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Chapter 1: Weed Science Research: Past, Present and Future Perspectives From Weed Research: Expanding Horizons, First Edition. Edited by Paul E. Hatcher and Robert J. Froud-Williams

Chapter 1: An introduction to cells and their organelles From Plant Cells and their Organelles, First Edition. Edited by William V. Dashek and Gurbachan S. Miglani

Chapter 1: Introduction From Applied Tree Biology, First Edition. By Andrew D. Hirons and Peter A. Thomas

Chapter 1: The Impact of Biotechnology on Plant Agriculture From Plant Biotechnology and Genetics: Principles, Techniques, and Applications, Second Edition. Edited by C. Neal Stewart, Jr

Chapter 1: An Introduction to Flowering Plants: Monocots and Eudicots From Flowering Plants: Structure and Industrial Products, First Edition. By Aisha Saleem Khan

Chapter 1: Recent Advances in Sexual Propagation and Breeding of Garlic From Horticultural Reviews, Volume 46, First Edition. Edited by Ian Warrington CONTENTS

Chapter 1: Weed Science Research: Past, Present 3 and Future Perspectives From Weed Research: Expanding Horizons, First Edition.

Chapter 1: An introduction to cells and their organelles 35 From Plant Cells and their Organelles, First Edition.

Chapter 1: Introduction 59 From Applied Tree Biology, First Edition.

Chapter 1: The Impact of Biotechnology on Plant Agriculture 72 From Plant Biotechnology and Genetics: Principles, Techniques, and Applications, Second Edition.

Chapter 1: An Introduction to Flowering Plants: Monocots 91 and Eudicots From Flowering Plants: Structure and Industrial Products, First Edition.

Chapter 1: Recent Advances in Sexual Propagation and Breeding 120 of Garlic From Horticultural Reviews, Volume 46, First Edition. 1

1

Weed Science Research: Past, Present and Future Perspectives Robert J. Froud‐Williams

‘Russets’, Harwell, Oxon, UK

Introduction

Plants popularly referred to as weeds have been described by Sir E.J. Russell (1958) as ‘The ancient enemy’. In his text on agricultural botany, Sir John Percival (1936) made the observation that the idea of uselessness was always present in the mind when weeds are being spoken of, while, in the editor’s preface to Weeds and Aliens by Sir Edward Salisbury (1961), weeds are likened to criminals – when not engaged in their nefarious activities both may have admirable qualities: ‘an aggressive weed in one environment may be a charming wild flower in another’. Our relationship with weeds certainly is as old as agriculture itself and the concept of weediness was recognised from biblical abstracts, for example the gospel according to St Matthew (Ch. 13 v. 7, the parable of the sower): ‘Other seed fell among thorns, which grew up and choked them’. Yet weed s cience as a discipline is less than one hundred years old, albeit Fitzherbert (1523) in his Complete Boke of Husbandry recognised the injurious effect of weeds on crop production: ‘Weeds that doth moche harme’ included kedlokes, coceledrake, darnolde, gouldes, dodder, haudoddes, mathe, dogfennel, ter, thystles, dockes and nettylles’. These are recognised today as corncockle, charlock, darnel, corn marigold, dodder, cornflower, mayweed, stinking mayweed, fumitory, thistles, docks and nettles, several of which are now greatly diminished in abundance. A major developmentCOPYRIGHTED in weed removal from MATERIAL within crops was achieved with the development of the seed drill by Jethro Tull c. 1701. Initially, the objective of this inven­ tion was to enable cereals to be sown in rows, whereby a horse‐drawn hoe could be used to pulverise the soil in the inter‐row. Tull conjectured that such ‘pulverisation’ would release nutrients beneficial to the crop, but coincidentally enabled weed removal, whereby ‘horse‐hoeing husbandry’ became standard practice, reducing weed competition and the necessity of fallow, a serendipitous discovery. Despite the efficacy of technological advances in weed control, weeds still exert great potential to reduce crop yields. Weeds are considered the major cause of yield loss in five crops (wheat, rice, maize, potato and soybean and a close second in cotton) (Oerke, 2006). Estimated potential losses due to weeds in the absence of herbicides were 23, 37,

Weed Research: Expanding Horizons, First Edition. Edited by Paul E. Hatcher and Robert J. Froud-Williams. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.

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40, 30, 37 and 36% for the six crops respectively, while weed control reduced these losses to 7.7, 10.2, 10.5, 8.3, 7.5 and 8.6%, albeit with considerable regional variation (Oerke, 2006). Efficacy of crop protection practices varied between geographic regions, but whereas efficacy of disease and pest control was only 32 and 39% respectively, e fficacy of weed control was almost 75%. The greater efficacy of weed control was attributed to the ability to employ both physical and chemical methods. Possible reasons for the apparent mismatch between weed control efficacy and actual yield losses were ascribed to changing cultural practices such as monoculture, multiple cropping, reduced rotation and tillage and the introduction of more vulnerable crop cultivars dependent on increased fertilisation. Weeds have a major impact on human activities for not only do they adversely affect economic crop yield indirectly through interspecific competition (see Bastiaans & Storkey, Chapter 2) directly as a result of parasitism (see Vurro et al., Chapter 11) and allelopathy, but also they affect human health and the well‐being of livestock through physical and chemical toxicity. Additionally they may negatively impact environmental quality and functionality, such as that posed by alien invasive species including aquatic weeds (see Bohren, Chapter 10). The objective of this preliminary chapter is one of scene setting. It seeks to associate ‘man’s’ controversy with weeds as a consequence of their detrimental as well as benefi­ cial relationships. Our changing perception of weeds is examined in terms of a shift in emphasis from that of pragmatic weed destruction to one of management and rational justification for their suppression. Agronomic practices greatly influence weed population dynamics and these are o utlined with particular attention to the UK weed floras. The history of weed science is explored as a discipline, together with a brief history of weed control technology includ­ ing the discovery and development of synthetic herbicides. The origins of the Weed Research Organization (WRO) are discussed, together with the subsequent formation of the European Weed Research Society. Weed science as a discipline originated at Rothamsted in England, the first agricul­ tural research institute to be established in the world, with the pioneering work of Winifred Brenchley on the classic long‐term continuous winter wheat experiment, Broadbalk, where she investigated the impact of various agronomic factors such as manuring, liming and fallow on the arable weed flora.

Factors Influencing the Weed Flora

Succession

The British flora is not an event, but a process that is continuing both with respect to accretions and diminutions (Salisbury, 1961). Vegetation is never static and weed popu­ lations are probably subject to greatest fluctuation as their habitat is continually dis­ turbed. Two types of change within plant communities may be recognised: fluctuating and successional. Arable plant communities are subject to fluctuations as a consequence of direct intervention. Weeds are fugitives of ecological succession; were it not for the activities of man they would be doomed to local extinction and relegated to naturally disturbed habitats such as dune and scree. Weeds have been described as the pioneers of secondary succession, of which the weedy arable field is a special case (Bunting, 1960).

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Successional change is less likely within ephemeral communities, although poten­ tially capable in systems of prolonged monoculture and non‐tillage. Two types of s uccessional change may be recognised – autogenic and allogenic. Autogenic succession occurs in response to changes within the habitat, as species better adapted to a chang­ ing habitat oust previous inhabitants. A classic example of autogenic succession is Broadbalk Wilderness, whereby climax vegetation was achieved 30 years after the a bandonment of an arable crop (Brenchley & Adam, 1915). Allogenic succession occurs in response to modified environmental factors such as fertiliser and herbicide input. Prior to the advent of selective herbicides in 1945, weeds were kept in check by a combination of rotation, cultivation and clean seed, the three tenets of good husbandry. Previously, weed control was strategic, but the availability of herbicides enabled a tacti­ cal approach. However, the realisation that some weed species are of beneficial value to the arable ecosystem rendered the pragmatic destruction of weeds other than those that were most intransigent less acceptable; maximisation of yield was not necessarily synonymous with maximisation of profit.

Clean Seed

The use of clean seed as a consequence of the development of threshing machinery was greatly assisted by improvements in seed screening and legislation such as the 1920 Seeds Act designed to reduce the number of impurities. Regular inspection by the Official Seed Testing Station (OSTS) provides testament to the merits of seed certifica­ tion. Early casualties of improved sanitation were the mimetic weeds such as Agrostemma githago L. (corncockle)*, a formerly characteristic weed of cereals which could be sepa­ rated by seed screening. Prior to 1930 it was a frequent grain contaminant, as witnessed by records of the OSTS; the last authenticated record of its occurrence was documented in 1968 (Tonkin, 1968). A further factor contributing to its demise was the fact that its seeds are of short persistency in soil and require continual replenishment for survival. A survey of cereal seed drills in 1973 indicated considerable contamination by weed seeds including wild oats (Avena spp.) and couch grass Elymus repens (L.) Gould) as well as Galium aparine L. (cleavers) and Polygonum spp. (Tonkin & Phillipson, 1973). EU legislation designed to reduce the incidence of weed seed impurities in crop seed has certainly reduced this as a source of infestation, with, for example, only a single wild oat seed permitted per 500‐g sample, provided that the next 500‐g sample is entirely free of contamination.

Rotation

The season of sowing is the greatest determinant of weed occurrence (Brenchley & Warington, 1930). Hence, in the 1960s when spring barley predominated, spring‐ germinating species were prolific, the most significant of which was Avena fatua L., but also a diverse array of broad‐leaved species, the periodicity of which is predominantly or entirely in the spring. The shift to autumn cropping in the 1980s disadvantaged spring‐germinating species as a consequence of crop competition. Avena fatua exhibits a bimodal pattern of germination such that it was not necessarily disadvantaged, but it is possible that the related Avena sterilis ssp. ludoviciana (Durieu) Gillet & Magne.,

* Botanical nomenclature follows Stace (1997).

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which is entirely autumnal in germination periodicity, may have supplanted it as the dominance of winter cropping continues. Previously, rotation for a spring‐sown crop would have detrimentally affected the incidence of Avena sterilis. The switch to autumn‐sown cereals sown increasingly earlier and established by minimal tillage has exacerbated the incidence of annual grass‐weeds, most notably Alopecurus myosuroides Huds. (Moss, 1980). Delayed drilling enables the use of stale seedbeds, thereby eliminating earlier weed emergence. It is of note that fallowing was introduced on the classic Broadbalk continuous winter wheat experiment as a response to the increasing problem posed by A. myosuroides (black‐grass) in the 1930s and 1940s (Moss et al., 2011). A deviant of rotation was fallow, designed to reduce the incidence of perennial weeds on heavy soils by means of repeated cultivation through desiccation and exhaustion of vegetative propagules. Indeed, prior to the advent of herbicides this was the favoured means of reducing infestations of perennial grass‐weeds, notably the five species of couch grass.

Fallow

Traditionally, perennial grass‐weeds proved intractable and control depended on the inclusion of rotation and fallowing to enable mechanical weed control. The develop­ ment of the non‐selective herbicide aminotriazole in 1955, providing both soil and foliar activity, offered opportunities for couch grass control in the uncropped situations of autumn stubble. Diquat and paraquat, introduced in 1957 and 1958 respectively, similarly allowed control of Elytrigia in non‐crop situations. Because of the limited translocated activity of diquat, it proved desirable to cultivate stubbles prior to treatment in order to frag­ ment rhizomes, thus alleviating apical dominance and enabling bud regeneration and regrowth. It was not until the advent of glyphosate in 1971 that a non‐selective foliar‐translocated herbicide no longer necessitated rhizome fragmentation. Its ability to be applied pre‐ harvest of cereals following crop senescence further enabled a reduction in the incidence of couch. Now in English farmland couch is not a problem. However, couch does remain a significant problem in Scotland owing to the delayed senescence of the crop, and the benefits of pre‐harvest application in wheat are disputed. Subsequently, the introduction of sulfosulfuron and propoxycarbazone‐sodium in 2002 for the selective control of couch and other grass‐weeds within crop situations has further contributed to the reduced incidence of these perennial grass‐weeds. The additional inclusion of winter oilseed rape as an alternative autumn‐sown crop resulted in considerable modification of the weed flora. By virtue of its optimal early sowing date, mid–late August, a number of late‐season germinating species became characteristic of the crop, including Sonchus spp. and Matricaria spp. (Froud‐Williams & Chancellor, 1987). Also, notable gaps in the herbicide arsenal enabled species such as Galium aparine and Geranium dissectum L. (cut‐leaved cranesbill) to proliferate, as well as unlikely candidates such as Lactuca serriola L. (prickly lettuce), Conium macu- latum L. (hemlock) and Sisymbrium officinale (L.) Scop. (hedge mustard). Hitherto, Papaver rhoeas L. (field poppy) that was highly susceptible to the phenoxyacetic acid herbicides in cereals became prominent in the absence of an effective treatment prior to

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the advent of metazachlor. The acreage of oilseed rape in the UK increased dramatically from c. 1000 ha in 1970 to 705,000 ha in 2011. One consequence of the expansion of oilseed rape was the legacy of feral rape as a roadside weed.

Cultivation

The transition from traditional systems of cultivation based on mouldboard ploughing to non‐inversion tillage, made possible by the advent of paraquat and glyphosate, exac­ erbated the incidence of grass‐weeds to the detriment of broad‐leaved weeds character­ istic of arable land. In particular this was exemplified by species such as Alopecurus myosuroides and Anisantha sterilis (L.) Nevski (barren brome), the latter particularly prevalent on shallow calcareous soils. A combination of straw burning and soil‐acting residual herbicides such as isoproturon and pendimethalin contributed to management of black‐grass, but during the 1970s suitable herbicides for brome management were lacking other than expensive combinations such as tri‐allate followed by a sequence of metoxuron. By comparison, inversion tillage with or without straw burning had prevented brome from becoming a significant problem prior to the uptake of minimal tillage and autumn cropping. That said, the incidence of Anisantha sterilis as a weed of cereals was documented in the 1960s (Whybrew, 1969).

Straw Burning

A further contributory factor enabling the adoption of non‐inversion tillage was the ability to remove previous straw residues by stubble burning. This had a sanitary effect, destroying a considerable number of weed seeds on the soil surface, albeit some impair­ ment of herbicide performance was observed with the phenylureas, most notably chlo­ rotoluron. However, the UK straw burning ban introduced in 1993 necessitated some return to traditional cultivation practices, as did the increasing threat of herbicide‐ resistant black‐grass. Since the mid‐1990s there has been a resurgence of non‐inversion tillage made possible through stubble incorporation and treatment with glyphosate. The overall effect of various agronomic practices on an individual weed species has been demonstrated in relation to black‐grass (Lutman et al., 2013). The greatest reduc­ tion was achieved by rotation with a spring‐sown cereal which reduced populations on average by 88%. Mouldboard ploughing prior to winter cropping reduced plant densi­ ties on average by 69% relative to non‐inversion tillage, while delaying drilling from September to October reduced densities by up to 50%. Increasing crop seed rate and selecting for more competitive cultivars reduced the number of reproductive heads by up to 15 and 22% respectively.

Soil Amelioration, Drainage and Fertiliser Use

Other characteristic cornfield weeds such as Chrysanthemum segetum L. (corn mari­ gold) have further suffered decline despite being relatively non‐susceptible to herbi­ cides, as a consequence of amelioration of soil conditions by liming. A weed more typical of the north and west of the British Isles, it is associated with sandy soils of low pH. Although it exhibits a bi‐modal pattern of germination in autumn and spring, the autumn‐emerging cohort is particularly prone to frost damage, and so it is more likely encountered in spring barley.

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Large‐scale soil drainage during the 1960s has resulted in decline of those species tolerant of a high water table, such as Gnaphalium uliginosum L. (marsh cudweed), Polygonum cuspidatum L. (amphibious bistort) and Polygonum hydropiper L. (water pepper). Consequently, many species have retreated to their climatic and geographic refugia (Holzner, 1978).

Nitrogen

Changes in the use of nitrogenous fertilisers have also had a considerable impact on those species that are least competitive, such as Legousia hybrida (L.) Delarbre (Venus’s looking glass), partly as a consequence of their inability to compete with nitrophilous species such as Galium aparine. It has been stated that the most effec­ tive means of weed suppression is a healthy vigorous crop. Studies at Broadbalk indicate that leguminous species such as Medicago lupulina L. are more prevalent on low nitrogen plots, as is also Equisetum arvense L., partly as a consequence of their tolerance or lack of suppression by nitrophilous species (Moss et al., 2004; Storkey et al., 2010). Conversely, Stellaria media (L.) Vill. (chickweed) showed a positive correlation with increasing nitrogen amount. Use of nitrogen in UK cereals increased dramatically between the 1960s and 1980s (Chalmers et al., 1990). Despite increased rates of nitrogen application this does not explain the demise of Lithospermum arvense L. (corn gromwell), which is nitrophilous and highly competitive and not excessively susceptible to herbicides. A major factor here has been the earlier drill­ ing date of cereals (Wilson & King, 2004). Species that are adversely affected by fer­ tiliser and herbicides have been shown to share characteristic traits of short stature, late flowering and large seed size (Storkey et al., 2010). Traits such as short stature and large seed size were shown to be of competitive advantage under conditions of low fertility. So too, Storkey et al. (2012) have shown a correlation between arable intensification and the proportion of rare, threatened or recently extinct arable plants within the European flora, with the greatest variance attributed to fertiliser use. Thus, the proportion of endangered species was positively related to increasing wheat yield. Despite the transitory effects of cultural practices on weed populations, herbicides* have most probably exerted the greatest impact on species diversity and abundance. This is further evident from depletion of arable weed seedbanks, which often exhibited − densities of between 30,000 and 80,000 m 2 in the pre‐herbicide era but have shown substantial reductions in recent years (Robinson & Sutherland, 2002).

Herbicides

The earliest attempts at chemical weed control involved inorganic salts and acids, perhaps the earliest example of which was the use of sodium chloride for total vegetation control, as occurred following the sacking of Carthage in 146 bc. During the latter half of the nineteenth century, inorganic salts were developed for selective weed control, for example, copper sulphate used selectively in France (1896) for control of charlock (Sinapis arvensis L.) in wheat (Smith & Secoy, 1976). Ferrous sulphate and sodium chlo­ rate were introduced between 1901 and 1919; the latter for total weed control in France,

* Herbicide chemical nomenclature follows Tomlin (2006).

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as reported in Timmons (2005). Ferrous sulphate is still used for moss control in lawns. Sulphuric acid introduced from 1930 for selective control of annual weeds in cereals was first used in France in 1911, but superseded by DNOC (4,6‐dinitro‐ortho‐cresol), developed as the first organic herbicide in 1932 and originally discovered to have insecticidal properties (Ivens, 1980) and used in early locust control. However, perhaps the earliest example of an organic herbicide was amurca derived from olive residue, used by the romans for weed control in olive groves (Smith & Secoy, 1976). Until 1945, chemical weed control was largely limited to the use of arsenical and c opper salts and sulphuric acid, the only organic substance being DNOC. Development of modern herbicides stems from the development of the growth regulator (hormone) herbicides during the 1940s following independent research of Imperial Chemicals Industry (ICI) and Rothamsted. ICI discovered the selective action of NAA (α‐naphthyl­ acetic acid), whilst the Rothamsted team demonstrated the selectivity of IAA (indole acetic acid) against clovers at low concentrations. Results of both groups were c ommunicated to Professor G.E. Blackman at the ARC Unit of Agronomy in Oxford, who led search for related structures of greater potency. Because of wartime secrecy, results were not disclosed until 1945. This research led to the development of MCPA (4‐chloro 2‐methyl phenoxy acetic acid) (Blackman, 1945) and of 2,4‐D (2‐4 dichloro‐ phenoxy acetic acid) independently in the USA. Following the advent of herbicides, methods of weed control departed considerably from hand hoeing and the use of steerage hoes. A survey of herbicide practice in four arable districts of eastern England in the cropping year 1959–60, of which about 80% of crops sown were cereals, indicated that herbicides were used on almost 80% of cereals in three of the areas (Lincolnshire Wolds, West Suffolk and Humber Warp) and 95% in the other (Isle of Ely). This compares with 56% usage on cereals in north‐west Oxfordshire 2 years previous (Church et al., 1962). MCPA was the most widely used herbicide, followed by mecoprop. By comparison, herbicide use in other arable crops ranged between 9 and 21%. Weeds that were targeted in these crops were Cirsium spp., Sinapis arvensis, Galium aparine, Stellaria media, Chenopodium album L. and Rumex spp. However, those species considered most intransigent were Avena spp., Persicaria maculosa Gray syn. Polygonum persicaria (L.), Tussilago farfara (L.), Stellaria media and Matricaria perforata Mérat. A comprehensive account of herbicide development prior to 1980 is provided by Ivens (1980). The recent history of weed communities has been one of acclimation to the intro­ duction of herbicides. Initially, the introduction of phenoxy‐acetic acids reduced the incidence of susceptible weeds such as Sinapis arvensis (charlock), only to find the niche vacated occupied by less susceptible species such as Galium aparine and Stellaria media, necessitating the introduction of phenoxy‐propionic acids such as mecoprop in 1957. So too were benzoic acids developed to address the incidence of Polygonum spp., while the hydroxybenzonitriles were introduced to target Matricaria spp. Following the introduction of the phenylurea herbicide isoproturon, Veronica persica (field speedwell) increased in prominence. Evidence for such a shift in weed floras is documented in studies conducted in Germany by Koch (1964) where depletion of weeds susceptible to DNOC resulted in increased occurrence of Alopecurus myosuroides, and that of Bachthaler (1967) where repeated application of phenoxy‐acetic acids over a 17‐year period displaced s usceptible species in favour of Matricaria spp., Polygonum spp. and Avena fatua.

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Likewise, Rademacher et al. (1970) observed a change in weed species dominance over a 12‐year period, while Hurle (1974) reported declines in the arable seedbank, particularly of Sinapis arvensis in response to repeated application of phenoxyacetic acids. However, in France, Barralis (1972) found little change in weed flora composi­ tion over 5 years. Similarly, Roberts and Neilson (1981) observed a progressive decline of Papaver rhoeas and Raphanus raphanistrum L. (wild radish) following application of simazine in maize, but substitution by Urtica urens L. (annual nettle) and Solanum nigrum L. (black nightshade). That said, other factors may contribute to fluctuations in weed populations, as indicated in a study by Chancellor (1979) where following application of a mixture of ioxynil, bromoxynil and dichlorprop to spring barley, most dicotyledonous species declined, whereas Papaver rhoeas decreased 92% on sprayed plots and by 91% on unsprayed plots. Conversely, Polygonum avicu- lare L. (knotgrass) increased by 67% on sprayed and by 189% on unsprayed plots. Such inexplicable dynamics have been reported for populations on Broadbalk (Warington, 1958). Despite the early success of discovering phenoxyalkanoic acid herbicides (hormone herbicides), row crops such as sugar beet benefited from the early discovery of c arbamates, for example, propham (IPC) in 1945. Chloridazon, a pyridazinone, was introduced in 1962, metamitron, a triazinone, and phenmedipham in 1965 and 1968 respectively. For use on mineral soils, lenacil was introduced in 1966. Likewise, h orticultural crops such as leeks and carrots benefited from the introduction of the substituted phenyl ureas monolinuron (1958) and linuron (1960), as did potatoes with regard to the latter. It is somewhat ironic that linuron use has been restricted in pota­ toes following EU legislation. Triazines became the mainstay of the horticultural fruit sector following the introduction of simazine in 1956, being applied to 62% of the black­ currant crop in 1962 (Davison, 1978). Usage in the amenity sector was revoked on 31 August 1993 and in the horticultural sector on 31 December 2007. Approval for the use of paraquat expired in July 2008. Inevitably, resistance to herbicides became an issue in the 1980s with resistance first appearing to the s‐triazines, notably simazine and atrazine. Resistance to the triazines had been predicted as a consequence of their persistency and, based on knowledge of selection pressure and ecological fitness, development of resistance could be foretold. Initially in the UK, resistance was confirmed in populations of Senecio vulgaris L. (groundsel) in geographically diverse locations, but with the common denominator of orchards and nurseries (Putwain, 1982). Resistance to s‐triazines involves a mutation of the chloroplast thylakoid membrane and is conferred by cytoplasmic inheritance involving maternal inheritance, and so is particularly likely to occur in inbreeding species such as Senecio vulgaris. Subsequently triazine resistance occurred in other weeds of fruit orchards, most notably Epilobium spp. The nature of resistance to the triazines somewhat misled subsequent conceptions concerning resistance to other herbicide classes such as the phenylureas, where resistance most commonly involves enhanced metabolism and was first evident in outcrossing Alopecurus myosuroides. Following the first reported incidence of resistance to chlorotoluron in 1982, resist­ ance to ACCase inhibitors and ALS inhibitors such as sulfonylureas is now well d ocumented in A. myosuroides, the latter often involving target site resistance. Furthermore, target site resistance has been documented in Stellaria media and Papaver rhoeas (see Moss, Chapter 7).

