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Plant Cloning: Macro-Propagation
Chapter 20
Plant Cloning: Macro-Propagation This chapter was previously published in Leakey, R.R.B., 2014. In: van Alfen, N., et al., (Eds.) Encyclopedia of Agriculture and Food Systems, vol. 4. Elsevier, San Diego, pp. 349 359, with permission of Elsevier
SUMMARY Techniques of macro-propagation have been used for millennia, however it was difficult to identify clear principles applying to all species. Research over the last 20-30 years has provided better understanding of the numerous pre- and post-severance interacting factors both morphological and physiological and led to greater clarity. This research has in particular helped to explain what was an apparently contradictory scientific literature based on experiments which did not adequately record details of the stockplant and its environment. The improved understanding has led to the use of macro-propagation in the domestication of new tropical tree crops.
INTRODUCTION Cloning is a process by which individual organisms are multiplied asexually—a process of vegetative regeneration or reproduction (Longman, 1993). Consequently, the individual plants forming a clone are genetically identical. Cloning can be both a natural and an artificial process. The natural process embraces the lateral spread of creeping plants by their shoots or roots, and the production of new plantlets from dispersed, separated, or fragmented plant parts. Vegetative regeneration is a common characteristic of undesirable and invasive weeds. It is often associated with organs that have evolved as part of a perennial life form involving specialized storage organs (e.g., tubers, bulbs, rhizomes). However, vegetative regeneration also has advantages in agriculture. Firstly some of the specialist storage organs are good sources of carbohydrates for human food and so become crops—potatoes, yams, cassava, onions, etc. In addition, the capacity to regenerate asexually can be used to multiply these crops without the alteration of their genetic characteristics during the segregation phase of sexual reproduction. This also applies to many trees and other plants which can be artificially regenerated by stem cuttings, grafting, budding, or marcotting (air layering). The development of clonal crops producing specialist storage organs will not be considered further here as the process just involves the selection of the best individuals from natural populations or from the progeny of breeding programs. Instead, we will examine how the artificial process of vegetative regeneration is used by agriculturalists, horticulturalists, and foresters in domestication programs (Gepts, 2014) to capture and multiply individual genotypes and so to produce cultivars and clones of crops which would not normally be multiplied clonally. This is especially important in trees as less progress has been made in tree crop domestication because of their relatively long generation times (approximately 3 20 years), irregularity in flowering and fruiting due to climate and physiological rhythms, predominantly outbreed nature with low heritability in many traits, and high genetic diversity of base populations. In this Encyclopedia two chapters examine the processes of vegetative regeneration. One involves the relatively new processes of micropropagation (Read and Preece, 2014) which have arisen from the development of modern biotechnology, and the other, this chapter, which involves the more traditional techniques of macropropagation.
Multifunctional Agriculture. DOI: http://dx.doi.org/10.1016/B978-0-12-805356-0.00020-9 © 2017 Elsevier Inc. All rights reserved.
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THE USE OF MACROPROPAGATION The use of cloning is not a new concept associated with the advent of biotechnology. It has been used mainly in perennial crops for thousands of years by agriculturalists and horticulturalists, and used by foresters for at least 800 years (Hartmann et al., 2010; Leakey, 2014c). This is because there are many instances when multiplication by vegetative means is a more appropriate strategy than multiplication by seed (Mudge and Brennan, 1999). It is the capacity to rapidly develop cultivars by clonal selection and propagation that is especially important in the domestication of food and nonfood crops from trees, shrubs, and woody vines (Leakey, 2012b,c). However, the techniques are not exclusively used to multiply woody plants, as many ornamental herbaceous plants are also propagated vegetatively because the techniques are simple, inexpensive and result in high rates of multiplication. This chapter focuses on woody perennials as they are the most challenging subjects for vegetative propagation. Vegetative propagation has the advantage that it captures nonadditive as well as additive gene effects, but it should be noted that vegetative propagation only replicates existing genetic traits within newly formed plantlets and does not in itself improve the genetic quality of the material being propagated. There are a number of important practical issues that need to be resolved when making the decision to use vegetative propagation (Table 20.1). These are associated with the use of appropriate technologies, techniques, and plant tissues (Table 20.2) to ensure the efficient and wise use of clones for improved and sustainable production
TABLE 20.1 When to use vegetative propagation as a tool in tree domestication. When elite trees have a rare combination of a few inherited traits When there are many desirable traits for simultaneous selection When high uniformity is needed to ensure profitability and to meet market specifications When the products have a high value that can justify the extra expense When the trees to be propagated are “shy” seeders and the material for propagation is scarce When the timescale required does not allow progress through the slower and less efficient process of breeding When seeds have a low level or short period of viability When the knowledge of proven traits is acquired through long-term experiments or the traditional knowledge of local people Extracted from Leakey, R.R.B., Simons, A.J., 2000. When does vegetative propagation provide a viable alternative to propagation by seed in forestry and agroforestry in the tropics and sub-tropics? In: Wolf, H., Arbrecht, J., (Eds.), Problem of Forestry in Tropical and Sub-tropical Countries The Procurement of Forestry Seed The Example of Kenya. Ulmer Verlag, Germany, pp. 67 81.
TABLE 20.2 Strategic opportunities to consider when using vegetative propagation to domesticate trees. What is the most appropriate level of technology to use? Which tissues are most appropriate—juvenile or mature? When using juvenile tissues, which is the best source? When using mature tissues, what are the best methods to use? How can an easy, sustainable and cost-effective approach be ensured? How can the best individuals for propagation be selected from broad and diverse wild populations? What are the opportunities for introducing new variation? How can clones be wisely used and deployed? How can a wide genetic base be maintained in clonal populations? Extracted from Leakey, R.R.B., Simons, A.J., 2000. When does vegetative propagation provide a viable alternative to propagation by seed in forestry and agroforestry in the tropics and sub-tropics? In: Wolf, H., Arbrecht, J., (Eds.), Problem of Forestry in Tropical and Sub-tropical Countries The Procurement of Forestry Seed The Example of Kenya. Ulmer Verlag, Germany, pp. 67 81.
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(Longman, 1993; Leakey and Simons, 2000) as part of a wise strategy for tree domestication (Leakey and Akinnifesi, 2008; Leakey, 2012c) for the delivery of agroforestry (Leakey, 2012b; Nair et al., 2014; Chapter 28 (Leakey, 2014a).
TECHNIQUES OF MACROPROPAGATION All techniques of macropropagation are dependent on the capacity of undifferentiated meristematic cells to divide and differentiate to form new shoots or roots (Hartmann et al., 2010). While it is sometimes possible to develop new shoots from root tissues, it is generally easier to develop new roots from shoot tissues.
GRAFTING AND BUDDING Grafting and budding techniques are especially important in the vegetative propagation of woody plants that have a long period (3 20 years) of juvenile vegetative growth before becoming sexually mature and capable of flowering and fruiting. This is because mature tissues of trees seem to be very difficult to propagate by the formation of roots at the base of a piece of stem (a stem cutting). Consequently, grafting and budding techniques are used in this situation as they don’t involve root formation. Instead they are dependent on the fusion of tissues from two different shoots (Hartmann et al., 2010). In this way grafting and budding form multiple copies of large mature trees on seedling rootstocks. Grafting and budding techniques involve the placement of a severed piece of shoot (a scion), or an axillary bud, from the chosen tree in immediate contact with similar tissues on the stump or rootstock of an unselected tree, such that the tissues grow together, fuse and the buds on the attached scion grow out to form a copy of the chosen tree. A number of well known techniques have been used and practiced for thousands of years—known as cleft grafts, approach grafts, whip/tongue grafts, and side veneer grafts (Hartmann et al., 2010). The success of all these techniques depends on the juxtaposed cambial cells producing callus to form a functional and strong graft union. The ability to achieve this is thought to be dependent on a combination of environmental, anatomical, physiological, and genetic factors. Genetically, grafting is most successful when the scion and rootstock are closely related—ideally scions of a mother plant grafted on her own seedling progeny. Successful grafts between species and between genera are less common. Even if a union is formed between these poorly related plants, there is the likelihood that there will be tissue rejection at a later date—even many years later—which results in the union breaking. Often this tissue incompatibility is seen as a differential in the growth rate between the rootstock and scion, with one having a larger diameter than the other. Graft incompatibility is thought to be biochemically mediated and to depend on recognition events between juxtaposed cells through their plasmodesmatal connections. One suggestion is that it involves differences in peroxidase activity across the union which may regulate lignification processes. A difference in the peroxidase banding patterns in both the scion and rootstock is thought to predict graft incompatibility and hence weak graft unions (Gulen et al., 2002). However, despite considerable recent experimentation the mechanisms remain elusive and general principles are hard to elucidate. Environmentally, probably the main causes of graft failure are nonoptimal temperature for cell division, loss of cell turgidity, movement at the scion/rootstock interface, and disease (Hartmann et al., 2010). Consequently, grafting should be done during the growing season and with protection from water stress and movement. However, almost nothing is known about the importance of the preseverance irradiance, the light quality, and the nutrient status of the severed shoot. These environmental conditions are important for the successful rooting of cuttings. Likewise, not much is known about the best pregrafting environment for rootstocks. Physiologically, the need for vigorous growth is widely recognized. However, in temperate trees success is often greatest when dormant scions that have been held in chilled storage are grafted to rootstocks already in active growth. This may reflect the reduced chance of water stress in the scion, although it may be associated with changes in the gibberellin and abscisic acid content of plant tissues. One problem that can arise with grafting is that the scion dies while the rootstock sprouts. If good observations are not made regularly, it can be difficult to know whether the shoots of a grafted plant are of the selected scion clone, or of the unselected rootstock seedling. Of course, if the latter occurs the grafting exercise has been a waste of time, effort, and money.
