, N., Morales, G., 2000. Producción del manzano (Malus sp. cv Anna) en el oriente Antioqueño con la abeja melifera, Apis mellifera L. (Hymenoptera: Apidae). Rev. Fac. Nal. Agron. Medellín. 53, 849–862. Boyle, R.M.D., Philogène, B.J.R., 1983. The native pollinators of an apple orchard: variations and signiﬁcance. J. Hortic. Sci. Biotechnol. 58, 355–364. Branscreidt, P., 1930. Zur Physiologic der Pollenkeirnung und ihrer experiinentellen Hceinﬂussung. Planta 11. Broothaerts, W., 2003. New ﬁndings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles. Theor. Appl. Genet. 106, 703–714. Broothaerts, W., Van Nerum, I., Keulemans, J., 2004a. Update on and review of the incompatibility (S-) genotypes of apple cultivars. HortScience 39, 943–947. Broothaerts, W., Keulemans, J., Nerum, I., 2004b. Self-fertile apple resulting from S-RNase gene silencing. Plant Cell Rep. 22, 497–501. Broothaerts, W., Janssens, G.A., Proost, P., Broekaert, W.F., 1995. cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Mol. Biol. 27, 499–511. Cain, J.C., 1971. Effects of mechanical pruning of apple hedgerows with a slotting saw on light penetration and fruiting. J. Am. Soc. Hortic. Sci. 96, 664–667. Calzoni, G.L., Speranza, A., 1982. Effect of methanol and gamma irradiation on enzymatic activity of apple pollen. Sci. Hortic. 17, 231–239. Calzoni, G.L., Speranza, A., Bagni, N., 1979. In vitro germination of apple pollens. Sci. Hortic. 10, 49–55. Campo Dall’Orto, F.A., Barbosa, W., Ojima, M., Ferraz, D.E., Campos, S.A., 1985. Análise do pólen em dezoito cultivares de macieira. Bragantia 41, 421–427. Currie, A.J., Noiton, D.A., Lawes, G.S., Bailey, D., 1997. Preliminary results of differentiating apple sports by pollen ultrastructure. Euphytica 98, 155–161. Dag, A., Stern, R.A., Shaﬁr, S., 2005. Honeybee (Apis mellifera) strains differ in apple (Malus domestica) pollen foraging preference. J. Apicult. Res. 44, 15–20. F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Dantas, A.Cd.M., Nunes, J.Cd.O., Brighenti, E., Ribeiro, L.G., Nodari, R.O., 2001. Efeito da polinizaçăo dirigida entre porta-enxertos de macieira (Malus sp.) na frutiﬁcaçăo efetiva e no desenvolvimento de frutos em Săo Joaquim-SC. Rev. Bras. Fruticult. 23, 497–503. Dantas, A.C.M., Morales, L.K.A., Pedrotti, E.L., Nodari, R.O., Guerra, M.P., 2002. Superação in vitro da dormência de embriões do porta-enxerto de macieira M9 (Malus pumilla Mill.). Rev. Bras. Fruticult. 24, 10–14. Darlington, C.D., Moffett, A.A., 1930. Primary and secondary chromosome balance in Pyrus. J. Gen. 22, 129. Das, B., Ahmad, N., Srivastava, K.K., Ranjan, P., 2011. Top working method and bloom density of pollinizers as productive determinant for spur type apple (Malus × domestica Borkh.) cultivars. Sci. Hortic. 129, 642–648. de Albuquerque, C., Denardi, F., de Mesquita Dantas, A., Nodari, R., 2011. The self-incompatible RNase S-alleles of Brazilian apple cultivars. Euphytica 181, 277–284. de Albuquerque, C.L., Denardi, F., Dantas A.Cd.M. Nodari, R.O., 2010a. Desenvolvimento de tubos polínicos em cruzamentos entre cultivares brasileiras de macieira. Pesqui. Agropecu. Bras. 45, 1324–1327. de Albuquerque, C.L., Denardi, F., Dantas, A.Cd.M., Nodari, R.O., 2010b. Número de anteras por ﬂor, grãos de pólen por antera e capacidade germinativa do pólen de diferentes cultivares de macieiras. Rev. Bras. Fruticult. 32, 1255–1260. Delaplane, K.S., Mayer, D.F., 2000. Crop Pollination by Bees. CABI, New York. De Witte, K., Keulemans, J., 1994. Restrictions of the efﬁciency of haploid plant production in apple cultivar Idared, through parthenogenesis in situ. Euphytica 77, 141–146. De Witte, K., Vercammen, J., van Daele, G., Keulemans, J., 1996. Fruit set, seed set and fruit weight in apple as inﬂuenced by emasculation, self-pollination and cross-pollination. Acta Hortic. 