The Biology of Malus domestica Borkh. (Apple)
4.1 Reproductive biology
M. domestica reproduces naturally by means of seed. Most cultivars rely on pollination to produce fertilized seeds but some cultivars can produce unfertilized (apomictic) seeds (Westwood 1993). Some cultivars can also reproduce vegetatively by root suckering (Hancock et al. 2008; Westwood 1993). The majority of cultivars cannot self-pollinate due to a multi-allelic S-locus (S-RNase) - mediated gametophytic self-incompatibility mechanism (Sassa et al. 1994). Because of this self-incompatibility, the majority of cultivars display high levels of allelic heterozygosity and thus when propagated from seed, are not true-to-type, in that they are extremely variable and generally bear fruits of poor size, appearance and quality (Webster and Wertheim 2003). Incompatibility genes are sufficiently disparate between cultivars that almost all cultivars are cross-fertile. Much study has gone into this area of research and compatibility lists are generally available (Kemp 1996). For consistent cropping, it is recommended that about 10% of an orchard area be devoted to pollinizer (pollen donor) cultivars. These pollinizer cultivars can be either another compatible apple cultivar or a specialized crabapple pollinizer cultivar (Maggs et al. 1971; Westwood 1993). Crabapples are commonly used as an alternative or additive source of pollen because they are heavy bloomers and provide a large source of compatible pollen. M. domestica flowers are predominantly insect-pollinated, mainly by bees when grown as a commercial crop (see Section 4.4). Mature pollen grains have 3 germinal furrows and are rugulate, having folds or wrinkles (Pratt 1988). Pollen grains are large and heavy resulting in very little wind pollination.
Flower development takes roughly 10 months, beginning with the transition from vegetative to reproductive development (late June in the Northern Hemisphere) and ending with anthesis (late April to the beginning of May) in the subsequent year (Dennis 2003; Kotoda et al. 2000). Flowering occurs in early spring when white to deep pink flowers develop in a cyme-like inflorescence of 4-6 flowers. The centre flower which opens first is often referred to as the "king bloom". Most flowers are borne terminally on spurs and less frequently, laterally on long shoots. Flowers borne on short spurs begin the transformation from vegetative buds to flower buds 4-6 weeks before lateral buds (Jackson 2003). The flowers are hermaphroditic with the ovary embedded in the floral cup and flower parts located above the ovary. A normal flower contains five carpels each with two ovules, five sepals, petals and styles and usually 20 stamens (Dennis 2003; Rieger 2006). Flowering can be affected by many biotic (endogenous phytohormones, previous year's crop load, pathogens and pests) and abiotic (light, water stress, nutrients, temperature and exogenously applied chemicals) factors as well as cultural practices including: grafting, pruning, scoring and/or ringing the base of the tree (Jackson 2003).
The stigma produces extracellular secretions which provide a moist environment for pollen deposition and germination (Jackson 2003). Once the pollen grains have germinated, the pollen tubes grow down the style into an ovule where fertilization of the egg cell (to form a zygote) and polar nuclei of the egg sac (to form the endosperm) occur (Dennis 2003). Pollen fertility of most apple cultivars is close to 100% but is reduced in some cultivars, such as 'McIntosh', by unknown factors and in others, such as 'Jonagold', by triploidy. The period of flowering during which viable pollen is produced varies depending on weather conditions and generally lasts from 7 to 30 days. The effective pollination time, the period during which the ovule is capable of being fertilized minus the time required for the pollen tube to grow from the stigma to the ovule, varies from 2 to 9 days (Pratt 1988). Thus the longevity of ovules is a limiting factor in fruit set.
