Propagation in vitro

Propagation in vitro provides a method of rapid propagation of clonal plant material. It has a number of specific uses in the production of apple and pear plants which extend and complement the traditional means by which these, both scions and rootstocks, are vegetatively propagated. It also provides the reliable and efficient regeneration systems from somatic tissues that are essential to the development of systems of transfer of individual genes in the process of genetic engineering.

Shoot culture involves the use of explants which may be nodal buds or shoot tips ranging from 0.3 mm to 1.0 cm. Explants are surface-sterilized, usually by washing in solutions of sodium or calcium hypochlorite. They are then cultured in a medium based on that of Murashige and Skoog (1962), containing mineral salts, sucrose, cytokinin and possibly some auxin and gibberellin, and solidified with agar. Sorbitol may be more effective than sucrose with some apple cultivars and phloridzin or its breakdown product phloroglucinol may increase shoot growth (Jones, 1993). Shoot cultures are maintained on the culture medium in illuminated growth rooms. Their axillary buds extend to give new shoots which are excised at approximately monthly intervals and transferred to a fresh medium where they in turn produce axillary shoots. Shoot culture lines may be multiplied indefinitely by sequential subculture.

Shoot cultures may become slow-growing with tightly-rolled translucent leaves. This condition is known as vitrification.

Introduction

Most of the water taken up by fruit trees is in response to evaporative loss (transpiration) through the leaf pores or stomata. This is a consequence of the need for stomata to be open to admit CO2 entry for photosynthesis.

Over any given period the fruit tree usually takes up more than 100 times as much water as it produces dry matter (Lenz, 1986). A cropping orchard in Washington State, USA requires up to 1 m of irrigation per year (Evans, 1982). This is 10 000 t ha−1 as compared with about 42.5 t of water in the fruits of a 50 t ha−1 crop. The close correlation between water use and crop yield that is generally observed is not a result of water use in the actual production of the crop. It is primarily a consequence of the close correlation between CO2 assimilation and water loss, as a result of the dependence of both of these on leaf area and stomatal behaviour.

However, the ability to keep stomata open for CO2 assimilation and to avoid consequent desiccation depends on the adequacy of the supply of water as well as mechanisms for controlling water stress. To this extent water supply is a truly limiting factor to crop production.

The water flux through the soil–tree–air system is under tension, i.e. negative pressure. Tree tissues and cells equilibrate with this tension and different aspects of growth, development and function respond to this in different ways.

Introduction

The use of dwarfing rootstocks for the control of tree vigour is a dominant feature of much of modern apple production and, to a lesser extent, pear production. The mechanism of vigour control by rootstocks has been studied for many years, partly in the hope that understanding the process will lead to its more effective utilization.

The effects of the rootstocks and interstocks are manifold, interactive and cumulative over years. Moreover, different mechanisms appear to be dominant in different species and even in different stock/scion cultivar combinations within a species. In practical terms the dwarfing effect of ‘M.9’ and its derivatives on apple scions and of quince rootstocks on pear scions are probably of the greatest importance so emphasis is given to these within the broader context.

Mechanisms of rootstock and interstock effects on vigour

The mechanisms of rootstock influence on tree vigour are best considered within the concept that the vigour of the composite tree reflects in an additive way the vigour of its components and that these interact. There is, as discussed earlier, a tendency to attainment of a functional equilibrium between roots and shoots. Roots supply shoots with nutrients and water, shoots supply roots with assimilates, and the roots and shoots appear to have specific roles in the production of the plant growth substances that control and coordinate activities in the plant.

Introduction

Apples and pears are subject to a large number of diseases and pests. Some are very obvious and may cause distinctive damage. Others are virtually symptomless other than leading to reduced growth and cropping.

The impact of the different diseases and pests may be reduced by confining susceptible cultivars to regions of climate unsuitable for the pathogen. It may also be reduced by the use of quarantine and ‘plant health’ procedures designed to ensure healthy planting material. Control of damage where conditions are such as to make this a serious threat may be achieved by chemical and biological control agents and by the deliberate breeding of resistant cultivars.

With the world-wide expansion of apple and pear growing, new pest and disease problems have arisen. Consumer fears of pesticide residues and concerns about ecological impacts have placed significant constraints on chemical control. Biological and ‘integrated’ control methods to reduce chemical inputs may require sophisticated localized monitoring of both pathogens and environmental factors. Genetic resistance may break down as new strains of disease organisms and pests evolve. Control of disease and pest incidence is thus very complex. Emphasis in this chapter is given to the pests and diseases of greatest importance in Europe, North America and Australasia and genetic resistance to these. Discussion of chemical and biological control and of specific pests and diseases prevalent in Asia, Africa and South America is more limited.