423, 177–183. Dennis, F.G., 1986. Apple. In: Monseline, S.P. (Ed.), Handbook of Fruit Set and Development. CRC Press, Boca Ratón, pp. 1–45. Dennis, F.J., 2003. Flowering, pollination and fruit set and development. In: Ferree, D.C. (Ed.), Apples Botany Production and Uses. CAB International, Cambridge, pp. 153–166. Díaz, M.D., 1993. Calidad de fruto, indices de corte y manejo de postcosecha de la manzana. In: Osorio, G.H., Ríos, L.J., Restrepo, H.J.F. (Eds.), Primer Simposio Internacional Sobre el Manzano. Memorias, Manigraf, Manizales, pp. 130–136. Di Sandro, A., Del Duca, S., Verderio, E., Hargreaves, A.J., Scarpellini, A., Cai, G., Cresti, M., Faleri, C., Iorio, R.A., Hirose, S., Furutani, Y., Coutts, I.G.C., Grifﬁn, M., Bonner, P.L.R., Seraﬁni-Fracassini, D., 2010. An extracellular transglutaminase is required for apple pollen tube growth. Biochem. J. 429, 261–271. Dreesen, R., Vanholme, B., Luyten, K., Van Wynsberghe, L., Fazio, G., Roldán-Ruiz, I., Keulemans, J., 2010. Analysis of Malus S-RNase gene diversity based on a comparative study of old and modern apple cultivars and European wild apple. Mol. Breed. 26, 693–709. Dzhangaliev, A.D., 2010. The Wild Apple Tree of Kazakhstan. Hort. Rev. John Wiley & Sons, Inc., pp. 63–303. Edwards, G.R., Notodimedjo, S., 1987. Defoliation, bending, and tip pruning of apple under tropical conditions. Acta Hortic. 199, 125–127. Endress, P.K., 1994. Diversity and Evolutionary Biology of Tropical Flowers. Cambridge University Press, Cambridge. Florin, R., 1927. Pollen production and incompatibilities in apples and pears. Mem. Hortic. Soc. NY 3. Forsline, P.L., Aldwinckle, H.S., 2004. Evaluation of Malus sieversii populations for disease resistance and horticultural traits. Acta Hortic. 663, 529–534. Forsline, P.L., Aldwinckle, H.S., Dickson, E.E., Luby, J.J., Hokanson, S., 2003. Collection, maintenance, characterization and utilization of wild apples of Central Asia. In: Janick, J., Forsline, P., Dickscon, E., Way, R., Thompson, M. (Eds.), Wild Apple and Fruit Trees of Central Asia. Hort. Rev., pp. 1–61. Foster, T., Johnston, R., Seleznyova, A., 2003. A morphological and quantitative characterization of early ﬂoral development in apple (Malus × domestica Borkh.). Ann. Bot. 92, 199–206. Free, J.B., 1960. The behavior of honeybees visiting ﬂowers of fruit trees. J. Anim. Ecol. 29, 239–385. Gardner, K.E., Ascher, J.S., 2006. Notes on the native bee pollinators in New York apple orchards. J. New York Entomol. Soc. 114, 86–91. Goldschmidt-Reischel, E., 1993. Use of ornamental apples for pollination of dessert cultivars. Acta Agric. Scand. B – Soil Plant Sci. 43, 176–180. Griggs, W.H., Vansell, G.H., Iwakiri, B.T., 1953. Pollen storage. Calif. Agric. 7, 12. Gruber, B., Eckel, K., Everaars, J., Dormann, C., 2011. On managing the red mason bee (Osmia bicornis) in apple orchards. Apidologie 42, 564–576. Guerrero-Prieto, V.M., Rascon-Chu, A., Romo-Chacon, A., Berlanga-Reyes, D.I., Orozco-Avitia, J.A., Gardea-Bejar, A.A., Parra-Quezada, R., Sanchez-Chavez, E., 2009. Short communication. Effective pollination period in ‘Redchief’ and ‘Golden Delicious’ apples (Malus domestica Borkh). Spanish J. Agric. Res. 7, 928–932. Guerrero Prieto, V.M., Romo Chacón, A., Orozco Avitia, J.A., Berlanga Reyes, D.I., Gardea Béjar, A.A., Parra Quezada, R.Á., 2006. Polinización en manzanos Red Delicious y Golden Delicious. Rev. Fitotec. Mex. 29, 41–45. Guevara, H., 1992. Comparative study of natural and artiﬁcial pollination of apple cv. ‘Anna’ in a high region of Costa Rica. Acta Hortic. 