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MARCOTTING OR AIR LAYERING Layering, the stimulation of roots on intact stems in contact with the ground is a natural feature of many plants, including some trees. This has been modified as an artificial process of vegetative propagation in two main ways—stooling and air layering (or marcotting). In the former, soil mounds are built up around the shoots emerging from coppiced stumps and then the rooted shoots are severed from the stump and planted. This typically captures the juvenile characteristics of the tree associated with the base of the tree trunk. Air layering is usually applied to mature (capable of flowering and fruiting) branches within the tree crown, usually a long way from the ground (Tchoundjeu et al., 2010). In this case, a ring of bark is removed to promote the accumulation of photosynthates. Then the exposed cambium is typically treated with an auxin rooting powder to promote rooting. It is then wrapped in black polythene enclosing damp compost, peat, or other rooting medium and left for some weeks or months to form roots (Fig. 20.1). This treated part of the branch should be close to the main stem. Once rooted the branch is severely pruned and detached from the tree and potted in a nursery. Typically air-layered shoots form roots on the underside of the stem, which means that when subsequently planted out the tree does not have a radially orientating root system and so is prone to fall over as the tree gets bigger. To avoid this it is preferable to air layer vertical shoots, such as those formed after pollarding a tree. Alternatively, if a nonvertical branch is used, it can be potted and managed as a stockplant from which to regularly harvest cuttings for subsequent repropagation. Rigorous studies are needed to determine the best environmental or physiological conditions for successful air layering, although it is affected by branch diameter/age and by season.
FIGURE 20.1 A marcott set on a vertical shoot from a decapitated branch of Dacryodes edulis. Courtesy of Roger Leakey.
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STEM CUTTINGS The vegetative propagation of plants by rooting stem cuttings is relatively easy in annual herbaceous species but becomes progressively more difficult as the subject becomes larger and more long-lived. Consequently, large trees provide the nursery manager with the ultimate test of skill, knowledge, and understanding. For this reason, this section focuses on some principles developed for the propagation of tree species. A wide range of physical facilities are available for the propagation of stem cuttings. These range from sophisticated, electronically controlled glasshouses with fogging equipment, through mist propagation benches to nonmist, polypropagators which are cheap and simple to build and use. The polypropagators are very effective and appropriate for use in remote locations without access to capital and reliable water or electricity services. All these facilities are designed to reduce the postseverance physiological stress (wilting and leaf abscission) resulting from water loss through transpiration by keeping the cuttings cool, moist, and turgid. Stem cuttings can come in many forms, but the two major groups are leafy softwood cuttings from relatively unlignified, young shoots which are dependent on current photosynthates for rooting, and leafless hardwood cuttings from older and more lignified shoots which depend on the mobilization of carbohydrate reserves stored within the stem tissues. The former are typically a short piece of stem—perhaps a single-node with its bud and leaf and the internode immediately below it (Fig. 20.2), while the latter is often a longer piece of stem with 2 10 nodes and internodes. It is leafless as it has already shed its leaves due to the onset of winter or a dry season. Typically, these large leafless cuttings are taken toward the end of the dormant season. Although widely practiced for hundreds of years, much of the literature on the rooting of cuttings has been anecdotal, based on the experience of individuals who have neither used standardized conditions nor adequately described
FIGURE 20.2 A single-node rooted cutting of Triplochiton scleroxylon. Courtesy of Roger Leakey.
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their protocols. This has left a confused literature with many contradictory statements about what determines success (Leakey, 2004). It is only in the last few decades that research has examined many of the factors influencing the rooting process under standardized conditions with the intention of identifying some key principles that ensure success. Furthermore, although much research has been focused on postseverance factors such as the use of auxin “rooting hormones” and the physical environment of the propagation system, much less, indeed very little, research has examined the effects of the preseverance environment of shoots on their stockplants or the effects of stockplant environment. Likewise, very little was known about the interactions between pre- and postseverance factors. This rest of this section examines five key sets of factors that play a crucial role in determining whether or not leafy cuttings rapidly form a good root system, and hence determine the success of macropropagation for the development of clonal approaches to agricultural production.
The Propagation Environment The act of severing a cutting from its stockplant creates a physiological shock. To ensure good rooting, the duration and severity of this shock has to be minimized (Mese´n et al., 1997a,b). Thus, the most important aspect of the propagation environment is that it minimizes the physiological stresses arising from: (1) the loss of water from the tissues due to transpiration, and (2) the loss of carbohydrate reserves due to tissue respiration. In addition the propagation environment should encourage photosynthesis in leafy cuttings, and promote meristematic activity (mitosis and cell differentiation) in the stem. These processes are linked to the need to transport assimilates and nutrients from the leaf to the base of the stem, and of water from the base of the stem to the leaf. To minimize these stresses the basic principles are to keep the cuttings well supplied with water at the cutting base, while also maintaining the leaves in an environment with high humidity (low vapor pressure deficit, VPD). The aerial environment in the propagation area is kept cool by shading, and the leaves themselves are cooled by the evaporation of moisture from the leaf surfaces. It is important that the level of shading does not restrict photosynthesis in the leaves (i.e., typically above 400 µmol m22 s21). Comparative studies have identified that one of the major advantages of the environment in nonmist polypropagators (Fig. 20.3) is their greater uniformity in both the moisture content of the rooting medium and the VPD across the beds, resulting in overall lower air and leaf temperatures (Newton and Jones, 1993). Mist systems have lower uniformity as a result of both the temporal and spatial distribution of moisture disbursed by intermittent bursts of pressurized mist from jets typically 0.5 1.0 m apart. In addition, peaks of VPD are associated with peaks in irradiance. Species vary in their sensitivity to VPD. This is a result of differences in leaf morphology which affect stomatal conductance, and is often associated with evolutionary adaptations to the physical environment found in the natural range of the species (e.g., rain forest vs woody savannah). The predominant use of mist and fogging systems by research teams has resulted in few studies on the relationship between photosynthesis and the rooting process due to the difficulty of measuring gas exchange in cuttings with wet leaves. However, studies of gas exchange during propagation have been made in nonmist polypropagators and these have illustrated the importance of photosynthesis for good rooting success, speed of rooting, and the number of roots produced per cutting (Hoad and Leakey, 1994, 1996). There is a general assumption that the successful rooting of cuttings is associated with a positive carbon balance (i.e., assimilate production . losses through respiration) and a concentration gradient within a cutting which enhances the transport of assimilates from the leaves toward the cutting base. However, little is known about the relationships between respiration losses and cutting origin, leaf area, stem length/diameter, or the rooting environment.
FIGURE 20.3 Left: Nonmist propagator with its lid open. Right: A cross-section showing the different layers of gravel and medium. The arrow marks the top of the water. Courtesy of Roger Leakey.
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In addition to the aerial environment of the propagator, the environment of the rooting medium is also important. However, the choice of medium is often based on availability of the materials or personal experience. Nevertheless, studies suggest that the medium should hold the cutting firm with its leaf held above the surface of the medium. In addition to providing moisture at the cutting base the medium should allow respiration from the tissues and prevent anoxia which encourages rotting and cutting mortality. To prevent this, the medium should have an air:water ratio that optimizes the oxygen diffusion rate vis a` vis the needs of tissue respiration. In practice this involves the use of various sized particles (sand and gravel) and a water-holding medium (compost, perlite, vermiculite, peat, coir, or other organic products) alone or in various mixtures. As in the relationship between leaf morphology and the aerial environment, there is some evidence that morphological and physiological adaptations to different environments may affect the optimum porespace. Root development and growth is generally enhanced by the cutting base being warmer (artificially enhanced by providing “bottom-heat”) than the leaves. This promotes meristematic activity at the cutting base, while the leaves remain cool and free from stress. The converse differential tends to promote the growth of buds creating competition for assimilates.