Introduction

Grafting is the art of connecting two pieces of living tissue together in such a way that they unite and grow as one. In apples and pears it is generally used to combine a scion (fruiting) cultivar with a rootstock. Budding is a special form of grafting in which the initial scionwood component is reduced to a single bud.

This art has been practised for thousands of years, Garner (1988) noting that grafting with detached scions was used by the Chinese before 2000 BC. It was described by writers in ancient Greece and Rome and very widely employed in western Europe in the Renaissance period and subsequently.

The main purposes of grafting are to assist in the propagation and perpetuation of clones that cannot readily be propagated by other asexual means, and to enable the production of composite trees from rootstocks and scions each of which possesses specific and distinct desirable attributes. It is also used to change scion cultivars in established orchards, to hasten the fruiting of seedling selections in breeding programmes and as a research tool in the study of physiological processes and viruses.

Formation of the graft union

The formation of the graft union can be considered as resulting from the wound-healing processes which take place on the cut surfaces of the rootstock and scion, operating in the context of close contact between surfaces. The union is accomplished entirely by cells that develop after the grafting operation. The sequence of events is as follows.

Introduction

The market price of fruits depends on their attractiveness and eating quality, which need to be retained for as long as possible after harvest to facilitate the matching of supply to demand.

This market price is of predominant importance in apple and pear production economics because many of the costs are fixed per kilogram and fruits of low perceived value do not meet their costs of production. Fruit quality is therefore a key determinant controlling the cultivars that are grown. Attainment of a specific size and, where appropriate, degree of red colour does not guarantee that the fruits are ready to eat, either immediately or after a period of storage. Good appearance after removal from store does not guarantee that eating quality has been maintained. Eating quality is based on developmental processes of maturation, ripening and senescence (Watada et al., 1984).

Maturation is the process leading to physiological or horticultural maturity. Physiological maturity is the stage of development when the fruit will continue ontogeny even if detached. Horticultural maturity is when the fruit meets the criteria laid down by consumers.

Ripening is the sum of the processes from the later stages of growth through to the early stages of senescence, that result in the attainment of the characteristic eating quality.

Senescence involves those processes following maturity that lead to death.

The history of apple and pear growing

Apples and pears in the wild and in prehistory

The genus Malus has, according to most authorities, 25 to 30 species and several subspecies of so-called crab apples. These species are found in the wild almost continuously throughout temperate Eurasia and North America. The primary centre of diversity appears to be within a region stretching from Asia Minor to the western provinces of China (Janick et al., 1996; Juniper et al., 1999, 2001). Forests of wild apples are still found in this region (Roach, 1985), with fruits ranging from small and unattractive to ones similar to the traditional cultivated eating apples.

There is evidence that the fruits of apples were collected as food by prehistoric man. Carbonized fruits dating from 6500 BC were found at çatal Hüyük in Anatolia and remains of both sour crab apples and a larger form, which may have been cultivated, were discovered in prehistoric lake dwellings in Switzerland. It seems likely that apples moved with human migration along the Old Silk Roads linking western China with the Near East and Danube valley even in Neolithic and Bronze Age times. These routes passed through Almaten (Alma Ata) in eastern Kazakhstan and the northern slopes of the Tien Shan Mountains, now thought to be the possible centre of origin of the domestic apple (Juniper et al., 2001).

Taxonomy

Apples and pears belong to the Rosaceae, subfamily Pomoideae, the pome fruits. Other members of this subfamily include quince and medlar. The flowers of the Rosaceae are actinomorphic with 5 sepals, 5 petals, numerous stamens and either one compound pistil or many simple pistils. The number of styles equals the number of carpels. The Pomoideae have mixed flower buds containing both leaf and flower initials, an epigynous ovary and 2–5 carpels. The Pomoideae have a basic chromosome number of 17 compared with 7–9 for the other subfamilies of Rosaceae.

The Malus (apple) inflorescence is determinate but the descriptive terminology is disputed (Pratt, 1988). It is variously described as a corymb, a corymbose raceme, a cyme and a false cyme. Pyrus (pear) inflorescences have been described as umbel-like simple corymbs (Clapham, Tutin and Warburg, 1952) and as racemes (Bell et al. 1996). Cydonia (quince) flowers are solitary.

The typical apple flower (see also Chapter 9) has 5 petals, varying from white to deep pink, 5 sepals, 20 stamens in three whorls (10 + 5 + 5) with yellow anthers, and a pistil which divides into five styles united at the base. The ovary has 5 locules, each usually containing 2 ovules giving a maximum seed content of 10 although some cultivars may have up to 30 (Janick et al., 1996). P. communis flowers typically have 5, usually white, petals, 5 sepals and 20–30 stamens with red or purple anthers.

Introduction

The configuration and productivity of the individual apple or pear tree is determined by its height, the number and length of its branches and the angle of these to the vertical.