310, 127–134. Hancock, J.F., Luby, J.J., Brown, S.K., Lobos, G.A., 2008. Apples. In: Hancock, J.F. (Ed.), Temperate Fruit Crop Breeding. Springer, Michigan, pp. 1–37. Harris, S.A., Robinson, J.P., Juniper, B.E., 2002. Genetic clues to the origin of the apple. Trends Genet. 18, 416–430. Hauagge, R., Bruckner, C.H., 2002. Macieira. In: Bruckner, C.H. (Ed.), Melhoramento de fruteiras de clima temperado. Viçosa, UFV, pp. 27–88. 201 Hegedus, A., 2006. Review of the self-incompatibility in apple (Malus × domestica Borkh., syn.: Malus pumila Mill.). Int. J. Hortic. Sci. 12, 31–36. Heo, S., Han, S., Kwon, S., Jun, J., Kim, M., Lee, H., 2011. Identiﬁcation of S-allele genotypes of Korean apple cultivars by using allele-speciﬁc polymerase chain reaction. Hortic. Environ. Biotechnol. 52, 158–162. Hopping, M.E., Jerram, E.M., 1980. Supplementary pollination of fruit trees. New Zeal. J. Agric. Res. 23, 509–515. Howlett, F.S., 1931. Factors affecting fruit set I Satyman Winesap. In: Ohio Agricultural Experimantal Station Bulletin No 483. Ignatov, A., Bodishevskaya, A., 2011. In: Kole, C. (Ed.), Malus Wild Crop Relatives: Genomic and Breeding Resources. Springer Heidelberg, Berlin, pp. 45–64. Imani, A., Barzegar, K., Piripireivatlou, S., Masomi, S.H., 2011. Storage of apple pollen and in vitro germination. Afr. J. Agric. Res. 6, 624–629. Jackson, J.E., 2003. Biology of Apples and Pears. Cambridge University Press, Cambridge. Janick, J., 1974. The apple in Java. HortScience 9, 13–15. Janick, J., Cummins, J.N., Brown, S.K., Hemmat, M., 1996. Apples. In: Janick, J., Moore, J.N. (Eds.), Fruit Breed, Volume I: Tree and Tropical Fruits. John Wiley & Sons, New York, pp. 1–77. Jefferies, C.J., Brain, P., 1984. A mathematical model of pollen-tube penetration in apple styles. Planta 160, 52–58. Jones, H.G., 1987. Repeat ﬂowering in apple caused by water stress or defoliation. Trees – Struct. Funct. 1, 135–138. Kendall, D.A., 1973. The viability and compatibility of pollen on insects visiting apple blossom. J. Appl. Ecol. 10, 847–853. Kendall, D.A., Smith, B.D., 1975. The foraging behavior of honeybees on ornamental Malus ssp. used as pollinizers in apple orchards. J. Appl. Ecol. 12, 465–471. Keogh, R.C., Robinson, A.P.W., Mullins, I.J., 2010. Pollination aware: the real value of pollination in Australia. Publication No. 10/081. Project No. PRJ-004588. p. 73. Keogh, R.C., Robinson, A.P.W., Mullins, I.J., 2010. Pollination Aware Case Study: Apple. Publication No. 10/109. p. 8. Kim, H., Park, J., Hirata, Y., Nou, I., 2008. Molecular characterization of new S-RNases (‘S-31’ and ‘S-32’) in apple (Malus × domestica Borkh.). J. Plant Biol. 51, 202–208. Kitahara, K., Soejima, J., Komatsu, H., Fukui, H., Matsumoto, S., 2000. Complete sequences of the S-genes, Sd- and Sh-RNase cDNA in apple. HortScience 35, 712–715. Knight, L.J., 1917. Physiological aspects of the self sterility of the apple. P. Am. Soc. Hortic. Sci. 14, 101–105. Ko, K., Brown, S.K., Norelli, J.L., Hrazdina, G., Aldwinckle, H.S., 2010. In vitro pollen functionality of attacin-transgenic Royal Gala apple plants and apples transformed with 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-antisense vector. Plant Biosyst. 144, 778–783. Kobel, F., Steinegger, P., Anliker, J., 1939. Weitere Untersuchungen über die Befruchtungsverhältnisse der Apfelund Birnsorten. Landw. Jahrb. Schweiz. 53, 160–191. Kotoda, N., Hayashi, H., Suzuki, M., Igarashi, M., Hatsuyama, Y., Kidou, S.