Postseverance Treatments Auxin Applications The application of auxin is considered to promote rooting by stimulation of cell differentiation, the promotion of starch hydrolysis and the attraction of sugars and nutrients to the cutting base, but a better understanding is needed of the mechanisms regulating auxin concentrations and pathways (Atangana et al., 2011a). Indole-3-butyric acid (IBA) is typically the most effective auxin. Auxins are not, however, a “cure-all” treatment and exogenous applications of auxin do not promote rooting in cuttings which are morphologically or physiologically dysfunctional. Thus for auxins to have their stimulatory effects, the cuttings should have been taken from shoots which are preconditioned by stockplant management to be physiologically active when in the propagator, free from water and respiratory stresses, and with the capacity to mobilize stored or current assimilates. A point of practical importance is that the stems of some species are hairy, while others are waxy. This affects the retention of applied auxin. In addition, uptake is affected by the cross-sectional area of the cutting base (i.e., stem diameter).
Leaf Area The rooting of softwood cuttings is typically dependent on the presence of a leaf. Studies using infrared gas analyzers to measure the rates of photosynthesis in nonmist polypropagators during the propagation process found that rooting ability was maximized in photosynthetically active cuttings. However, a large photosynthetically active leaf is also actively losing water by transpiration and can suffer water stress—so closing its stomata or shedding its leaf. This then prevents further photosynthesis. Conversely, small leaved cuttings with inadequate assimilate production rapidly decline in their carbohydrate (sugars and starch) content due to respiration losses and hence cannot support root development. Successful rooting therefore requires an optimal leaf area which balances the positive effects of photosynthesis and the negative effects of transpiration (Leakey, 2004). The leaf area associated with this balance varies depending on the adaptations in leaf morphology of different species to different environments. Such variation is also determined by leaf age (node position) and the position of a shoot with the stockplant. In addition to leaf area, the correct balance between photosynthesis and transpiration will also be determined by the photosynthetic efficiency of the leaf due to the light environment of the propagator (level of irradiance) and the cutting’s water relations (affected by the aerial environment of the propagator and the air:water ratio of the medium). Studies of these important aspects of propagation have found a relationship between rooting ability and the content of refluxextracted soluble carbohydrates, which confirm the importance of assimilate production throughout the rooting process. The determination of the optimum leaf area is therefore a very important aspect of developing a successful propagation protocol, especially in difficult-to-root species. In general, rooting experiments focus on the factors enhancing rooting success. However, to learn more about the processes affecting rooting, there is much that could be learned from paying more attention to the causes of rooting failure. For example, more information is needed about how and when cutting death can be attributed to: water and heat stress, leaf abscission, photoinhibition, negative carbon balance, etc. In addition, some cuttings neither die nor root.
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Other related reasons for the failure of cuttings to root can be the death of the leaf due to microbial infection, anoxia and rotting, necrosis, bleaching, or leaf abscission. These problems can arise due to the use of old shoots with senescent or photosynthetically inactive leaves that are past their compensation point (photosynthetic activity vis a` vis senescence); water stressed or starch-filled. The most common symptoms are leaf shedding, leaf rot, and stem rot. Gaining an understanding of these causes of cutting death and hence the failure of the propagation process can be as important as determining how to achieve good rooting. Despite the importance of photosynthesis for the rooting of leafy softwood cuttings, there is some evidence that some species are also able to mobilize and use stored reserves of carbohydrates to contribute to the rooting process. This may reflect differences in stem anatomy and perhaps also adaptations to seasonally harsh environments.
Cutting Length The stem length of a cutting is another important variable affecting rooting success (Leakey, 2004). It is important in two ways. Firstly it affects the depth of insertion into the rooting medium, as well as the height of the leaf above the surface of the rooting medium, and secondly it inherently affects the capacity of the cutting to root. This is something we will investigate later. Both the depth of insertion and the height of the leaves above the medium can be important in terms of providing uniform conditions for water uptake and preventing competition between cuttings for light. It is also desirable to prevent the leaves from touching the medium and getting saturated by water droplets. Cuttings can be cut to a constant length (these may vary in the number of nodes and leaves present), or can be cut to the length determined by a chosen number of nodes. The simplest cutting is a single-node cutting. It has one internode and generally has one leaf and one bud. In this case the internode will generally vary in length depending on its position within the stem (Fig. 20.4). As a general rule, basal internodes are shorter than more apical ones, this reflecting the vigor of growth at the time the node was formed in the terminal bud. We will examine this within stem variability later. For practical purposes it is probably best to use a constant length close to the optimum, although this may not result in the availability of greatest number of cuttings.
Stockplant Factors: Cutting Origin and Environment There are two major sources of variation attributable to the stockplant: (1) Within-shoot factors, and (2) Between shoot factors. Both of these are subject to the stockplant environment and so can be influenced by stockplant management.
Within-Shoot Factors We saw earlier that single-node cuttings vary in their internode length (Fig. 20.4). This variation is associated with gradients in numerous other variables which basically run from the bottom to the top of the stem in parallel with chronological age, such as leaf size, leaf water potential, leaf carbon balance, leaf senescence, internode diameter, stem
FIGURE 20.4 The node-to-node variation in the length of the internode and the petiole in cuttings from the top two shoots of a Triplochiton scleroxylon stockplant (uppermost nodes on the left). Courtesy of Roger Leakey.
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FIGURE 20.5 The gradient of variation in rooting ability within a shoot: the long uppermost cuttings root best, whereas the short basal cuttings die.
lignification, nutrient and stem carbohydrate content, and respiration (Leakey, 2004). Thus no two cuttings are identical and, as a consequence, no two cuttings have the same rooting ability (Fig. 20.5). This is because all these variables affect the physiological processes in the cutting. A number of studies have used this node-by-node variation as an experimental variable to examine some of the key factors affecting rooting ability. For example, a comparison of the normal gradient in cutting length with an inverse gradient showed the importance of cutting length while a comparison with standard length cuttings showed the importance of cutting diameter/volume. It seems that the stem volume of the cutting determines the storage capacity of the cutting for current assimilates. In this connection, there are interactions between stem volume and leaf area. The stockplant environment and/or stockplant management also interact with node position in ways that help to elucidate the effects of different treatments. Studies of this sort have shed light on the relative importance of carbohydrates and nutrients in the rooting process. Between Shoot Factors As plants grow and increase in size they branch and become more complex. This creates competition between shoots for assimilates and nutrients, as well as causing mutual shading between leaves. This competition is made more complex by the processes of correlative inhibition and the development of dominance between shoots which are mediated by growth regulators as well as by the plant’s environment, especially light and nutrients (Leakey, 2004). To maintain stockplants in a good condition for easy rooting they need to be pruned back hard to leave a short stump of about 20 cm. So the simplest form of stockplant has just been cut back once and then sprouts from its uppermost remaining buds. A comparison of the rooting ability of these different shoots has found that cuttings from the uppermost one roots best and those from lower shoots root progressively less well. However, if such stockplants are cut back to different heights (say between 20 and 100 cm) then usually the taller stockplants produce more shoots. In this case the rooting ability of cuttings from the top shoot declines with increasing height and a relationship is found between the number of shoots and the percentage of cuttings rooted (Fig. 20.6). This implies that competition between the shoots reduces rooting ability and the removal of the lower shoots increases the rooting ability of cuttings from upper shoots. Further studies to test the competition hypothesis in plants of the same height have found that other factors also seem to be involved. For example, the lower shaded shoots can root very much better than less shaded shoots, especially under conditions of high soil nitrogen. Additionally, reorienting the stockplant so that its stem is not vertical
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FIGURE 20.6 Effects on the rooting success of cuttings from the top shoot of stockplants pruned to different heights. Cuttings from short stockplants with few shoots root best.
(e.g., 45 degrees or 90 degrees from the vertical) alters the location of vigorous growth and the fast-growing lower basal shoots become the most easily rooted. Thus in addition to competition there are effects of shoot position and of stockplant environment. When studies tried to elucidate these interactions between shoot position and intershoot competition in two-shoot stockplants of the same height, it was found that basal shaded shoots had a higher rooting ability than upper shoots. However, if these basal and upper shoots were under conditions with the same light environment (irradiance) the differences between shoot positions were eliminated (Fig. 20.7). This finding led to experiments in controlled-environment growth chambers to investigate the role of light (Hoad and Leakey, 1994, 1996).
STOCKPLANT ENVIRONMENT As seen previously, both nutrients and shade seem to affect the rooting ability of cuttings from shoots on relatively simple stockplants, but these complex preconditioning processes are poorly understood. It is clear that both the amount of light (irradiance) and the spectral quality of light (red:far-red ratio)—both features of shade—are important and independently affect the rooting ability of cuttings. Shade light is also commonly associated with shoot etiolation and hence with long internodes, and with large thin leaves. These affects are then further complicated by an interaction with soil nutrients which promote shoot growth (Leakey and Storeton-West, 1992). The light/nutrient interaction affects photosynthetic processes. The most dramatic of these interactions seems to be the combination of high irradiance and low nutrients which results in short starch-filled stems, the inhibition of photosynthesis and very poor rooting (Fig. 20.8). At the other extreme, low irradiance, and high nutrients are associated with active photosynthesis, low starch, and good rooting. These preconditioning effects of irradiance, light quality, and nutrients on morphology are also associated with physiological differences in the stems and leaves of the shoots preseverance (Hoad and Leakey, 1994, 1996). Physiologically, shade reduces the tendency of the top shoot to dominate and reduce the growth of other shoots
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FIGURE 20.7 Effects of shoot position on the rooting success of cuttings from stockplants with apical and basal shoots. The effects of shade were examined by adjusting the height of the pots.