The size, density and arrangement of the branch and shoot framework determine the leaf area and light capture, and hence potential photosynthesis. They also determine the number of fruit buds and fruits.

Manipulation of shoot growth begins in the nursery, with the objective of producing trees with numerous lateral branches capable of bearing fruits in their early years in the orchard. It continues throughout the life of the tree with emphasis in the early years on branch initiation, development and training, followed by emphasis on the renewal of fruiting wood and ensuring adequate penetration of light into the canopy.

The stages in the development of an apple tree and the key elements of its above-ground structure are shown in Figure 6.1. Other tree forms may be used but the essentials are the same for both apples and pears.

Buds

All shoots of apple and pear scions arise from buds. The first in the life of the tree is the bud which is inserted into the rootstock stem by budding or is present on scionwood grafted on the rootstock. The buds on the orchard tree can be on the long (extension) shoots or the short (spur) shoots and may be terminal, i.e. at the end of the shoot, or lateral, i.e. in the axil of a leaf.

The special characteristics of apple and pear production: setting the scene for their scientific study

Apples and pears are among the oldest of the world's fruit crops, figuring in both the Bible and the tales of Homer. They are by far the most important of the deciduous tree fruits, are widely grown in temperate and, increasingly, in tropical regions, and figure prominently in world trade.

The fruits of apples and pears are primarily grown for the fresh fruit market, which is much more remunerative than that for processing. O'Rourke (1994) noted that in the United States a thousand tons of apples qualifying for fresh sale would, on average, generate more than three times the revenue of a thousand tons sold for juice and that even within the fresh fruit category the most desirable fruits may sell for three or four times the price of the least desirable fruits. Moreover, apples and pears for fresh consumption, and also even in some processed forms, are marketed by cultivar name to a much greater extent than has been traditional for other fresh fruits and vegetables, and the different cultivars command different prices. The culture of apples and pears is, therefore, directed towards the production of fruits of named cultivars and to the production of fruits of high perceived quality within each cultivar. In general the cultivars do not come true-to-type when grown from seed and the necessary uniformity is achieved by clonal propagation.

Biology of Apples and Pears is written for undergraduates and postgraduate students of horticultural science, for scientists from other disciplines who need a core reference book on these crops, and for fruit growers and technical advisers.

There is already a vast amount of published work on apple and pear biology and production. However, much of this literature is difficult to interpret and apply in the context of rapid changes in areas of production – hence environmental constraints – and of cultivars with basic differences in physiology. This book addresses these issues directly by emphasising responses to environment, and cultural and storage technology at the cultivar level.

Biology of Apples and Pears deals with the biology of their eating quality and its retention as well as of their tree growth and cropping. It also emphasises the factors underlying the dramatic change from orchards of large trees to modern high-density orchards of dwarfed trees, and also those underlying modern techniques of fruit tree irrigation and nutrition.

Throughout the book the results of research on apple and pear biology are linked to the relevant current concepts in more basic seciences. The numerous references, therefore, cover both crop-specific and basic-science research papers and reviews.

Given the breadth of coverage, from anatomy and histology through physiology to biotechnology and disease, pest and environmental stress resistance there must inevitably be questions of relative emphasis.

Introduction

In field crops grown from seed the root system develops, in general, in an uncomplicated way so as fully to exploit the soil to a depth characteristic of the crop. In crop physiological analysis, and for irrigation scheduling, the root system is considered to be defined fully in terms of rooting depth and density, the roots usually being considered to be evenly distributed.

Root development and distribution is much more complex in apple and pear trees as grown commercially. It involves, and relates to, very distinctive technologies of propagation, tree establishment, and nursery and orchard soil management and irrigation. Prior to consideration of the biological basis of these technologies it is best to consider the general anatomy and structure of the roots of the orchard tree.

The essential root system of the mature tree consists, as a rule, of an underground rootstock stem from which arises a system of permanent, thickened, scaffold roots spreading almost horizontally, usually less than 50 cm from the surface, and numerous more or less vertical ‘sinkers’ descending as a rule to either an impermeable layer or a water table (Rogers and Head, 1966; Atkinson, 1980). These woody ‘skeletal’ roots are long-lived, provide anchorage and form the framework which bears the fine (fibrous) roots. New roots can emerge from the underground rootstock stem, from coarse roots (>2 mm diameter) or from fine roots. They are initially white and most are ephemeral with only a proportion thickening and becoming perennial roots.