-I., Igasaki, T., Nishiguchi, M., Yano, K., Shimizu, T., Takahashi, S., Iwanami, H., Moriya, S., Abe, K., 2010. Molecular Characterization of FLOWERING LOCUS T-Like Genes of Apple (Malus × domestica Borkh.). Plant Cell Physiol. 51, 561–575. Koutinas, N., Pepelyankov, G., Lichev, V., 2010. Flower induction and ﬂower bud development in apple and sweet cherry. Biotech. Biotechnol. Equip. 24, 1549–1558. Kozma, P., Nyéki, J., Soltész, M., Szabó, Z., 2003. Floral biology, pollination and fertilisation in temperate zone fruit species and grape. Budapest, Akadémiai Kiadò. Kron, P., Husband, B., 2006. The effects of pollen diversity on plant reproduction: insights from apple. Sex. Plant Reprod. 19, 125–131. Kron, P., Husband, B.C., Kevan, P.G., 2001a. Across- and along-row pollen dispersal in high-density apple orchards: Insights from allozyme markers. J. Hortic. Sci. Biotechnol. 76, 286–294. Kron, P., Husband, B.C., Kevan, P.G., Belaoussoff, S., 2001b. Factors affecting pollen dispersal in high-density apple orchards. HortScience 36, 1039–1046. Kubo, K-I., Entani, T., Takara, A., Wang, N., Fields, A.M., Hua, Z., Toyoda, M., Kawashima, S.-I., Ando, T., Isogai, A.T., Kao, T.-h., Takayama, S., 2010. Collaborative non-self recognition system in S-RNAse-based self-incompatibility. Science 330, 796–799. Kuhn, E.D., Ambrose, J.T., 1984. Pollination of ‘Delicious’ apple by megachilid bees of the genus Osmia (Hymenoptera: Megachilidae). J. Kans. Entomol. Soc. 57, 169–180. Kvaale, E., 1927. Abortive and sterile apple pollen. Mem. Hort. Soc. NY 3. Langridge, D.F., Jenkins, P.T., 1970. The role of honeybees in pollination of apples. Aust. J. Exp. Agric. Anim. Husb. 10, 366–368. Larsen, A., Kjær, E., 2009. Pollen mediated gene ﬂow in a native population of Malus sylvestris and its implications for contemporary gene conservation management. Conserv. Genet. 10, 1637–1646. Larsen, P., Tung, S.M., 1950. Growth-promoting and growth-retarding substances in pollen from diploid and triploid apple varieties. Bot. Gaz. 111, 436–447. Lecuyer, M.P., Zhang, Y.X., Tellier, M., Lespinasse, Y., 1991. In vitro pollen tube division of irradiated and non-irradiated apple pollen. Agronomie 11, 483–489. Long, S., Li, M., Han, Z., Wang, K., Li, T., 2010. Characterization of three new S-alleles and development of an S-allele-speciﬁc PCR system for rapidly identifying the S-genotype in apple cultivars. Tree Genet. Genomes 6, 161–168. Lopriore, G., 1897. Azione dei raggi X sul protoplasma della cellula vegetal vivente. Estr. del Nuova [Rassegna], Catania. Bot. Ztrbl. 73, 451–452. Lenucci, M., Piro, G., Miller, J.G., Dalessandro, G., Fry, S.C., 2005. Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins? Phytochemistry 66, 2581–2594. 202 F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Lerner, R., Hurst, P., 2002. Pollination of Fruits and Nuts, http://www. hort.purdue.edu/ext/ho-174.pdf Li, M., Zhu, K., Bai, S., Liu, Z., Li, T., 2011. Isolation and S-genotyping application of S-allelic polymorphic MdSLFBs in apple (Malus domestica Borkh.). Mol. Breed. 28, 171–180. Li, T.Z., Katoh, N., Miyair, K., Okun, T., 2007. S-RNase is secreted from transmitting tract cells into the intercellular spaces after pollen tubes enter the style in apple (Malus pumila Mill.). J. Hortic. Sci. Biotechnol. 