FIGURE 20.8 The effects of growing stockplants at two levels of light (low and high irradiance) and two levels of nutrients (low and high). This affects the rates of photosynthesis and the starch content of cuttings. Starch-filled cuttings with low rates of photosynthesis rooted very poorly. Modified from Leakey, R.R.B., Storeton-West, R., 1992. The rooting ability of Triplochiton scleroxylon K. Schum. cuttings: the interactions between stockplant irradiance, light quality and nutrients. For. Ecol. Manage., 49, 133 150.
(i.e., promotes codominance), lowers the rates of preseverance net photosynthesis, lowers leaf chlorophyll concentration, but stimulates higher rates of net photosynthesis per unit of chlorophyll. Preseverance conditioning thus actively promotes postseverance photosynthesis during the rooting process. The shoots are therefore said to be physiological “young,” while shoots with low vigor and inhibited photosynthesis are physiologically “old.” A mechanistic model of carbohydrate dynamics during the rooting process can be used to gain further insights into the rooting process (Dick and Dewar, 1992) and to define key principles for the development of robust rooting protocols.
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TABLE 20.3 The importance of stockplant management for successful vegetative propagation. Standard hedged stockplant
Standard hedged stockplant
Preconditioned (far-red light and fertilizers) hedged stockplant
No knowledge of stockplant management
Knowledge that rooting is best from upper shoots
Knowledge that preconditioning improves physiological activity and rooting capacity
No. of cuttings harvested
31
16
45
No. of cuttings rooted
16
16
40
Percentage of cuttings rooted
52%
100%
89%
Number of plants established
16
16
40
Note the lack of relationship between rooting percentage and the numbers of plants established—this reflecting the understanding of the importance of stockplant management (which shoots to use and the role of stockplant environment. Adapted from Leakey, R.R.B., 2004. Physiology of vegetative reproduction. In: Burley, J., Evans, J., Youngquist, J.A., (Eds.), Encyclopedia of Forest Sciences. Academic Press, London, pp. 1655 1668.
Stockplant Management The major importance of all of the previous morphological and physiological stockplant factors makes it clear that stockplant management (a combination of regular pruning, fertilizer use, and light management) should be a key component of any macropropagation protocol. To retain physiological youth, stockplants are often managed as hedges. Using nitrogen-fixing tree/shrub species to shade these hedges then maximizes the rooting success by provision of desirable light and soil nutrient environments. One of the issues which has created much of the confusion in the scientific literature of vegetative propagation is the very common (even ubiquitous) use of percentage rooting as the measure of success. Rooting percentage is, however, affected by the standards of stockplant management, use of the best shoots (the shoots with optimal morphology and highest physiological activity, Table 20.3). Contradictory results arise in the literature when authors do not provide the relevant information in the description of their experimental protocols.
Phase Change The vegetative propagation literature has numerous references to what is called “Phase Change” or ontogenetic aging. This literature basically attributes the loss of rooting ability as perennial plants age and get larger to their gradual transition from the juvenile phase of vegetative growth to a phase of sexual maturity. The propagation of mature tissues is one of the major constraints to many tree improvement programs focusing on cultivar development through macropropagation. This loss of rooting ability with increasing size and structural complexity is perhaps not surprising given the already mentioned effects of stockplant factors in even small and simple stockplants. Nevertheless, the importance of phase change vis a` vis rooting ability is an unresolved aspect of vegetative propagation (Leakey, 2004). The phase change hypothesis assumes that plants (and trees in particular) gradually progress from juvenility (sexually immature and easy-to-root) to maturity (sexually mature and capable of flowering and fruiting associated with low rooting ability) over time. In trees, maturity may not be achieved for 3 20 years. It is generally recognized that the best way to return a tree to the juvenile state is to cut it down and to allow coppice shoots to grow from the stump. These coppice shoots are young and vigorous and cuttings from them can usually be rooted easily. Studies have suggested however that there is a decline in rooting ability with the increasing diameter of the stump. It is not known whether this is due to some aspect of the aging process or to stockplant factors like those mentioned for seedlings above. For example, large stumps generally produce more shoots than smaller stumps. This loss of rooting ability could therefore be due to increased intershoot competition.
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There are some inconsistencies between the concept of phase change and observation. Originally the hypothesis was based on work in ivy (Hedera helix) which has various leaf shapes. It was assumed that the normal palmate leaves of climbing ivy vines were characteristic of the juvenile easy rooting phase, while the ovate leaves of flowering shoots were assumed to be characteristic of the difficult-to-root mature phase. Observation of large ivy plants suggests a different interpretation as palmate leaves can be seen to be associated with main stems (the climbing vine), while ovate leaves are associated with free-standing branches on which flowers are formed. The association of flowering with branches is common in many woody plants, and it is not unusual for branches and main stems to differ in the arrangement of their leaves and buds. Certainly in ivy it is common to see old and large vines climbing a cliff or dead tree with palmate leaves tens of meters above the ground and flowering branches with ovate leaves all the way up the vine. A second way in which the hypothesis is not supported is the evidence that ontogenetically mature plants (with the capacity to flower and form fruits) propagated by cuttings, grafts or marcotts can be easily repropagated by cuttings when they are used as small managed stockplants. Although often plagiotropic, such stockplants have the vigor of juvenile seedlings and coppice shoots. This suggests the poor rooting ability of “mature” shoots can be attributed to “physiological aging” rather than to “ontogenetic aging.” In other words, the difficulty in rooting crown shoots in large mature trees is due to a severe case of the combination of undesirable morphological and physiological factors resulting from within and between stockplant factors and their interaction with the stockplant environment (Dick and Leakey, 2006). A start has been made to examining this experimentally within the crown of mature trees (Pauku et al., 2010).
Genetic Variation in Rooting Ability Early in this chapter we saw that there are genetic differences in the ease of propagation between species and even between provenances and clones within species. In severe cases this has led to the conclusion that some species are “impossible to root.” We have also seen that species vary in the leaf and stem morphology, and in their adaptations and responses to environmental factors. There is now good evidence that species previously thought to be “impossible to root” can now be preconditioned to be easier to root by good stockplant management based on an understanding of the morphological and physiological factors affecting rooting ability. Thus the apparent genetic differences in rooting ability can instead be attributed to genetic differences in the growth and development of shoots. The concept that inherent genetic differences in rooting ability are not critically important is also supported by the use of stepwise regression in the analysis of experimental data from rooting studies. Such analysis commonly finds that the factors explaining much of the variance are cutting length, leaf abscission, leaf area, etc., rather than clone or provenance (Dick et al., 1999). TABLE 20.4 The analogy between athletic ability and rooting ability in the vegetative propagation of tree stem cuttings. Sporting ability
Rooting ability
Young
Juvenile
Physiologically fit—lean, well-prepared, strong
Physiologically young—high rates of photosynthesis—long internodes, large leaf area, chronologically young node position
Genetic morphology—muscles, body build, etc.
Genetic morphology—stem and leaf anatomy
Preparation in gym—physiologically fit and full of energy
Preconditioning by stockplant management —physiologically active
Highly competitive and ready for action
Preconditioned to contain low starch/high soluble sugars and free from competing shoots
Use of performance-enhancing drugs
Use of auxins as rooting hormone
Stress-free Olympic village
Stress-free propagation environment
Different sports—refine training regime
Different species—refine propagation regime
Adapted from Leakey, R.R.B., 2012b. Living with the Trees of Life UK, 200 pp.
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Contrary to this, some studies to detect quantitative trait loci affecting vegetative propagation have, however, reported that phenotypic variation has a meaningful genetic component.
CONCLUSIONS There are many facets to developing a robust approach to macropropagating plants, especially by the rooting of stem cuttings. Over the last 15 20 years much progress has been made to gain a good understanding of the numerous interacting factors (stockplant environment 3 stockplant management 3 topophytic variables 3 node position 3 nursery management 3 postseverance treatments 3 propagation environment) by studying them in tree species. This understanding now provides some general principles that can be applied to the propagation of new species, and especially those considered to be difficult to ltotaoatpropagate. Leakey (2012b) has likened many of the factors that determine the success of propagation by stem cuttings to those that determine the success of an athlete competing in the Olympic Games (Table 20.4). This analogy has been found to help people to understand the key principles. The greatest challenge remaining is to improve the design and reporting of experiments so that the quality of the literature is improved by researchers adequately describing the material they used, as well as the pre and postseverance environments. This should remove the contradictory results in the literature created by people not using comparable material in terms of the physiology and morphology of the tissues used. Then there is a need for more extensive studies of the importance of stockplant management and the stockplant environment across a wider range of species.