Fruit reach maturity 120-150 days after bloom for most cultivars and weigh about 150-350 grams (Rieger 2006). Fruit development can be divided into three stages: (i) the first 25 days when petals fall, fruit growth is rapid, embryo in the seed develop slowly and growth is predominated by cell division, (ii) the next 50 days (up to 75 days after petal fall) when the embryo develops rapidly and the fruit approaches its final size with growth predominated by cell enlargement, and (iii) the last roughly 14 days (up to 90 days after petal fall) during which the seed testa turns brown and the fruit enlarges slightly, ripens and, in some cultivars, falls (Pratt 1988). In cultivation, about 1 to 5% of apple flowers develop into mature fruit. The others fail because of lack of pollination, competition between fruits or cultural practices (i.e., thinning to promote fruit size and quality and discourage biennial bearing). Biennial bearing results from heavy cropping in one year which acts to inhibit flower bud initiation and reduce flowering in the second year (Jackson 2003). Fruit is picked between early August and late November in north temperate zones and can be stored for up to a year depending on the cultivar. A large, mature, cultivated apple tree can produce in the order of 2000 fruits per year, potentially yielding 10,000 seeds, and may live for 50 years or longer, theoretically producing 500,000 seeds in its lifetime.
Commercial apple trees are composed of genetically distinct portions. The rootstock constitutes the root system including a small portion of the lower trunk, while the scion comprises the majority of the above-ground tissues, including the fruit bearing portion of the tree. Grafting or budding techniques are typically the process by which the scion is joined to the rootstock (Webster and Wertheim 2003). As mentioned in section 4.1, because cultivars are of a heterozygous nature, fruits resulting from open pollinations bear highly variable seeds and this precludes the propagation of apple cultivars by seed. Instead, the cultivation and maintenance of M. domestica cultivars with desirable characteristics relies on vegetative propagation methods such as grafting, layering or other clonal methods rather than seeds (Dennis 2003; Rom and Carlson 1987). The principal reason for the use of rootstocks is for the clonal propagation of desirable cultivars (Webster and Wertheim 2003). There are also other important factors conferred to the grafted tree from the rootstock. Rootstocks can affect the vigour of vegetative growth, fruit size, fruit growth, precocity and yield of the scion portion of the tree. The rootstock can also play a role in the susceptibility of a tree to biotic and abiotic stresses (Webster and Wertheim 2003). As cultivated apple is a composite, some cultivars are bred based on selection of their qualities to the fruiting scion portion and others are developed focusing on the important attributes of desirable rootstock.
Some cultivars are available in various strains, which differ from the original cultivar in growth habit, fruit color or time to maturity. Strains are thought to occur from natural bud mutations in established cultivars that are selected and propagated for improved characteristics. Some cultivars have many strains, for example approximately 250 different strains of Red Delicious have been described and cultivated (Penn State University 2013).
In modern breeding programs, major objectives include increasing the quality or marketability of fruit, reducing production costs and increasing resistance to pests (Hancock et al. 2008; Laurens 1999).
There are several characteristics of M. domestica that inhibit rapid genetic improvement of cultivars, most notably: a long juvenile period, large tree size, and self-incompatibility, in conjunction with high allelic heterozygosity and inbreeding depression (Brown and Maloney 2003). The traditional breeding method that is most commonly used is a modified backcross, where a different recurrent parent is used in each generation of backcrossing. This process is laborious and time consuming; for example, it took several decades to successfully introgress a scab resistance trait from crabapple into a commercial cultivar (Gessler and Pertot 2012). Some traits, for instance fruit quality, are affected by many environmental and genetic factors, making breeding efforts towards their development and improvement even more difficult (Brown 2012; Kumar et al. 2012a). Marker-assisted selection is a strategy used in modern breeding programs that employs the use of genetic markers associated with a specific trait to inform the breeding process with the goal of integrating that specific marker/trait into new cultivars. There are several advantages to using a marker-assisted approach over a phenotypic evaluation, namely removing the environmental influences from the screening process and early screening (typically at the seedling stage) which translates into savings in overall breeding costs (i.e., land area requirements, maintenance, field evaluations) (Khan et al. 2012). While marker-assisted selection has been effective in integrating large-effect loci (e.g., red-fleshed fruit) into apple breeding programs (Chagné et al. 2007), it has proved difficult to develop the many traits associated with multiple small-effects genes, so-called quantitative or polygenic traits (Kumar et al. 2012a).