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Consequences of Changing Practices

Changing Weed Floras

Various means are available to determine the changing status of weed floras and of the priorities in weed research. For example, reference to reports of the WRO indicates those weeds that were considered of prime economic importance for control. So too, surveys can provide information on weed species occurrence at any particular time, whereas pesticide usage survey data provide an alternative indication as to which weeds were being targeted. Weed surveys carried out early in the life of the crop provide infor­ mation on those weeds present during establishment, whereas surveys conducted prior to harvest indicate those species inadequately controlled (see Krähmer and Bàrberi, Chapter 3). Chancellor (1976a,b) mapped changes in the weed flora of individual fields at the former WRO and was able to relate them to rotational and herbicide inputs. In one field the most dominant species Chrysanthemum segetum declined from 46% of the weed population in 1961 to only 6.8% in 1966, and only a single seedling of this species s urvived by 1976, this reduction being possibly attributed to liming and herbicide use. Likewise, in another field, similar declines of C. segetum and Raphanus raphanistrum were documented. However, other species, notably Polygonum aviculare, occupied the niche vacated. Chancellor also monitored changes in the weed flora over twenty years following the ploughing‐up of permanent pasture in 1960 whereby those species c haracteristic of grassland (with the exception of Trifolium repens L. still present after 20 years) were all eliminated within 15 years. Ranunculus bulbosus L. survived 14 years, Rumex obtusifolius L. 12 years and Plantago lanceolata L. 8 years (Chancellor, 1986). Weed surveys also provide an invaluable guide to the status of individual species, as demonstrated by the Botanical Society of the British Isles in which, despite its suscepti­ bility to herbicides, Sinapis arvensis was the most frequently occurring species, being recorded in 60% of tetrads assessed in the early 1970s (Chancellor, 1977). By compari­ son, Ranunculus arvensis L. was one of the least frequently recorded, this being attrib­ uted to its susceptibility to herbicides and lack of fecundity. Another example is that afforded by Sutcliffe and Kay (2000). This study surveyed weeds in 156 arable fields in Oxfordshire and Berkshire during the 1960s and then the same fields were re‐surveyed in 1997. Of the species common in the 1960s, those that had increased most were Alopecurus myosuroides and Galium aparine, as well as Anisantha sterilis which was absent in the 1960s. In addition, Cirsium vulgare (Savi) Ten (spear thistle), Geranium dissectum and Papaver rhoeas were more prevalent, indicative of the increased acreage of oilseed rape. Conversely, of those species considered to be rare arable weeds, their incidence had been reduced by 1997, most notably of Silene noctiflora L. (night flowering catchfly), perhaps indicative of its preference for spring‐sown crops. However, the continued presence of species such as Kickxia spp. is testament to the role of the seed­ bank in species survival. The decline of arable plants since 1930 is fully documented by Smith (1988). Comparable surveys have been undertaken elsewhere in Europe, most notably in Scandinavia. Thus, Andreasen et al. (1996) surveyed weeds in Danish arable fields between 1967 and 1970 and again in 1987 to 1989. While the dominant species were similar in both surveys, several of the less common species had declined considerably,

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most notably Silene noctiflora in spring barley crops. Surprisingly, G. aparine was also less frequent, but Stellaria media had increased in grass leys. A number of studies have been conducted since the 1960s in Finland (Mukula et al., 1969; Raatikainen et al., 1978; Kallio‐Mannila, 1986; Salonen et al., 2001). Thus, Kallio‐ Mannila observed that species most common in the 1960s remained so in the 1980s but at reduced frequency. Species typical of Finnish spring cereals were Spergula arvensis L., Stellaria media, Galeopsis spp., Chenopodium album and Viola arvensis Murray. Despite the use of herbicides, these same species remained dominant in the 1990s (Salonen et al., 2001). Changes in the weed flora have been monitored over 60 years in Hungary between 1947 and 2008 where, in recent years, agriculture has undergone great transformations (Novák et al., 2010). A survey conducted between 2007 and 2008 indicated that whereas Matricaria perforata remained the dominant weed species in wheat, the incidence of Galium aparine had decreased. Conversely, Ambrosia artemisiifolia L. (ragweed) had increased to become the second most frequent species in wheat. Some authors have attempted to quantify reasons for the changing status of weeds under various agronomic situations, such as organic vs. conventional farming systems (Hald, 1999a; Eyre et al., 2011), crop rotation (Hald 1999b; Eyre et al., 2011) and cultiva­ tion regime (Tuesca et al., 2001; Semb Tørreson & Skuterud, 2002), as well as chemical inputs including fertiliser (Boström & Fogelfors, 2002) and combinations of various factors (Andersson & Milberg, 1998; Bàrberi, 2002). In southern Europe a number of surveys have been undertaken in Spain indicating correlations between weed species occurrence and crop phenological development and management practices (Saavedra et al., 1989; Hidalgo et al., 1990), as well as environ­ mental factors including soil texture (Saavedra et al., 1990). Elsewhere, Andreasen et al. (1991) have characterised the relationship between weed distribution in Danish arable fields with respect to soil properties, indicating that soil amelioration with lime and nitrogen has contributed to less diverse weed communities. Evaluation of seedbank composition obtained from the farm‐scale evaluations of genetically modified (GM) herbicide‐tolerant crops conducted in the UK between 2000 and 2002 enables comparison with previous studies and indicates that whereas some taxa have declined in relative frequency, others have increased. Thus, notably both Chenopodium album and Polygonum aviculare have decreased in rank order relative to surveys undertaken in the 1960s, 1970s and 1980s, perhaps indicative of the switch to autumn cropping, whereas Sonchus spp. and Matricaria have increased, both of which are associated with oilseed rape. Indeed, Brassica napus L., although not previously recorded, ranked tenth (Heard et al., 2005). Epilobium spp., not previously recorded, ranked twelfth and Alopecurus myosuroides twentieth, indicative of non‐inversion tillage. In the UK, cleavers (Galium aparine) appeared to make a significant increase during the early 1980s, partly as a consequence of reduced tillage intensity and the fact that it was neither well controlled in winter cereals nor winter oilseed rape. In both cereals and rape it is competitive, interferes with harvesting and is likely to result in contamination of the harvested crop. The population dynamics of Galium aparine in winter wheat in relation to tillage regime indicated an annual 23‐fold increase in the seedling p opulation after shallow tine cultivation, relative to only four‐fold increase with inversion tillage in the absence of herbicides (Wilson & Froud‐Williams, 1988).

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Thus, the soil seedbank was depleted more rapidly following tine cultivation such that 83% of seedlings were derived from seed shed the previous year relative to only 14% after ploughing. Hence, had a suitable herbicide been available the infestation would have been depleted most rapidly with non‐inversion tillage. A plethora of herbicides have been developed to contain cleavers in both winter cereals and oilseed rape, including amidosulfuron, carfentrazone +/– mecoprop, cinidon‐ ethyl, fluroxypyr, florasulam, florasulam +/– fluroxypyr, florasulam + pyroxsulam and picolinaflen + pendimethalin in cereals as an alternative to mecoprop, and in oilseed rape metazachlor + quinmerac, dimethenamid‐p + metazachlor and clomazone +/– metazachlor, and despite varying levels of efficacy Galium aparine is now a much less important weed in either crop than in the 1980s. In surveys conducted in cereals and oilseed rape prior to harvest, Galium aparine was the most frequently occurring broad‐leaved species (Froud‐Williams & Chancellor, 1987). Surveys by Whitehead and Wright (1989) prior to herbicide application indi­ cated a similar weed flora composition, but with Stellaria media, Matricaria spp., Veronica persica Poir. and Lamium purpureum L. (red dead nettle) of greater frequency, a legacy of their seedbank accumulation in cereal rotations. A survey conducted in 1981 and repeated in 1982 provided information on the status of grass‐weeds in cereal crops in southern central England (Froud‐Williams & Chancellor, 1982; Chancellor & Froud‐Williams, 1984). The incidence of both wild oats and couch grass had undoubtedly decreased since earlier surveys reported by Phillipson (1974) and Elliott et al. (1979), but interestingly both black‐grass and rough‐stalked meadow grass had increased in prominence. The primary purpose of these surveys was to assess the incidence of Bromus sterilis, which was recorded as the fifth most com­ monly occurring grass‐weed. In addition to B. sterilis, other bromes were identified, most significantly Bromus commutatus Schrad. (meadow brome). A subsequent survey by Cussans et al. (1994) provided further evidence of the increased incidence of bromes including B. diandrus Roth. (great brome) and B. secalinus L. (rye brome). The relative importance of the latter has recently been highlighted by Cook et al. (2012) possibly as a consequence of a herbicide‐deflected succession. In the earlier surveys of Froud‐ Williams and Chancellor, Lolium multiflorum Lam. (Italian rye‐grass) ranked sixth. It is alleged that L. multiflorum infests at least 14% of cereal fields, having increased in importance as a result of ACCase resistance. Since the 1960s remarkable changes have occurred in the horticultural industry as production methods have intensified to the extent that crops formerly considered as market garden commodities, such as onions, carrots, peas and brassicas, are now grown at a field scale. Despite the need to extend field vegetable production into for­ mer arable areas, the hierarchy of weeds appears to have changed little between the early 1950s and the mid‐1970s (Davison & Roberts, 1976). Nonetheless, species hith­ erto considered as arable weeds have become more prevalent, such as Viola arvensis and Veronica persica. The reduced interval between rotations has enabled volunteer oilseed rape and groundkeeper potatoes to become more problematic. The transition of carrot production from fenland soils to the Breckland sands made possible by irriga­ tion has enabled Reseda lutea L. (wild mignonette) to become predominant in this crop and in parsnips. Mayweeds which increased in lettuce production did so as a consequence of the introduction of chlorpropham in 1958 to which they were not susceptible.

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Despite the great armoury of herbicides, recent EU legislation has resulted in the loss of many actives, particularly so for minority crops. For example, EU Directive 91/414 resulted in the loss of trietazine for use in vining peas, metoxuron in carrots, monolinu­ ron in onions and leeks and desmetryne in brassicas. Despite gaining successful deroga­ tions for fomesafen and terbutryn in legumes, approval did not extend beyond 2007. The loss of propachlor and cyanazine will contribute further to the intransigence of Matricaria spp. in onions, as will Fumaria officinalis L. (fumitory) with the loss of p rometryn (see Tei & Pannacci, Chapter 12). So too the Water Framework Directive could result in the loss of clopyralid, metazachlor, carbetamide and propyzamide in horticultural brassicas, with implications for weed control. Recent change from risk‐ to hazard‐based criteria for assessing herbicides for registration (EU1107/2009), and in particular endocrine disruption, is likely to result in considerably more loss of actives, as is the Sustainable Use Directive (Stark, 2011). A consequence of the Sustainable Use Directive (2009/128EC) is the promotion of low pesticide input management including non‐chemical methods, and hence a return in some situations such as row crops to physical weed control (see Melander et al., Chapter 9). Changing agronomic practices in sugar beet in the UK, such as the introduction of monogerm seed enabling the crop to be planted to a stand, appear to have aggravated the incidence of weed beet, as the non‐necessity of singling reduced the need for hand‐ hoeing. A reduction in the interval between successive crops also enabled regeneration from groundkeepers and from the soil seedbank. Thus the incidence of weed beet increased from negligible numbers in the early 1970s to a maximum in the mid–late 1980s before declining in the early 1990s (Longden, 1993). Infestations subsequently increased because of lax management, such that 60% of the crop was infested by 2001 (May, 2001). The expansion of oilseed rape has made it virtually impossible to prevent volunteer oilseed rape from occurring in subsequent sugar beet crops, albeit susceptible to triflu­ sulfuron. Although GM herbicide‐tolerant crops are currently not approved for release in the UK, the potential for glyphosate‐resistant oilseed rape volunteers in glyphosate‐ resistant beet cannot be ignored. Similarly, changes in fruit production systems have influenced weed species composi­ tion (Jones, 1984). As residual herbicides lost their potency, late‐germinating species became prevalent, most notably Convolvolus arvensis L., Hypericum perforatum L. and Malva sylvestris L. Additionally, Heracleum sphondylium L. has become associated with bush and cane fruit as a consequence of lack of soil disturbance (Banwell, 1972). Increased use of glyphosate in fruit crops to overcome weed issues in the absence of alternative products has led to the appearance of resistant weed biotypes in other EU countries but not so far in the UK. By comparison, weeds of grassland appear to be relatively unchanged since earlier surveys, with Cirsium arvense (L.) Scop. dominant on beef farms and Rumex obtusifo- lius L. (broad‐leaved dock) on dairy farms, despite being listed as notifiable weeds under the Weeds Act of 1959, as is also Senecio jacobea L. (ragwort) which appears to be rampant. In the uplands, Pteridium aqilinum (L.) Kuhn (bracken) is still widespread, with implications for its control with the possible revocation of asulam. Asulam failed to gain approval under EU Plant Protection Product Directive 91/414 after appeal in 2011. Since then there have been repeated annual emergency authorisations for its use on bracken in the UK during part of the year, and this is continuing into 2017.

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Episodic Decline

Weeds often exhibit cycles of episodic decline, as exemplified by Sisymbrium irio L. following the Great Fire of London, hitherto of notable occurrence, hence its common name of London rocket. In a comparative study of brome species, Mortimer et al. (1993) showed that Bromus interruptus (Hack) Druce (interrupted brome) is less ecologically fit to survive in arable environments than other bromes. Thus, relative to B. sterilis, B. mollis and B. commutatus it has a greater likelihood that its seeds will be harvested with the crop and hence removed by seed screening; for those seeds that are disseminated, germination is synchronous and they are likely to suffer premature germination and fail to establish through moisture stress. But for the actions of the former curator of Edinburgh Royal Botanic Gardens, Philip Smith, it would have become extinct. It has since been re‐introduced as part of arable reversion. Another example of episodic weed occurrence is that of Phalaris paradoxa L. (awned canary grass) which occurred spo­ radically in England during the early 1980s (Thurley & Chancellor, 1985), its occurrence attributed allegedly to contaminated seed imports. Freckleton and Watkinson (2002) examined the population dynamics of twelve arable weed species over a 12‐year period, based on observations reported by Thurston (1968) for Broadbalk which showed both increases and decreases in abundance irrespective of whether herbicides were applied or not, and concluded that annual variation in popula­ tion size was driven by exogenous factors. Nonetheless, in the instance of Papaver rhoeas, intermittent population crash remains unexplained. However, García de León et al. (2014a) have hypothesised that species with similar resource requirements are able to co‐exist because they differ temporally in their demands relative to climatic conditions. Based on relative abundance data over 21 years on Broadbalk, despite having similar nutri­ ent requirements, P. rhoeas and Matricaria perforata responded differentially to climate, the latter species favoured by higher spring temperatures whereas the former was not. Likewise in central Spain, García de León et al. (2014b) examined the relative impor­ tance of endogenous (density dependence) related factors and exogenous factors (tillage, rotation and climatic variables) on the population dynamics of seven weed species in cereal–legume rotations. Whereas endogenous factors were the main determinant driver under zero‐tillage than minimum tillage, while under the latter, temperature negatively affected the population dynamics of Descurania sophia (L.) Webb ex. Prantl. but had the converse effect on Atriplex patula L.

Weed Spatial Distribution

As indicated by Krähmer and Bàrberi in Chapter 3, weed mapping has also enabled the changing status of weeds to be documented, including that of invasive alien species and also herbicide‐resistant biotypes, the latter being continually updated and archived by Ian Heap at www.weedscience.org (Heap, 2017). Weed mapping, whether at a global or national level, may contribute to vegetation management policy decisions, while at a farm or individual field scale may contribute to decision support and site‐specific weed management. Weed mapping is closely associated with weed population dynamics and spatial distribution, the latter greatly influenced by cultural practices including tillage implements, harvesting machinery and crop spatial arrangement. Spatial aggregation of weeds has been shown to be influenced by direction of agricul­ tural field operations such that weed patches are often elliptical with row direction

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(Colbach et al., 2000). Stability of weed spatial pattern in maize was positively correlated for those species of greatest intrinsic rate of increase, for example Chenopodium album and Echinochloa crus‐galli (L.) Beauv. (barnyard grass), but whereas Echinochloa crus‐galli lacked stability in space, Chenopodium lacked stability in time (Heitjing et al., 2007). Wind‐ disseminated species showed no spatial correlation. Distance of seed dispersal can differ depending on harvesting equipment employed (Howard et al., 1991; Rew et al., 1996; Gonzàlez‐Andújar et al., 2001; Barroso et al., 2006; Heitjing et al., 2009). So too, seed dis­ persal can result in both horizontal and vertical distribution within the soil seedbank and differs depending on the type of tillage implement employed (Dessaint et al., 1991; Howard et al., 1991; Grundy et al., 1999; Marshall & Brain, 1999; Woolcock & Cousens, 2000). Other agencies are implicated in weed seed dispersal post‐dissemination and include invertebrates and vertebrates as well as inanimate forces such as wind and rain splash. Consequently, seed incorporation within the soil seedbank contributes to seed persis­ tence and also imposition of dormancy mechanisms which contribute to seed survival. However, considerable losses of seed may occur between dispersal and potential incor­ poration and thereby regulate seedbank density (Westerman et al., 2003). Predation of weed seeds is seasonal (Maucheline et al., 2005) and is greater in systems of reduced tillage (Menalled et al., 2007; Baraibar et al., 2009) and in less intensive or organic s ystems (Navntoft et al., 2009), while presence and identity of vegetation present also influences predation (O’Rourke et al., 2006; Williams et al., 2009). The roles of seed dormancy and seed predation are discussed in Chapters 4 and 5 of this volume respectively. The recognition that seeds of many weed species exhibit cyclic changes of dormancy whereby germination outside of these periods is not possible has enabled both an understanding of periodicity of germination and the ability to predict weed seedling emergence (Bouwmeester & Karssen, 1993; Vleeshouwers et al., 1995; Cirujeda et al., 2006). That cyclic changes of dormancy are regulated by temperature has enabled greater accuracy of such predictions, and so affect judicial timing of weed removal either by chemical or physical means (Grundy & Mead, 2000).

History of Weed Science in the UK and Origins of the Weed Research Organization

The Weed Research Organization (WRO) was established in 1960 as a successor to the ARC Unit of Agronomy, which under the leadership of Professor G.E. Blackman had contributed to the development of 4‐chloro 2‐methyl phenoxyacetic acid (MCPA). During its brief existence, 1960–1985, WRO had two directors: the first, Dr E.K. Woodford, remained in post until 1964, when he was succeeded by Professor J.D. Fryer. The institute had been deliberately named Weed Research rather than Weed Control Organization so as to allay public concern, yet a major role of the institute was inde­ pendent evaluation of the burgeoning number of new herbicide discoveries being made by the agri‐chemical industry. By 1965 the work of the establishment was split between applied aspects of weed control and the more scientific underpinning of the knowledge relating to herbicide chemistry and weed biology. WRO was the acknowledged centre of excellence for publicly funded weed science in the UK. Its remit ranged from weed control in both arable and horticultural crops, as well as grassland and aquatic and

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uncropped land. Aquatic weeds are inadequately discussed in this chapter, but their invasive potential is referred to in Chapter 13 and their suitability as candidates for biological control in Chapter 8. In addition, WRO provided an advisory role in conjunc­ tion with the Agricultural Development Advisory Service (ADAS, previously National Agricultural Advisory Service (NAAS)), as well as a tropical weeds unit. The significant role provided by this latter unit is not adequately articulated here (see, for example, Baker & Terry, 1991; Parker & Riches, 1993) as, with the exception of Cyperus spp. and Sorghum halepense, tropical perennial grass‐weeds and sedges are not encountered within Europe, nor parasitic weeds, excluding Orobanche spp., which are fully explored by Vurro et al. (in Chapter 11). Aquatic weeds are no longer represented as a separate working group by the society, but are considered among invasive species and suitable candidates for biological control (see Bohren, Chapter 10, and Shaw & Hatcher, Chapter 8). Initial studies at WRO were largely focused on the intractable perennial grass‐weeds Elytrigia repens and Agrostis gigantea Roth., necessitating both evaluation of herbicides and understanding of their biology in relation to agronomic practices. Early studies indicated the benefits of a competitive crop canopy as provided by spring barley and of defoliation on regenerative capacity. An element of collaboration was already in exist­ ence between WRO and Rothamsted with regard to understanding the seed biology and regenerative strategies of these species. During the late 1960s the threat posed by wild oats had increased significantly, neces­ sitating investigation in 1968 of its competitive relationships with spring‐sown cereals. It is of interest to note that Alopecurus myosuroides was already recognised as an increasing problem in winter cereals by 1966, but that increased seed rates and reduced row‐widths could reduce its competitive ability and number of inflorescences. However, at that time little was known of its population biology. The situation concerning wild oats had become so severe that in 1973 a national cam­ paign for its eradication was announced. Wild oats were estimated to infest 500,000 ha of arable land at that time. This situation precipitated a survey of wild oats in 1973 and a subsequent investigation in 1977 in which a random survey was made of 2250 fields throughout the UK (Elliott et al., 1979). It was estimated that wild oats were present in 67% of the cereal crops in England, 37% in Scotland, 16% in Northern Ireland and 13% in Wales. Additionally, black‐grass was estimated to infest 22% of the cereal acreage, but was largely restricted to eastern and south‐eastern England. In parallel with the increased concern regarding wild oats, the agri‐chemical industry sought to discover potential candidate herbicides with renewed vigour. Of particular note were the aralanalines benzoylprop‐ethyl, flamprop‐isopropyl and flamprop‐methyl introduced in 1969, 1972 and 1974 respectively. Flamprop‐isopropyl was eventually succeeded by flamprop‐m‐isopropyl. The introduction of difenzoquat in 1973 and diclofop‐methyl in 1975 further contributed to wild oat control, albeit diclofop‐methyl found a particular niche against black‐grass. Previously, reliance for wild oat control had been placed on barban (introduced in 1958) and chlorfenprop‐methyl (introduced in 1968), both with a limited window of opportunity for application. A notable achieve­ ment was the development of the rouging glove with a herbicide‐impregnated pad to reduce the time required to physically remove wild oats from the field. In subsequent years the spray window was extended following discovery of the s pecific graminicide aryloxyphenoxypropionates such as fenoxaprop‐ethyl in 1982

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(re‐formulated as fenoxaprop‐P‐ethyl in 1990) and clodinafop‐propargyl in 1990, the cylohexanediones, for example, tralkoxydim in 1987 and latterly pinoxaden, a phenyl pyrazole introduced in 2006, and pyroxsulam, a triazopyrimidine with aceto‐lactate synthase (ALS) activity in 2009. Although herbicide discovery was beyond the remit of WRO, herbicides were evalu­ ated and opportunities to enhance their efficacy proposed through the use of additives and more efficient spray delivery systems. Greater emphasis on understanding weed biology and in particular population dynamics further contributed to the management of wild oats. The development of an annual seed cycle enabled its biology to be exploited. For Avena sterilis ssp. ludoviciana, the periodicity of germination of which is restricted to autumn, rotation with a spring‐ sown crop proved an effective strategy. Subsequent understanding of the biology of Alopecurus myosuroides and of Anisantha sterilis further contributed to their management. Additional weed research was undertaken at the Rothamsted Experimental Station following the pioneering work of Brenchley and Warington; National Vegetable Research Station (NVRS) (now University of Warwick); Scottish Horticultural Research Institute (now James Hutton Institute after the merger of Scottish Crop Research Institute (SCRI) and Macaulay Land Use Research Institute); Grassland Research Institute (now Institute of Grassland and Environment Research); Norfolk Agricultural Station (subsequently Morley Research); Processors and Growers Research Organisation; various Scottish Agricultural Colleges (now amalgamated with James Hutton Institute); and Department of Agriculture Northern Ireland and various Experimental Husbandry Farms of NAAS (subsequently ADAS), in particular Boxworth. Notable achievements of these other organisations included the determination of weed seedling periodicity and the longevity of seeds in soil conducted by H.A. Roberts at the former NVRS and of critical periods of competition both at NVRS and SCRI, the latter by H.M. Lawson. Research at WRO was instrumental in understanding population dynamics and weed demographic studies. Perhaps somewhat surprisingly, WRO lacked a remit to investigate herbicide resist­ ance, this being considered ‘near market’ and hence more appropriate for commercial industry in collaboration with universities and polytechnic colleges. It was not until resistance to chlorotoluron was identified in 1982 that it was deemed as appropriate. At its peak, about 100 members of staff were employed at WRO. However, the decision in 1984 to close the institute and redeploy approximately 50 members of staff between Long Ashton Research Station (LARS) and Broom’s Barn Experimental Station resulted in a substantial reduction in publicly funded weed science. Subsequently, with the clo­ sure of LARS in 2003 the remaining staff complement were transferred to Rothamsted Research, remarkable in that during the 1930s–1960s some weed biology had been conducted here with regard to Broadbalk investigations. This group has now dimin­ ished from 16 to 4. Previously, Burnside (1993) had likened weed science to that of a ‘step child’. Cuts in the funding of weed science appear to be disproportionate and cur­ rently as few as ten principal investigators are engaged in full‐time publicly funded weed research in England. This is further compounded by losses within the university provision of weed science education in the UK and the average age of weed scientists, as outlined by Froud‐Williams and Moss at a meeting of the Association of Applied Biologists in 2008 on ‘The future of weed science in the UK’.

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Despite the closure of WRO, a new conceptual approach has been taken in weed sci­ ence since the mid‐1980s based on a systems approach, with particular emphasis on reduced inputs and environmental impact. Many of these programmes involved ADAS in collaboration with other partner organisations. These include Low Input Farming and the Environment (LIFE) initiated at LARS, Integrated Farming Systems (IFS), Seeking Confirmation about Results at Boxworth (SCARAB), and Towards a Low Input System Minimising Agro‐Chemicals and Nitrogen (TALISMAN) and Sustainable Arable Farming for an Improved Environment‐Enhancing Biodiversity (SAFFIE). Subsequently, studies designed to predict the implications of GM crops were initiated, notably Botanical and Rotational Effects of Genetically Herbicide Tolerant Crops (BRIGHT) and the Farm Scale Evaluations (FSE). More recently there has been collabo­ rative involvement between publicly funded and private sector research through the sustainable arable LINK programme such as Farm4Bio. All of these investigations have involved monitoring of weed populations. However, the subsequent privatisation of ADAS had the effect of reducing the amount of applied research conducted by this organisation (Pray, 1996). As with publicly funded weed research, the private sector also underwent considera­ ble re‐organisation as a consequence of mergers and consolidation into fewer agri‐ chemical companies. In the 1960s a plethora of companies existed in the UK, including Atlas Interlates, Boots, Ciba‐Geigy, Fisons, ICI, May and Baker, PBI and Shell. Currently, as a result of various mergers, the agri‐chemical industry is composed of relatively few companies – BASF, Bayer Crop Science, Dow, Dupont, Monsanto and Syngenta being most noteworthy. A more comprehensive account concerning the evolution of the agri‐chemical industry is provided by Copping (2003).