Courses 5 Training videos by Edinburgh Center for Tropical Forests (DVD)
Relevant Websites ICRAF website tree_domestication
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Plant Cloning: Macro-Propagation This chapter was previously published in Leakey, R.R.B., 2014. In: van Alfen, N., et al., (Eds.) Encyclopedia of Agriculture and Food Systems, vol. 4. Elsevier, San Diego, pp. 349 359, with permission of Elsevier
SUMMARY Techniques of macro-propagation have been used for millennia, however it was difficult to identify clear principles applying to all species. Research over the last 20-30 years has provided better understanding of the numerous pre- and post-severance interacting factors both morphological and physiological and led to greater clarity. This research has in particular helped to explain what was an apparently contradictory scientific literature based on experiments which did not adequately record details of the stockplant and its environment. The improved understanding has led to the use of macro-propagation in the domestication of new tropical tree crops.
INTRODUCTION Cloning is a process by which individual organisms are multiplied asexually—a process of vegetative regeneration or reproduction (Longman, 1993). Consequently, the individual plants forming a clone are genetically identical. Cloning can be both a natural and an artificial process. The natural process embraces the lateral spread of creeping plants by their shoots or roots, and the production of new plantlets from dispersed, separated, or fragmented plant parts. Vegetative regeneration is a common characteristic of undesirable and invasive weeds. It is often associated with organs that have evolved as part of a perennial life form involving specialized storage organs (e.g., tubers, bulbs, rhizomes). However, vegetative regeneration also has advantages in agriculture. Firstly some of the specialist storage organs are good sources of carbohydrates for human food and so become crops—potatoes, yams, cassava, onions, etc. In addition, the capacity to regenerate asexually can be used to multiply these crops without the alteration of their genetic characteristics during the segregation phase of sexual reproduction. This also applies to many trees and other plants which can be artificially regenerated by stem cuttings, grafting, budding, or marcotting (air layering). The development of clonal crops producing specialist storage organs will not be considered further here as the process just involves the selection of the best individuals from natural populations or from the progeny of breeding programs. Instead, we will examine how the artificial process of vegetative regeneration is used by agriculturalists, horticulturalists, and foresters in domestication programs (Gepts, 2014) to capture and multiply individual genotypes and so to produce cultivars and clones of crops which would not normally be multiplied clonally. This is especially important in trees as less progress has been made in tree crop domestication because of their relatively long generation times (approximately 3 20 years), irregularity in flowering and fruiting due to climate and physiological rhythms, predominantly outbreed nature with low heritability in many traits, and high genetic diversity of base populations. In this Encyclopedia two chapters examine the processes of vegetative regeneration. One involves the relatively new processes of micropropagation (Read and Preece, 2014) which have arisen from the development of modern biotechnology, and the other, this chapter, which involves the more traditional techniques of macropropagation.
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THE USE OF MACROPROPAGATION The use of cloning is not a new concept associated with the advent of biotechnology. It has been used mainly in perennial crops for thousands of years by agriculturalists and horticulturalists, and used by foresters for at least 800 years (Hartmann et al., 2010; Leakey, 2014c). This is because there are many instances when multiplication by vegetative means is a more appropriate strategy than multiplication by seed (Mudge and Brennan, 1999). It is the capacity to rapidly develop cultivars by clonal selection and propagation that is especially important in the domestication of food and nonfood crops from trees, shrubs, and woody vines (Leakey, 2012b,c). However, the techniques are not exclusively used to multiply woody plants, as many ornamental herbaceous plants are also propagated vegetatively because the techniques are simple, inexpensive and result in high rates of multiplication. This chapter focuses on woody perennials as they are the most challenging subjects for vegetative propagation. Vegetative propagation has the advantage that it captures nonadditive as well as additive gene effects, but it should be noted that vegetative propagation only replicates existing genetic traits within newly formed plantlets and does not in itself improve the genetic quality of the material being propagated. There are a number of important practical issues that need to be resolved when making the decision to use vegetative propagation (Table 20.1). These are associated with the use of appropriate technologies, techniques, and plant tissues (Table 20.2) to ensure the efficient and wise use of clones for improved and sustainable production
TABLE 20.1 When to use vegetative propagation as a tool in tree domestication. When elite trees have a rare combination of a few inherited traits When there are many desirable traits for simultaneous selection When high uniformity is needed to ensure profitability and to meet market specifications When the products have a high value that can justify the extra expense When the trees to be propagated are “shy” seeders and the material for propagation is scarce When the timescale required does not allow progress through the slower and less efficient process of breeding When seeds have a low level or short period of viability When the knowledge of proven traits is acquired through long-term experiments or the traditional knowledge of local people Extracted from Leakey, R.R.B., Simons, A.J., 2000. When does vegetative propagation provide a viable alternative to propagation by seed in forestry and agroforestry in the tropics and sub-tropics? In: Wolf, H., Arbrecht, J., (Eds.), Problem of Forestry in Tropical and Sub-tropical Countries The Procurement of Forestry Seed The Example of Kenya. Ulmer Verlag, Germany, pp. 67 81.
TABLE 20.2 Strategic opportunities to consider when using vegetative propagation to domesticate trees. What is the most appropriate level of technology to use? Which tissues are most appropriate—juvenile or mature? When using juvenile tissues, which is the best source? When using mature tissues, what are the best methods to use? How can an easy, sustainable and cost-effective approach be ensured? How can the best individuals for propagation be selected from broad and diverse wild populations? What are the opportunities for introducing new variation? How can clones be wisely used and deployed? How can a wide genetic base be maintained in clonal populations? Extracted from Leakey, R.R.B., Simons, A.J., 2000. When does vegetative propagation provide a viable alternative to propagation by seed in forestry and agroforestry in the tropics and sub-tropics? In: Wolf, H., Arbrecht, J., (Eds.), Problem of Forestry in Tropical and Sub-tropical Countries The Procurement of Forestry Seed The Example of Kenya. Ulmer Verlag, Germany, pp. 67 81.
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(Longman, 1993; Leakey and Simons, 2000) as part of a wise strategy for tree domestication (Leakey and Akinnifesi, 2008; Leakey, 2012c) for the delivery of agroforestry (Leakey, 2012b; Nair et al., 2014; Chapter 28 (Leakey, 2014a).
TECHNIQUES OF MACROPROPAGATION All techniques of macropropagation are dependent on the capacity of undifferentiated meristematic cells to divide and differentiate to form new shoots or roots (Hartmann et al., 2010). While it is sometimes possible to develop new shoots from root tissues, it is generally easier to develop new roots from shoot tissues.
GRAFTING AND BUDDING Grafting and budding techniques are especially important in the vegetative propagation of woody plants that have a long period (3 20 years) of juvenile vegetative growth before becoming sexually mature and capable of flowering and fruiting. This is because mature tissues of trees seem to be very difficult to propagate by the formation of roots at the base of a piece of stem (a stem cutting). Consequently, grafting and budding techniques are used in this situation as they don’t involve root formation. Instead they are dependent on the fusion of tissues from two different shoots (Hartmann et al., 2010). In this way grafting and budding form multiple copies of large mature trees on seedling rootstocks. Grafting and budding techniques involve the placement of a severed piece of shoot (a scion), or an axillary bud, from the chosen tree in immediate contact with similar tissues on the stump or rootstock of an unselected tree, such that the tissues grow together, fuse and the buds on the attached scion grow out to form a copy of the chosen tree. A number of well known techniques have been used and practiced for thousands of years—known as cleft grafts, approach grafts, whip/tongue grafts, and side veneer grafts (Hartmann et al., 2010). The success of all these techniques depends on the juxtaposed cambial cells producing callus to form a functional and strong graft union. The ability to achieve this is thought to be dependent on a combination of environmental, anatomical, physiological, and genetic factors. Genetically, grafting is most successful when the scion and rootstock are closely related—ideally scions of a mother plant grafted on her own seedling progeny. Successful grafts between species and between genera are less common. Even if a union is formed between these poorly related plants, there is the likelihood that there will be tissue rejection at a later date—even many years later—which results in the union breaking. Often this tissue incompatibility is seen as a differential in the growth rate between the rootstock and scion, with one having a larger diameter than the other. Graft incompatibility is thought to be biochemically mediated and to depend on recognition events between juxtaposed cells through their plasmodesmatal connections. One suggestion is that it involves differences in peroxidase activity across the union which may regulate lignification processes. A difference in the peroxidase banding patterns in both the scion and rootstock is thought to predict graft incompatibility and hence weak graft unions (Gulen et al., 2002). However, despite considerable recent experimentation the mechanisms remain elusive and general principles are hard to elucidate. Environmentally, probably the main causes of graft failure are nonoptimal temperature for cell division, loss of cell turgidity, movement at the scion/rootstock interface, and disease (Hartmann et al., 2010). Consequently, grafting should be done during the growing season and with protection from water stress and movement. However, almost nothing is known about the importance of the preseverance irradiance, the light quality, and the nutrient status of the severed shoot. These environmental conditions are important for the successful rooting of cuttings. Likewise, not much is known about the best pregrafting environment for rootstocks. Physiologically, the need for vigorous growth is widely recognized. However, in temperate trees success is often greatest when dormant scions that have been held in chilled storage are grafted to rootstocks already in active growth. This may reflect the reduced chance of water stress in the scion, although it may be associated with changes in the gibberellin and abscisic acid content of plant tissues. One problem that can arise with grafting is that the scion dies while the rootstock sprouts. If good observations are not made regularly, it can be difficult to know whether the shoots of a grafted plant are of the selected scion clone, or of the unselected rootstock seedling. Of course, if the latter occurs the grafting exercise has been a waste of time, effort, and money.