The genome of M. domestica was recently sequenced (Velasco et al. 2010). This has led to an increase in trait-marker linkage efforts. Of note, large-scale, multi-year projects aimed at bridging the gap between molecular genetic research and breeding (e.g., RosBREED, Fruit Breedomics Consortium) are currently underway (Fruit breedomics consortium 2012; Iezzoni et al. 2010), and significant implications for genetic improvement have already been reported. Chagné et al. (2012) used genomic sequence data from 27 globally important apple cultivars (produced by RosBREED) for the development of a broad coverage single nucleotide polymorphism (SNP) array (Chagné et al. 2012), and assembly of other SNP arrays has also been reported (Khan et al. 2012). Development of such tools allows for novel methods of genetic improvement, such as Genomic Selection, a statistical approach to estimate breeding potential. Genomic Selection has been effective at directing breeding for complex polygenic traits (Jannink et al. 2010) and has recently been successfully used to direct fruit quality trait breeding in apple (Kumar et al. 2012b).
Recent developments using transgenic approaches are addressing some of the breeding bottlenecks. Successful reduction of the juvenile period was reported using a transgenic approach (Flachowsky et al. 2007). Integration and constitutive expression of the mads4 gene from silver birch resulted in a significant decrease in age at flowering, with transformed tissue flowering in tissue culture and a generational turnover of one year. The usefulness of this early-flowering trait in expediting plant breeding was further demonstrated by using a marker-assisted approach to increasing resistance to pyramid fire blight, apple scab and powdery mildew over a very short two year span (Flachowsky et al. 2011). This technology is currently being implemented as a tool to reduce trait introgression time for cultivar development by the Fruit Breedomics Consortium (Fruit breedomics consortium 2012). Another recent targeted gene transfer approach to breeding involved the development of marker-free scab resistance events in the non- resistant cultivar 'Gala' (Vanblaere et al. 2011). This study is significant because the research was designed using a cisgenic approach, the first successful report of such an approach in any crop. Cisgenic differs from transgenic recombinant DNA technology, in that the entirety of the introduced genes (including the flanking regions, promoter, introns, exons and terminator sequences in sense orientation) are derived from a crossable donor plant, in this case from M. floribunda, a species of crabapple that shows natural resistance to some strains of the apple scab fungus (Vanblaere et al. 2011).
4.3 Cultivation and use as a crop
The majority of trees planted in commercial apple orchards are produced in specialized fruit tree nurseries. However, there are some growers who produce their own nursery trees. Rootstocks may be chosen that reduce the vigour of the scions, although the ultimate size of a cultivated apple tree at maturity is also influenced by the inherent vigour of the scion as well as environmental conditions and pruning (Webster and Wertheim 2003). Trees are generally considered standard (100%), semi-dwarf (60 to 85% of standard size) or dwarf (20 to 50% of standard size) (Parker 1993). The advantages of dwarf trees are that they can be planted at higher densities, trained, pruned and harvested from the ground, have greater pesticide application efficiency and produce fruit earlier than standard trees, which is appealing to growers as there is an increased economic benefit from earlier production (Parker and Unrath 1998). In Canada, there has been a shift towards planting high density commercial apple orchards over the last decade (AAFC 2012). There is more initial cost and labour associated with high density orchards, as the smaller branch framework of the dwarf trees cannot support the heavy crop. To overcome this, a number of different support/training systems have been developed including wooden props, super spindle, slender spindle, vertical axis and vertical trellis (Parker and Unrath 1998; Sanders 1994; van Dalfsen 1989) . Although the general trend in recent years is high density planting of dwarf trees, some semi-dwarf and standard trees are still planted in areas where the climate and soil conditions are unsuited to dwarfing rootstock (Webster and Wertheim 2003). In Canada, apple trees are typically planted in the spring, at densities of 150 to 250 trees/acre for standard trees to densities of 500 to 2000+ trees/acre for dwarf trees, depending on the rootstock and the type of training system used (Parker and Unrath 1998; Sanders 1994; van Dalfsen 1989) . In addition to rootstock and training system, there are a number of other factors that are considered when planting an orchard, including main cultivar and pollinating cultivar(s), soil type and fertility, irrigation, soil drainage, and the spacing between rows of trees as well as between trees in a row.