Origins of the European Weed Research Society

The origins of the European Weed Research Society (EWRS) date back to 1950 with the formation of the ARC Unit of Agronomy at Oxford. Here a conference was organised in 1951 for overseas weed specialists on developments in weed control, but was also attended by many from western Europe. A further opportunity for liaison and collabo­ ration between these delegates was provided by the First British Weed Control Conference at Blackpool in 1953. This was further reinforced by subsequent meetings, and in 1958 a meeting was held in Ghent, Belgium, to discuss the need for international co‐operation and resulted in the formation of an International Research Group on Weed Control, with Dr Wybo van der Zweep from Wageningen, The Netherlands, as its secretary. The group held its first meeting in 1959 under the presidency of Professor Rademacher at Stuttgart‐Hohenheim. In 1960 the Unit of Agronomy organised a c onference in Oxford, where the European Weed Research Council (EWRC) came into being. One of the key objectives of the council was to launch a dedicated journal for the dissemination of weed research, edited initially by John Fryer with support from Harold Roberts. Papers were submitted in English, French and German, with the latter two languages being edited by Dr Longchamp of INRA, Versailles, and Professor Rademacher of Stuttgart‐Hohenheim. In 1964 John Fryer succeeded Ken Woodford as director of WRO such that Harold Roberts of the NVRS took on the role of editing Weed Research. The organisation of the EWRC required the election of a president, a vice president and a president elect, all of whom would serve 2 years of duty in each post.

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Representatives were nominated from each member country, but the main burden of responsibility for organising the council’s activities fell to the secretary, Wybo van der Zweep. By 1973, when John Fryer was elected president, it was clear that some re‐organisation was inevitable as the activities of the council became more diverse, with increasing involvement from the growing agri‐chemical industry and the formation of specialised subject groups. Consequently, a draft constitution was written to replace the council and approved by the Executive Committee to enable wider participation of what now became the European Weed Research Society. A major change was that the appointment of president and vice president should alternate between industry and the public sector. Thus, the EWRS formally came into being on 3 December 1975 at a s ymposium on the ‘status, biology and control of grass‐weeds’ held in Paris and at which the first general assembly of the new society was held (Van der Zweep & Hance, 2000). I am grateful to John Fryer for providing much background information on the forma­ tion of EWRS. A more complete account of his memoirs is held by the archive reposi­ tory of the EWRS Secretariat, together with details of former secretaries and presidents of the society. A comprehensive account of weed science as a discipline is provided by Fryer (1978).

Weed Research (Journal): Origin of Papers and Discipline

In the year after WRO was founded, the official journal of the EWRS was launched. It is of interest to review the changing nature of the subject matter published since the 1960s in this journal. Initially, emphasis was on physiological aspects of herbicide activity and weed physiology, with subsequent emphasis on soil relationships, including effects on soil microorganisms. The ratio of weed control to weed biology was roughly 2:1 during the period 1961–1980. Within the society are represented a number of working groups, enabling specialist activity in the various sectors of weed science. These groups are interactive and organise workshops, for example, physical and cultural weed control, and joint experiments between member countries, such as crop–weed interactions and germination and early growth (Schutte et al., 2014), as well as the collation of information on weed control technologies between member countries, such as publications by the weed management systems in vegetables working group (see, for example, Tei & Pannacci, Chapter 12).

Changing Attitudes to Weeds

That attitudes to weeds have changed is evident from the shift of emphasis in weed l iterature. Early titles of weed text books ranged from Weed Suppression by Long and Brenchley (1934) to Weed Control Handbook edited by Woodford (1958) and Weed Destruction (Evans, 1962). The Weed Control Handbook which ran to eight editions was subsequently superseded by the Weed Management Handbook in 2002. A greater eco­ logical dimension is apparent from Ecological Management of Agricultural Weeds (Liebmann et al., 2001), Weeds and Weed Management on Arable Land: An Ecological Approach (Håkanssson, 2003) and Non‐Chemical Weed Management (Upadhyaha & Blackshaw, 2007). Not all weeds are harmful to the crop in which they occur. Several species such as Veronica hederifolia L. (ivy‐leaved speedwell) senesce before a cereal crop matures and so may not pose a legitimate target for control. That weeds differ in their competitive

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abilities was recognised by Wilson et al. (1995) who devised a competitive ‘pecking order’ based on crop equivalents. Economic thresholds at which a financial return from direct intervention can be gained have been established for a number of species (Wilson & Wright, 1990). However, density is not a good predictor because of sequential flushes of germination resulting in a range of plant size. Hence ground cover or leaf area became a better predictor of competitive ability (Lutman et al., 1996; Storkey et al., 2003). For an overview of the evolution of a modelling approach to characterising crop–weed interactions see Chapter 2, this volume. That weeds may provide a beneficial role in food webs has recently been acknowl­ edged (Marshall et al., 2003), seeds often providing a source of seed to granivorous birds or attracting arthropods that are important constituents in the diet of insectivorous birds. Thus, for example, Polygonum aviculare which attracts sawfly larvae and Polygonum beetle is of beneficial value to the grey partridge, a farmland bird considered to be in decline. The arable flora is recognised as having suffered the greatest decline in species diver­ sity in recent years (Preston et al., 2002). In reflection of the endangered status of many of our arable plants, the former British Agrichemicals Association (Crop Protection Association) instigated the Wild Flower Project, with Adonis annua L. (pheasant’s eye) as its emblem. Reasons for the decline of various species were investigated by Wilson (1990). Remarkably, some such as Scandix pecten‐veneris L. (shepherds purse) exhib­ ited a recovery in some areas under set‐aside management. However, despite section 8 of Broadbalk never receiving herbicides, Galium tricornutum Dandy (corn cleavers) has failed to recover more than a few individuals per year between 1991 and 2002 and then only on section 9 (Moss et al., 2004).

Set‐Aside and Agri‐Environment

Set‐aside was introduced in 1988 as a means of reducing excess arable production. Initially land could be managed as rotational or permanent set‐aside following natural regeneration or by means of a sown cover. Although it had been predicted that such a scheme would offer environmental benefits, including increased biodiversity, perma­ nent set‐aside enabled ecological succession, becoming dominated by perennial grass‐ weeds, thereby excluding floristically rich annual species (Boatman et al., 2011). Previously it had been suggested that rotational set‐aside would benefit endangered arable plant species, although no such evidence was obtained from a sample of set‐aside sites of differing duration. However, Clarke and Froud‐Williams (1989) had shown that on light soils species such as Legousia hybrida could benefit from rotational set‐aside, whereas on heavier soils Kickxia spp. could be favoured. Neve et al. (1996) concluded that conservation of rare arable species needed specific management to cater for their particular requirements. Similarly, Rew et al. (1992) showed that species diversity declined with distance from the field boundary and that species more typical of the arable flora occurred to the greatest extent distant from the boundary. Critchley et al. (2004) concluded that the type of vegetation associated with individual agri‐environment schemes could be predicted. Thus, for example, naturally regener­ ated field margins were dominated by perennial species, including Arrhenatherum elatius (L.) P. Beauv. ex. J. & C. Presl., Agrostis stolonifera L., Poa trivialis L. and Cirsium arvense, whereas sown margins were characterised by the constituent species

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Festuca rubra L., Phleum pratense L. and Dactylis glomerata L. Conversely, uncropped cultivated margins contained arable weeds, notably Sonchus asper (L.) Hill, Polygonum aviculare L., Poa annua L., Galium aparine and Veronica spp. The biodiversity benefits of uncropped field margins have been exemplified by Walker et al. (2007), who recorded greatest diversity including rare arable plants Centaurea cyanus L., Fumaria purpurea Pugsley, Scandix pecten‐veneris L. and Silene gallica L. Under Common Agriculture Policy Reform, Environmental Stewardship Schemes have been replaced by Countryside Stewardship, with some possible implications for enhancing weed biodiversity under Greening Measures, as well as more effective man­ agement of pernicious weeds such as black‐grass through the inclusion of fallow and the three crop rule that involves a return to inclusion of spring‐sown crops. The biodiversity value of weeds is addressed by Gerowitt et al. (Chapter 5).

Weeds, Climate and Invasive Aliens

Clearly, many of our weed species were at the limit of their geographic and ecological range, as evident by the necessity of repeated introductions of species such as Centaurea cyanus L. (cornflower). However, climate change has the potential to result in a modi­ fied weed spectra as a result of either changing cropping practices, such as an increased acreage of maize, or changing distribution of crops and their associated weed flora. The introduction of game cover crops has been associated with weed impurities often more frequently encountered in warmer climates, such as Echinochloa spp. and Amaranthus spp. (Hanson & Mason, 1985). Of particular concern is the potential for introduction of Ambrosia artemisiifolia, an allergenic species currently spreading rapidly throughout central Europe (Gerber et al., 2011). Conversely, increasingly cold winters may have contributed to the spread of Cochlearia danica L., which has spread along arterial routes as a consequence of salting of our highways. In contrast to marked reductions of species such as Caucalis platycarpos L. which declined by an index factor of –7.86, Cochlearia danica increased by a factor of +3.31 between 1979 and 1999 (Preston et al., 2002). Invasive species have been much associated with non‐crop habitats such as riparian corridors and aquatic habitats (Sheppard et al., 2006). These seem likely to increase in number and distribution as such areas are rarely subject to vegetation management. Of particular note are the injurious species listed in the Wildlife and Countryside Act of 1981 – Reynoutria japonica Houtt and Heracleum mantegazzianum Sommier & Levier, the latter posing a health hazard. Impatiens glandulifera Royle appears to be spreading at an alarming rate and displacing native vegetation, while aquatic species such as Hydrocotyle ranunculoides L. and Ludwigia grandiflora (Michx.) Greuter & Burdet (water primrose) threaten our aquatic habitats. That said, competition for pollinator services between native Lythrum salicaria L. (purple loosestrife) and the invasive alien L. grandiflora appeared to be unaffected by L. grandiflora, nor was seed‐set diminished, and at high canopy density of the invasive, greater numbers of pollinators were present on L. salicaria (Stiers et al., 2014). While herbicides are unlikely to receive approval for use in these areas, particularly after the revocation of tryclopyr and imazapyr in riparian corridors and diquat in aquatic habitats, an opportunity may be afforded for biological control, as currently being evaluated for Reynoutria japonica with the plant psyllid Aphalora itadori

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(Shaw et al., 2011). The current status of biological control of weeds within Europe is discussed by Shaw and Hatcher (Chapter 8). Until now, the challenges to the adoption of bio‐control seemed too immense. Possible reasons for the slow uptake of biological control are outlined and include the economic implications of product development for use in intensive agriculture relative to the proven efficacy of herbicides and ownership issues within extensive and non‐crop situations. Nonetheless, the authors consider that the tide is ‘turning’ as registration of new herbicide products declines and legislation removes those deemed to be detrimental to the environment. In particular, optimism is expressed for non‐native species as bio‐control targets, especially within aquatic and riparian habitats such as Azolla filiculoides Lam. and Impatiens glandulifera respectively.

Future Directions (Quo Vadis?)

Environmental Weed Management

Since the initial discovery of synthetic herbicides, remarkable innovations have occurred both in the discovery of active ingredients and in the manner in which they are applied. Initially herbicide doses could be measured in kilogrammes or litres per hectare, whereas many are now applied at a fraction of that dose. Consequently, the tonnage of active ingredient applied has fallen dramatically. Likewise, spraying systems have made substantial advances since the earliest hydrau­ lic sprayers that were dependent on high volume rates and were wasteful because of the wide range of droplet size. Indeed, spraying systems have achieved a high degree of sophistication, enabling the possibility of site‐specific weed control (Christensen et al., 2009). Custom‐built self‐propelled vehicles are now available that do not need to be trailed or tractor mounted and achieve high work rates because of increased forward speed. Application technology has also been advanced greatly with developments such as air induction nozzles designed to reduce spray drift, whilst forward and backward facing trajectories such as the ‘Defy’ jet increase the efficiency of spray coverage. Likewise, the necessity to seek more environmentally benign alternative methods of weed control as a consequence of the Sustainable Use Directive which promotes low input, integrated management has resulted in an increased use of physical weed con­ trol. However, while this may appear to be a return to mechanical weeding as witnessed in the pre‐herbicide era, current technology has resulted in fast, efficient flexi‐tine weeders and vision guidance steerage systems of value in row crops, such as sugar beet for tackling weed beet (Hatcher & Melander, 2003). The future is likely to be more reli­ ant on remote sensing and robotics as a means of controlling specific weeds that are considered harmful, but at the same time sparing those species of biodiversity value. Andújar et al. (2011) have demonstrated the ability to discriminate weeds based on ultrasonic sensors by means of height differential, achieving 81% discrimination for grass‐weeds and 99% for broad‐leaved weeds. Thus, site‐specific weed management has the potential to reduce environmental pollution, increase selectivity, reduce cost and enhance biodiversity. For high value horticultural crops, thermal weeding, laser technology and electrocution may substitute for the greatly diminished herbicide arse­ nal available for use in minority crops, whereas in non‐crop urban situations, steam, hot water or foam may be employed.

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An area of research that deserves further investigation is the potential to utilise plant traits of increased competitive ability with regard to varietal selection, including allel­ opathy (Seavers & Wright, 1999). Some success has already been achieved in varietal selection for use in organic farming systems (Drews et al., 2009) and Andrew et al. (2015) have argued as to the necessity to screen potential new cultivars for traits of tolerance or suppression.

Evolutionary Genetics and the Role of Molecular Ecology

The tools provided by advances in molecular biology have been quickly adopted by weed scientists in various sectors (O’Hanlon et al., 2000). Such examples include the ability to accurately identify taxa, of absolute importance in developing biological con­ trol programmes. So too, the origins of individual populations can be assessed to deter­ mine the extent of seed dispersal (Rutledge et al., 2000), pollen flow and outcrossing (hybridisation) within a species (Green et al., 2001). This has particular application for assessing the risk of introducing GM crops and their potential impact on non‐GM crop production. Powles (2008) has warned of the implications of total reliance on glypho­ sate for weed control in transgenic crops, such that without diversification of weed management this technology could be rendered obsolete as weeds resistant to glypho­ sate assume dominance. Contrary to expectation, the introduction of GM crops in the USA has actually resulted in an increase in the number and volume of herbicides applied as a direct result of glyphosate resistance occurring in weed species (Benbrook, 2012). Glyphosate resistance within Lolium spp. has now been reported within Europe (Collavo & Sattin, 2014). The use of molecular markers has found particular application in iden­ tifying mechanisms of herbicide resistance, in particular target site resistance (see, for example, Moss, Chapter 7). A potential solution to this problem is through RNA inter­ ference (RNAi) technology whereby genes conferring herbicide resistance may be silenced (Shaner & Beckie, 2014). However, such technology is beyond the scope of this chapter and the current remit of the EWRS working groups.

Is there a Need for a Change of Emphasis?

A quiet revolution has already started as emphasis has shifted to weed management rather than pragmatic weed control. Alternative weed management strategies may need to be sought as the discovery of new active herbicide modes of action diminish and tougher legislation restricts which herbicides may be registered and in what situations. Recent concerns affecting herbicide registration include their potential risk for endo­ crine disruption. Restrictions are likely to be further imposed where there is a risk of water contamination or that the subsequent cost of water purification is uneconomic. Consequently, greater reliance may be placed on physical methods of weed control, possibly involving autonomous machinery and remote sensing to identify weed infes­ tations and enable site‐specific weed control. Organic and sustainable systems of f arming could become attractive, but these are not without their environmental impacts. To date, the introduction of GM herbicide‐tolerant crops in the UK would appear somewhat distant, particularly as the main proponent Monsanto has virtually with­ drawn from the UK. However, the recent decision to allow individual EU member states to grow GM crops could ease their adoption within Europe, albeit at present this is likely to be confined to GM maize resistant to European corn borer.

0003060919.INDD 22 05/04/2017 6:29:10 PM Conclusion 23

The realisation that not all weeds are necessarily detrimental to crop production could enable a greater degree of tolerance of weeds and their co‐existence, especially those of biodiversity value. In some areas where endangered species seek refuge, s pecific agri‐environment schemes could be introduced to maintain their populations. Nonetheless, this would require careful habitat and vegetation prescriptive management. Of increasing concern is the threat posed by invasive species, because of not simply their impact on the native flora, but also their environmental and health impact. Given that herbicide development is unlikely to be targeted for use in non‐crop situations, the potential of biological control offers considerable promise (Sheppard et al., 2006; Shaw & Hatcher, Chapter 8). Screening of natural products with herbicidal activity could enable suitable analogues to be developed following serendipitous discovery, as with the example of mesotrione following the isolation of leptospermone from Callistemon citrinus (Lee et al., 1997). Mesotrione, an inhibitor of hydroxyl‐phenyl‐pyruvate dioxygenase (HPPD), was sub­ sequently marketed as Callisto for selective broad‐leaved weed control in maize. Alternatively, weed‐suppressive mulches with allelopathic activity need to be investi­ gated further (Yang et al., 2004; Dhima et al., 2006). An alternative approach currently under investigation is the potential use of RNAi to suppress allelopathic traits in weed species (Fang & Zhihui, 2015). An understanding of the mechanisms whereby weed seed mortality is enhanced in weed‐suppressive soils should be explored further (Davis et al., 2006, 2008). Implications of climate change and weed adaptation require greater consideration as the nature and distribution of crops changes in response to a changing climate. Decision support s ystems will be required to integrate weed population dynamics models so as to deliver effective weed management in both an economic and environmentally sound manner (see Tørresen et al., Chapter 4).

Conclusion

So to conclude, weed science is a multidisciplinary subject that draws on an eclectic source of technologies to advance our understanding of weeds. Since the formation of the European Weed Research Society in 1950, great advances have been achieved, signifi­ cantly in weed biology and the control of weeds. Within the former, advances in seed science technology have enabled a greater understanding of the mechanisms regulating seed dormancy and germination, both in terms of its periodicity and proportion of the population at any given time based on meteorological conditions during seed maturation and subsequently in the soil seedbank. This together with demographic studies and popu­ lation dynamics has enabled construction of life history cycles, thereby identifying the potential ‘Achilles heel’. An awareness of the critical period of competition and of a hierar­ chy of competitiveness coupled with an ability to predict potential yield losses based on a combination of thresholds and modelling have enabled deployment of decision support systems. An awareness of biodiversity value based on functional traits has permitted a less prescriptive approach to weed management rather than pragmatic control. Of course, the recognition that herbicide discovery revolutionised weed control cannot be overstated; but so too the further development of chemicals, including those

0003060919.INDD 23 05/04/2017 6:29:11 PM 24 Weed Science Research: Past, Present and Future Perspectives

based on natural products of greater potency at reduced dose rates while achieving selectivity, is of great environmental value. There is a pressing need to discover novel target sites for increased herbicide efficacy (Duke, 2012). However, as pointed out by Gressel (2010), the incentive to develop alternative herbicide modes of action has been stifled by the ubiquitous use of glyphosate, particularly within transgenic crop situations. Nonetheless, concern for the environmental fate of herbicides will necessitate an alternative approach and the next cohort of weed scientists will have the task of achieving this objective.

Acknowledgements

I am indebted to Professor J.D. Fryer for providing me with his memoirs relating to the founding of WRO and the formation of the EWRS.

References

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Fryer JD (1978) Status of weed science – a world’s perspective. Weed Science 26, 560–566. Garcia de León D, Storkey J, Moss SR & Gonzàlez‐Andújar JL (2014a) Can the storage effect hypothesis explain weed co‐existence on the Broadbalk long‐term fertiliser experiment? Weed Research 54, 445–456. Garcia de León D, Freckleton RP, Lima M et al. (2014b) Identifying the effect of density dependence, agricultural practices and climate variables on the long‐term dynamics of weed populations. Weed Research 54, 556–564. Gerber E, Schaffner U, Gassmann A, Hinz HL, Seier M & Müller‐Schärer H (2011) Prospects for biological control of Ambrosia artemisiifolia in Europe: learning from the past. Weed Research 51, 559–573. Gonzàlez‐Andújar JL, Plant RE & Fernandez‐Quintanilla C (2001) Modelling the effect of farmers’ decisions on the population dynamics of winter wild oat in an agricultural landscape. Weed Science 49, 414–422. Green J, Barker JHA, Marshall EJP et al. (2001) Microsatellite analysis of the inbreeding grass weed barren brome (Anisantha sterilis) reveals genetic diversity within and between farm scales. Molecular Ecology 10, 1035–1045. Gressel J (2010) Global advances in weed management. Journal of Agricultural Science, Cambridge 149, 47–53. Grundy AC & Mead A (2000) Modelling weed emergence as a function of meteorological records. Weed Science 48, 594–603. Grundy AC, Mead A & Burston S (1999) Modelling the effect of cultivation on seed movement with application to the prediction of weed seedling emergence. Journal of Applied Ecology 36, 663–678. Håkansson S (2003) Weeds and Weed Management on Arable Land: An Ecological Approach. CABI, Wallingford. Hald AB (1999a) Weed vegetation (wild flora) of long established organic versus conventional cereal fields in Denmark. Annals of Applied Biology 134, 307–314. Hald AB (1999b) The impact of changing the season in which cereals are sown on the diversity of the weed flora in rotational fields in Denmark. Journal of Applied Ecology 36, 24–32. Hanson CG & Mason JL (1985) Bird seed aliens in Britain. Watsonia 15, 237–252. Hatcher PE & Melander B (2003) Combining cultural, physical and biological methods: prospects for integrated non‐chemical weed management strategies. Weed Research 43, 303–322. Heap I (2017) International Survey of Herbicide Resistant Weeds. www.weedscience.org. Heard MS, Hawes C & Champion GT (2005) Weeds in fields with contrasting conventional and genetically modified herbicide tolerant crops. II. Effects on individual species. Philosophical Transactions of the Royal Society London B 358, 1833–1846. Heitjing S, van der Werf W, Stein A & Kropff MJ (2007) Are weed patches stable in location? Application of an explicitly two dimensional methodology. Weed Research 47, 381–395. Heitjing S, van der Werf W & Kropff MJ (2009) Seed dispersal by forage harvester and rigid‐tine cultivator in maize. Weed Research 49, 153–163. Hidalgo B, Saaverdra M & Garcia‐Torres L (1990) Weed flora of dryland crops in the Córdoba region (Spain). Weed Research 30, 309–318. Holzner W (1978) Weed species and weed communities. Vegetatio 38, 13–20.

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Howard CL, Mortimer AM, Gould P, Putwain PD, Cousens, R & Cussans GW (1991) The dispersal of weeds: seed movement in arable agriculture. Proceedings of Brighton Crop Protection Conference – Weeds, Brighton, 821–828. Hurle K (1974) Effect of long‐term weed control measures on viable weed seeds in the soil seedbank. Proceedings of the 12th British Weed Control Conference, London, 1145–1152. Ivens GW (1980) Weed science and technology. In: Perspectives in World Agriculture, 181–206. CAB, Farnham Royal, Slough. Jones A (1984) Current and future weed problems in fruit crops. Aspects of Applied Biology 8, Weed Control in Fruit Crops, 1–7. Kallio‐Mannila K (1986) Changes in the composition of the weed flora of spring cereal fields in recent decades. Memoranda Societas Fauna Flora Fennica 67, 107–110. Koch W (1964) Some observations on changes in weed populations under continuous cereal cropping and with different methods of weed control. Weed Research 4, 351–356. Lee DL, Prysbill M, Cromartie TH et al. (1997) The discovery and structural requirements of inhibitors of p‐hydroxy phenylpyruvate dioxygenase. Weed Science 45, 601–609. Liebmann M, Mohler CL & Staver CP (2001) Ecological Management of Agricultural Weeds. Cambridge University Press, Cambridge. Long H & Brenchley WE (1934) Weed Suppression by Fertilizers and Chemicals. Crosby Lockwood, London. Longden PC (1993) Weed beet: a review. Aspects of Applied Biology 35, 185–194. Lutman PJW, Risiott R & Osterman HP (1996) Investigation into alternative methods to predict the competitive effects of weeds on crop yields. Weed Research 34, 167–175. Lutman PJW, Moss SR, Cook S & Welham SJ (2013) A review of the effects of crop agronomy on the management of Alopecurus myosuroides. Weed Research 53, 299–313. Marshall EJP & Brain P (1999) The horizontal movement of seeds in arable soil by different soil cultivation methods. Journal of Applied Ecology 36, 443–454. Marshall EJP, Brown VK, Boatman ND, Lutman PJW, Squire GR & Ward LK (2003) The role of weeds in supporting biological diversity within crop fields. Weed Research 43, 77–89. Maucheline AL, Watson SJ, Brown VK & Froud‐Williams RJ (2005) Post‐dispersal seed predation of non‐target weeds in arable crops. Weed Research 45, 157–164. May M (2001) Weed beet – how to get the most from control methods. British Sugar Beet Review 69, 38–43. Menalled FD, Smith RG, Dauer JT & Fox TB (2007) Impact of agricultural management on carabid communities and weed seed predation. Agriculture, Ecosystems and Environment 118, 10–16. Mortimer AM, Putwain PD & Howard C (1993) The abundance of brome grasses in arable agriculture – comparative population studies of four species. Proceedings of the Brighton Crop Protection Conference – Weeds, Brighton, 505–514. Moss SR (1980) A study of populations of black‐grass (Alopecurus myosuroides) in winter wheat as influenced by seed shed in the previous crop, cultivation system and straw disposal method. Annals of Applied Biology 94, 121–126. Moss SR, Storkey J, Cussans JW, Perryman SAH & Hewitt MV (2004) The Broadbalk long‐term experiment at Rothamsted: what has it told us about weeds? Weed Science 52, 864–873.