82, 433–438. Looney, N.E., Pharis, R.P., Noma, M., 1985. Promotion of ﬂowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4 . Planta 165, 292–294. McLaughlin, J.M., Greene, D.W., 1984. Effects of BA, GA4+7 , daminozide on fruit set, fruit quality, vegetative growth, ﬂower initiation, and ﬂower quality of ‘Golden Delicious’ apple. J. Am. Soc. Hortic. Sci. 109, 34–39. Maeta, Y., Tezuka, H., Susuki, K., 1992. Utilization of the Brazilian stingless bee, Nannotrigona testaceicornis as a pollinator of strawberries. Honeybee Sci. 13, 71–78. Maggs, D.H., Martin, G.J., Needs, R.A., 1971. The spread of cross-pollination in a solid block of Granny Smith apples. Aust. J. Exp. Agric. Anim. Husb. 11, 113–117. Marcucci, C.M., Ragazzini, D., Sansavini, S., 1984. The effects of gamma and laser rays on the functioning of apple pollen in pollination and mentor pollen experiments. J. Hortic. Sci. 59, 57–61. Marcucci, M.C., Visser, T., Tuyl, J.M., 1982. Pollen and pollination experiments. VI. Heat resistance of pollen. Euphytica 31, 287–290. Matsumoto, S., Komori, S., Kitahara, K., Imazu, S.J.S., 1999. S-genotypes of 15 apple cultivars and self-compatibility of ‘Megumi’. J. Jpn. Soc. Hortic. Sci. 68, 236–241. Matsumoto, S., Furusawa, Y., Kitahara, K., Soejima, J., 2003. Partial genomic sequences of S6-, S12-, S13-, S14-, S17-, S19-, and S21-RNase of apple and their allele designations. Plant Biotechnol. 20, 323–329. Matsumoto, S., Kitaharah, K., Komatsu, H., Abe, K., 2006. Cross-compatibility of apple cultivars possessing S-RNase alleles of similar sequence. J. Hortic. Sci. Biotechnol. 81, 934–936. Matsumoto, S., Eguchi, T., Maejima, T., Komatsu, H., 2008. Effect of distance from early ﬂowering pollinizers ‘Maypole’ and ‘Dolgo’ on ‘Fiji’ fruit set. Sci. Hortic. 117, 151–159. Matsumoto, S., Abe, A., Maejima, T., 2009. Foraging behavior of Osmia cornifrons in an apple orchard. Sci. Hortic. 121, 73–79. Matsumoto, S., Maejima, T., 2010. Several new aspects of the foraging behavior of Osmia cornifrons in an apple orchard. Psyche. Matsumoto, S., Soejima, J., Maejima, T., 2012. Inﬂuence of repeated pollination on seed number and fruit shape of ‘Fuji’ apple. Sci. Hortic. 137, 131–137. Mayer, D.F., Johansen, C.A., Lunden, J.D., 1985. Bee pollination of tree fruits. Paciﬁc Northwest Extension Publication, PNW 0282. pp. 57–70. McClure, B., Cruz-García, F., Romero, C., 2011. Compatibility and incompatibility in S-RNase-based systems. Ann. Bot. 108, 647–658. McGregor, S.E., 1976. Insect Pollination of Cultivated Crop Plants. United States Department of Agriculture, Washington. Milutinovic, M., Surlan-Momirovic, G., Nikolic, D., 1996. Relationship between pollinizer distance and fruit set in apple. Acta Hort. 423, 91–94. Mimida, N., Ureshino, A., Tanaka, N., Shigeta, N., Sato, N., Moriya-Tanaka, Y., Iwanami, H., Honda, C., Suzuki, A., Komori, S., Wada, M., 2011a. Expression patterns of several ﬂoral genes during ﬂower initiation in the apical buds of apple (Malus × domestica Borkh.) revealed by in situ hybridization. Plant Cell Rep. 30, 1485–1492. Mimida, N., Kidou, S-I., Iwanami, H., Moriya, S., Abe, K., Voogd, C., Varkonyi-Gasic, E., Kotoda, N., 2011b. Apple FLOWERING LOCUS T proteins interact with transcription factors implicated in cell growth and organ development. Tree Physiol. 31, 555–566. Minamikawa, M., Kakui, H., Wang, S., Kotoda, N., Kikuchi, S., Koba, T., Sassa, H., 2010. Apple S locus region represents a large cluster of related, polymorphic and pollen-speciﬁc F-box genes. Plant Mol. Biol. 74, 143–154. Modlibowska, I., 1945. Pollen tube growth and embryo-sac development in apples and pears. J. Pomol. Hortic. Sci. 21, 57–89. Montalti, P., Filiti, N., 1984. Mentor pollen effect on the in-vivo germination of selfincompatible apple pollen. Sci. Hortic. 