Trees must transition from a juvenile phase to an adult phase in order to flower and bear fruit. There are several physiological and morphological characteristics that define the juvenile phase including: glabrous, lobed leaves, creeping stems, thorny stems, semi-evergreen nature (in deciduous species), ease of rooting of stems, and less ribonucleic acid in their tissues (Westwood 1993). Mature grafts or buds are typically used for scion propagation and the subsequent vegetative growth consists of mature phase tissue (Westwood 1993). However, newly planted nursery trees undergo a non-flowering phase known as the vegetative adult phase (Westwood 1993). The type of rootstock used has a direct effect on the precocity of the scion portion, though the exact mechanism by which this process occurs is still uncertain (Webster and Wertheim 2003). Scions on dwarfing rootstocks begin to bear fruit approximately 2 to 4 years after planting, scions on semi-dwarfing rootstocks at approximately 2 to 6 years after planting and scions on vigorous rootstocks at approximately 6 to 10 years after planting (Parker 1993). Although no commercial crop is produced during the adult vegetative phase, there are still a number of agronomic issues that must be properly managed to ensure good tree growth including nutrition (through fertigation or soil application), pest control, irrigation, weed control and pruning (Sanders 1994).
M. domestica is a very labour intensive, highly managed crop, especially when the orchard has reached maturity and is ready for commercial production. Table 1 outlines the highlights of a typical management schedule for apple production in Canada.
|Time of Year||Action|
Winter - dormancy (December to late April)
Spring- green tip to fruit set (late March to May)
Summer - fruit growth
Fall - harvest period
The average life expectancy of a commercial high-density apple orchard is less than 20 years, although many orchards do maintain an adequate level of production beyond this age (Nova Scotia Department of Agriculture 2009; H. Ardiel, pers. comm.). The life expectancy of a commercial orchard is dependent on a number of factors, such as tree health, apple cultivar, soil quality, environmental location (i.e., heat units, winter injury), market opportunities etc. Changes in consumer preferences for certain apple varieties may also play a role in the decision to replant an orchard.
Fresh fruit is the primary use of cultivated apple in Canada and worldwide (AAFC 2011; Jackson 2003; O'Rourke 2003). Once harvested, the fruit can be stored in controlled atmosphere storage for up to a year (Janick et al. 1996). Apples are also used for juice and juice concentrate, alcoholic cider, fresh apple slices, pie filling, apple sauce, fruit leather, dehydrated fruit bars and other products. By-products of manufacturing, such as pomace left over from juice production, may be fed to livestock, wild animals or used as a food ingredient in, for example, baked goods, for extraction of ester flavours, etc. The apple fruit is notable for its nutritional qualities. It is a natural snack food with low fat content and sugar content of 11-16%; it is also a good source of potassium and soluble fibre, including pectin and other complex carbohydrates and phenol antioxidants (Vinson et al. 2001). There is evidence that regular consumption of apples as part of a healthy diet may aid in the maintenance of good health and prevention of chronic disease (Boyer and Liu 2004).
In Canada, apple fruit from commercial orchards is marketed locally, nationally and internationally. In 2010, the total marketed production of apples in Canada was 336, 834 metric tonnes (mt) (AAFC 2012). In 2010, Canada exported 25, 969 mt of fresh apple fruit to various countries including the United States (81%), United Kingdom (7%), Mexico (6%) and Taiwan (5%) (AAFC 2012). In 2010, Canada imported 191, 714 mt of fresh apple fruit from the United States (79%), Chile (13%), New Zealand (4%), China (2%) and South Africa (1%).