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Moss SR, Orson JH & Froud‐Williams RJ (2011) Fallowing for control of annual grass weeds: what are the issues? Aspects of Applied Biology 106, Crop Protection in Southern Britain, 17–22. Mukula J, Raatikainen M, Lallukka R & Raatikainen T (1969) Composition of weed flora in spring cereals in Finland 1887–1965. Annales of Agriculturae Fenniae 8, 59–110. Navntoft S, Wratten SD, Kristensen K & Esbjerg P (2009) Weed seed predation in organic and conventional fields. Biological Control 49, 11–16. Neve P, Mortimer AM & Putwain PD (1996) Management options for the establishment of communities of rare arable weeds on set‐aside. Aspects of Applied Biology 44, 257–262. Novák R, Istávan D, Szentey L & Karamán J (2010) Arable Weeds of Hungary. The Fifth National Weed Survey (2007–2008). Ministry of Agriculture and Rural Development, Budapest. Oerke E‐C (2006) Crop losses to pests. Journal of Agricultural Science, Cambridge 144, 31–43. O’Hanlon PC, Peakall R & Briese DT (2000) A review of new PCR‐based genetic markers and their utility to weed ecology. Weed Research 40, 239–254. O’Rourke ME, Heggenstaller AH, Liebman M & Rice ME (2006) Post‐dispersal weed seed predation by invertebrates in conventional and low‐external‐input crop rotation systems. Agriculture, Ecosystems & Environment 116, 280–288. Parker C & Riches CR (1993) Parasitic Weeds of the World: Biology and Control. CABI, Wallingford. Percival J (1936) Agricultural Botany: Theoretical and Practical. Duckworth, London. Phillipson A (1974) Survey of the presence of wild oats and black‐grass in parts of the United Kingdom. Weed Research 14, 123–135. Powles SB (2008) Evolution in action: glyphosate‐resistant weeds threaten world crops. Outlooks on Pest Management 19, 256–259. Pray CE (1996) The effect of privatizing agricultural research in Great Britain: an interim report on PBI and ADAS. Food Policy 21, 305–318. Preston C, Pearman DA & Dines TD (2002) New Atlas of the British and Irish Flora. Oxford University Press, Oxford. Putwain PD (1982) Herbicide resistance in weeds – an inevitable consequence of herbicide use. Proceedings of the British Crop Protection Conference – Weeds, London, 719–728. Raatikainen M, Raatikanen T & Mukula J (1978) Weed species, frequencies and densities in winter cereals in Finland. Annales of Agriculturae Fenniae 17, 115–142. Rademacher B, Koch W & Hurle K (1970) Changes in the weed flora as a result of continuous cropping of cereals and the annual use of the same weed control measures since 1956. Proceedings of the 10th British Weed Control Conference, Brighton, 1–6. Rew LJ, Wilson PJ, Froud‐Williams RJ & Boatman ND (1992) Changes in vegetation composition and distribution within set‐aside land. British Crop Protection Council Monograph 50, 79–84. Rew LJ, Froud‐Williams RJ & Boatman ND (1996) Dispersal of Bromus sterilis and Anthriscus sylvestris seed within arable field margins. Agriculture Ecosystems & Environment 59, 107–114. Roberts HA & Neilson JE (1981) Changes in the soil seedbank of four long‐term crop/ herbicide experiments. Journal of Applied Ecology 18, 661–668.

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Robinson RA & Sutherland WJ (2002) Post war changes in arable farming and biodiversity in Great Britain. Journal of Applied Ecology 39,157–176. Russell EJ (1958) Weeds, the ancient enemy. Agriculture 65, 5–8. Rutledge J, Talbot RE & Sneller CH (2000) RAPD analysis of genetic variation among propanil resistant and susceptible Echinochloa crus‐galli populations in Arkansas. Weed Science 48, 669–674. Saavedra M, Garcia‐Torres L, Hernadez‐Bermejo E & Hidalgo B (1989) Weed flora of the Middle Valley of the Guadalquivir, Spain. Weed Research 29, 167–179. Saavedra M, Garcia‐Torres L, Hernadez‐Bermejo E & Hidalgo B (1990) Influence of environmental factors on the weed flora in crops in the Guadalquivir Valley. Weed Research 30, 363–374. Salisbury EJ (1961) Weeds and Aliens. Collins, London. Salonen J, Hyvonen T & Jalli H (2001) Weeds in spring cereal fields in Finland – a third survey. Agricultural and Food Science in Finland 10, 347–364. Schutte BJ, Tomasek BJ, Davis AS et al. (2014) An investigation to enhance understanding of the stimulation of weed seedling emergence by soil disturbance. Weed Research 54, 1–12. Seavers GP & Wright KJ (1999) Crop canopy development and structure influence weed suppression. Weed Research 39, 319–328. Semb Tørreson K & Skuterud R (2002) Plant protection in spring cereal production with reduced tillage. IV. Changes in the weed flora and weed seedbank. Crop Protection 21, 179–193. Shaner DL & Beckie HJ (2014) The future for weed control and technology. Pest Management Science 70, 1329–1339. Shaw RH, Tanner R, Djeddour D & Cortat G (2011) Classical control of Fallopia japonica in the United Kingdom: lessons for Europe. Weed Research 51, 551–558. Sheppard AW, Shaw RH & Sforza R (2006) Top 20 environmental weeds for classical biological control in Europe: a review of opportunities, regulations and other barriers to adoption. Weed Research 46, 93–117. Smith A (1988) Endangered Species of Disturbed Habitats. Nature Conservancy Council, Peterborough. Smith AE & Secoy DM (1976) Early chemical control of weeds in Europe. Weed Science 24, 594–597. Stace C (1997) New Flora of the British Isles. Cambridge University Press, Cambridge. Stark G (2011) EU pesticide legislation – an update. Aspects of Applied Biology 106, 259–262. Stiers I, Coussemont K & Triest L (2014) The invasive plant Ludwigia grandiflora affects pollinator visitants to a native plant at high abundances. Aquatic Invasions 9, 357–367. Storkey J, Cussans JW, Lutman PJW & Blair A (2003) The combination of a simulation and an empirical model of crop weed competition to estimate yield loss from Alopecurus myosuroides in winter wheat. Field Crops Research 84, 291–301. Storkey J, Moss SR & Cussans JW (2010) Using assembly theory to explain changes in a weed flora in response to agricultural intensification. Weed Science 58, 39–46. Storkey J, Meyer S, Still KS & Leuschiner C (2012) The impact of agricultural intensification and land‐use change on the European arable flora. Proceedings of the Royal Society B 279, 1421–1429.

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0003060919.INDD 32 05/04/2017 6:29:11 PM CHAPTER 1 An introduction to cells and their organelles

William V. Dashek Retired Faculty, Adult Degree Program, Mary Baldwin College, Staunton, VA, USA

Cells

Parenchyma, chlorenchyma, collenchyma, and sclerenchyma are the four main plant cell types (Figure 1.1, Evert, 2006). Meristematic cells, which occur in shoot and root meristems, are parenchyma cells. Chlorenchyma cells contain chloroplasts and lack the cell wall thickening layers of collenchyma and scleren- chyma. Certain epidermal cells can be specialized as stomata that are important in gas exchange (Bergmann and Sack, 2007). The diverse cell types (Zhang et al., 2001; Yang and Liu, 2007) are shown in Table 1.1. Photomicrographs of certain of these cell types can be found in Evert (2006), Fahn (1990), Beck (2005), Rudall (2007), Gunning (2009), MacAdam (2009), Wayne (2009), Beck (2009), Assmann and Liu (2014) and Noguchi et al. (2014).

How do cells arise? Cells arise by cell divisions (see Chapter 8 for mitosis and meiosis) in shoot and root (Figures 1.2 and 1.3) meristems (Table 1.2, Lyndon, 1998; McManus and Veit, 2001; Murray, 2012). The shoot apex is characterized by a tunica–corpus organiza- tion (Steeves and Sussex, 1989). The tunica gives rise to the protoderm and its derivative, the epidermis. In contrast, the corpus provides the procambium which yields the primary xylem and phloem. In addition, the ground tissue derives from the corpus originating the pith and cortex. Following divisions, cells can differenti- ate into tissues (Table 1.3) and organs of the mature plant body (Leyser and Day, 2003; Sachs, 2005; Dashek and Harrison, 2006). The leaf primodium arises on the apex (MicolCOPYRIGHTED and Hake, 2003). The mature MATERIAL angiosperm leaf consists of palisade cells and spongy mesophyll cells sandwiched between the upper and the lower epider- mis (Figure 1.4). The epidermis possesses guard cells with associated stomata that function in gas exchange. KNOX genes affect meristem maintenance and suitable patterning of organ formation (Hake et al., 2004). In dissected leaves, KNOX genes are expressed in leaf primordia (Hake et al., 2004). Hake et al. (2004) suggest that

Plant Cells and their Organelles, First Edition. Edited by William V. Dashek and Gurbachan S. Miglani. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd.

1

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Primary phloem bres

Epidermis

CO Cortex

Phloem

Vascular par cambium

Secondary xylem

A

Figure 1.1 Plant cell types: Left: parenchyma (par) and collenchyma (co). Right: sclerenchyma. Source: Evert (2006). Reproduced with permission of John Wiley & Sons.

KNOX genes may be important in the diversity of leaf form. Extensive discussions of leaf development occur in Sinha (1999), Micol and Hake (2003) and Efroni et al. (2010). Under appropriate stimuli the vegetative apex can be converted to a floral apex (Figure 1.5). Photoperiod (Mazumdar, 2013), such as short days and long days and combinations of the two, is one such stimulus (Glover, 2007; Kinmonth‐Schultz et al., 2013). This induction results in the production of florigen (Turck et al., 2008), the flowering hormone (Zeevaart, 2006). While early reports suggest that florigen is an mRNA species (Huang et al., 2005), a more recent inves- tigation indicates that florigen is a protein complex (Yang et al., 2007; Taoka et al., 2013). Taoka et al. state that florigen protein is encoded by the gene, Flowering Locus T, in Arabidopsis species (Shresth et al., 2014). It is believed that florigen is induced in leaves and that it moves through the phloem to the shoot apex. Plant hormones (see Appendix A) can influence floral development (Howell, 1998). Gibberellins (Blázquez et al., 1998), auxins, and jasmonic acid can affect petal development. In contrast, auxin can influence gynoecium development. The ABC model has been proposed for regulating the development of floral parts (Soltis et al., 2006). The A gene expression is responsible for sepals, while the petals are the result of co‐expression of A and B genes. The B and C genes are responsible for stamen development and carpels require C genes. In certain plants, vernalization (low temperature) can induce flowering in certain plants (Kemi et al., 2013). A diagram of the mature angiosperm plant body is presented in Figure 1.6. Plant

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Table 1.1 Plant cell types.

Cell types Characteristics References

Epidermal cells Unspecialized cells; one layer of cells in thickness; outer covering of various plant parts; Evert (2006) variable in shape but often tabular Examples Guard cells Specialized epidermal cells; crescent shaped; contain chloroplasts; form defines stomatal pore Wille and Lucas (1984) Subsidiary cells Cells which subtend the stomatal guard cells http://anubis.ru.ac.za/Main/ ANATOMY/guardcells.html Trichomes An outgrowth of an epidermal cell; can be unicellular or multicellular Callow (2000) Parenchyma cells Isodiametric, thin‐walled primary cell wall; in some instances may have secondary walls; not Evert (2006) and Sajeva and highly differentiated; function in photosynthesis, secretion, organic nutrient and water Mauseth (1991) storage; regeneration in wound healing Examples Transfer cells Specialized parenchyma cells; plasmalemma greatly expanded; irregular extensions of cell Dashek et al. (1971) and Offler et al. wall into protoplasm; transfer dissolved substances between adjacent cell; occur in pith (2003) and cortex of stems and roots; photosynthetic tissues of leaves; flesh of succulent fruits; endosperm of seeds Collenchyma cells Lamellar or plate collenchyma, with thickenings on the tangential walls Angular collenchyma, with thickenings around the cell walls Present in aerial portions of the plant body Vascular cells Evert (2006) Phloem Sieve cells Sieve elements Companion cells Specialized parenchyma cells; possess numerous plasmodesmatal connections Oparka and Turgeon (1999) Albuminous cells in Absence of starch; cytoplasmic bridges with sieve cells; dense protoplasm, abundance of Alosi and Alfieri (1972) and gymnosperms polysomes, highly condensed euchromatin and abundant mitochondria Sauter et al. (1976) Xylem Tracheids Long tapering cell with lignified secondary wall thickenings; can have pits in walls; devoid of Tyree and Zimmerman (2002) Vessels protoplasm at maturity; not as specialized as vessels; widespread Fukuda (2004) and Evert (2006) 10/22/2016 6:36:26AM (Continued ) 0002796388.indd 4

Table 1.1 (Continued)

Cell types Characteristics References

Specialized cells – Hydathodes Consist of terminal tracheids epithem, thin‐walled chloroplast‐deficient cells, a sheath with Lersten and Curtis (1996), (modified parts of leaves and water pores; guttation discharge of liquid containing various dissolved solutes from a leaf’s https://www.biosci.utexas.edu/ and leaf tips or margins) interior Maeda and Maeda (1988)

Laticifer cells Cells or a series of cells which produce latex Fahn (1990), Pickard (2008) and Botweb.uwsp.Edu Simple Single-celled Compound and Union of cells compound in origin and consist of longitudinal chains of cells; wall separating articulated cells remain intact, can become perforated or entirely removed Salt glands Modified trichomes, two‐celled and positioned flat on the surface in rows parallel to the leaf Evert (2006), Tan et al. (2010), Oross surface; occur in Poaceae; et al. (1985) and Thomson et al. (1988) Cap cell – large nucleus and expanded cuticle Naidoo and Naidoo (1998) Basal cell – numerous and large extensive partitioning invaginations of plasmalemma Nectaries Found in nectarines; produce nectar, usually at the base of a flower Fahn (1990), Nicolson and Nepi (2005) and Paiva (2009) Idioblasts Crystal‐containing cells Lersten and Horner (2005) Example Raphides Produce needle‐shaped crystals Mucilage cell Occur in a large number of dicots, common in certain cacti; slimy mucilage prevents http://www.sbs.utexas.edu/masuetl/ evaporation of water by binding to water; a parenchyma cell whose dictyosomes produce weblab/webchap9secretory/9.1‐2.html, mucilage as in seed coats; cell walls are cellulosic and unlignified Western et al. (2000) and Arsovskia et al. (2010) Oil cells Specialized cells appear like large parenchyma cells; can occur in vascular and ground tissues Rodelas et al. (2008), http:brittanica. of stem, and leaf cell wall has three distinct layers; cavity is formed after the inner wall layer com and Lersten et al. (2006) has been deposited Druses Spherical aggregates of prismatic crystals Lersten and Horner (2005) Cells in non‐angiosperms Bryophytes Gemmae One to many cells http://buildingthepride.com/faculty/ pgdavison/bryology_links.htm

10/22/2016 6:36:26AM Hydroids Water‐conducting cells http://www.Biology‐online.org Leptoids – Pteridophytes Organic compound‐conducting cells; sporogenous cells present in sporangia of sori An introduction to cells and their organelles 5

Figure 1.2 Angiosperm shoot meristem section. Source: Alison Roberts. Reproduced with permission of University of Rhode Island.

Figure 1.3 Angiosperm root meristem section. Source: Alison Roberts. Reproduced with permission of University of Rhode Island.

development is discussed in Fosket (1999), Moore and Clark (1995), Greenland (2003), Leyser and Day (2003) and Rudall (2007).

What is the composition of cells? Certain plant components exhibit polar growth, for example, the tip growth of pollen tubes (Hepler et al., 2001). The tubes elongate via the fusion of Golgi‐ derived vesicles with the plasmalemma and subsequent deposition of the vesi- cles’ contents into the cell wall (Taylor and Hepler, 1997; Parton et al., 2001 and others as reviewed in Malho (2006a, 2006b)). In 2007, Dalgic and Dane (2005) published a diagram depicting the now known tube‐tip structural elements and physiological processes that facilitate tube elongation. The diagram represents a

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Table 1.2 Meristems and their derivatives.*

Meristems Derivatives

Primary Protoderm Epidermis From tunica (Evert, 2006) Procambium (provascular) Primary xylem and phloem From corpus (Evert, 2006) Vascular cambium Ground Ground tissue: pith and cortex Lateral Vascular cambium Fusiform initials Secondary xylem Secondary phloem Ray initials (Evert, 2006) Ray cells Cork cambium Phellogen Replaces the epidermis when cork cambium initiates stem girth Periderm (Evert, 2006) increase; composed of ‘boxlike’ cork cells which are dead at maturity; protoplasm secretes suberin; some cork cells that are loosely packed give rise to lenticels which function in gas exchange between the air and the stem’s interior. http://www.Biology‐online. org, Evert (2006), http://www.vebrio.Sceince.vu.nl.en/virut Phelloderm Parenchyma cells produced on the inside by the cork cambium

* Meristems are discussed by Steeves and Sussex (1989).

Table 1.3 Plant tissues.

Tissue system

Meristematic Ground Vascular Dermal

significant advance over the early studies of pollen tubes as it assigns function to ultrastructural components, for example, signalling molecules, the Rho family of GTPases and phosphatidylinositol 4,5 bisphosphate appear to be localized in the apical plasma membrane. Besides pollen tubes, root hairs exhibit polar growth.

Cell organelles – an introduction

Organelles are required for plant growth, development and function (Sadava, 1993; Gillham, 1994; Herrmann, 1994, Agrawal, 2011). These organelles (Figure 1.7) are the loci for a myriad of physiological and biochemical processes (Tobin, 1992; Daniell and Chase, 2004 – see individual chapters). There are many diagrams of a generalized plant cell. Some of these are available at www.explorebiology.com, http://www.daviddarling.info/images/plant_cell.jpg

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Figure 1.4 SEM of a pecan leaf. Diagram of a leaf’s interior is available at http://pics4learning. com. Source: Reproduced with permission of Asaf Gal.

Includes the shoot meristem and t

Floral meristems generate oral organs, i.e., sepals, petals, stamens and carpels he owers Inorescence meristem

Leaf primordia Genes occur in oral meristems oral in occur Genes

Both day length and temperature regulate owering

Flowering is regulated by a protein hormone named origen Homeotic genes encode proteins proteins encode genes Homeotic

Figure 1.5 Schematic of the floral meristem.

and http://micromagnet.fsu.edu. The organelle contents of plant and animal cells in common and those unique to plant cells are depicted in Table 1.4. The dimensions of plant organelles are presented in Table 1.5. A plant organelle data- base (PODB) has been reviewed by Mano et al. (2008). To enter a plant cell, molecules must traverse both the cell wall and the fluid mosaic plasmalemma (Singer and Nicolson, 1972; Leshem et al., 1991; Larsson and Miller, 1990). In contrast to the fluid mosaic model (Figure 1.8) of the plasmalemma,

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Starch or sugar C H O Photosynthesis, 6 12 6 storage organ respiration and photorespiration Sugars O2 CO2 H2O vapour Sugars

starch H2O

Sugars

starch or sugar storage organ

Respiration, no photorespiration O2 H2O and CO2 minerals enter through root hairs

Figure 1.6 Diagram of angiosperm plant body. Source: From http://www.msu.edu/course/ te/8021/science08plants/foods.html.

Figure 1.7 Electron micrograph of a plant cell and its organelles. Source: Reproduced with permission of H.J. Horner.

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Table 1.4 Comparison of organelle contents of plant and animal cells.*

Organelle Animal cell Plant cell

Cell wall Absent Present Centrioles Present Absent Endoplasmic reticulum Present Present Glyoxysomes Absent Present Golgi apparatus Present Present Microfilaments Present Present Mitochondrion Present Present Nucleus Present Present Peroxisomes Present Present Plastids Absent Present Protein bodies Absent Present Spindle Present Present Vacuoles Sometimes small Present (mature cell – large central)

* Early discussions of plant cell organelles occur in Hongladarom et al. (1964), Pridham (1968), Reid and Leech (1980) and Tobin (1992).

Table 1.5 Dimensions of subcellular organelles.

Organelles Dimension

Chloroplast 4–6 µm in diameter Golgi apparatus Individual cisternae, 0.9 µm Coated vesicles 50–280 µm in diameter Microbodies 0.1–2.0 µm in diameter Microtubules 0.5–1.0 µm in diameter Mitochondria 1–10 µm Nuclear envelope pores 30–100 µm in diameter Nucleus 5–10 µm in diameter Peroxisome 0.2–0.7 µm Plasmodesmata 2–40 µm in diameter Primary wall 1–3 µm Protein bodies 2–5 µm in diameter Vacuoles 30–90% of cell volume

the picket–fence model proposes the accumulation of membrane protein anchored in an actin network beneath the membrane (Kusumi et al., 2012). The plasmalemma is composed of water, protein and lipids. There are both integral and peripheral proteins (Leshem et al., 1991). The integral proteins may be simple (classical α‐helical structure that traverses the membrane only once) or complex (globular – composed of several α‐helical loops which may span the membrane several times). Peripheral proteins can be easily isolated by altering

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Fluid mosaic model of the plasmalemma Consists of a lipid bilayer in which globular proteins are embedded; There are two types of proteins: integral and peripheral. Oliogsaccharides (2–20 monosaccharides) can be attached to the integral proteins. Phospholipids from the bilayer with a polar head on the outside and non-polar tails on the inside.

Fence model of the plasmalemma There is a membrane skeleton with skeleton-anchored proteins and transmembrane proteins projected outwards into the cytoplasm. Cytoplasmic domains of proteins collide with the actin skeleton, yielding temporary con nement of the transmembrane proteins. The membrane can contain lipid rafts and related caveolae invaginations. The rafts are combinations of proteins and the lipids which may function in signalling. sphingolipids are prevalent in the rafts.

Picket model of the plasmalemma Phospholipids can also be con ned by the membrane skeleton. Some investigators combine the fence and picket models.

Figure 1.8 Top: Fluid mosaic model of the plasmalemma. Middle: Fence model of the plasmalemma. Bottom: picket model of the membrane.

the ionic strength or pH of the encasing medium. The transport proteins are pumps, carriers or chemicals (see section on membrane transport). The lipids are electro-negative and anionic phospholipids, sphingolipids (Figure 1.9), chloroplast‐ specific glycerolipids and sterols (Table 1.6). Lipid rafts are specialized phase domains containing sterols and sphingolipids which may be important in signal transitions (Gray, 2004; Furt et al., 2007; Grennan, 2007; Mongrand et al., 2004). Caveolae, which give rise to clathrin‐ coated vesicles (Brodsky et al., 2001), are anchored multifunctional platforms in lipids (Van Deurs et al., 2003; Patel and Insel, 2009). The organization of the caveolae (Bastani and Parton, 2010) in the plasma- lemma and clathrin‐coated vesicles (Samaj et al., 2005) is presented in Figure 1.10. The current discussion focuses on membrane transport mechanism. Plants can internalize certain molecules by endocytosis via invaginations of the plasmalemma yielding clathrin‐coated vesicles (Figure 1.11, Holstein, 2003) which become the endosome (Low and Chandra, 1994; Battey et al., 1999; Šamaj et al., 2006). Proteins involved in clathrin‐dependent endocytosis appear to be clathrin, adaptor proteins and two adaptins (Pearse and Robinson, 1990; Šamaj et al., 2006). Plant endocytosis and endosomes (Contento and Bassham, 2012) seem to be significant in auxin‐mediated cell–cell communication, gravity responses, stomatal move- ments, cytokinesis and cell wall morphogenesis (Šamaj et al., 2006).

Ion channels

Plasma membranes contain potassium (K+), calcium (Ca++) and anion channels (Roberts, 2006). Voltage‐gated ion channels are transmembrane ion channels acti- vated by changes in electrical potential. Gating is the precise control of ion channel opening (Krol and Trebacz, 2000). An example of an ion channel is the K+ the

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Phosphatidic acid Phosphatidylethonolamine Leeithin O O O O O R1 O O R1 O O R1 CH R2 R2 O R2 O 3 O O O O + NH2 N O P OH OOP O P O CH3 CH3 OH OH OH

Phosphatidylserine Phosphatidylinositol 1-Lysoleeithin O O O R1 O O O O R1 O H CH R2 O O R2 O O HO O 3 O OH + OO OH O P O OH N P P OO CH3 CH3 OH NH2 OH OH HO OH OH CH3 H3C N CH3 + CH2 + O CH2 + O P O O + P OO O O + O O P O H2C CH CH2 H2C CH CH O OO 2 OO O CC O Phosphatidylcholine H2C CH CH OOCC 2 (leeithin) HO OH Glycerol-3 phosphate

Phosphatidate (a)

Alpha-spinasterol H3C Brassicasterol

29 sterol (C29H48O) H C 28 sterol (C28H46O) 3 CH3 isolated from `Phytolacca´ CH3 H synthesized by oilseed rape CH3 CH3 and several unicellular algae H H H HO H H H HO http;//www.3dmet.dna.affec.go.jp/bin2/show_data.e?acc-B02450 http;//en.wikipedia.org/wiki/Brassicasterol

H H HO (b)

Figure 1.9 Structures of (a) phospholipids and (b) sphingolipids.

inwardly potassium channel. This type of channel possesses a positive charge in the cell. Stomatal pore movements are mediated by a rise in intracellular K+ and anion contents of guard cells (Schroeder and Hagiwara, 1989). Another example is the adenosine triphosphate (ATP) binding cassette transporter or ABC trans- porter. These transport toxic substances from the cell or into the vacuole. These

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Table 1.6 Composition of certain cellular membranes.