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MARCOTTING OR AIR LAYERING Layering, the stimulation of roots on intact stems in contact with the ground is a natural feature of many plants, including some trees. This has been modified as an artificial process of vegetative propagation in two main ways—stooling and air layering (or marcotting). In the former, soil mounds are built up around the shoots emerging from coppiced stumps and then the rooted shoots are severed from the stump and planted. This typically captures the juvenile characteristics of the tree associated with the base of the tree trunk. Air layering is usually applied to mature (capable of flowering and fruiting) branches within the tree crown, usually a long way from the ground (Tchoundjeu et al., 2010). In this case, a ring of bark is removed to promote the accumulation of photosynthates. Then the exposed cambium is typically treated with an auxin rooting powder to promote rooting. It is then wrapped in black polythene enclosing damp compost, peat, or other rooting medium and left for some weeks or months to form roots (Fig. 20.1). This treated part of the branch should be close to the main stem. Once rooted the branch is severely pruned and detached from the tree and potted in a nursery. Typically air-layered shoots form roots on the underside of the stem, which means that when subsequently planted out the tree does not have a radially orientating root system and so is prone to fall over as the tree gets bigger. To avoid this it is preferable to air layer vertical shoots, such as those formed after pollarding a tree. Alternatively, if a nonvertical branch is used, it can be potted and managed as a stockplant from which to regularly harvest cuttings for subsequent repropagation. Rigorous studies are needed to determine the best environmental or physiological conditions for successful air layering, although it is affected by branch diameter/age and by season.
FIGURE 20.1 A marcott set on a vertical shoot from a decapitated branch of Dacryodes edulis. Courtesy of Roger Leakey.
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STEM CUTTINGS The vegetative propagation of plants by rooting stem cuttings is relatively easy in annual herbaceous species but becomes progressively more difficult as the subject becomes larger and more long-lived. Consequently, large trees provide the nursery manager with the ultimate test of skill, knowledge, and understanding. For this reason, this section focuses on some principles developed for the propagation of tree species. A wide range of physical facilities are available for the propagation of stem cuttings. These range from sophisticated, electronically controlled glasshouses with fogging equipment, through mist propagation benches to nonmist, polypropagators which are cheap and simple to build and use. The polypropagators are very effective and appropriate for use in remote locations without access to capital and reliable water or electricity services. All these facilities are designed to reduce the postseverance physiological stress (wilting and leaf abscission) resulting from water loss through transpiration by keeping the cuttings cool, moist, and turgid. Stem cuttings can come in many forms, but the two major groups are leafy softwood cuttings from relatively unlignified, young shoots which are dependent on current photosynthates for rooting, and leafless hardwood cuttings from older and more lignified shoots which depend on the mobilization of carbohydrate reserves stored within the stem tissues. The former are typically a short piece of stem—perhaps a single-node with its bud and leaf and the internode immediately below it (Fig. 20.2), while the latter is often a longer piece of stem with 2 10 nodes and internodes. It is leafless as it has already shed its leaves due to the onset of winter or a dry season. Typically, these large leafless cuttings are taken toward the end of the dormant season. Although widely practiced for hundreds of years, much of the literature on the rooting of cuttings has been anecdotal, based on the experience of individuals who have neither used standardized conditions nor adequately described
FIGURE 20.2 A single-node rooted cutting of Triplochiton scleroxylon. Courtesy of Roger Leakey.
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their protocols. This has left a confused literature with many contradictory statements about what determines success (Leakey, 2004). It is only in the last few decades that research has examined many of the factors influencing the rooting process under standardized conditions with the intention of identifying some key principles that ensure success. Furthermore, although much research has been focused on postseverance factors such as the use of auxin “rooting hormones” and the physical environment of the propagation system, much less, indeed very little, research has examined the effects of the preseverance environment of shoots on their stockplants or the effects of stockplant environment. Likewise, very little was known about the interactions between pre- and postseverance factors. This rest of this section examines five key sets of factors that play a crucial role in determining whether or not leafy cuttings rapidly form a good root system, and hence determine the success of macropropagation for the development of clonal approaches to agricultural production.
The Propagation Environment The act of severing a cutting from its stockplant creates a physiological shock. To ensure good rooting, the duration and severity of this shock has to be minimized (Mese´n et al., 1997a,b). Thus, the most important aspect of the propagation environment is that it minimizes the physiological stresses arising from: (1) the loss of water from the tissues due to transpiration, and (2) the loss of carbohydrate reserves due to tissue respiration. In addition the propagation environment should encourage photosynthesis in leafy cuttings, and promote meristematic activity (mitosis and cell differentiation) in the stem. These processes are linked to the need to transport assimilates and nutrients from the leaf to the base of the stem, and of water from the base of the stem to the leaf. To minimize these stresses the basic principles are to keep the cuttings well supplied with water at the cutting base, while also maintaining the leaves in an environment with high humidity (low vapor pressure deficit, VPD). The aerial environment in the propagation area is kept cool by shading, and the leaves themselves are cooled by the evaporation of moisture from the leaf surfaces. It is important that the level of shading does not restrict photosynthesis in the leaves (i.e., typically above 400 µmol m22 s21). Comparative studies have identified that one of the major advantages of the environment in nonmist polypropagators (Fig. 20.3) is their greater uniformity in both the moisture content of the rooting medium and the VPD across the beds, resulting in overall lower air and leaf temperatures (Newton and Jones, 1993). Mist systems have lower uniformity as a result of both the temporal and spatial distribution of moisture disbursed by intermittent bursts of pressurized mist from jets typically 0.5 1.0 m apart. In addition, peaks of VPD are associated with peaks in irradiance. Species vary in their sensitivity to VPD. This is a result of differences in leaf morphology which affect stomatal conductance, and is often associated with evolutionary adaptations to the physical environment found in the natural range of the species (e.g., rain forest vs woody savannah). The predominant use of mist and fogging systems by research teams has resulted in few studies on the relationship between photosynthesis and the rooting process due to the difficulty of measuring gas exchange in cuttings with wet leaves. However, studies of gas exchange during propagation have been made in nonmist polypropagators and these have illustrated the importance of photosynthesis for good rooting success, speed of rooting, and the number of roots produced per cutting (Hoad and Leakey, 1994, 1996). There is a general assumption that the successful rooting of cuttings is associated with a positive carbon balance (i.e., assimilate production . losses through respiration) and a concentration gradient within a cutting which enhances the transport of assimilates from the leaves toward the cutting base. However, little is known about the relationships between respiration losses and cutting origin, leaf area, stem length/diameter, or the rooting environment.
FIGURE 20.3 Left: Nonmist propagator with its lid open. Right: A cross-section showing the different layers of gravel and medium. The arrow marks the top of the water. Courtesy of Roger Leakey.
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In addition to the aerial environment of the propagator, the environment of the rooting medium is also important. However, the choice of medium is often based on availability of the materials or personal experience. Nevertheless, studies suggest that the medium should hold the cutting firm with its leaf held above the surface of the medium. In addition to providing moisture at the cutting base the medium should allow respiration from the tissues and prevent anoxia which encourages rotting and cutting mortality. To prevent this, the medium should have an air:water ratio that optimizes the oxygen diffusion rate vis a` vis the needs of tissue respiration. In practice this involves the use of various sized particles (sand and gravel) and a water-holding medium (compost, perlite, vermiculite, peat, coir, or other organic products) alone or in various mixtures. As in the relationship between leaf morphology and the aerial environment, there is some evidence that morphological and physiological adaptations to different environments may affect the optimum porespace. Root development and growth is generally enhanced by the cutting base being warmer (artificially enhanced by providing “bottom-heat”) than the leaves. This promotes meristematic activity at the cutting base, while the leaves remain cool and free from stress. The converse differential tends to promote the growth of buds creating competition for assimilates.
Postseverance Treatments Auxin Applications The application of auxin is considered to promote rooting by stimulation of cell differentiation, the promotion of starch hydrolysis and the attraction of sugars and nutrients to the cutting base, but a better understanding is needed of the mechanisms regulating auxin concentrations and pathways (Atangana et al., 2011a). Indole-3-butyric acid (IBA) is typically the most effective auxin. Auxins are not, however, a “cure-all” treatment and exogenous applications of auxin do not promote rooting in cuttings which are morphologically or physiologically dysfunctional. Thus for auxins to have their stimulatory effects, the cuttings should have been taken from shoots which are preconditioned by stockplant management to be physiologically active when in the propagator, free from water and respiratory stresses, and with the capacity to mobilize stored or current assimilates. A point of practical importance is that the stems of some species are hairy, while others are waxy. This affects the retention of applied auxin. In addition, uptake is affected by the cross-sectional area of the cutting base (i.e., stem diameter).