23, 337–343. Monteverde, E.E., 1989. El cultivo del manzano en Venezuela. Fonaiap Divulga 31. Namikawa, I., 1923. Growth of pollen tubes in self-pollinated apple ﬂowers. Bot. Gaz. 76, 302–310. Naor, A., Flaishman, M., Stern, R., Moshe, A., Erez, A., 2003. Temperature effects on dormancy completion of vegetative buds in apple. J. Am. Soc. Hortic. Sci. 128, 636–641. Nicoll, M.F., Chapman, G.P., James, D.J., 1987. Endosperm responses to irradiated pollen in apples. Theoret. Appl. Genet. 74, 508–515. Orozco Corral, A.L., Valverde Flores, M.I., 2010. Polinización artiﬁcial por aspersió en manazanos cv. Gala y Golden Delicious en Chihuahua. www.unifrut.com.mx/ archivos/simposiums/2010/v7.pdf Palmer-Jones, T., Clinch, P.G., 1966. Observations on the pollination of apple trees (Mallus sylvestris Mill.) I variety Cox’s Orange Pippin. New Zeal. J. Agric. Res. 9, 191–196. Palmer-Jones, T., Clinch, P.G., 1967. Observation of the pollination of apple trees (Mallus sylvestris Mill.) II varieties Granny Smith, Sturmer, Jonathan and Cox’s Orange Pippin. New Zeal. J. Agric. Res. 10, 143–149. Palmer-Jones, T., Clinch, P.G., 1968. Observation of the pollination of apple trees (Mallus sylvestris Mill) III varieries Granny Smith, Kid’s Orange Red, and Golden Delicious. New Zeal. J. Agric. Res. 11, 149–154. Pereira-Lorenzo, S., Ramos-Cabrer, A.M., Fischer, M., 2009. Breeding apple (Malus × domestica Borkh). In: Jain, S.M., Priyadarshan, P.M. (Eds.), Breeding Plantation Tree Crops: Temperate Species. Springer, Berlin, pp. 33–81. Petri, J.L., 1993. Fisiología pre y postcosecha del manzano. In: González, O.H., Restrepo, H.J.F., Rios, L.J. (Eds.), Primer simposio internacional sobre el manzano memorias. Manigraf, Manizales, pp. 114–124. Petri, J.L., Hawerroth, F.J., Leite, G.B., 2008. Fenologia de espécies silvestres de macieira como polinizadora das cultivares gala e fuji. Rev. Brasil. Fruticult. 30, 868–874. Pharis, R.P., Looney, N.E., Mander, L.N., 1992. Promotion of ﬂowering of fruit trees. United States Patent. Patent Number 5,085,683. Phillips, M., 2005. The Apple Grower: A Guide for the Organic Orchardist, 2nd ed. Chelsea Green Publishing Company, London. Pratt, C., 1988. Apple ﬂower and fruit: morphology and anatomy. Hort. Rev. 10, 273–307. Racskó, J., Miller, D.D., 2010. Intra-inﬂorescence pattern of the opening of apple (Malus domestica Borkh.) ﬂowers. Int. J. Plant Rep. Biol. 2, 65–77. Ramírez, F., Davenport, T.L., 2010. Mango (Mangifera indica L.) ﬂowering physiology. Sci. Hortic. 126, 65–72. Ramírez, F., Davenport, T.L., 2012. Reproductive Biology (Physiology) – the case of mango. In: Valavi, S.G., Rajmohan, K., Govil, J.N., Peter, K.V., Thottappilly, G. (Eds.), The Mango. Studium Press, LLC, pp. 56–81. Ramírez, H., Hoad, G.V., 1981. Effects of growth substances on fruit-bud initiation in apple. Acta Hortic. 120, 131–136. Robbie, F.A., Atkinson, C.J., 1994. Wood and tree age as factors inﬂuencing the ability of apple ﬂowers to set fruit. J. Hortic. Sci. Biotechnol. 69, 609–623. Roberts, R.H., 1945. Blossom structure and setting of ‘Delicious’ and other apple varieties. Proc. Am. Soc. Hortic. Sci. 46, 90–97. Robinson, J.P., Harris, S.A., Juniper, B.E., 2001. Taxonomy of the genus Malus Mill. (Rosaceae) with emphasis on the cultivated apple, Malus domestica Borkh. Plant Syst. Evol. 226, 35–58. Roldán, A., Arango, X., Aristizábal, M., 1999. Polinización artiﬁcial en áboles de manzana (Malus domestica Borkh.) cv Anna. Fitopatología 25. Rohozinski, J., Edwards, G.R., Hoskyns, P., 1986. Effects of brief exposure to nitrogenous compounds on ﬂoral initiation in apple trees. Physiol. Végétale 24, 673–677. Rybin, V.A., 1926. Cytological investigations of the genus Malus (Preliminary account). Bull. Appl. Bot. 16, 187. Saito, A., Fukasawa-Akada, T., Igarashi, M., Sato, T., Suzuki, M., 2007. Selfcompatibility of 3 