Chemical composition

Fatty acyl groups in membrane lipids 16:0, 16:1, t‐16:1, 16:3, 18:0, 18:1, 18:2, α18:3, δ18:3, 18:4, 22:0, 22:1, 24:0, 24:1 Electroneutral phospholipids Phosphatidylcholine, phosphatidylethanol, phosphatidylethanolamine Anionic phospholipids Phosphatidylserine, phosphatidylglycerol, phosphatidylinositides Lyo‐phospholipids Cerebrosides Sphingolipids Galactolipids, sulpholipids Chloroplast‐specific glycerolipids Diphosphatidylglycerol and monophosphatidylglycerol Mitochondrial phospholipids Sterols Sitosterol Campesterol Stigmasterol Unusual sterols Cycloartenol Cholesterol, minute quantities Sterol glycosides Lanosterol Pathogenic fungal membranes Water Extramembrane water Membrane is a bilayer sandwiched between two layers of water Water located within the bilayer which is attached to or in approximate contact with the expanses of membrane constituents Proteins May cross the membrane once or several times and are Integral proteins linked either electrostatically or by means of biophysical lipophilicity to the inner domains of the bilayer Simple integral proteins Classic α‐helical structure that traverses the membrane only once Complex integral proteins Globular – comprised of several α‐helical loops that may span the membrane several times Peripheral proteins Associated with only leaflet–easily isolated by altering ionic strength or pH of the encasing medium Transport proteins Pumps, carrier and channel

Source: From Leshem et al. (1991).

transporters are composed of four core domains, two cytosolic nucleotide‐binding proteins and two transmembrane domains (Malmstrom, 2006). Besides cation channels there are anion channels regulated by voltage, but their activity is also influenced by Ca++, ATP, phosphorylation or membrane stretching (Tyerman, 1992). Anion plasma membrane channels function as efflux channels when they are open.

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Normal ta-Golgi Protein with longer network membrane Cholesterol Cytosol transmembrane domain

Lumen Glycolipids Protein with short transmembrane Lectins GPI-achored domain cannot (a) protein enter lipid raft

Integral proteins excluded from transport vesicles Exoplasmic face

Cytosolic face Fibrous Assembly clathrin particle coat

GTP Dynamin GDP

Clathrin-coated vesicle

(b)

Figure 1.10 Depictions of a (a) lipid raft, (b) caveolae and a clathrin‐coated vesicle. Source: Reproduced with permission of Caveolae and Clathrin Vesicle.

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Vacuole Late endosome/ PVC ARA6 δ-TIP AtSYP21 AtSYP22 Intermediate endosome ARA7 RHA1 AtVAMP727 VSR AtALEU

AtSKD1 ERC/ ? PCR? TGN AtSYP61 PM ATPase GNOM Golgi PM receptor BFA

PIN1+NPA–binding protein SVP – a syntaxin GNOM – Plant-speci c protein that participates in ADP-ribosylation ESCRT – protein endosomal sorting complex RHA – a member of the Rab GTPases function in traf cking pathways ARA6 – a member of the Rab GTPases SYP – a SNARE component of the late endosome VSR – vacuolar sorting receptor SKD – vacuolar protein suppressor Ubiquitylation – signal that regulates the cell surface expression

Figure 1.11 Diagram of plant endocytosis. Source: Reproduced with permission of M. Otegui, University of Wisconsin.

Proton pumps

The transport of a substance against its electro channel gradient requires energy generated by ATP‐proton pumps (Briskin and Hanson, 1992; Evert, 2006). One such pump is the V‐ATPase found in both the plasmalemma and the tonoplast (Barkla and Pantoja, 1996; Vinay et al., 2009). The H+‐ATPase in the plasma- lemma is the P‐ATPase which forms electrochemical gradients (Elmore and Coaker, 2011). Mitochondria and chloroplast membranes possess F‐ATPases.

Water channels

Aquaporins are channel proteins which exist in the plasmalemma in intracellular spaces (Maurel et al., 2008). These proteins permit water to move freely but exclude ions and metabolites (Chrispeels and Maruel, 1994; Muller et al., 2007),

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providing for buffering osmotic fluctuations in the cytosol. Aquaporins are major intrinsic membrane proteins which are composed of four subunits, each of which comprises six transmembrane‐spanning helices. Aquaporins are encoded by multiple gene families (Johansson et al., 1998).

Carriers

Carriers are unitransporters and co‐transporters (Evert, 2006). Unitransporters transport only one solute from one side of the membrane to the other. On the contrary, co‐transporters transfer one solute with the simultaneous or sequential transfer of another solute. A thorough discussion of membrane transport processes occurs in Malmstrom (2006). Organelle structure and function can be influenced by a variety of environ- mental parameters which affect plant growth. A discussion of parameters is pre- sented because of the increasing pollution of the earth’s atmosphere and ecosystem. In addition, global climate change is a current issue of urgent con- cern (Dashek and McMillin, 2009). Both major and minor elements are required for growth and development (Table 1.7). Metals and metalloids at elevated levels can result from mining (Dashek and McMillin, 2009). What effects do these levels have on the structure and function of cellular organelles? (See Lepp, 1981; Medioini et al., 2008; Yusuf et al., 2011; see also Table 1.8.)

Elevated levels of SO2, CO2, NO2 and O3 (Treshow and Anderson, 1989) can occur in the atmosphere as a result of industrial and contemporary activities. Table 1.9 presents the effects of certain gases (Bell and Treshow, 2002) on the structure and function of organelles. Of special interests are the increasing levels

Table 1.7 Major and minor elements required for plant growth and development.

Element mg/kg Minor or major

Nitrogen N 15 000 Major Potassium K 10 000 Major Calcium Ca 5 000 Major Magnesium Mg 2 000 Major Phosphorus P 2 000 Major Sulfur S 1 000 Major Chlorine Cl 100 Minor Iron Fe 100 Minor Boron B 20 Minor Manganese Mn 50 Minor Zinc Zn 20 Minor Copper Cu 6 Minor Molybdenum Mo 0.1 Minor

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Table 1.8 Toxic metals and metalloids.

Metal or Toxic level effects References metalloid

Aluminium Affects root cells of plasmalemma Mossor‐Pietraszewska (2001) Arsenic Pale green to yellow lesions on leaves and Treshow and Anderson (1989) necrosis of leaves Defoliation Impaired nitrogen metabolism Needle abscission Cadmium General chlorosis Treshow and Anderson (1989), Reduced photosynthesis Saadati et al. (2012) and Khateeb Reduced transpiration; toxic effects – changes (2014) in proline levels; changes in lipid peroxidation and seed germination Copper Interference with normal metabolic reactions Treshow and Anderson (1989) Blocks specific enzymatic reactions and Shah et al. (2001) Chromium Contamination Treshow and Anderson (1989) Can promote white dead patches on leaves and Antonovics et al. (1971) Lead Condensation of nuclear chromatin; decrease Rout and Das (2003) and in germination of two Brassica cultivars Hosseini et al. (2007) Nickel Dilution of nuclear membrane Seregin and Kozhernikova (2006) Zinc Disruption of cortical cell Rout and Das (2009)

Table 1.9 Effects of environmental pollutants on organelles.

Elevated CO2

Stomatal openings reduce as CO2 increases Woodward et al. (1991) Affects both primary and secondary meristems Pritchard et al. (1999) of shoots and roots; alternation of leaf size and anatomy; increased branching and stem diameter Increase in the number of mitochondria and Griffin et al. (2001) amount of chloroplast stroma thylakoid membranes Stomatal densities decrease in two species of Lammertsmaa et al. (2011) Spartia Acid rain Leaching of nutrients on tree needles; damages Godbold and Hüttermann surfaces of needles and leaves and reduces a (1994), Schulze et al. (2000) tree’s ability to withstand cold and White and Terninko (2003) Nitric oxide Necrotic lesions, marginal chlorosis Lamattina and Polacco (2007) Ozone and its Changes in metabolism Roshchina and Roshchina (2003) derivatives

of CO2 in the atmosphere, which many scientists believe causes global warming (Dashek and McMillin, 2009). Table 1.10 offers the effects of sublethal and lethal temperatures on organelles. Franklin and Wigge (2014) discuss the effects of temperature on plant development. Other environmental parameters which can

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Table 1.10 Effects of temperature or subcellular organelles.*

Temperature Effect Reference

Sublethal Swollen chloroplasts and loss of chlorophyll in Elodea leaves Quinn (1988) Lethal Plasmolysis of Elodea and soybean leaves; disintegration of Daniell et al. cellular membranes (1969)

* Other effects of elevated temperature are on photosynthetic activities (Weis and Berry, 1988) and the plant immune response (Franklin and Wigge, 2014).

Biotic stress – chemical, humidity, mechanical, radiations, temperature and water

Abiotic stress – competition, herbivory, infection

ROS levels increase superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen, Nitric oxide

involving cell wall, chloroplasts, mitochondria, phagosomes

Oxidative damage

Programmed cell death

Figure 1.12 Reactive oxygen species (ROS) and plant cell death.

affect organelle structure and function are radiation (Parida et al., 2002; Mokobia and Anomohanran, 2005; Borzouei et al., 2012) and salinity (Bennici and Tani, 2009; Kumar et al., 2013).

Cell death

In certain mammalian systems, there appear to be two apoptotic pathways: extrinsic and intrinsic. Whereas the extrinsic pathway involves death receptor liquids, in the intrinsic pathway a variety of factors act upon mitochondria to promote loss of mitochondrial membrane potential. Whether these two path- ways are significant in plant apoptosis remains to be established with certitude. Programmed cell death in plants (Bryant et al., 2000) is viewed as a normal phase of development (Gray, 2004; Lam, 2008). These authors state that little is known about how plant cell death occurs and is regulated. However, reactive oxygen species (ROS) seem to be involved (Karuppanapandian et al., 2011 – Figure 1.12). Fragmentation of nuclear DNA, involvement of Ca++, alterations in protein phosphorylation, increases in nuclear heterochromatin and involve- ment of ROS seem to occur. Beers et al. (2000) conclude that proteases may possess a role in programmed cell death. In animals, caspases are significant components of programmed cell death. Although caspase attributes have been

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detected in plants, a role for these proteases in plant cell death is unclear (Lam, 2006). Lastly, van Doorn (2011) distinguished between ureolytic and non‐ ureolytic cell death. Whereas the former involves tonoplast rupture and subse- quent destruction of the cytoplasm, the latter includes tonoplast rupture but not cytoplasmic destruction. Finally, aspects of plant cells can be found in the following general plant cell biology textbooks: Batra (2009), Dashek and Harrison (2006), Gupta (2004), Pandian (2008), Pickett‐Heaps and Pickett‐Heaps (1994) and Wayne (2009).

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Further reading

Alberts, B., Johnson, A., Lewis, J., et al. (2014) Molecular Biology of the Cell, 6th edition, Garland Science, New York. Buchanan, B. and Gruissem, W. (2015) Biochemistry and Molecular Biology of Plants, Wiley, Chichester, UK. Mano, S., Miwa, T., Nishikawa, S., Mimura, T. and Nishimura, M. (2011) The plant organelle databases 2 (PODB2): An updated resource containing movie data of plant organelle dynamics, Plant Cell Physiology, 52 (2), 244–253. Mano, S., Nakamura, T., Kondo, M. et al. (2014) The plant organelles database 3 (PODB3) Update 2014: Integrating electron micrographs and new options for plant organelle research, Plant Cell Physiology, 55 (1), e1, doi: 10.1093/pcp/pct140. Nick, P. and Opatrny, Z. (2014) Applied Plant Cell Biology: Cellular Tools and Approaches for Plant Biotechnology, Springer, New York, NY. Plopper, G., Sharp, D. and Sikorski, E. (2015) Lewin’s Cells, 3rd edition, Jones & Bartlett, New York.

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1

Introduction

Value of Trees Globally

The three trillion trees around the world (Crowther et al. 2015) are hugely important to us and to the well‐being of our planet (Figure 1.1). Their value is usually described in terms of ecosystem services – what trees and forests can do to help us humans. A detailed list of ecosystem services provided by trees and forests would fill this book (the UK National Ecosystem Assessment 2011 provides a very good summary) so, by way of illustration, here are just three major services. One of the major services is storing carbon. Forests hold around 45% of the carbon stored on land (i.e. not including the reserves held in oceans) which amounts to 2780 Gt of carbon (Giga has nine zeros; i.e. billions). This is about 3.3 times the amount already in the atmosphere (829 Gt). Carbon dioxide in the atmosphere has increased from 280 ppm in pre‐industrial times to 404 ppm at the time of writing, an increase of 42%. If all the world’s trees died and decomposed to release their carbon into the atmosphere, the atmospheric level of carbon dioxide would rise to 1700 ppm (>600% pre‐industrial) with catastrophic effects on our world (UNEP 2008), so global carbon storage in trees and forests is a hugely important service. Forests also help to determine weather patterns. This is partly by forests evaporating large amounts of water, producing clouds that release rain downwind. Furthermore, it has recently been discovered that a chemical released by trees, pinene (one of the monoterpenes), can help ‘seed’ clouds by acting as nuclei for water to condense around, and so help clouds to form and rain to fall (Kirby et al. 2016). It seems plausible that other volatile organic compounds (VOCs) emitted by trees have a similar effect. Trees and forests are also beneficial by acting as sponges, slowing the journey of rainfall to the ground and helpingCOPYRIGHTED to improve soil structure, MATERIALboth of which encourage water to sink into the soil rather than run off the surface. This delays water discharge to streams and rivers, helping to reduce flooding and soil erosion. Most of the world’s biodiversity is held in forests. Tropical forests, which cover 7% of land surface, hold more than 60% of the world’s species of terrestrial animals and plants (Bradshaw et al. 2009), and all the world’s forests hold more than 80% of species (Balvanera et al. 2014).

Applied Tree Biology, First Edition. Andrew D. Hirons and Peter A. Thomas. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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Figure 1.1 Forests are globally important to mankind for storing carbon, helping to determine weather patterns and providing a habitat for a vast range of life. This scene is of the temperate forest in Robert H. Treman State Park, New York.

Value of Urban Trees

On a smaller scale, urban trees and woodlands also have an important role in our well‐ being, but for slightly different reasons. Fundamentally, urban trees make our towns and cities better places to live. Quite apart from making urban areas look more appeal- ing, trees can provide a sense of place and time. They help provide outdoor recreation opportunities and make the urban environment more pleasant. Economic benefits of urban trees include higher property values; reduced energy costs of buildings; and reduced expenditure on air pollution removal and storm water infrastructure (Roy et al. 2012; Mullaney et al. 2015). There are also many environmental benefits, the most important of which are summarised in Expert Box 1.1. With more than half of the world’s population now living in cities, one of the most important contributions that trees and green spaces make is to our health. There is a growing body of information that shows that exposure to trees and green spaces improves wellness and our sociability (Wolf and Robbins 2015). Studies have also shown that the positive health impact of trees is independent of access to green space in general. For example, in Sacramento, California, higher tree cover within 250 m of home was associated with better general health, partially mediated by lower levels of obesity and better neighbourhood social cohesion (Ulmer et al. 2016). There is also a body of

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information that shows that psychological benefits of trees can affect the physiology of our bodies by reducing pulse rate and levels of cortisol, a major stress hormone (Ochiai et al. 2015). This works even when looking at pictures of trees. There is also a physio- logical response because chemicals released by some trees affect us directly. For example, Ikei et al. (2015) found that oil from the Hinoki cypress Chamaecyparis obtusa, widely used in fragrances in soap, toothpaste and cosmetics in Japan, positively affects brain activity and induces a feeling of ‘comfortableness’. This is the basis for shinrin‐ yoku (forest‐air breathing or forest bathing), a popular form of relaxation in Japan, walking through wooded areas or standing beneath a tree and slowly breathing (Figure 1.2). The same monoterpenes that cause cloud formation are known to reduce tension and mental stress, reducing aggression and depression and increasing feelings of well‐being. Even a short lunchtime walk of 1.8 km through green areas can improve sleep patterns that night (Gladwell et al. 2016). Moreover, the physiological effects stay with us. A study by Li (2010) found that a 3‐day forest visit had positive effects on the immune system up to 30 days later. The loss of trees from urban environments has also been demonstrated to have negative outcomes for human health. Over 100 million ash Fraxinus spp. trees have been lost in the north‐eastern USA since 2002 as a result of the emerald ash borer (EAB), an invasive beetle. This huge loss of trees has been linked to increased human mortality as a result of higher levels of cardiovascular and respiratory diseases (Donovan et al. 2013). Social costs, such as an increase in crime, have also been associated with the loss of trees

Figure 1.2 A sign encouraging people to breathe in the air in a forest in northern Honshu Island, Japan. This shinrin‐yoku (forest‐air breathing) is a popular form of relaxation in Japan.

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caused by EAB (Kondo et al. 2017). Consequently, there is a growing body of evidence that the presence of trees in and around our urban environments provides major public health and societal benefits. However, in some cases, the much‐championed value of urban trees is perhaps not all that is claimed. Examples of this include oxygen production and carbon sequestration (the locking‐up of carbon). It is true that trees produce an abundance of oxygen. For example, urban forests in the USA have been estimated to produce enough oxygen (61 Mt of it) annually to keep two‐thirds of the US population breathing (Nowak et al. 2007). However, given the enormous reserves of oxygen in the atmosphere, this is a fairly minor benefit of urban trees. Another benefit of urban trees that is often over‐ played is their role in mitigating carbon emissions. Roland Ennos, Expert Box 1.1, points out that Greater London’s 8.4 million trees are estimated to store 2.4 million tonnes of carbon (t C) and sequester about 77 200 t C each year (Rogers et al. 2015). This amounts to about 3% of the city’s annual carbon emissions or, to put it another way, enough to cover the city’s emissions for about 12 days. London’s trees sequester only about 0.2% of annual carbon emissions. This is not to disparage carbon sequestration in urban trees, but just to put it into perspective; urban trees are very valuable to us but planting them will not be a solution for climate change or even offset the carbon emissions of our towns and cities to any great extent. In this regard, conservation of the world’s forests is of much greater significance. Although trees are overwhelmingly beneficial for our landscapes and for us, they can also create problems, particularly if they are inappropriately planted, the wrong species is selected for the site or the site is poorly designed with respect to tree development. Trees can get too big for their location; they can conflict with buildings, utilities and sightlines. At certain times of year, pollen from trees can contribute to discomfort amongst those with hay‐fever; litter from flowers, fruit and leaves can create slip haz- ards or block drains. Tree roots sometimes cause damage to pavements, making them uneven, and they may exacerbate damage to pipes by exploiting them as a source of water and nutrition. Occasionally, in dry years, certain species growing on shrinkable clay soils can extract enough water to cause subsidence damage to built structures. Trees may also pose a risk to persons or property if they are structurally unstable or develop extensive decay. But should these potential problems prevent us keeping and planting urban trees? Emphatically not. Even though many of the problems associated with trees in urban landscapes can be linked to poor planning, design and workmanship, the tree is invariably blamed. Despite the evidence for the benefits of trees, widespread loss of trees from our urban environ- ments is often reported. In the USA it has been estimated that four million urban trees are lost per year (Nowak and Greenfield 2012) and a similar trend can be seen across Europe. More insidiously, even where the total number of trees is not appreciably declining, the size of the tree is changing. In the UK, the number of large trees, such as London plane Platanus × acerifolia, is declining while the smaller hawthorns Crataegus spp., cherries Prunus spp., whitebeams and rowans Sorbus spp. and birch Betula spp. are increasingly common (Trees and Design Action Group 2008). This reduces the ben- efits derived from the urban forest. Larger trees intercept more rainfall (Xiao and McPherson 2002) and reduce temperatures more than small trees (Gratani and Varone 2006; Gómez‐Muñoz et al. 2010), especially when they have denser crowns (Sanusi et al. 2017). It is therefore vital that we strive to provide opportunities and the right conditions for large trees across our landscapes.

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Managing Trees

Forests have survived for millions of years without us ‘managing’ them, so why is it necessary to look after trees at all? The answer is threefold. First, in a healthy mature forest, the next generation of trees is established from thousands of seeds. Most of these are eaten, develop in unsuitable growing conditions or are out‐competed by other species. The fact that only a fraction of these seeds ever develop into mature trees is insignificant to the bigger picture of a forest: such losses are just part of a forest’s natural ecology. However, in parks and gardens the success of each individual tree is tightly coupled to the success of the planting scheme. We need to actively manage the selection, planting and establishment of the tree to ensure that each tree can make a long‐term contribution to the landscape. Secondly, whilst stable forest environments represent ideal conditions, many trees in gardens, parks and streets have to cope with human‐induced problems or conflicts imposed on them by our built environments. Trees often occupy space that is shared with humans; this erodes the quality of the environment for the tree. In some cases, even our admiration of trees or desire to be amongst them is detrimental to the tree. Visitors to parks and gardens, drawn by the appeal of the landscape, can cause high levels of soil compaction; buried utilities lead to excavation of rooting environments; the need for safe roads and paths in winter leads to high levels of salt being applied close to trees; the list goes on. Trees and their environment need managing so that these conflicts (and others) are not detrimental to tree health. Thirdly, normal patterns of tree development mean that trees can become too large for their position, or their condition can decline over time to such an extent that they endanger people or property. In these cases, trees need managing to control their size and safety. If the many benefits from trees are to be realised, we must do what we can to ensure the health and longevity of trees across our landscapes and provide well‐designed space for new trees. A fundamental requisite for these aims is a sound understanding of tree biology. Conditions for trees within our towns and cities are highly variable. It is wrong to think of the urban environment as being always hostile to trees: there are many parks and gardens that provide excellent conditions for tree growth which may well exceed the quality of the tree’s natural habitat. However, many sites provide very challenging conditions for trees. Soils may be infertile and compacted; sealed surfaces can restrict water infiltration and limit soil aeration; and the rooting environment may need to be shared with utilities. Above ground, branches are removed to reduce conflict with buildings, traffic, cables (Figure 1.3) and sightlines, particularly given the rise in the number of CCTV cameras. Natural processes are also disrupted, leaves are swept off to some remote location far away from the roots that they were intended to nourish. Most of these constraints, however, can be ameliorated with a little informed foresight. If we expect trees to add value to our landscapes, then it is vital that we seek to emu- late the forest environment wherever possible in the design and construction of plant- ing sites. Applying the concept of forest mimicry, mimicking the way that trees work in their natural environment, and being aware of the tree’s biology is crucial. An apprecia- tion of the conditions that trees naturally thrive in and an understanding of the tree’s biology make the difference between successful management that promotes tree health and interventions that simply accelerate tree decline. In this way it is possible to develop

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(a) (b)

Figure 1.3 (a) An ash tree Fraxinus sp. conflicting with overhead wires in northern Japan. This tree now requires intensive management if it is to persist on this site. (b) A mature oak Quercus sp. in Atlanta, USA that has had to endure decades of pruning because it was planted in an unsuitable location. Source: (b) Courtesy of Lukas Ball.

sustainable landscapes with trees that provide communities with a link to their past, as well as a vision of their future. An excellent example of how understanding tree biology can positively influence our management of trees relates to tree pruning practice. For much of the twentieth cen- tury, pruning guidelines recommended taking the branch back so that the final cut was flush with the tree’s stem. By studying decay behind pruning wounds and looking at the process of natural branch shedding, Alex Shigo was able to promote the idea of natural target pruning (Shigo 1989). This transformed our approach to tree pruning and has been of immeasurable benefit to trees as they are now able to seal pruning wounds and restrict the development of decay more effectively (see Chapters 3 and 9). Applying tree biology to practice can also lead to improved rooting environments; more effective management of water and nutrient resources; improved tree establishment rates; and more accurate assessments of tree condition. Further, understanding how trees grow in different environments greatly assists our ability to anticipate their likely limitations and tolerances when we place them in human landscapes. Such variable growing conditions across our parks, gardens and hard landscapes means that a highly prescriptive book on tree management would be left wanting. Instead, our approach is to give the reader an understanding of tree biology and ecology so that this can be used to better inform management decisions, whether in a small garden, a large public park, a street or a courtyard.

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The chapters are divided into key themes: tree growth and development (Chapter 2); leaves and crowns (Chapter 3); roots (Chapter 4); the next generation of trees (Chapter 5); tree water relations (Chapter 6); tree carbon relations (Chapter 7); tree nutrition (Chapter 8); interactions with other organisms (Chapter 9) and, environmental challenges for trees (Chapter 10).

Expert Box 1.1 The Environmental Benefits of Urban Trees Roland Ennos Trees are, of course, marvellous organisms – you would not be reading this book if you did not realise that – and there is no doubt that urban trees beautify surroundings that would otherwise look bare and soulless. However, many claims are also made about the environmental benefits of urban trees: that they reduce traffic noise; absorb pollution; take up and store carbon; provide shade and cooling; and help prevent flooding. These claims all seem plausible, but it is only recently that experimental investigations have tested these claims and started to quantify the environmental benefits of trees.

Noise Reduction You might expect trees to be as good at shielding noise as they are at visual screening. However, experiments have shown that trees are actually poor at reducing noise levels (Fang and Ling 2003). The structures of trees, even their trunks, are simply too narrow to affect sound waves – particularly the long waves that carry the deep hum of traffic noise – and sound simply goes right through them. However, trees do help reduce the nuisance of traffic noise in other ways. They shield us from seeing passing vehicles, so helping reduce our awareness of them, and they make their own, more restful rustling noise in the wind, further distracting us from the noise of traffic. There is even evidence that people drive more slowly in tree‐lined streets, lowering the noise their vehicles make.