4.4 Gene flow during commercial fruit production
Insects, most notably honeybees but also bumblebees, other wild bees and to a lesser extent some flies, are the primary vectors for pollination in commercial apple orchards as apple pollen is heavy and not easily carried by the wind (Dennis 2003; Jackson 2003). In apple orchards, the majority of honeybee foraging flights are between flowers on the same tree and secondarily between adjacent trees in the same row and to a lesser extent across rows (Free 1966; Free and Spencer-Booth 1964). Growers typically rent honey bees while an orchard is in bloom, and it is recommended that they be placed at a density of four or five strong colonies per hectare in a mature orchard (Dennis 2003).
Within a commercial orchard, it is recommended that a small percentage (about 10%) of the trees are of a different, compatible cultivar to ensure sufficient availability of suitable pollen required for fruit set (Westwood 1993). Spacing of these pollinizer trees in the orchard can vary, being planted as entire rows, interspersed trees or grafts. If pollinizer trees are unable to produce an adequate supply of pollen, pollen may alternatively be introduced by placing flowering branches, also known as bouquets, from other cultivars throughout the orchard.
Pollen transfer has been studied both for optimizing the layout of an orchard (Kron et al. 2001; Maggs et al. 1971; Wertheim 1991) and to assess the potential for gene flow in Malus spp. (Larsen and Kjær 2009; Reim et al. 2006; Soejima 2007; Tyson et al. 2011) . Maggs et al. (1971) found that in a solid-block planted orchard of the 'Granny Smith' cultivar supplied with flowering bouquets of a compatible pollen source, the maximum pollination range was roughly 12 m from the pollen source. Using the 'Baskatong' cultivar, which carries a dominant gene for red leaf colour, Wertheim (1991) monitored gene flow in apple orchards by observing the percentages of red leafed seedlings borne from trees at increasing distances from the 'Baskatong' pollinizer. Findings from this study suggest that the majority of cross-pollination occurs within 15 m from the pollinizer tree. A similar approach, using red leaf colour as a marker to monitor gene flow in an orchard-like scenario, was taken by Reim et al. (2006) who determined that 69% of seeds fertilized by pollen donor plants occurred within the first 10 m and 91% of seeds fertilized by the pollen donor plants occurred within 60 m of the pollen donor plants. Also using a red-leaf screening approach to monitor gene flow in an apple orchard setting, Soejima (2007) determined that roughly 65% of seeds fertilized by pollen donor plants occurred within 8 m and roughly 95% of seeds fertilized by pollen donors occurred within 60 m of the pollen donor tree.
In a survey of a wild population of the crabapple M. sylvestris, Larsen and Kjaer (2009) used microsattelite loci data in conjunction with spatial distances of the individual trees in the population to monitor pollen movement in a natural environment. These authors found that successful pollination occurred mostly between nearby trees, with a median distance of about 23 m (Larsen and Kjær 2009). Kron et al. (2001) used allozyme markers to determine parentage of seeds in an effort to track pollen flow in orchards. An allozyme marker from the cultivar 'Idared' was chosen to screen seeds from open pollinated fruit resulting on other cultivars at increasing distances from the 'Idared' cultivar rows. Pollen dispersal generally declined with increasing distance, with 50% of the total seeds sired by 'Idared' occurring within the first four rows, or roughly 20m (Kron et al. 2001).