Leaf Area The rooting of softwood cuttings is typically dependent on the presence of a leaf. Studies using infrared gas analyzers to measure the rates of photosynthesis in nonmist polypropagators during the propagation process found that rooting ability was maximized in photosynthetically active cuttings. However, a large photosynthetically active leaf is also actively losing water by transpiration and can suffer water stress—so closing its stomata or shedding its leaf. This then prevents further photosynthesis. Conversely, small leaved cuttings with inadequate assimilate production rapidly decline in their carbohydrate (sugars and starch) content due to respiration losses and hence cannot support root development. Successful rooting therefore requires an optimal leaf area which balances the positive effects of photosynthesis and the negative effects of transpiration (Leakey, 2004). The leaf area associated with this balance varies depending on the adaptations in leaf morphology of different species to different environments. Such variation is also determined by leaf age (node position) and the position of a shoot with the stockplant. In addition to leaf area, the correct balance between photosynthesis and transpiration will also be determined by the photosynthetic efficiency of the leaf due to the light environment of the propagator (level of irradiance) and the cutting’s water relations (affected by the aerial environment of the propagator and the air:water ratio of the medium). Studies of these important aspects of propagation have found a relationship between rooting ability and the content of refluxextracted soluble carbohydrates, which confirm the importance of assimilate production throughout the rooting process. The determination of the optimum leaf area is therefore a very important aspect of developing a successful propagation protocol, especially in difficult-to-root species. In general, rooting experiments focus on the factors enhancing rooting success. However, to learn more about the processes affecting rooting, there is much that could be learned from paying more attention to the causes of rooting failure. For example, more information is needed about how and when cutting death can be attributed to: water and heat stress, leaf abscission, photoinhibition, negative carbon balance, etc. In addition, some cuttings neither die nor root.
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Other related reasons for the failure of cuttings to root can be the death of the leaf due to microbial infection, anoxia and rotting, necrosis, bleaching, or leaf abscission. These problems can arise due to the use of old shoots with senescent or photosynthetically inactive leaves that are past their compensation point (photosynthetic activity vis a` vis senescence); water stressed or starch-filled. The most common symptoms are leaf shedding, leaf rot, and stem rot. Gaining an understanding of these causes of cutting death and hence the failure of the propagation process can be as important as determining how to achieve good rooting. Despite the importance of photosynthesis for the rooting of leafy softwood cuttings, there is some evidence that some species are also able to mobilize and use stored reserves of carbohydrates to contribute to the rooting process. This may reflect differences in stem anatomy and perhaps also adaptations to seasonally harsh environments.
Cutting Length The stem length of a cutting is another important variable affecting rooting success (Leakey, 2004). It is important in two ways. Firstly it affects the depth of insertion into the rooting medium, as well as the height of the leaf above the surface of the rooting medium, and secondly it inherently affects the capacity of the cutting to root. This is something we will investigate later. Both the depth of insertion and the height of the leaves above the medium can be important in terms of providing uniform conditions for water uptake and preventing competition between cuttings for light. It is also desirable to prevent the leaves from touching the medium and getting saturated by water droplets. Cuttings can be cut to a constant length (these may vary in the number of nodes and leaves present), or can be cut to the length determined by a chosen number of nodes. The simplest cutting is a single-node cutting. It has one internode and generally has one leaf and one bud. In this case the internode will generally vary in length depending on its position within the stem (Fig. 20.4). As a general rule, basal internodes are shorter than more apical ones, this reflecting the vigor of growth at the time the node was formed in the terminal bud. We will examine this within stem variability later. For practical purposes it is probably best to use a constant length close to the optimum, although this may not result in the availability of greatest number of cuttings.
Stockplant Factors: Cutting Origin and Environment There are two major sources of variation attributable to the stockplant: (1) Within-shoot factors, and (2) Between shoot factors. Both of these are subject to the stockplant environment and so can be influenced by stockplant management.
Within-Shoot Factors We saw earlier that single-node cuttings vary in their internode length (Fig. 20.4). This variation is associated with gradients in numerous other variables which basically run from the bottom to the top of the stem in parallel with chronological age, such as leaf size, leaf water potential, leaf carbon balance, leaf senescence, internode diameter, stem
FIGURE 20.4 The node-to-node variation in the length of the internode and the petiole in cuttings from the top two shoots of a Triplochiton scleroxylon stockplant (uppermost nodes on the left). Courtesy of Roger Leakey.
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FIGURE 20.5 The gradient of variation in rooting ability within a shoot: the long uppermost cuttings root best, whereas the short basal cuttings die.
lignification, nutrient and stem carbohydrate content, and respiration (Leakey, 2004). Thus no two cuttings are identical and, as a consequence, no two cuttings have the same rooting ability (Fig. 20.5). This is because all these variables affect the physiological processes in the cutting. A number of studies have used this node-by-node variation as an experimental variable to examine some of the key factors affecting rooting ability. For example, a comparison of the normal gradient in cutting length with an inverse gradient showed the importance of cutting length while a comparison with standard length cuttings showed the importance of cutting diameter/volume. It seems that the stem volume of the cutting determines the storage capacity of the cutting for current assimilates. In this connection, there are interactions between stem volume and leaf area. The stockplant environment and/or stockplant management also interact with node position in ways that help to elucidate the effects of different treatments. Studies of this sort have shed light on the relative importance of carbohydrates and nutrients in the rooting process. Between Shoot Factors As plants grow and increase in size they branch and become more complex. This creates competition between shoots for assimilates and nutrients, as well as causing mutual shading between leaves. This competition is made more complex by the processes of correlative inhibition and the development of dominance between shoots which are mediated by growth regulators as well as by the plant’s environment, especially light and nutrients (Leakey, 2004). To maintain stockplants in a good condition for easy rooting they need to be pruned back hard to leave a short stump of about 20 cm. So the simplest form of stockplant has just been cut back once and then sprouts from its uppermost remaining buds. A comparison of the rooting ability of these different shoots has found that cuttings from the uppermost one roots best and those from lower shoots root progressively less well. However, if such stockplants are cut back to different heights (say between 20 and 100 cm) then usually the taller stockplants produce more shoots. In this case the rooting ability of cuttings from the top shoot declines with increasing height and a relationship is found between the number of shoots and the percentage of cuttings rooted (Fig. 20.6). This implies that competition between the shoots reduces rooting ability and the removal of the lower shoots increases the rooting ability of cuttings from upper shoots. Further studies to test the competition hypothesis in plants of the same height have found that other factors also seem to be involved. For example, the lower shaded shoots can root very much better than less shaded shoots, especially under conditions of high soil nitrogen. Additionally, reorienting the stockplant so that its stem is not vertical
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FIGURE 20.6 Effects on the rooting success of cuttings from the top shoot of stockplants pruned to different heights. Cuttings from short stockplants with few shoots root best.
(e.g., 45 degrees or 90 degrees from the vertical) alters the location of vigorous growth and the fast-growing lower basal shoots become the most easily rooted. Thus in addition to competition there are effects of shoot position and of stockplant environment. When studies tried to elucidate these interactions between shoot position and intershoot competition in two-shoot stockplants of the same height, it was found that basal shaded shoots had a higher rooting ability than upper shoots. However, if these basal and upper shoots were under conditions with the same light environment (irradiance) the differences between shoot positions were eliminated (Fig. 20.7). This finding led to experiments in controlled-environment growth chambers to investigate the role of light (Hoad and Leakey, 1994, 1996).
STOCKPLANT ENVIRONMENT As seen previously, both nutrients and shade seem to affect the rooting ability of cuttings from shoots on relatively simple stockplants, but these complex preconditioning processes are poorly understood. It is clear that both the amount of light (irradiance) and the spectral quality of light (red:far-red ratio)—both features of shade—are important and independently affect the rooting ability of cuttings. Shade light is also commonly associated with shoot etiolation and hence with long internodes, and with large thin leaves. These affects are then further complicated by an interaction with soil nutrients which promote shoot growth (Leakey and Storeton-West, 1992). The light/nutrient interaction affects photosynthetic processes. The most dramatic of these interactions seems to be the combination of high irradiance and low nutrients which results in short starch-filled stems, the inhibition of photosynthesis and very poor rooting (Fig. 20.8). At the other extreme, low irradiance, and high nutrients are associated with active photosynthesis, low starch, and good rooting. These preconditioning effects of irradiance, light quality, and nutrients on morphology are also associated with physiological differences in the stems and leaves of the shoots preseverance (Hoad and Leakey, 1994, 1996). Physiologically, shade reduces the tendency of the top shoot to dominate and reduce the growth of other shoots
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FIGURE 20.7 Effects of shoot position on the rooting success of cuttings from stockplants with apical and basal shoots. The effects of shade were examined by adjusting the height of the pots.