Absorption of Pollution Trees reduce pollution, particularly the particulate pollutants produced by the engines of motor vehicles, by intercepting them with their leaves. Rainwater then washes the particles off on to the ground and down the drain. Modelling and experimental studies have suggested that this process could reduce the concentration of particulates by 5–20% in a typical city (McDonald et al. 2007). However, as trees are not adapted to absorb pollution, this reduction is not very great, and depends very much on wind‐speeds and the fine details of the airflow around the city. In some cases, trees can reduce wind‐speeds so much that they keep particulates trapped in urban streets, and increase pollution lev- els. Some species of tree – especially willows, poplars and oaks – emit volatile organic compounds (VOCs) from their leaves which react with nitrogen oxides from vehicle emis- sions to produce harmful ozone (Donovan et al. 2011). Trees’ anti‐pollution credentials are therefore fairly weak.

Carbon Storage and Sequestration There is no doubt that trees are important stores of carbon, and growing trees actively take up the greenhouse gas carbon dioxide. They use it to make sugars in the process of

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photosynthesis and lay down and store the carbon in the form of wood. A hectare of urban forest typically stores around 10–30 tonnes of carbon above‐ground, while the roots store a further 2–5 tonnes. A growing stand of trees will take up and sequester a further 0.5–0.9 tonnes per hectare per year (McPherson et al. 2013). Over a whole town, this builds up to a large quantity. Over Greater London, for instance, trees store around 2.4 million tonnes of carbon and take up a further 72 thousand tonnes every year (Rogers et al. 2015). However, these benefits need to be put into perspective. Some of the sequestered carbon is lost because of the death and removal of old and diseased trees, while a further percentage is counterbalanced by the carbon emitted from the power tools used for management operations. The amounts sequestered are also far smaller than the amounts of carbon dioxide emitted by vehicles and by the heating systems of buildings in the city, which exceed 10 million tonnes annually. It is really only the huge areas of forest in the countryside that make a real contribution to removing carbon from the atmosphere.

Shade and Cooling If some of their other environmental benefits seem disappointing, trees really do have major shading and cooling benefits. The leaves of trees are adapted to intercept and absorb light, so they do it extremely well. Moreover, to allow carbon dioxide to enter for photosynthesis, the leaves have to keep their stomata open during the day, and that allows large quantities of water to evaporate from them. This cools down the leaves and the air surrounding them, just as the evaporation of sweat from our bodies keeps us cool in hot weather. At a local level, the cooling benefits of trees are largely a result of the shade that they provide. Radiation from the sun is reduced by up to 90% under the canopy of a tree, and this shading cools the roads and pavements beneath (Figure EB1.1). Tarmac can reach temperatures of 50–60 °C in the sun on a hot summer’s day but in the shade of a tree it can be kept below 30 °C (Ennos et al. 2014). Both effects greatly improve the comfort of people, because how hot we feel depends far more on the radiation balance with our surroundings than on air temperature. So, although a single tree has a negligible effect on the air tempera- ture around it, a person in tree‐shade will take up far less short‐wave radiation from the sun and emit much more long‐wave radiation to the surroundings; all this means that we can actually feel 10–15 °C cooler. The shading effect is also important in helping make buildings more habitable and cheaper to run in hot summer weather. Tree Figure EB1.1 Infrared image showing shading, particularly by trees situated on the people resting in the cool shade provided by park trees. The red in the background western and eastern sides of buildings, can shows an area of tarmac, while the yellow reduce wall temperatures by up to 30 °C in shows grass in the sun. Source: Courtesy of sunny weather. Studies in the USA and Canada Roland Ennos.

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3000 May July

2000

1000 Energy loss (W/tree)

0 Crataegus Sorbus Prunus Pyrus Malus

Figure EB1.2 The relative cooling performance of five small street trees in Manchester, UK. Source: Ennos et al. (2014). Licensed Open Government Licence v3.0, http://www.nationalarchives. gov.uk/doc/open-government-licence/version/3/.

have shown that optimally positioned trees can reduce air conditioning costs by around 30% (Akbari et al. 1997). Conversely, the effect that trees can have on the wind – reducing wind speeds and turbulence – can help reduce heating costs, particularly at high lati- tudes such as Canada and Scandinavia. In northern Europe, air conditioning is rare, and here trees provide natural air conditioning; they keep our buildings cooler in summer, helping to improve thermal comfort and prevent the deaths seen in heatwaves such as those that occurred in the summer of 2003. Unfortunately, the effectiveness of trees in this role has not been quantified. On a larger scale – the entire city – the cooling effect of trees is actually brought about by the evaporation of water from their leaves, which cools the air around them. This is important because in periods of hot, calm, sunny weather, cities heat up far more than the surrounding countryside, an effect known as the urban heat island. City streets absorb a little more of the sun’s radiation, and the heat gets trapped within steep urban canyons. The result is that cities heat up more during the day, by up to 2 °C, and remain up to 7 °C warmer at night than the surrounding countryside. Experiments have shown that trees can reverse this effect to some extent by using one‐third to half of the energy hitting the leaves to evaporate water, rather than heat up the air (Ennos et al. 2014). However, the cooling effectiveness of trees is very variable; fast‐growing species with a dense canopy, such as Callery pears Pyrus calleryana, can be four times as effective as sparsely leafed cherry Prunus ‘Umineko’ (Figure EB1.2). Trees also need to be actively growing and transpiring to provide cooling, and hence need well‐watered and aerated soil. We showed, for instance, that Pyrus calleryana trees growing in compaction‐resistant ‘Amsterdam structural soil’ can produce five times the cooling of trees of the same species in conventional topsoil within a well‐used pavement (Figure EB1.3). The basic principle seems to be that the healthier and faster‐growing a tree is, the more cooling it will provide. It is relatively easy to determine how much cooling individual trees are providing – you just have to measure the amount of water they are losing. However, the overall effect of trees on the city temperature is harder to gauge because it is simply not possible to compare two cities that are identical apart from their vegetation. Many studies have sought to overcome this problem by comparing the temperature of city parks with their

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9000 Pavements Grass verges Amsterdam soil 7500

6000

4500

3000

Energy loss (W/tree) 1500

0 July August

Figure EB1.3 The relative cooling performance of Callery pears Pyrus calleryana growing in conventional soil pits within pavements; in grass verges; and in compaction‐resistant Amsterdam structural soil in Manchester, UK. Source: Ennos et al. (2014). Licensed Open Government Licence v3.0, http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/.

surrounding streets. Unfortunately, these measurements always give disappointing results: a park is typically only 1 °C cooler (Bowler et al. 2010). The problem is that winds blow cool air out of parks and warm air into them, so the cooling effect is spread over the city. To get better estimates of the cooling effects of trees, it would be more effective to incorporate their evaporative cooling into a large meteorological model of the city. Unfortunately, because of the complexity of cities, this is incredibly difficult and expensive to do.

Flood Prevention Urban trees also have important benefits in helping reduce flood risk. During rainstorms, the leaves of trees intercept some of the rain before it reaches the ground and, of the rainfall that does get through or that drips down from the canopy, much of it infiltrates into the soil. This reduces the run‐off – the rain that is diverted into the drains, and which could result in surface flooding – by around 50% (Wang et al. 2008). Trees perform better than areas of grass for two reasons: first, the canopy prevents 1–2 mm of rain from even reaching the soil; secondly, the tree roots dry out the soil and break it up, increasing the rate at which water can permeate into it by a factor of up to 60. City‐wide, the effects of trees are usually estimated to be relatively small, around 5–6%, because of the relatively low cover of trees, which is typically in the region of 15–20% of the urban area (Gill et al. 2007). However, some experiments performed in Manchester suggested that these effects might be underestimates; even water that falls outside the canopy of a tree can enter its planting hole and be diverted away from the drains (Ennos et al. 2014). There is also great potential to increase the anti‐flooding benefits of trees by growing them within sustainable urban drainage systems (SUDS) schemes. These schemes divert rainfall from buildings and roads into grassy swales and infiltration basins, and so are more effective at reducing flooding than simply adding areas of vegetation. Given the likely effects of cli- mate change, using trees within such schemes could have two main benefits: they would help irrigate trees automatically, helping them survive the long summer droughts that are forecast; and they would help absorb and drain the water diverted into the area, so reducing waterlogging during the increasingly larger storms that are predicted to occur.

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Uses and Limitations As we have seen therefore, the environmental benefits of trees are at last starting to be properly understood and quantified. We are even starting to be able to measure their benefits in monetary terms. The i‐Tree software developed in the USA uses the results of tree surveys to estimate the cash worth of the carbon storage, pollution removal and flood prevention benefits of trees. i‐Tree surveys certainly demonstrate the great benefits of trees. The London survey, for instance, came up with a figure of £132 m (Treeconomics 2015). However, this software leaves out many benefits and does not account for the great variability in the performance of trees, both within and between species. Finally, of course, trees also cause environmental problems. Fast‐growing species such as poplar and willow, which provide the most cooling and flood prevention, also emit the most volatile organic compounds; they are also more likely to cause building subsidence because they use more water, causing clay soils to dry out and shrink in the summer. Some limes (Tilia spp.) tend to be unpopular in cities because aphids suck sap from their leaves and secrete a sticky sap that drips on to cars. People can also object to trees reduc- ing light levels and dropping their leaves in autumn. The key to deploying trees as envi- ronmental engineers is to grow the right tree in the right place, maximising the benefits they supply, while keeping everyone happy.

References

Akbari, H., Kurn, D.M., Bretz, S.E. and Hanford, J.W. (1997) Peak power and cooling energy savings of shade trees. Energy and Buildings, 25: 139–148. Balvanera, P., Siddique, I., Dee, L., Paquette, A., Isbell, F., Gonzalez, A., et al. (2014) Linking biodiversity and ecosystem services: Current uncertainties and the necessary next steps. BioScience, 64, 49–57. Bowler, D.E., Buyung‐Ali, L., Knight, T.M. and Pullin, A.S. (2010) Urban greening to cool towns and cities: A systematic review of the empirical evidence. Landscape and Urban Planning, 97: 147–155. Bradshaw, C.J.A., Sodhi, N.S. and Brook, B.W. (2009) Tropical turmoil: A biodiversity tragedy in progress. Frontiers in Ecology and the Environment, 7, 79–87. Crowther, T.W., Glick, H.B., Covey, K.R., Bettigole, C., Maynard, D.S., Thomas, S.M., et al. (2015) Mapping tree density at a global scale. Nature, 525: 201–205. Donovan, G.H., Butry, D.T., Michael, Y.L., Prestemon, J.P., Liebhold, A.M., Gatziolis, D., et al. (2013) The relationship between trees and human health: Evidence from the spread of the emerald ash borer. American Journal of Preventive Medicine, 44: 139–145. Donovan, R., Owen, S., Hewitt, N., Mackenzie, R. and Brett, H. (2011)The Development of an Urban Tree Air Quality Score (UTAQS): Using the West Midlands, UK Conurbation as a Case Study Area. VDM Verlag Dr. Müller, Düsseldorf, Germany. Ennos, A.R., Armson, D. and Rahman, M.A. (2014) How useful are urban trees? The lessons of the Manchester Research Project. In: Johnston, M. and Percival, G. (eds). Trees, People and the Urban Environment II. Institute of Chartered Foresters, Edinburgh, UK, pp. 62–70. Fang, C.F. and Ling, D.L. (2003) Investigation of the noise reduction provided by tree belts Landscape and Urban Planning, 63: 187–195.

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Gill, S., Handley, J.F., Ennos, A.R. and Pauleit, S. (2007) Adapting cities for climate change: The role of the green infrastructure. Built Environment, 33: 97–115. Gladwell, V.F., Kuoppa, P., Tarvainen, M.P. and Rogerson, M. (2016) A lunchtime walk in nature enhances restoration of autonomic control during night‐time sleep: Results from a preliminary study. International Journal of Environmental Research and Public Health, 13: article 280. Gómez‐Muñoz, V.M., Porta‐Gándara, M.A. and Fernández, J.L. (2010) Effect of tree shades in urban planning in hot‐arid climatic regions. Landscape and Urban Planning, 94: 149–157. Gratani, L. and Varone, L. (2006) Carbon sequestration by Quercus ilex L. and Quercus pubescens Willd. and their contribution to decreasing air temperature in Rome. Urban Ecosystems, 9: 27–37. Ikei, H., Song, C. and Miyazaki, Y. (2015) Physiological effect of olfactory stimulation by Hinoki cypress (Chamaecyparis obtusa) leaf oil. Journal of Physiological Anthropology, 34: article 44. Kirby, J., Duplissy, J., Sengupta, K. et al. (2016) Ion‐induced nucleation of pure biogenic particles. Nature, 533: 521–526. Kondo, M.C., Han, S., Donovan, G.H. and MacDonald, J.M. (2017) The association between urban trees and crime: Evidence from the spread of the emerald ash borer in Cincinnati. Landscape and Urban Planning, 157: 193–199. Li, Q. (2010) Effect of forest bathing trips on human immune function. Environmental Health and Preventive Medicine, 15: 9–17. McDonald, A.G., Bealey, W.J., Fowler, D., Dragosits, U., Skiba, U., Smith, R.I., et al. (2007) Quantifying the effect of urban tree planting on concentrations and depositions of PM10 in two UK conurbations Atmospheric Environment, 41: 8455–8467. McPherson, E.G., Xiao, Q.F. and Aguaron, E. (2013) A new approach to quantify and map carbon stored, sequestered and emissions avoided by urban forests. Landscape and Urban Planning, 120: 70–84. Mullaney, J., Lucke, T. and Trueman, S.J. (2015) A review of benefits and challenges in growing street trees in paved urban environments. Landscape and Urban Planning, 134: 157–166. Nowak, D.J. and Greenfield, E.J. (2012) Tree and impervious cover change in US cities. Urban Forestry and Urban Greening, 11: 21–30. Nowak, D.J., Hoehn, R. and Crane, D.E. (2007) Oxygen production by urban trees in the United States. Arboriculture and Urban Forestry, 33: 220–226. Ochiai, H., Ikei, H., Song, C., Kobayashi, M., Miura, T., Kagawa, T., et al. (2015) Physiological and psychological effects of a forest therapy program on middle‐aged females. International Journal of Environmental Research and Public Health, 12: 15222–15232. Rogers, K., Sacre, K., Goodenough, J. and Doick, K. (2015) Valuing London’s Urban Forest: Results of the London i‐Tree Eco Project. Treeconomics, London, UK. Roy, S., Byrne, J. and Pickering, C. (2012) A systematic quantitative review of urban tree benefits, costs, and assessment methods across cities in different climatic zones. Urban Forestry and Urban Greening, 11: 351–363. Sanusi, R., Johnstone, D., May, P. and Livesley, S.J. (2017) Microclimate benefits that different street tree species provide to sidewalk pedestrians relate to differences in Plant Area Index. Landscape and Urban Planning, 157: 502–511.

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0003304933.INDD 13 10/06/2017 4:40:03 PM CHAPTER 1 The Impact of Biotechnology on Plant Agriculture

GRAHAM BROOKES PG Economics Ltd, Frampton, Dorchester, UK

1.0. CHAPTER SUMMARY AND OBJECTIVES

1.0.1. Summary Since the first stably transgenic plant produced in the early 1980s and the first commercialized transgenic plant in 1994, biotechnology has revolutionized plant agriculture. In the United States, between 80 and 90% of the maize (corn), soybean, cotton, and canola crops are transgenic for insect resistance, herbicide resistance, or both. Biotechnology has been the most rapidly adopted technology in the history of agriculture and continues to expand in much of the developed and developing world.

1.0.2. Discussion Questions 1. What biotechnology crops are grown and where? 2. Why do farmers use biotech crops? 3. How has the adoption of plant biotechnology impacted the environment?

1.1. INTRODUCTION The technology of geneticCOPYRIGHTED modification (GM, also standsMATERIAL for “genetically modified”), which consists of genetic engineering and also known as genetic transformation, has now been utilized globally on a widespread commercial basis for 18 years; and by 2012, 17.3 million farmers in 28 countries had planted 160 million hectares of crops using this technology. These milestones provide an opportunity to critically assess the impact of this technology on global agriculture. This chapter therefore examines specific global socioeconomic impacts on farm income and environmental impacts with respect to pesticide usage and greenhouse gas (GHG) emissions of the technology. Further details can be found in Brookes and Barfoot (2014a, b).

Plant Biotechnology and Genetics: Principles, Techniques, and Applications, Second Edition. Edited by C. Neal Stewart, Jr. © 2016 John Wiley & Sons, Inc. Published 2016 by John Wiley & Sons, Inc.

1

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1.2. CULTIVATION OF BIOTECHNOLOGY (GM) CROPS

Although the first commercial GM crops were planted in 1994 (tomatoes), 1996 was the first year in which a significant area of crops containing GM traits were planted (1.66 million hectares). Since then, there has been a dramatic increase in plantings, and by 2012 the global planted area reached over 160.4 million hectares. Almost all of the global GM crop area derives from soybean, maize (corn), cotton, and canola (Fig. 1.1). In 2012, GM soybean accounted for the largest share (49%) of total GM crop cultivation, followed by maize (32%), cotton (14%), and canola (5%). In terms of the share of total global plantings to these four crops accounted for by GM crops, GM traits accounted for a majority of soybean grown (73%) in 2012 (i.e., non‐GM soybean accounted for 27% of global soybean acreage in 2012). For the other three main crops, the GM shares in 2012 of total crop production were 29% for maize, 59% for cotton, and 26% for canola (i.e., the majority of global plantings of maize and canola continued to be non‐GM in 2012). The trend in plantings of GM crops (by crop) from 1996 to 2012 is shown in Figure 1.2. In terms of the type of biotechnology trait planted, Figure 1.3 shows that GM herbicide‐tolerant soybeans dominate, accounting for 38% of the total, followed by insect‐ resistant (largely Bt) maize, herbicide‐tolerant maize, and insect‐resistant cotton with respective shares of 26, 19, and 11%. It is worth noting that the total number of plantings by trait produces a higher global planted area (209.2 million hectares) than the global area by crop (160.4 million hectares) because of the planting of some crops containing the stacked traits of herbicide tolerance and insect resistance (e.g., a single plant with two biotech traits). In total, GM herbicide‐tolerant (GM HT) crops account for 63%, and GM insect‐resistant (GM IR) crops account for 37% of global plantings. Finally, looking at where biotech crops have been grown, the United States had the largest share of global GM crop plantings in 2012

c

c

c

Figure 1.1. Global GM crop plantings in 2012 by crop (base area: 160.4 million hectare). (Sources: ISAAA, Canola Council of Canada, CropLife Canada, USDA, CSIRO, ArgenBio.)

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1 1 1 1 2 21 22222222221211212 Figure 1.2. Global GM crop plantings by crop 1996–2012. (Sources: ISAAA, Canola Council of Canada, CropLife Canada, USDA, CSIRO, ArgenBio.)

H H .2 . B 1.

H .

H 1.

H 2.1

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Figure 1.3. Global GM crop plantings by main trait and crop: 2012. (Sources: Various, including ISAAA, Canola Council of Canada, CropLife Canada, USDA, CSIRO, ArgenBio.)

(40%: 64.1 million hectares), followed by Brazil (37.2 million hectares: 23% of the global total) and Argentina (14%: 23.1 million hectares). The other main countries planting GM crops in 2012 were India, Canada, and China (Fig. 1.4). In 2012, there were also additional GM crop plantings of papaya (395 hectares), squash (2000 hectares), alfalfa (425,000 hectares), and sugar

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I O

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Figure 1.4. Global GM crop plantings 2012 by country. (Sources: ISAAA, Canola Council of Canada, CropLife Canada, USDA, CSIRO, ArgenBio.)

beet (490,000 hectares) in the United States, of papaya (5000 hectares) in China and of sugar beet (13,500 hectares) in Canada.

1.3. WHY FARMERS USE BIOTECH CROPS

The primary driver of adoption among farmers (both large commercial and small‐scale subsistence) has been the positive impact on farm income. The adoption of biotechnology has had a very positive impact on farm income derived mainly from a combination of enhanced productivity and efficiency gains (Table 1.1). In 2012, the direct global farm income benefit from GM crops was $18.8 billion. This is equivalent to having added 5.6% to the value of global production of the four main crops of soybean, maize, canola, and cotton, a substantial impact. Since 1996, worldwide farm incomes have increased by $116.6 billion, directly because of the adoption of GM crop technology. The largest gains in farm income in 2012 have arisen in the maize sector, largely from yield gains. The $6.7 billion additional income generated by GM IR maize in 2012 has been equivalent to adding 6.6% to the value of the crop in the GM crop‐growing countries, or adding the equivalent of 3% to the $226 billion value of the global maize crop in 2012. Cumulatively since 1996, GM IR tech­ nology has added $32.3 billion to the income of global maize farmers. Substantial gains have also arisen in the cotton sector through a combination of higher yields and lower costs. In 2012, cotton farm income levels in the GM‐adopting countries increased by

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Table 1.1. Global Farm Income benefits from Growing GM Crops 1996–2012 (Million US $) Farm income benefit in Farm income benefit 2012 as percentage of in 2012 as Increase in Increase in total value of production percentage of total farm income farm income of these crops in GM value of global Trait 2012 1996–2012 adopting countries production of crop GM herbicide‐tolerant 4,797.9 37,008.6 4.4 4.0 soybeans GM herbicide‐tolerant 1,197.9 5,414.7 1.2 0.5 maize GM herbicide‐tolerant 147.2 1,371.6 0.4 0.3 cotton GM herbicide‐tolerant 481.0 3,664.4 4.9 1.3 canola GM insect‐resistant 6,727.8 32,317.2 6.6 3.0 maize GM insect‐resistant 5,331.3 36,317.2 13.1 11.2 cotton Others 86.3 496.7 N/A N/A Total 18,769.4 116,590.4 6.8 5.6

Notes: All values are nominal. Others = Virus resistant papaya and squash and herbicide‐tolerant sugar beet. Totals for the value shares exclude “other crops” (i.e., relate to the four main crops of soybeans, maize, canola, and cotton). Farm income calculations are net farm income changes after inclusion of impacts on yield, crop quality, and key variable costs of production (e.g., payment of seed premia, impact on crop protection expenditure). N/A = not applicable.

$5.5 billion; and since 1996, the sector has benefited from an additional $37.7 billion. The 2012 income gains are equivalent to adding 13.5% to the value of the cotton crop in these countries, or 11.5% to the $47 billion value of total global cotton production. This is a substantial increase in value‐added terms for two new cotton seed technologies. Significant increases to farm incomes have also resulted in the soybean and canola sectors. The GM HT technology in soybeans has boosted farm incomes by $4.8 billion in 2012, and since 1996 has delivered over $37 billion of extra farm income. In the canola sector (largely North American) an additional $3.66 billion has been generated (1996–2012). Overall, the economic gains derived from planting GM crops have been of two main types: (a) increased yields (associated mostly with GM IR technology) and (b) reduced costs of production derived from less expenditure on crop protection (insecticides and herbicides) products and fuel. Table 1.2 summarizes farm income impacts in key GM‐adopting countries highlighting the important farm income benefit arising from GM HT soybeans in South America (Argentina, Bolivia, Brazil, Paraguay, and Uruguay), GM IR cotton in China and India, and a range of GM cultivars in the United States. It also illustrates the growing level of farm income benefits being obtained in South Africa, the Philippines, Mexico, and Colombia from planting GM crops. In terms of the division of the economic benefits, it is interesting to note that farmers in devel­ oping countries derived in 2012 (46.2%) relative to farmers in developed countries (Table 1.3). The vast majority of these income gains for developing country farmers have been from GM IR cotton and GM HT soybean.1

1 The author acknowledges that the classification of different countries into “developing” or “developed” status affects the distribution of benefits between these two categories of country. The definition used here is consistent with the definition used by others, including the International Service for the Acquisition of Agri‐Biotech Applications (ISAAA) (see the review by James (2012)].

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Table 1.2. GM Crop Farm Income benefits During 1996–2012 in Selected Countries (Million US $) GM HT GM HT GM HT GM HT GM IR GM IR soybeans maize cotton canola maize cotton Total United States 16,668.7 3752.3 975.8 268.3 26,375.9 4,046.7 52,087.7 Argentina 13,738.5 766.7 107.0 N/A 495.2 456.4 15,563.8 Brazil 4,825.6 703.4 92.5 N/A 2,761.7 13.3 8,396.5 Paraguay 828 N/A N/A N/A N/A N/A 828.0 Canada 358 81.3 N/A 3368.8 1,042.9 N/A 4,851.0 South Africa 9.1 4.1 3.2 N/A 1,100.6 34.2 1,151.2 China N/A N/A N/A N/A N/A 15,270.4 15,270.4 India N/A N/A N/A N/A N/A 14,557.1 14,557.1 Australia N/A N/A 78.6 27.3 N/A 659.6 765.5 Mexico 5.0 N/A 96.4 N/A N/A 136.6 238.0 Philippines N/A 104.7 N/A N/A 273.6 N/A 378.3 Romania 44.6 N/A N/A N/A N/A N/A 44.6 Uruguay 103.8 N/A N/A N/A 17.6 N/A 121.4 Spain N/A N/A N/A N/A 176.3 N/A 176.3 Other EU N/A N/A N/A N/A 18.8 N/A 18.8 Colombia N/A 1.7 18.1 N/A 47.4 15.4 826.6 Bolivia 432.2 N/A N/A N/A N/A N/A 432.2 Burma N/A N/A N/A N/A N/A 215.4 215.4 Pakistan N/A N/A N/A N/A N/A 725.1 725.1 Burkina Faso N/A N/A N/A N/A N/A 186.9 186.9 Honduras N/A N/A N/A N/A 6.9 N/A 6.9

Notes: All values are nominal. Farm income calculations are net farm income changes after inclusion of impacts on yield, crop quality, and key variable costs of production (e.g., payment of seed premia, impact on crop protection expenditure). N/A = not applicable. US total figure also includes $491 million for other crops/traits (not included in the table). Also not included in the table is $5.5 million extra farm income from GM HT sugar beet in Canada.

