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Apple pollinitation Review

Scientia Horticulturae 162 (2013) 188–203
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Apple pollination: A review
Fernando Ramírez a,∗ , Thomas Lee Davenport 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
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.
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] (T.L. Davenport).
0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
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
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
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
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
F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203
Table 1
Pollen irradiation effects.
Erovan, Golden Delicious
R1-49 and X6677
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
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
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.,
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
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
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
F. Ramírez, T.L. Davenport / Scientia Horticulturae 162 (2013) 188–203
8. Self incompatibility, semi compatibility and
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
Table 2
Self incompatible, pseudo compatible and compatible alleles. Data represents most up to date corrected information.
Self incompatible alleles
20 cultivars
137 diploid and 14 triploid cultivars
Northern Spy
Ralls Janet
Golden Delicious
Seven cultivars
York Imperial
Stark Earliblazel
20 cultivars
Daiane, Imperatriz and Princesa
Joaquina and Fred Hough
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
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)
Williams and Maier (1977)
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
Doud Golden Delicious
Sweden Spartan
Sweden Alpha 68A
Welday Jonathan
S2 S9
S2 S2 S3 S3
S9 S9 S10 S10
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
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
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
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.,
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.,
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
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
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.
Special thanks to L. Marien for her valuable support.
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