apple cultivars and identiﬁcation of S-allele genotypes in their self-pollinated progenies. Hortic. Res. (Japan) 6, 27–32. Sakurai, K., Brown, S.K., Weeden, N., 2000. Self-incompatibility alleles of apple cultivars and advanced selections. Hortscience 35, 116–119. Samish, R.M., Lavee, S., 1962. The chilling requirement of fruit trees. In: Ducolot SA (Ed.), Proceedings of the 16th International Horiulture Congress. Gembloux, Belgim, pp. 372–388. Sanders, R., 2010. The Apple Book. Frances Lincoln Limited, London. Sanzol, J., Herrero, M., 2001. The effective pollination period in fruit trees. Sci. Hortic. 90, 1–17. Sassa, H., Kakui, H., Miyamoto, M., Suzuki, Y., Hanada, T., Ushijima, K., Kusaba, M., Hirano, H., Koba, T., 2007. S Locus F-box brothers: multiple and pollen-speciﬁc F-box genes with S haplotype-speciﬁc polymorphisms in apple and Japanese pear. Genetics 175, 1869–1881. Schneider, D., Stern, R.A., Goldway, M., 2005. A comparison between semi- and fully compatible apple pollinators grown under suboptimal pollination conditions. HortScience 40, 1280–1282. Schneider, D., Stern, R.A., Eisikowitch, D., Goldway, M., 2002. The relationship between ﬂoral structure and honeybee pollination efﬁciency in ‘Jonathan’ and ‘Topred’ apple cultivars. J. Hortic. Sci. Biotechnol. 77, 48–51. Schwarz, H., 1994. Propagación: especies y variedades. In: Sarmiento, A.N. (Ed.), Frutales caducifolios. ICA, Bogotá, pp. 41–54. Sedgley, M., 1990. Flowering of deciduous perennial fruit crops. Hortic. Rev. 12, 223–264. Sekita, N., 2001. Managing Osmia cornifrons to pollinate apples in Aomori Prefecture, Japan. Acta Hortic. 561, 303–307. Sestili, S., Ficcadenti, N., 1996. Irradiated pollen for haploid production. In: Mohan Jain, S., Sopory, S.K., Veilleux, R.E. (Eds.), Invitro Haploid Production in Higher Plants: Volume 1. Fundamental Aspects and Methods. Kluwer Academic Publishing, Dordrecht, pp. 263–274. Shefﬁeld, C.S., Smith, R.F., Kevan, P.G., 2005. Perfect syncarpy in apple (Malus × domestica ‘Summerland McIntosh’) and its implications for pollination, seed distribution and fruit production (Rosaceae: Maloideae). Ann. Bot. 95, 583–591. Shefﬁeld, C.S., Kevan, P.G., Westby, S.M., Smith, R.F., 2008. Diversity of cavitynesting bees (Hymenoptera: Apoidea) within apple orchards and wild habitats in the Annapolis Valley, Nova Scotia, Canada. Can. Entomol. 140, 235–249. Shefﬁeld, C.S., Kevan, P.G., Smith, R.F., Rigby, S.M., Rogers, R.E.L., 2003. Bee species of Nova Scotia, Canada, with new records and notes on bionomics and ﬂoral relations (Hymenoptera: Apoidea). J. Kans. Entomol. Soc. 76, 357–384. Somerville, D., White, B., 2005. Pollination of apples by honeybees. Agnote, DAI-132. NSW Department of Primary Industry. Speranza, A., Calzoni, G.L., 1980. Compounds released from incompatible apple pollen during in vitro germination. Z. Pﬂanzenzucht. 97, 95–102. F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203 Speranza, A., Calzoni, G.L., Cresti, M., Ciampolini, F., 1982. Effects of gamma irradiation on in vitro germination and ultrastructure of apple pollen. Env. Exp. Bot. 22, 339–347. Stern, R.A., Eisikowitch, D., Dag, A., 2001. Sequential introduction of honeybee colonies and doubling their density increases cross-pollination, fruit-set and yield in ‘Red Delicious’ apple. J. Hortic. Sci. Biotechnol. 76, 17–23. Stösser, R., Hartmann, W., Anvari, S.F., 1996. General aspects of pollination and fertilization of pome and stone fruit. Acta Hortic. 423, 15–22. Stott, K.G., 1972. Pollen germination and pollen-tube characteristics in a range of apple cultivars. J. Hortic. Sci. Biotechnol. 