Table 1.3. GM Crop Farm Income benefits, 2012: Developing Versus Developed Countries (Million US $) Developed Developing GM HT soybeans 2,955.4 1842.5 GM HT maize 654.0 543.9 GM HT cotton 71.4 75.8 GM HT canola 481.0 0 GM IR maize 5,327.5 1400.3 GM IR cotton 530.7 4800.7 GM virus‐resistant papaya and squash and GM HT sugar beet 86.3 0 Total 10,106.3 8663.2

Note: Developing countries = All countries in South America, Mexico, Honduras, Burkina Faso, India, China, the Philippines, and South Africa.

Examination of the cost farmers pay for accessing GM technology relative to the total gains derived shows that across the four main GM crops, the total cost was equal to about 23% of the total farm income gains (Table 1.4). For farmers in developing countries, the total cost is equal to about 21% of total farm income gains, while for farmers in developed countries the cost is about 25% of the total farm income gain. Although circumstances vary between countries, the higher share of total technology gains accounted for by farm income gains in developing countries, relative to the farm income share in developed countries, reflects factors such as weaker provision and enforcement of intellectual property rights in developing countries and the higher average

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Table 1.4. Cost of accessing GM Technology Relative to Total Farm Income benefits (US Millions) 2012 Total benefit of Farm Total benefit of Cost of Farm income technology to income technology to technology: gain: farmers and seed Tech costs: gain: all farmers and seed developing developing supply chain: all farmers farmers supply chain countries countries developing countries GM HT soy 1528.1 4,797.9 6,326.0 998.7 1842.5 2,841.2 GM HT maize 1059.4 1,197.9 2,257.3 364.5 543.9 908.4 GM HT cotton 295.0 147.2 442.2 22.2 75.8 98.0 GM HT canola 161.2 481.0 642.2 N/A N/A N/A GM IR maize 1800.8 6,727.8 8,528.6 512.3 1400.3 1,912.6 GM IR cotton 720.7 5,331.3 6,052.0 422.7 4800.7 5,223.4 Others 76.2 86.3 162.5 N/A N/A N/A Total 5641.4 18,769.4 24,410.8 2320.4 8663.2 10,983.6

N/A = not applicable. Cost of accessing technology based on the seed premiums paid by farmers for using GM technology relative to its conventional equivalents.

level of farm income gain on a per‐hectare basis derived by developing country farmers relative to developed country farmers. In addition to the tangible and quantifiable impacts on farm profitability presented earlier, there are other important, more intangible (difficult to quantify) impacts of an economic nature. Many studies on the impact of GM crops have identified the factors listed later in the text as being impor­ tant influences for the adoption of the technology.

1.4. GM’S EFFECTS ON CROP PRODUCTION AND FARMING

Based on the yield impacts used in the direct farm income benefit calculations discussed earlier and taking account of the second soybean crop facilitation in South America, GM crops have added important volumes to global production of maize, cotton, canola, and soybeans since 1996 (Table 1.5). The GM IR traits, used in maize and cotton, have accounted for 97.1% of the additional maize production and 99.3% of the additional cotton production. Positive yield impacts from the use of this technology have occurred in all user countries (except for GM IR cotton in Australia2) when compared to average yields derived from crops using conventional technology (i.e., application of insecticides and seed treatments). The average yield impact across the total area planted to these traits over the 17 years since 1996 has been +10.4% for maize and +16.1% for cotton. As indicated earlier, the primary impact of GM HT technology has been to provide more cost‐ effective (less‐expensive) and easier weed control, as opposed to improving yields. The improved weed control has, nevertheless, delivered higher yields in some countries. The main source of additional production from this technology has been via the facilitation of no‐tillage production system, shortening the production cycle and how it has enabled many farmers in South America to plant a crop of soybeans immediately after a wheat crop in the same growing season. This second crop, additional to traditional soybean production, has added 114.3 million tonnes to soybean production in Argentina and Paraguay between 1996 and 2012 (accounting for 93.5% of the total GM‐related additional soybean production).

2 This reflects the levels of Heliothis/Helicoverpa (boll and bud worm pests) pest control previously obtained with intensive insecticide use. The main benefit and reason for adoption of this technology in Australia has arisen from significant cost savings (on insecticides) and the associated environmental gains from reduced insecticide use.

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Table 1.5. additional Crop Production arising from Positive Yield effects of GM Crops 1996–2012 additional 2012 additional production production (million tonnes) (million tonnes) Soybeans 122.3 12.0 Maize 231.4 34.1 Cotton 18.2 2.4 Canola 6.6 0.4 Sugar beet 0.6 0.15

Note: GM HT sugar beet has been commercialized only in the United States and Canada since 2008.

1.5. HOW THE ADOPTION OF PLANT BIOTECHNOLOGY HAS IMPACTED THE ENVIRONMENT

Two key aspects of environmental impact of biotech crops examined later are decreased insecticide and herbicide use, and the impact on carbon emissions and soil conservation.

1.5.1. Environmental Impacts from Changes in Insecticide and Herbicide Use Usually, changes in pesticide use with GM crops have traditionally been presented in terms of the volume (quantity) of pesticide applied. While comparisons of total pesticide volume used in GM and non‐GM crop production systems can be a useful indicator of environmental impacts, it is an imperfect measure because it does not account for differences in the specific pest control programs used in GM and non‐GM cropping systems. For example, different specific chemical products used in GM versus conventional crop systems, differences in the rate of pesticides used for efficacy, and differences in the environmental characteristics (mobility, persistence, etc.) are masked in general comparisons of total pesticide volumes used. To provide a more robust measurement of the environmental impact of GM crops, the analysis presented in the following text includes an assessment of both pesticide active‐ingredient use and the specific pesticides used via an indicator known as the environmental impact quotient (EIQ). This universal indicator, developed by Kovach et al. (1992) and updated annually, effec­ tively integrates the various environmental impacts of individual pesticides into a single field value per hectare. This index provides a more balanced assessment of the impact of GM crops on the environment as it draws on all of the key toxicity and environmental exposure data related to individual products, as applicable to impacts on farmworkers, consumers, and ecology, and provides a consistent and comprehensive measure of environmental impact. Readers should, however, note that the EIQ is an indicator only and, therefore, does not account for all environ­ mental issues and impacts. The EIQ value is multiplied by the amount of pesticide active ingredient (AI) used per hectare to produce a field EIQ value. For example, the EIQ rating for glyphosate is 15.3. By using this rating multiplied by the amount of glyphosate used per hectare (e.g., a hypothetical example of 1.1 kg applied per hectare), the field EIQ value for glyphosate would be equivalent to 16.83/hectare. In comparison, the field EIQ/hectare value for a commonly used herbicide on corn crops (atrazine) is 22.9/hectare. The EIQ indicator is therefore used for comparison of the field EIQ/hectare values for conven­ tional versus GM crop production systems, with the total environmental impact or load of each system, a direct function of respective field EIQ/hectare values, and the area planted to each type of production (GM vs. non‐GM). The EIQ methodology is used in the following to calculate and compare typical EIQ values for conventional and GM crops and then aggregate these values to a national level. The level of pesticide

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use in the respective areas planted for conventional and GM crops in each year was compared with the level of pesticide use that probably would otherwise have occurred if the whole crop, in each year, had been produced using conventional technology (based on the knowledge of crop advisers). This approach addresses gaps in the availability of herbicide or insecticide usage data in most countries and differentiates between GM and conventional crops. Additionally, it allows for compar­ isons between GM and non‐GM cropping systems when GM accounts for a large proportion of the total crop planted area. For example, in the case of soybean in several countries, GM represents over 60% of the total soybean crop planted area. It is not reasonable to compare the production practices of these two groups as the remaining non‐GM adopters might be farmers in a region characterized by below‐average weed or pest pressures or with a tradition of less intensive production systems, and hence, below‐average pesticide use. GM crops have contributed to a significant reduction in the global environmental impact of production agriculture (Table 1.6). Since 1996, the use of pesticides was reduced by 503 million kg of AI, constituting an 8.8% reduction, and the overall environmental impact asso­ ciated with pesticide use on these crops was reduced by 18.7%. In absolute terms, the largest environmental gain has been associated with the adoption of GM IR technology. GM IR cotton has contributed a 25.6% reduction in the volume of AI used and a 28.2% reduction in the EIQ indicator (1996–2012) due to the significant reduction in insecticide use that the technology has facilitated, in what has traditionally been an intensive user of insecticides. Similarly, the use of GM IR technology in maize has led to important reductions in insecticide use, with associated environmental benefits. The volume of herbicides used in GM maize crops also decreased by 203 million kg (1996–2012), a 9.8% reduction, whilst the overall environmental impact associated with herbicide use on these crops decreased by a significantly larger 13.3%. This highlights the switch in herbicides used with most GM HT crops to AIs with a more environmentally benign profile than the ones generally used on conventional crops.

Table 1.6. Impact of Changes in the Use of Herbicides and Insecticides from Global Cultivation of GM Crops, Including environmental Impact Quotient (eIQ), 1996–2012 Percentage change Change in Change in field in environmental mass of active EIQ (in terms Percentage impact associated ingredient of million field change in AI with herbicide and Area GM trait used EIQ/ use on GM insecticide use on 2012 Trait (million kg) hectare units) crops GM crops (million hectare) GM herbicide‐tolerant −4.7 −6,654 −0.2 −15.0 79.1 soybeans GM herbicide‐tolerant −203.2 −6,025 −9.8 −13.3 38.5 maize GM herbicide‐tolerant −15.0 −509 −16.7 −26.6 8.6 canola GM herbicide‐tolerant −18.3 −460 −6.6 −9.0 4.4 cotton GM insect‐resistant −57.6 −2,215 −47.9 −45.1 42.3 maize GM insect‐resistant −205.4 −9,256 −25.6 −28.2 22.1 cotton GM herbicide‐tolerant +1.3 −2 +29.3 −2.0 0.51 sugar beet Totals −503.1 −25,121 −8.8 −18.7

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Table 1.7. Changes in environmental Impact Quotient (eIQ) form GM Crops and associated Changes in associated Insecticide and Herbicide Use in 2012: Developing versus Developed Countries Change in field EIQ (in terms of Change in field EIQ (in terms of million field EIQ/hectare units): million field EIQ/hectare units): developed countries developing countries GM HT soybeans −4,773.9 −1,880.2 GM HT maize −5,585.9 −438.8 GM HT cotton −351.0 −109.3 GM HT canola −509.1 0 GM IR maize −1,574.4 −640.8 GM IR cotton −805.5 −8,451.0 GM HT sugar beet −2 0 Total −13,601.8 −11,520.1

Important environmental gains have also arisen in the soybean and canola sectors. In the soybean sector, herbicide use decreased by 4.7 million kg (1996–2012) and the associated environmental impact of herbicide use on this crop area decreased, from a switch to more environmentally benign herbicides (−15%). In the canola sector, farmers reduced herbicide use by 15 million kg (a 16.7% reduction) and the associated environmental impact of herbicide use on this crop area fell by 26.6% (from switching to more environmentally benign herbicides). In terms of the division of the environmental benefits associated with less insecticide and herbi­ cide use for farmers in developed countries relative to farmers in developing countries, Table 1.7 shows a 54 : 46% split of the environmental benefits (1996–2012), respectively, in developed (54%) and developing countries (46%). About three‐quarters (73%) of the environmental gains in developing countries have been from the use of GM IR cotton. It should, however, be noted that in some regions where GM HT crops have been widely grown, some farmers have relied too much on the use of single herbicides, such as glypho­ sate, to manage weeds in GM HT crops and this has contributed to the evolution and spread of weed resistance. There are currently 31 weed species recognized as exhibiting resistance to glyphosate worldwide, of which several are not associated with glyphosate‐tolerant crops (www.weedscience.org). For example, there are currently 14 weeds recognized in the United States as exhibiting resistance to glyphosate, of which two are not associated with glyphosate tolerant crops. In the United States, the affected area is currently within a range of 15–40% of the total area annually devoted to maize, cotton, canola, soybeans, and sugar beet (the crops in which GM HT technology is used). In recent years, there has also been a growing consensus among weed scientists of a need for changes in the weed management programs in GM HT crops, because of the apparent increase of evolution glyphosate‐resistant weeds. Growers of GM HT crops are increasingly being advised to be more proactive and include other herbicides (with different and complementary modes of action) in combination with glyphosate in their integrated weed management systems, even where instances of weed resistance to glyphosate have not been found. This proactive, diversified approach to weed management is the principal strategy for avoiding the emergence of HR weeds in GM HT crops. It is also the main way of tackling weed resistance in conventional crops. A proactive weed management program also generally requires using less herbicide, has a better environmental profile, and is more economical than a reactive weed management program. At the macrolevel, the adoption of both reactive and proactive weed management programs in GM HT crops has already begun to influence the mix, total amount and overall environmental pro­ file of herbicides applied to GM HT soybeans, cotton, maize, and canola, and this is reflected in the data presented in this chapter.

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1.5.2. Impact on GHG Emissions The reduction in the levels of GHG emissions from GM crops are from the following two principal sources:

1. GM crops contribute to a reduction in fuel use from less frequent herbicide or insecticide applications and a reduction in the energy use in soil cultivation. For example, Lazarus (2012) estimated that one pesticide spray application uses 0.84 l of fuel per hectare, which is equivalent to 2.24 kg/hectare of carbon dioxide emissions. In this analysis, we used the conservative assumption that only GM IR crops reduced spray applications and ultimately GHG emissions. In addition to the reduction in the number of herbicide applications, there has been a shift from conventional tillage to no‐/reduced tillage (NT) and herbicide‐ based weed control systems, which has had a marked effect on tractor fuel consumption. The GM HT crop where this is most evident is GM HT soybean and where the GM HT soybean and maize rotation is widely practiced, for example in the United States. Here, adoption of the technology has made an important contribution to facilitating the adoption of NT farming (CTIC 2002, American Soybean Association 2001). Before the introduction of GM HT soybean cultivars, NT systems were practiced by some farmers using a number of herbicides and with varying degrees of success. The opportunity for growers to con­ trol weeds with a nonresidual foliar herbicide as a “burndown” preseeding treatment, followed by a postemergent treatment when the soybean crop became established, has made the NT system more reliable, technically viable, and commercially attractive. These technical advantages, combined with the cost advantages, have contributed to the rapid adoption of GM HT cultivars and the near‐doubling of the NT soybean area in the United States (and also a ≥sevenfold increase in Argentina). In both countries, GM HT soybean crops are estimated to account for 95% of the NT soybean crop area. Substantial growth in NT production systems has also occurred in Canada, where the NT canola area increased from 0.8 to 8 million hectares a (equal to about 90% of the total canola area) between 1996 and 2012 (95% of the NT canola area is planted with GM HT cultivars). The area planted to NT in the US cotton crop increased from 0.2 to 1 million hectare 1996–2005 (86% of which is planted to GM HT cultivars), although the NT cotton area has not risen above about 25% of the total crop. The fuel savings used in this chapter are drawn from a review of literature including Jasa (2002), CTIC (2002), University of Illinois (2006), USDA Energy Estimator (USDA 2013b), Reeder (2010), and the USDA Comet‐VR model (USDA 2013a). It is assumed that the adoption of NT farming systems in soybean production reduces cultivation and seedbed preparation fuel usage by 27.12 l/hectare compared with traditional conventional tillage and in the case of RT (mulch till) cultivation by 10.39 l/ hectare. In the case of maize, NT results in a saving of 24.41 l/hectare and 7.52 l/hectare in the case of RT compared with conventional intensive tillage. These are conservative esti­ mates and are in line with the USDA Energy Estimator for soybeans and maize. The adoption of NT and RT systems in respect of fuel use therefore results in reductions of carbon dioxide emissions of 72.41 kg/hectare and 27.74 kg/hectare respectively for soybeans and 65.17 kg/hectare and 20.08 kg/hectare for maize. 2. The use of NT3 farming systems that utilize less plowing increases the amount of organic carbon in the form of crop residue that is stored or sequestered in the soil. This carbon

3 NT farming means that the ground is not plowed at all, while reduced tillage means that the ground is disturbed less than it would be with traditional tillage systems. For example, under an NT farming system, soybean seeds are planted through the organic material that is left over from a previous crop such as corn, cotton, or wheat. NT systems also significantly reduce soil erosion, and hence deliver both additional economic benefits to farmers, enabling them to cultivate land that might otherwise be of limited value and environmental benefits from the avoidance of loss of flora, fauna, and landscape features.

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sequestration reduces carbon dioxide emissions to the environment. Rates of carbon seques­ tration have been calculated for cropping systems using normal tillage and reduced tillage, and these were incorporated in our analysis on how GM crop adoption has significantly

facilitated the increase in carbon sequestration, ultimately reducing the release of CO2 into the atmosphere. Of course, the amount of carbon sequestered varies by soil type, cropping system, and ecoregion.

Drawing on the literature and models referred to earlier, the analysis presented in the following text has several assumptions by country and crop. For the United States, the soil carbon sequestered by tillage system for maize in continuous rotation with soybeans is assumed to be a net sink of 250 kg of carbon/hectare/year based on NT systems store 251 kg of carbon/hectare/year, RT systems store 75 kg of carbon/hectare/, and CT systems store 1 kg of carbon/hectare/year. For the United States, the soil carbon sequestered by tillage system for soybeans in a continuous rotation with maize is assumed to be a net sink of 100 kg of carbon/hectare/year based on NT systems release 45 kg of carbon/hectare/year, RT systems release 115 kg of carbon/hectare/year, and CT systems release 145 kg of carbon/hectare/year. For Argentina and Brazil, soil carbon retention is 275 kg carbon/hectare/year for NT soybean cropping and CT systems release 25 kg carbon/hectare/year (a difference of 300 kg carbon/hectare/ year). Table 1.8 summarizes the impact on GHG emissions associated with the planting of GM crops

between 1996 and 2012. In 2012, the permanent CO2 savings from reduced fuel use associated with GM crops was 2111 million kg. This is equivalent to removing 900,000 cars from the road for a year.

Table 1.8. Impact of GM Crops on Carbon Sequestration Impact in 2012; Car equivalents Permanent carbon Permanent fuel Potential additional Soil carbon sequestration dioxide savings savings: as average soil carbon savings: as average arising from reduced family car equivalents sequestration family car equivalents Crop/trait/ fuel use (million kg removed from the savings (million kg removed from the road country of carbon dioxide) road for a year (‘000s) of carbon dioxide) for a year (‘000s) US: GM HT 210 93 1,070 475 soybeans Argentina: GM 736 327 11,186 4,972 HT soybeans Brazil GM HT 394 175 5,985 2,660 soybeans Bolivia, Paraguay, 156 69 2,365 1,051 Uruguay: GM HT soybeans Canada: GM HT 203 90 1,024 455 canola US: GM HT corn 210 93 2,983 1,326 Global GM IR 45 20 0 0 cotton Brazil IR corn 157 69 0 0 Total 2,111 936 24,613 10,939

Notes: Assumption: an average family car produces 150 g of carbon dioxide per km. A car does an average of 15,000 km/year and therefore produces 2,250 kg of carbon dioxide/year.

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The additional soil carbon sequestration gains resulting from reduced tillage with GM crops

accounted for a reduction of 24,613 million kg of CO2 emissions in 2012. This is equivalent to removing nearly 10.9 million cars from the roads per year. In total, the carbon savings from reduced fuel use and soil carbon sequestration in 2012 were equal to removing 11.88 million cars from the road (equal to 41% of all registered cars in the United Kingdom).

1.6. CONCLUSIONS

Crop biotechnology has, to date, delivered several specific agronomic traits that have overcome a number of production constraints for many farmers. This has resulted in improved productivity and profitability for the 17.3 million adopting farmers who have applied the technology to 160 million hectares in 2012. During the past 17 years, this technology has made important positive socioeconomic and environmental contributions. These have arisen even though only a limited range of GM agronomic traits have so far been commercialized, in a small range of crops. Crop biotechnology has delivered economic and environmental gains through a combination of their inherent technical advances and the role of the technology in the facilitation and evolu­ tion of more cost effective and environment‐friendly farming practices. More specifically the following: The gains from the GM IR traits have mostly been delivered directly from the technology (yield improvements, reduced production risk and decreased use of insecticides). Thus, farmers (mostly in developing countries) have been able to both improve their productivity and economic returns, whilst also practicing more environment‐friendly farming methods; The gains from GM HT traits have come from a combination of direct benefits (mostly cost reductions to the farmer) and the facilitation of changes in farming systems. Thus, GM HT tech­ nology (especially in soybeans) has played an important role in enabling farmers to capitalize on the availability of a low cost, broad‐spectrum herbicide (glyphosate) and, in turn, facilitated the move away from conventional to low‐/no‐tillage production systems in both North and South America. This change in production system has made additional positive economic con­ tributions to farmers (and the wider economy) and delivered important environmental benefits, notably reduced levels of GHG emissions (from reduced tractor fuel use and additional soil carbon sequestration). Both IR and HT traits have made important contributions to increasing world production levels of soybeans, corn, cotton, and canola. In relation to GM HT crops, however, overreliance on the use of glyphosate by some farmers, in some regions, has contributed to the evolution and spread of HR weeds. As a result, farmers are increasingly adopting a mix of reactive and proactive weed management strategies incorporating a mix of herbicides. Despite this, the overall environmental and economic gain from the use of GM crops has been, and continues to be, substantial. Overall, there is a considerable body of evidence, in the peer‐reviewed literature, and sum­ marized in this chapter, that quantifies the positive economic and environmental impacts of crop biotechnology. The analysis in this chapter therefore provides insights into the reasons why so many farmers around the world have adopted and continue to use the technology. Readers are encouraged to read the peer‐reviewed papers cited, and the many others who have published on this subject (and listed in the references section of the two main papers from Brookes and Barfoot that provided the background information for this chapter) and to draw their own conclusions.

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LIFE BOX 1.1. NORMAN E. BORLAUG Norman E. Borlaug (1914–2009) Nobel Laureate, Nobel Peace Prize, 1970; Recipient of the Congressional Gold Medal, 2007.

Dr. Borlaug initiated three innovations that greatly increased Mexico’s wheat yields. First, he and his Mexican technicians crossed thou­ sands of varieties to find a select few that were resistant to rust disease. Next, he carried out a “shuttle breeding” program to cut in half the time it took to do the breeding work. He harvested seed from a summer crop that was grown in the high altitudes near Mexico City, flew to Obregon to plant the seed for a winter crop at sea level. Seed from that crop was flown back to near Mexico City and planted Norman borlaug. Courtesy of Norman Borlaug. for a summer crop. Shuttle breeding not only worked against the advice of fellow scientists, The following text is excerpted from the but serendipitously the varieties were widely book by biographer Leon Hesser, The Man adapted globally because it had been grown at Who Fed the World: Nobel Peace Prize different altitudes and latitudes and during Laureate Norman Borlaug and His Battle to different day lengths. End World Hunger, Durban House Dallas, Texas (2006): But, there was a problem. With high levels of fertilizer in an attempt to increase yields, From the day he was born in 1914, Norman the plants grew tall and lodged. For his third Borlaug has been an enigma. How could a innovation, then, Borlaug crossed his rust‐ child of the Iowa prairie, who attended a resistant varieties with a short‐strawed, one‐teacher, one‐room school; who flunked heavy tillering Japanese variety. Serendipity the university entrance exam; and whose squared. The resulting seeds were respon­ highest ambition was to be a high school sci­ sive to heavy applications of fertilizer ence teacher and athletic coach, ultimately without lodging. Yields were six to eight achieve the distinction as one of the hundred times higher than for traditional varieties in most influential persons of the twentieth Mexico. It was these varieties, introduced in century? And receive the Nobel Peace Prize India and Pakistan in the mid‐1960s, which for averting hunger and famine? And could stimulated the Green Revolution that took he eventually be hailed as the man who those countries from near‐starvation to self‐ saved hundreds of millions of lives from sufficiency. For this remarkable achieve­ starvation—more than any other person in ment, Dr. Borlaug was awarded the Nobel history? Peace Prize in 1970. Borlaug, ultimately admitted to the University In 1986, Borlaug established the World Food of Minnesota, met Margaret Gibson, his wife Prize, which provides $250,000 each year to to be, and earned B.S., M.S., and Ph.D. recognize individuals in the world who are degrees. The latter two degrees were in plant deemed to have done the most to increase the pathology and genetics under Professor E. C. quantity or quality of food for poorer people. Stakman, who did pioneering research on the A decade later, the World Food Prize Found­ plant disease rust, a parasitic fungus that ation added a Youth Institute as a means to feeds on phytonutrients in wheat, oats, and get young people interested in the world food barley. Following 3 years with DuPont, problem. High school students are invited to Borlaug went to Mexico in 1944 as a member submit essays on the world food situation. of a Rockefeller Foundation team to help Authors of the 75 best papers are invited increase food production in that hungry to read them at the World Food Prize nation where rust diseases had taken their toll Symposium in Des Moines in mid‐October on wheat yields. each year. From among these, a dozen are

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sent for 8 weeks to intern at agricultural One of Borlaug’s dreams, through genetic research stations in foreign countries. By the engineering, is to transfer the rice plant’s summer of 2007, approximately 100 Youth resistance to rust diseases to wheat, barley, Institute interns had returned enthusiastically and oats. He is deeply concerned about a from those experiences, and all are on track recent outbreak of rust disease in sub‐Saharan to become productively involved. This is an Africa which, if it gets loose, can devastate answer to Norman Borlaug’s dream. wheat yields in much of the world. Borlaug has continually advocated increasing As President of the Sasakawa Africa Associ­ crop yields as a means to curb deforestation. ation (SAA) since 1986, Borlaug has In addition to his being recognized as having demonstrated how to increase yields of wheat, saved millions of people from starvation, it rice, and corn in sub‐Saharan Africa. To focus could be said that he has saved more habitat on food, population and agricultural policy, than any other person. Jimmy Carter initiated Sasakawa‐Global 2000, a joint venture between the SAA and the When Borlaug was born in 1914, the world’s Carter Center’s Global 2000 program. population was 1.6 billion. During his life­ time, population has increased four times, to Norman Borlaug has been awarded more 6.5 billion. Borlaug is often asked, “How than 50 honorary doctorates from institutions many more people can the Earth feed?” His in 18 countries. Among his numerous other usual response: “I think the Earth can feed awards are the U.S. Presidential Medal of 10 billion people, IF, and this is a big IF, we Freedom (1977); the Rotary International can continue to use chemical fertilizer and Award (2002); the National Medal of Science there is public support for the relatively new (2004); the Charles A. Black Award for genetic engineering research in addition to contributions to public policy and the public conventional research.” understanding of science (2005); the Congressional Gold Medal (2006); and the To those who advocate only organic fertil­ Padma Vibhushan, the Government of India’s izer, he says, “For God’s sake, let’s use all second highest civilian award (2006). the organic materials we can muster, but don’t tell the world that we can produce The Borlaug family includes son William, enough food for 6.5 billion people with daughter Jeanie, five grandchildren, and organic fertilizer alone. I figure we could four great grandchildren. Margaret Gibson produce enough food for only 4 billion with Borlaug, who had been blind in recent years, organics alone.” died on March 8, 2007 at age 95.