Leaf anatomy and morphology

Leaf primordia are initiated by periclinal divisions in layers 2 or 3 of the tunica on the flanks of apical meristems (Pratt, 1990). They develop into protuberances flattened on the adaxial side. The base of the leaf primordium is an intercalary meristem and forms the petiole. The leaf blade develops concomitantly from two layers of cells derived from marginal and sub-marginal initials which produce a marginal meristem on the lateral flanks of the midrib. The superficial cells of the marginal meristems divide anticlinally to form epidermal cells. The sub-epidermal cells on the adaxial (upper) side become the first row of palisade cells and those of the abaxial side form the spongy mesophyll. Cells between these two layers form the central mesophyll and the smaller vascular bundles. On the adaxial side sub-epidermal and central meristematic cells differentiate into one to three or more layers of palisade cells depending on leaf type and environmental conditions. Each mature palisade cell is surrounded by air space continuous with that in the spongy mesophyll, except in the vicinity of a vein.

At maturity (Figure 7.1) the adaxial cuticle consists of a layer of wax and cutin. The abaxial epidermal cells have thinner cuticles than the adaxial ones. They are variable in thickness and shape except for the paired, kidney-shaped guard cells which are nearly constant in size within a cultivar and surround the pores or stomates through which gas exchange with the external air takes place.

Introduction

Aspects of nutrient uptake by roots and of mineral nutrition in relation to vegetative growth and fruiting have been discussed in earlier chapters. Fruit mineral content in relation to storage and eating quality was considered in Chapter 10 and calcium uptake, transport and effects on cell structure and metabolism were discussed in particular detail because of its dominant rôle with respect to fruit firmness and the incidence of some pre- and post-harvest physiological disorders. In the present chapter more general aspects of the uptake, transport and redistribution of nutrients are dealt with.

Nutrient requirements

A first approximation of the necessary supply of major elements for apple and pear tree growth is obtained by measurement of the mineral content of well-grown and productive trees. Relevant figures from Washington State, USA are given in Table 11.1. These are for an old-style orchard at maturity, with a similar cropping level but most probably more vegetative dry matter than many modern orchards on dwarfing rootstocks. Most of the nutrients removed from the soil and not returned to it are in the fruits so the need to replace nutrients is largely a function of crop yield. Where yields are much higher, e.g. in South Africa and, especially, New Zealand the replacement needs will be much greater. Losses by leaching can be appreciable under conditions of high rainfall or irrigation on appropriate soil types.

The defining characteristics of apple and pear flowers and fruits were summarized in Chapter 2 and the morphology and anatomy of apple flowers and fruits comprehensively reviewed by Pratt (1988). Flowers are initiated and develop within the buds borne terminally on fruiting spurs (short shoots) and terminally or laterally on long shoots. These buds, as discussed in Chapter 6, consist essentially of a shortened shoot axis with a ‘leaf formation’ inserted at each node. These ‘leaf formations’ in apple commonly consist of about nine bud scales, three transition leaves, six true leaves and three bracts (cf. Figure 6.2, p. 159). Flower primordia may form at the apex and in the axils of the bracts and the three uppermost leaves (Abbott, 1970). Alternatively, the buds may fail to develop far enough to produce flowers and remain vegetative.

Juvenility

Seedling apple and pear trees usually remain vegetative for several years after seed germination. During this ‘juvenile’ phase they readily form adventitious roots, as discussed in Chapter 3, are often thorny and have a distinctive leaf shape and, especially, cannot be induced to flower (Zimmerman, 1972). They emerge from this juvenile state after reaching a minimum size, characteristic of the cultivar, measured as height or number of main stem nodes. The duration of the juvenile phase can be shortened by growing the seedling tree under conditions which speed up its growth or by specific horticultural practices such as grafting the seedling on to a dwarfing rootstock.

Introduction

With the introduction of plants and plant products comes associated plant diseases. Diseases are particularly insidious because they may show few or no symptoms on the hosts with which they have evolved. However, native species that are susceptible to these new diseases are likely to have very low resistance or tolerance. Introduced plant diseases have probably had stronger impacts on native plant communities than have their original plant hosts. In some cases, introduced diseases have changed the species composition of forests in a major way.

It is often difficult to identify the origins of plant diseases. A survey of potato, rhododendron, citrus, wheat, Douglas fir and kudzu found that an average of 13% of their pathogens were non-indigenous (Schoulties, cited in OTA 1993). Among forest pests that have been introduced on imported live plants are dogwood anthracnose, Discula destructiva, Melampsora fungus on larch and poplar, and pine pitch canker, Fusarium subglutinans, on Monterey pine. Green wood used in packing and green logs and wood chips are potential carriers for plant disease organisms. Imports of raw wood products from distant sources are increasing and so are the risks of introducing new forest diseases (Campbell 2001). One fascinating case is the introduction of the fungus Cryptococcus neoformans gattii to coastal British Columbia, Canada. This is a saprophyte of tropical eucalyptus trees that has now established on Douglas fir, cedar and alder trees.