FIGURE 20.8 The effects of growing stockplants at two levels of light (low and high irradiance) and two levels of nutrients (low and high). This affects the rates of photosynthesis and the starch content of cuttings. Starch-filled cuttings with low rates of photosynthesis rooted very poorly. Modified from Leakey, R.R.B., Storeton-West, R., 1992. The rooting ability of Triplochiton scleroxylon K. Schum. cuttings: the interactions between stockplant irradiance, light quality and nutrients. For. Ecol. Manage., 49, 133 150.
(i.e., promotes codominance), lowers the rates of preseverance net photosynthesis, lowers leaf chlorophyll concentration, but stimulates higher rates of net photosynthesis per unit of chlorophyll. Preseverance conditioning thus actively promotes postseverance photosynthesis during the rooting process. The shoots are therefore said to be physiological “young,” while shoots with low vigor and inhibited photosynthesis are physiologically “old.” A mechanistic model of carbohydrate dynamics during the rooting process can be used to gain further insights into the rooting process (Dick and Dewar, 1992) and to define key principles for the development of robust rooting protocols.
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TABLE 20.3 The importance of stockplant management for successful vegetative propagation. Standard hedged stockplant
Standard hedged stockplant
Preconditioned (far-red light and fertilizers) hedged stockplant
No knowledge of stockplant management
Knowledge that rooting is best from upper shoots
Knowledge that preconditioning improves physiological activity and rooting capacity
No. of cuttings harvested
31
16
45
No. of cuttings rooted
16
16
40
Percentage of cuttings rooted
52%
100%
89%
Number of plants established
16
16
40
Note the lack of relationship between rooting percentage and the numbers of plants established—this reflecting the understanding of the importance of stockplant management (which shoots to use and the role of stockplant environment. Adapted from Leakey, R.R.B., 2004. Physiology of vegetative reproduction. In: Burley, J., Evans, J., Youngquist, J.A., (Eds.), Encyclopedia of Forest Sciences. Academic Press, London, pp. 1655 1668.
Stockplant Management The major importance of all of the previous morphological and physiological stockplant factors makes it clear that stockplant management (a combination of regular pruning, fertilizer use, and light management) should be a key component of any macropropagation protocol. To retain physiological youth, stockplants are often managed as hedges. Using nitrogen-fixing tree/shrub species to shade these hedges then maximizes the rooting success by provision of desirable light and soil nutrient environments. One of the issues which has created much of the confusion in the scientific literature of vegetative propagation is the very common (even ubiquitous) use of percentage rooting as the measure of success. Rooting percentage is, however, affected by the standards of stockplant management, use of the best shoots (the shoots with optimal morphology and highest physiological activity, Table 20.3). Contradictory results arise in the literature when authors do not provide the relevant information in the description of their experimental protocols.
Phase Change The vegetative propagation literature has numerous references to what is called “Phase Change” or ontogenetic aging. This literature basically attributes the loss of rooting ability as perennial plants age and get larger to their gradual transition from the juvenile phase of vegetative growth to a phase of sexual maturity. The propagation of mature tissues is one of the major constraints to many tree improvement programs focusing on cultivar development through macropropagation. This loss of rooting ability with increasing size and structural complexity is perhaps not surprising given the already mentioned effects of stockplant factors in even small and simple stockplants. Nevertheless, the importance of phase change vis a` vis rooting ability is an unresolved aspect of vegetative propagation (Leakey, 2004). The phase change hypothesis assumes that plants (and trees in particular) gradually progress from juvenility (sexually immature and easy-to-root) to maturity (sexually mature and capable of flowering and fruiting associated with low rooting ability) over time. In trees, maturity may not be achieved for 3 20 years. It is generally recognized that the best way to return a tree to the juvenile state is to cut it down and to allow coppice shoots to grow from the stump. These coppice shoots are young and vigorous and cuttings from them can usually be rooted easily. Studies have suggested however that there is a decline in rooting ability with the increasing diameter of the stump. It is not known whether this is due to some aspect of the aging process or to stockplant factors like those mentioned for seedlings above. For example, large stumps generally produce more shoots than smaller stumps. This loss of rooting ability could therefore be due to increased intershoot competition.
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There are some inconsistencies between the concept of phase change and observation. Originally the hypothesis was based on work in ivy (Hedera helix) which has various leaf shapes. It was assumed that the normal palmate leaves of climbing ivy vines were characteristic of the juvenile easy rooting phase, while the ovate leaves of flowering shoots were assumed to be characteristic of the difficult-to-root mature phase. Observation of large ivy plants suggests a different interpretation as palmate leaves can be seen to be associated with main stems (the climbing vine), while ovate leaves are associated with free-standing branches on which flowers are formed. The association of flowering with branches is common in many woody plants, and it is not unusual for branches and main stems to differ in the arrangement of their leaves and buds. Certainly in ivy it is common to see old and large vines climbing a cliff or dead tree with palmate leaves tens of meters above the ground and flowering branches with ovate leaves all the way up the vine. A second way in which the hypothesis is not supported is the evidence that ontogenetically mature plants (with the capacity to flower and form fruits) propagated by cuttings, grafts or marcotts can be easily repropagated by cuttings when they are used as small managed stockplants. Although often plagiotropic, such stockplants have the vigor of juvenile seedlings and coppice shoots. This suggests the poor rooting ability of “mature” shoots can be attributed to “physiological aging” rather than to “ontogenetic aging.” In other words, the difficulty in rooting crown shoots in large mature trees is due to a severe case of the combination of undesirable morphological and physiological factors resulting from within and between stockplant factors and their interaction with the stockplant environment (Dick and Leakey, 2006). A start has been made to examining this experimentally within the crown of mature trees (Pauku et al., 2010).
Genetic Variation in Rooting Ability Early in this chapter we saw that there are genetic differences in the ease of propagation between species and even between provenances and clones within species. In severe cases this has led to the conclusion that some species are “impossible to root.” We have also seen that species vary in the leaf and stem morphology, and in their adaptations and responses to environmental factors. There is now good evidence that species previously thought to be “impossible to root” can now be preconditioned to be easier to root by good stockplant management based on an understanding of the morphological and physiological factors affecting rooting ability. Thus the apparent genetic differences in rooting ability can instead be attributed to genetic differences in the growth and development of shoots. The concept that inherent genetic differences in rooting ability are not critically important is also supported by the use of stepwise regression in the analysis of experimental data from rooting studies. Such analysis commonly finds that the factors explaining much of the variance are cutting length, leaf abscission, leaf area, etc., rather than clone or provenance (Dick et al., 1999). TABLE 20.4 The analogy between athletic ability and rooting ability in the vegetative propagation of tree stem cuttings. Sporting ability
Rooting ability
Young
Juvenile
Physiologically fit—lean, well-prepared, strong
Physiologically young—high rates of photosynthesis—long internodes, large leaf area, chronologically young node position
Genetic morphology—muscles, body build, etc.
Genetic morphology—stem and leaf anatomy
Preparation in gym—physiologically fit and full of energy
Preconditioning by stockplant management —physiologically active
Highly competitive and ready for action
Preconditioned to contain low starch/high soluble sugars and free from competing shoots
Use of performance-enhancing drugs
Use of auxins as rooting hormone
Stress-free Olympic village
Stress-free propagation environment
Different sports—refine training regime
Different species—refine propagation regime
Adapted from Leakey, R.R.B., 2012b. Living with the Trees of Life UK, 200 pp.
Towards the Transformation of Tropical Agriculture, CAB International, Wallingford,
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Contrary to this, some studies to detect quantitative trait loci affecting vegetative propagation have, however, reported that phenotypic variation has a meaningful genetic component.
CONCLUSIONS There are many facets to developing a robust approach to macropropagating plants, especially by the rooting of stem cuttings. Over the last 15 20 years much progress has been made to gain a good understanding of the numerous interacting factors (stockplant environment 3 stockplant management 3 topophytic variables 3 node position 3 nursery management 3 postseverance treatments 3 propagation environment) by studying them in tree species. This understanding now provides some general principles that can be applied to the propagation of new species, and especially those considered to be difficult to ltotaoatpropagate. Leakey (2012b) has likened many of the factors that determine the success of propagation by stem cuttings to those that determine the success of an athlete competing in the Olympic Games (Table 20.4). This analogy has been found to help people to understand the key principles. The greatest challenge remaining is to improve the design and reporting of experiments so that the quality of the literature is improved by researchers adequately describing the material they used, as well as the pre and postseverance environments. This should remove the contradictory results in the literature created by people not using comparable material in terms of the physiology and morphology of the tissues used. Then there is a need for more extensive studies of the importance of stockplant management and the stockplant environment across a wider range of species.
Courses 5 Training videos by Edinburgh Center for Tropical Forests (DVD)
Relevant Websites ICRAF website tree_domestication
Narrated
PowerPoint
lectures
www.worldagroforestry.org/Units/training/downloads/