Monitoring for beta-glucuronidase (GUS) activity in seeds borne from trees located at increasing distances from a row of transgenic GUS-expressing 'Gala' trees was used to track pollen flow in an orchard setting (Tyson et al. 2011). These authors found that 95% of the transgenic seeds observed were found within 15 m of the transgenic row of apples (Tyson et al. 2011). Taken together, these data suggest that the majority of cross-pollination in commercial apple orchards occurs between receptive flowers and proximate pollen sources; however some of these studies have also observed pollen flow at much greater distances. Pollen dispersal distances were reported up to 40 m (Wertheim 1991), 86 m (Kron et al. 2001), 104 m (Reim et al. 2006), 137 m (Tyson et al. 2011) and 150 m (Soejima 2007) in orchard settings, and almost 300m (Larsen and Kjær 2009) in natural settings. In-hive transfer of viable pollen between bees foraging in geographically distant areas may explain long-range pollen flow (DeGrandi-Hoffman et al. 1986).
In all of these aforementioned studies, distance from the pollen source was a significant factor affecting pollination. Other important factors include weather, pollinator presence, cultivar compatibility and flowering synchrony (Kron et al. 2001). Tyson et al. (2011) have developed a mechanistic model, informed from the transgenic-GUS pollen flow data, to predict bee-vectored pollen transfer. The authors further use the model to examine the effect of buffer rows and isolation distances on outcrossing rates. The model demonstrates that the level of outcrossing is affected by the relative sizes of the transgenic and conventional orchards. As the size of the conventional orchard becomes smaller relative to the transgenic orchard, the isolation distance required to limit the frequency of outcrossing is increased. Furthermore, incorporation of buffer rows between the two orchard types generally reduces the isolation distance required in order to limit outcrossing frequency (Tyson et al. 2011).
4.5 Cultivated Malus domestica as a volunteer weed
M. domestica is not regarded as a weedy species although seedlings can be persistent and the species has escaped cultivation and naturalized in both the U.S. and Canada (Brouillet et al. 2010+; CFIA and NRCan/CFS 2011+; Kartesz 1999; Scoggan 1979; Stover and Marks 1998; USDA-NRCS 2012). Generally, volunteer plants originating from seed in apple orchards are very rare due to the perennial nature of the crop and associated orchard management practices, including herbicide treatment of the tree row and mowing of the alley between rows. In a study conducted in New York state, M. domestica was reported to be a successional species in abandoned pasture but not in abandoned cultivated fields (Stover and Marks 1998).
4.5.1 Cultural/mechanical control
M. domestica, both in commercial orchards and in feral populations, can be removed by cutting at the base of the stump. The roots must be killed by physical removal of the stump in order to prevent regeneration through suckering.
4.5.2 Chemical control
Similarly to the cultural method mentioned above, apple trees can be removed by cutting at the base of the stump. In order to eliminate re-growth, a herbicide is applied to the exposed cambium area of the stump.
Typically in commercial orchards, herbicide is applied to the tree row to control weeds that would compete with the tree roots for moisture and nutrients. This herbicide application would also control any volunteer apple that may have germinated. There are different herbicide options, comprising different modes of action, available for use in commercial apple production (for a more detailed list, refer to Table 13 in AAFC (2011)).
4.5.3 Integrated weed management
Integrated weed management (IWM) incorporates mechanical, chemical and cultural weed control methods to achieve optimal crop yields (Swanton and Weise 1991). As volunteer apple trees are not a concern in commercial production, IWM methods have not been developed for control.
4.5.4 Biological control
Volunteer apple trees are not a concern in commercial production, thus methods of biological control for apple have not been developed.
4.6 Means of movement and dispersal
The main means of movement and dispersal of apple seeds in natural settings is through frugivorous mammals, such as bears, foxes and deer (Myers et al. 2004; Willson 1993), as well as birds (Witmer 1996). The seeds are small enough to sometimes avoid mastication during consumption by white-tailed deer and can pass unaffected through the digestive system and remain germinable (Myers et al. 2004). White-tailed deer travel a range of many hectares on a daily basis and are considered dispersers of low numbers of apple seeds (Myers et al. 2004; Williams and Ward undated) (see Figure 2). Aside from the natural means of movement and dispersal, apple seeds are dispersed by humans as a result of the sale of marketable fruit.
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