47, 191–198. Torchio, P.F., Asensio, E., 1985. The introduction of the European bee, Osmia cornuta Latr, into the US as a potential pollinator of orchard crops, and a comparison of its manageability with Osmia lignaria propinqua Cresson (Hymenoptera, Megachilidae). J. Kansas Entomol. Soc. 58, 42–52. Torchio, P.F., Asensio, E., Thorp, R.W., 1987. Introduction of the European bee, Osmia cornuta, into California almond orchards (Hymenoptera, Megachilidae). Environ. Entomol. 16, 664–667. Tränkner, C., Lehmann, S., Hoenicka, H., Hanke, M.-V., Fladung, M., Lenhardt, D., Dunemann, F., Gau, A., Schlangen, K., Malnoy, M., Flachowsky, H., 2010. Overexpression of an FT-homologous gene of apple induces early ﬂowering in annual and perennial plants. Planta 232, 1309–1324. Tromp, J., 1980. Flower-bud formation in apple under various day and night temperature regimes. Sci. Hortic. 13, 235–243. Tupý, J., 1959. Callose formation in pollen tubes and incompatibility. Biol. Plant. 1, 192–198 [Prague]. Van Marrewijk, G.A., 1993. Flowering biology and hybrid varieties. In: International Course on Applied Plant Breeding. IAC, The Netherlands. Visser, T., Verhaegh, J.J., 1980. Pollen and pollination experiments. II. The inﬂuence of the ﬁrst pollination on the effectiveness of the second one in apple. Euphytica 29, 385–390. Visser, T., Oost, E.H., 1981. Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage. Euphytica 30, 65–70. 203 Waite. M.B., 1865. The pollination of pear ﬂowers. Bulletin 5, Div. Veg. Path. U.S. Dept. Agric. pp. 110; pls. XII. 1894. Pollination of Pomaceous Fruits. Warmund, M.R., 2002. Pollinating Fruits Crops. Horticultural Guide. University of Missoury, Columbia. Watson., J.C., Wolf, A.T., Ascher, J.S., 2011. Forested landscapes promote richness and abundance of native bees (Hymenoptera: Apoidea: Anthophila) in Wisconsin apple orchards. Environ. Entomol. 40, 621–632. Wei, S-G., Wang, R., Smirle, M.J., Xu, H-L., 2002. Release of Osmia excavata and Osmia jacoti (Hymenoptera: Megachilidae) for apple pollination. Can. Entomol. 134, 369–380. Wilkie, J.D., Sedgley, M., Olesen, T., 2008. Regulation of ﬂoral initiation in horticultural trees. J. Exp. Bot. 59, 3215–3228. Williams, R.R., 1966. Pollination studies in fruit trees. III. The effective pollination period for some apple and pear varieties. Report of Long Ashton Research Station 1966, 136–238. Williams, R.R., 1970. In: Williams, R.R., Wilson, D. (Eds.), Towards Regulated Cropping: A Report of Recent Fruit-set Experiments in British Orchards. Grower Books, London, pp. 57–61. Williams, R.R., Maier, M., 1977. Pseudo-compatibility after self-pollination of the apple ‘Cox’s Orange Pippin’. J. Hortic. Sci. and Biotech. 52, 475–484. Yamane, H., Tao, R., 2009. Molecular basis of self-(in)compatibility and current status of S-genotyping in Rosaceous Fruit Trees. J. Jpn. Soc. Hortic. Sci. 78, 137–157. Yoder, K., Yuan, R., Combs, L., Byers, R., McFerson, J., Schmidt, T., 2009. Effects of temperature and the Combination of Liquid Lime Sulfur and Fish Oil on Pollen Germination, Pollen Tube Growth, and Fruit Set in Apples. HortScience 44, 1277–1283. Yuda, E., Utsunomiya, N., Kubota, N., 1991. Seed formation by self-pollination of ‘Rome Beauty’ apple in East Java. Jpn. J. Trop. Agric. 35, 289–293. Zhang, Y.X., Lespinasse, Y., 1991. Pollination with gamma-irradiation pollen and development of fruits, seeds and parthenogenetic plants in apple. Euphytica 54, 101–109. Zhou, Z.Q., 1999. The apple genetic resources in China: the wild species and their distributions, informative characteristics and utilisation. Genetic Resources and Crop Evolution 46, 599–609.