LIFE BOX 1.2. MARY‐DELL CHILTON Mary‐Dell Chilton, Scientific and Technical Principal Fellow, Syngenta Biotechnology, Inc.; World Food Prize Laureate (2013); Winner of the Rank Prize for Nutrition (1987), and the Benjamin Franklin Medal in Life Sciences (2001); Member, National Academy of Sciences.

I entered the University of Illinois in the I found a new love: in a course called “The fall of 1956, the autumn that Sputnik flew Chemical Basis of Biological Specificity” over. My major was called the “Chemistry I learned about the DNA double helix, the Curriculum,” and was heavy on science and genetic code, bacterial genetics, mutations, light on liberal arts. When I entered graduate and bacterial transformation. I was hooked! school in 1960 as an organic chemistry major, I found that I could stay in the chemistry still at the University of Illinois, I took a department (where I had passed prelims, a minor in microbiology (we were required to grueling oral exam) and work on DNA under minor in something…). To my astonishment, guidance of a new thesis advisor, Ben Hall, a

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microbes, biochemistry, genetics, protein chemistry, natural products chemistry (in collaboration with Scott), and plant tissue culture. The multifaceted nature of the problem bound us together. My first task was to write a research grant application to raise funds for my own salary. My DNA hybridization proposal was funded. Grant money flowed in the wake of Sputnik. Our primary objective was to determine whether DNA transfer from the bacterium to the plant cancer cells was indeed the basis of the disease, as some believed and others disputed. We disputed this continually amongst ourselves, often switching sides! This was the start of a study that has extended over my entire career. While we hunted for bacterial DNA, com­ petitors in Belgium discovered that virulent strains of Agrobacterium contained enor­ mous plasmids (circular DNA molecules) Mary‐Dell Chilton in the Washington which we now know as Ti (tumor‐inducing) University (St. Louis) greenhouse in 1982 plasmids. Redirecting our analysis, we with tobacco, the white rat of the plant found that gall cells contained not the whole kingdom. Courtesy of Mary‐Dell Chilton. Ti plasmid but a sector of it large enough to encompass 10–20 genes. Further studies in several laboratories world­ professor in physical chemistry. When Hall wide showed that this transferred DNA, took a new position in the Department of T‐DNA, turned out to be in the nuclei of the Genetics at the University of Washington, plant cells, attached to the plant’s own chro­ I followed him. This led to a new and fasci­ mosomal DNA. It was behaving as if it were nating dimension to my education. My thesis plant genes, encoding messenger RNA and was on transformation of Bacillus subtilis by proteins in the plant. Some proteins brought single‐stranded DNA. about the synthesis of plant growth hormones As a postdoctoral fellow with Dr. Brian that made the plant gall grow. Others caused McCarthy in the microbiology department at the plant to synthesize, from simple amino the University of Washington, I did further acids and sugars or keto acids, derivatives work on DNA of bacteria, mouse, and finally called “opines,” some of which acted as maize. I became proficient in all of the then‐ bacterial hormones, inducing conjugation of current DNA technology. During this time, the plasmid from one Agrobacterium to I married natural products chemist Prof. another. The bacteria could live on these Scott Chilton, and we had two sons to whom opines, too, a feat not shared by most other I was devoted. But that was not enough. It bacteria. Thus, a wonderfully satisfying was time to start my career! biological picture emerged. We could envi­ sion Agrobacterium as a microscopic genetic Two professors (Gene Nester in microbi­ engineer, cultivating plant cells for their own ology and Milt Gordon in biochemistry) and benefit. I (initially as an hourly employee) launched a collaborative project on Agrobacterium At that time, only a dreamer could imagine the tumefaciens and how it causes the plant possibility of exploiting Agrobacterium to put cancer “crown gall.” In hindsight, it was no genes of our choice into plant cells for crop accident that we three represented at least improvement. There were many obstacles to three formal disciplines (maybe four or five, overcome. We had to learn how to manipulate if you count my checkered career). Crown genes on the Ti plasmid, how to remove the gall biology would involve us in plants, bad ones that caused the plant cells to be

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tumorous, and how to introduce new genes. incompatibility: while I was good at engi­ We had to learn what defined T‐DNA on the neering genes into (dicotyledonous) tobacco plasmid. It turned out that Agrobacterium plants, the company’s main seed business determined what part of the plasmid to transfer was (monocotyledonous) hybrid corn seed. by recognizing a 25 base pair repeated Nobody knew whether Agrobacterium could sequence on each end. One by one, as a result transfer T‐DNA. This problem was solved of research by several groups around the and maize is now transformable by either world, the problems were solved. The Miami Agrobacterium or the “gene gun” technique. Winter Symposium in January 1983 marked Our company was first to the market with the beginning of an era. Presentations by Bt maize. Belgian, German and two US groups, The company underwent mergers and spi­ including mine at Washington University in noffs, arriving at the new name of Syngenta a St. Louis, showed that each of the steps in few years ago. My role also evolved. After genetic engineering was in place, at least for 10 years of administration, I was allowed to (dicotyledonous) tobacco and petunia plants. leave my desk and go back to the bench. Solutions were primitive by today’s standards; I began working on “gene targeting,” which but, in principle, it was clear that genetic means finding a way to get T‐DNA inserts to engineering was feasible; Agrobacterium could go where we want them in the plant chromo­ be used to transform a number of dicots. somal DNA, rather than random positions it I saw that industry would be a better setting goes of its own accord. than my university lab for the next step: har­ Transgenic crops now cover a significant nessing the Ti plasmid for crop improvement. fraction of the acreage of soybeans and corn. When a Swiss multinational company, CIBA– In addition, transgenic plants serve as a Geigy, offered me the task of developing research tool in plant biology. Agrobacterium from scratch an agricultural biotechnology has already served us well, both in agricul­ lab to be located in North Carolina where I ture and in basic science. New developments had grown up, it seemed tailor made for me. in DNA sequencing and genomics will surely I joined this company in 1983. CIBA–Geigy lead to further exploitation of transgenic and I soon found that we had an important technology for the foreseeable future.

LIFE BOX 1.3. ROBERT T. FRALEY Robert Fraley, Chief Technology Officer, Monsanto Co.; World Food Prize Laureate (2013); National Academy of Science Award for the Industrial Application of Science (2008); National Medal of Technology from President Clinton (1998).

When I think back to my childhood on our family farm in central Illinois, I remember bailing hay and walking soybean fields to pull weeds. These pretty common farm jobs provided me with a perspective and the motivation to find better solutions to help farmers, like my dad, fight their most diffi­ cult problems. I am particularly grateful for my experience on our family farm because I learned firsthand both how challenging farming really is and how farmers continu­ Robert T. Fraley. Courtesy of Robert T. Fraley. ally adopt new and improved innovations.

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It’s humbling to remember life as a young to spread and our team became recognized as farm boy, and then look at my career which key contributors to the worldwide scientific progressed to pioneering research on gene and agriculture communities. This was very transfer in plants and the development of humbling and led to an experience I will Roundup Ready® soybeans and other bio­ never forget, receiving the National Medal of tech innovations. Although, from a very Technology from President Clinton in 1998. young age I knew I wanted to pursue a career Looking back on all this though, we didn’t do in research, I had no idea then where science a great job of communicating directly with and innovation would take me, allowing me consumers and because of that, years later, to travel the globe, interact with so many we continue to work to address common mis­ interesting people, in a career I truly enjoy. perceptions about how food is grown and if it Growing up in a rural setting, I attended a is safe, nutritious, and sustainable. As a sci­ very small high school. In fact, I was the only entist, I was comfortable letting the evidence student in my high school biology and physics speak for itself. Although not joining the classes. While a bit intimidated by how much conversation with consumers earlier is my one‐on‐one time I had with my science greatest regret, I am pleased that we have teacher, for me, this was an opportunity to since engaged in this dialogue and continue grow, ask questions and absorb a new world to find common ground. of science and information. After graduating Throughout my career at Monsanto, I’ve held from high school, I received my Bachelor in several roles within the Technology organiza­ Science at the University of Illinois, which tion. My current role as Chief Technology established a sound foundation for my future. Officer continues to excite me because I am I continued my education at the U of I where not only leading a team of the top scientists in I earned my Ph.D. in microbiology and bio­ the ag industry, but I have the privilege of chemistry. I then spent 2 years of postdoc­ talking with farmers and seeing the process toral fellowship research at University of from beginning (in the lab) to end (on our cus­ California, San Francisco, where I studied tomers’ fields). One opportunity that has been ways to introduce genes into plant and animal especially rewarding for me in the last couple cells using liposomes. This was the period of years is engaging with broad audiences and where I became focused on how biotech­ furthering the dialogue with consumers, as nology could be used to improve crops. well as partners like the Gates Foundation, In 1981, Dr. Ernie Jaworski hired me to join a Clinton Global Initiative and Conservation small, but talented team of scientists at International. I see the opportunity to join the Monsanto. It was exciting to work with this conversation with new and diverse groups as team. I valued our collaborative efforts to an important step in the right direction. address some of agriculture’s greatest chal­ As I look back, the recognition that means the lenges. Ironically though, our research started most to me as a scientist is the World Food by using Agrobacterium to introduce new Prize. The acknowledgment that biotech­ genes into the petunia, not your traditional nology has made an important contribution to crop! Looking back, this was a great decision world food security was very rewarding and because we were able to quickly prove the my close relationship with Dr. Norman science. The petunia became the first Borlaug made this award even more special genetically engineered plant, and it laid the and personal. I have always admired Dr. foundation for many innovations in agricul­ Borlaug and the impact his scientific leader­ ture, including plants with resistance to pests, ship provided. He emphasized the significance increased crop yields and protection against of food security and always impressed on me drought, and other environmental conditions. the need to think globally and forward for As we advanced the research and technology, future generations. This is particularly critical we developed solutions to help farmers today, as we face one of mankind’s greatest address challenges on their farms. We shared challenges. By 2050, our global population our research results and safety analyses with will swell to 9.5 billion people, so we will the scientific community, regulatory bodies need to produce more food in the next around the world and our farmer customers. 30–35 years than we have in the entire history Excitement supporting the science continued of the world. Dr. Borlaug said, “Food is the

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moral right of all who are born into this agriculture globally. I believe that we can not world,” and by using agriculture effectively, only meet the challenge of food security but we can address poverty, hunger, and overcome also sustainably increase production to the some of our biggest obstacles. Dr. Borlaug’s point where we can reduce farming’s foot­ leadership and mentorship continue to have a print around the world. It is a very exciting great impact on me, my career, and my time to be involved in agriculture and I world view. encourage all who are interested in science to consider career opportunities in this industry. The agriculture industry holds great growth The innovation and developments that create potential and is at the center of so many sustainable solutions for farmers can lead to of today’s challenges—mitigating climate fulfilling and rewarding careers. change, environmental impacts, growing population, changing diets, and food produc­ Roundup Ready is a registered trademark tion demand. Continued innovation, both in of Monsanto Technology, LLC. © 2015 biology and data science, can transform Monsanto Company.

REFERENCES

American Soybean Association (2001): Conservation Tillage Study. Available at: http://www.soygrowers.com/ ctstudy/ctstudy_files/frame.htm (accessed on September 8, 2015). Brookes G, Barfoot P (2014a): Economic impact of GM crops: the global income and production effects 1996–2012. GM Crops Food 5: 1, 65–75. Available at: www.landesbioscience.com (accessed on September 8, 2015). Brookes G, Barfoot P (2014b): Key global environmental impacts of GM crop use 1996–2012. GM Crops Food 5: 2, 1–12. Available at: www.landesbioscience.com (accessed on September 8, 2015). Conservation Tillage and Plant Biotechnology (CTIC) (2002): How New Technologies Can Improve the Environment by Reducing the Need to Plough. Available at: http://www.ctic.purdue.edu/CTIC/Biotech.html (accessed on September 8, 2015). James C (2012): Global Status of Transgenic Crops: 2013, Brief Number 46. International Service for the Acquisition of Agri‐Biotech Applications (ISAAA). Available at: http://www.isaaa.org/resources/publications/ briefs/46/default.asp (accessed on September 8, 2015). Jasa P (2002): Conservation Tillage Systems. University of Nebraska. Available at: agecon.okstate.edu/isct/ labranza/jasa/tillagesys.doc (accessed on September 8, 2015). Kovach J, Petzoldt C, Degni J, Tette J (1992): A Method to Measure the Environmental Impact of Pesticides. New York’s Food and Life Sciences Bulletin. NYS Agricultural Experiment Station, Cornell University, Geneva, NY, p 139, 8 pp annually updated. Available at: http://www.nysipm.cornell.edu/publications/EIQ. html (accessed on September 8, 2015). Lazarus WF (2012): Machinery Cost Estimates May 2012. University of Minnesota Extension Service. Available at: http://www.minnesotafarmguide.com/news/regional/machinery‐cost‐estimates/pdf_a5a9623c‐636a‐11e3‐ 8546‐0019bb2963f4.html (accessed on September 8, 2015). Reeder R (2010): No‐Till Benefits Add Up with Diesel Fuel Savings. Available at: http://www.thelandonline.com/ currentedition/x1897235554/No‐till‐benefits‐add‐up‐with‐diesel‐fuel‐savings (accessed on September 8, 2015). University of Illinois (2006): Costs and Fuel Use For Alternative Tillage Systems. Available at: www.farmdoc. uiuc.edu/manage/newsletters/fefo06 07/fefo06 07.html (accessed on September 8, 2015). USDA (2013a): An Online Tool for Estimating Carbon Storage in Agroforestry Practices (COMET‐VR). Available at: http://www.cometvr.colostate.edu/ (accessed on September 8, 2015). USDA (2013b): Energy Estimator: Tillage. Available at: http://ecat.sc.egov.usda.gov (accessed on September 8, 2015).

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1

An Introduction to Flowering Plants: Monocots and Eudicots

There is no doubt about it that plants are main producers of ecosystem and important in every aspect of our daily lives. Many products which are used in food, nutraceutical, pharmaceutical, textile, cosmetics, perfumery, coffee, tea and beverage industries are in fact derived from plants. They are biosynthesized in different parts of plants and are known as natural products or secondary metabolites. Many of these compounds are defensive in nature which are produced during primary metabolic activities in plants. Many pigments in flowering plants are also secondary metabolites which are crucial for their pollination. Secondary metabolites include alkaloids, flavonoids, betalains, glyco­ sides, tannins, terpenoids and saponins. They will be introduced in the next chapter. This book deals with flowering plants, that is, angiosperms as they make one of the abundant group of plants of economic importance. However, before discussing major products of angiosperms, their biosynthesis and applications, it is important to discuss what are angiosperms? How did they evolve? What is their body organization and what kind of cells they have? So in the next section, a brief introduction of angiosperms and their classification is discussed.

1.1 An Introduction to Major Group of Angiosperms: Monocots, Eudicots and Basal Angiosperms

All plants are considered to be a group of related organisms which are capable to synthesize their own sugars during photosynthesis, possess the cell wall, and generally with the differentiation of their bodies in roots, stems, leaves, flowers or flower‐ like structures. But recent trends in molecular phylogenetics have shown that they are not as much closelyCOPYRIGHTED related as thought before. In fact,MATERIAL plants can be best described as ‘a group of different organisms which evolved independently during course of evolution and share similar characteristics like ability to synthesize their own food within their chloroplasts, have chlorophyll a as a necessary photosynthetic pigment and possess the cell wall which largely comprises of cellulose’. Their body is differentiated in vegetative and reproductive organs (spore or seed‐producing structures) and are therefore classified in one kingdom plantae. Division within kingdom plantae is based either on the presence or absence of vascular tissues (xylem and phloem) or spore‐producing structures. Bryophytes like liverworts, hornworts and mosses are non‐vascular spore producing plants while pteridophytes are vascular plants which produce spore, for

Flowering Plants: Structure and Industrial Products, First Edition. Aisha Saleem Khan. © 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.

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example, ferns, horsetails and clubmossses. Other two major groups are seed‐producing plants, that is, gymnosperms which produce seeds which are not enclosed within their ovaries, and angiosperms or flowering plants, in which seeds develop within carpels and are covered by ovary wall. Angiosperms also known as flowering plants are the largest monophyletic group of seed producing plants which have evolved many efficient ways of survival over the period of time. They are unique from other group of plants due to the development of endosperm (nutritive tissue around embryo within seeds), flowers with carpels and stamens having two pairs of pollen sacs and phloem for transportation of sugars. Their fossils are over 135 million years old. Angiosperms are considered to be close relatives of living gymnosperms but some recent evidence suggested that seed ferns represent sister group to angiosperms. They are relatively evolved group of plants as compared with gymnosperms as they possess several mechanisms which ensure successful asexual and sexual reproduction, one of the main reason which makes them one of the abun­ dant group of seed plants. Although monocots (angiosperms with one cotyledons) and dicots (angiosperms with two cotyledons) are referred as two main groups of angiosperms but modern classifica­ tion which is based on molecular evidences have characterized angiosperms as core and basal angiosperms according to their monophyletic origin (descendants of common ancestors) and facts provided by molecular data including studies from DNA sequences from chloroplasts gene rbcL. Therefore, modern system of plant taxonomy, that is, Angiosperms Phylogeny Group (APG) system is a molecular‐based systematics which retains order and families of Linnean systems and includes groups which are monophy­ letic. APG I was published in 1998 which was followed by APG II in 2003 (Chase et al., 2003) and APG III in 2009 (Bremer et al., 2009) and then APG IV in 2016. However, further development in molecular techniques, advancement in techniques related to metabolomics and proteomics is exploring the molecular phylogenetics which will form foundation of evidence‐based classification of flowering plants. Evolutionary evidences suggest that basal angiosperms which are characterized by absence of xylem vessels are primitive, however, some recent phylogenetic analysis reported that Amborella trichopoda is sister to all extant angiosperms and is at the base of angiosperms phylogenetic tree. They are composed of only few species which include many aquatic plants like water lilies (Figure 1.1), Amborella and star anise. Core angio­ sperms are represented by monocots and core eudicots. They include three major groups including monocots, eudicots and magnoliids, and the latter group was once considered to be dicots but now it is placed in a separate group. Important magnoliids include plants like avocado, black pepper, magnolia, nut‐meg, bay leaf, tuliptree or yellow poplar. Eudicots also known as true dicots, composed of more than 75% of angiosperms and are characterized by their monophyletic origin and presence of tricolpate pollens ( having three apertures). This group of angiosperms represents abundant clade of angiosperms. Figure 1.2 shows a cladogram of flowering plants based on information from APG I, II and III. A cladogram represents an evolutionary diagram which is used to explain evolutionary relationships within a group of related organisms which share common ancestors. Orders of basal angiosperms (Amborellales, Nymphaeales and Austrobaileyales) represent primitive groups whereas core eudicots are represented as advanced or modern group of flowering plants. Magnoliids like Laureales, Magnoliales, Canellales and Piperales are evolved with monocots. Eudicots represent abundant group of flowering plants, among which core eudicots include two highly evolved and

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Figure 1.1 (a‐b) Basal (a) angiosperms, (a) Nymphaea alba from family Nymphaeaceae, (b) Magnolia sp. is another basal angiosperm which belongs to family Magnoliaceae.

(b)

diverse clades which evolved separately are asterids (lamiids and campanulids) and rosids (fabids and malvids) (based on APG III) which are classified on the basis of their tendency to produce fused or free petals (Figures 1.3 and 1.4). Evolutionary traits, apo­ morphies, which are important in classification are represented where the origin of a clade takes place. Eudicots represent group of many economically important plants like members of family Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Malvaceae, Rosaceae and Solanaceae. Other main group of flowering plants, that is, monocots represent one of the highly evolved clade with monophyletic origin (Figure 1.5). They are characterized by presence of only one cotyledon, non‐woody stem, fibrous roots, long and slender leaves with par­ allel venation and scattered vascular bundles. They produce inconspicuous, mostly non‐ fragrant flowers with floral parts in multiple of three often which are arranged to form a spikelet in case of grasses. Table 1.1 shows comparison of monocots and eudicots. Commelinid clade represents most derived group of angiosperms which includes many plants from Arecales, Commelinales, Poales and Zingiberales. Monocots include palms, orchids and grasses which evolved about 60 millions years ago and are composed of almost 10,000 species. Fossils of palms and members from arum family are the oldest known monocots which are reported to found in rocks almost 100 millions years old. Monocots include many economically important plants which make our staple food like all cereals and grasses are monocots. They are important source of biofuel and bioenergy.

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Angiosperms (owering plants)

Core eudicots (75% owering plants) Basal Monocots angiosperms Magnoliids (25%) Rosids Asterids

Monocotyledons Austrobaileyales Ceratophyllales Trochodendrales Caryophyllales Chloranthaceae Ranunculales Malvids Amborellale Nymphaeales Saxifragales GunneralesDillenales Santalales Laurales Lamiids ProtealesSabialesBuxales Vitales Fabids Campanulids Canellales (Daisy) Magnoliales s Piperales

Free petals Fused petals (Brassica)

(Grass ower)

Flower in multiple of 3 Pollen monocolpate No vascular cambium Scattered vascular bundles Parallel venation Vascular bundles Tricolpate pollen vascular bundles Vascular cambium present, vascular bundles not scattered Reticulate venation

Seed with one cotyledon

Sieve tubes with companion cells for conducting carbohydrates Companion cell Ovules with 2 integuments Presence of endosperm in ovule Phloem (as seive tube member) Male gametophyte with 3 nuclei Stamen with 2 lacteral theca Female gametophyte with 8 nuclei Carpels Flowers 1/12/2017 3:55:31PM Figure 1.2 A cladogram of angiosperms based on information from the Angiosperms Phylogeny Group (APG III, 2009) (Bremer et al., 2009). An Introduction to Flowering Plants: Monocots and Eudicots 5

(a) (b)

(c) (d)

(e)

Figure 1.3 (a‐e) Rosids (fabids and malvids) are characterized by the presence of free petals (a) Quisqualis indica, (b) Chamelaucium uncinatum, (c) Millettia peguensis is an economically important plant with insecticidal properties and antiviral activities, (d) Tropaleum majus is an ornamental member of family Tropaeolaceae, and (e) Rosa sp. which belongs to Rosaceae is one of the popular ornamental and medicinal shrub.

Before describing the functional products of angiosperms, their biosynthesis and industrial uses, it is important to revise and update knowledge about angiosperms cell. So the following section will be dealing with the structure of an angiosperm cell along with some updates.

1.2 Plant Cell: Revisions and Few Updates

All plant cells are surrounded by the cell wall which not only protects them but also gives them definite shape. A lipoprotein bilayer, that is, plasma membrane is present next to the cell wall and regulates the movement of molecules in and out of plant cells.

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(a) (b)

(c) (d)

Figure 1.4 (a‐d) Asterids (lamiids and campanulids) are core eudicots which are differentiated from other eudicots due to the presence of fused petals (a) Petunia hybrid, (b) Daisy, (c) Lycopersicon esculentum, (d) Duranta erecta.

(a)

Figure 1.5 (a‐e) Monocots are composed of many economically important plants: (a) Wheat (Triticum aestivum) is a major cereal, bioenergy crop and a staple food worldwide.

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(b) (c)

(d)

(e)

Figure 1.5 (Cont’d) (b) Arum lily (Zentedeschia aethiopica) showing spadix (in yellow) and spathe, (c) Epipremnum aureum is a popular house plant which removes many indoor pollutants, such as formaldehyde, xylene and benzene, (d) Bambusa sp., (e) Canna indica also known as canna lily is used in constructed wetland for the removal of organic pollutants and heavy metals.

Plasma membrane encloses many membrane‐bound structures, that is, organelles which are present within a fluid cytosol which is the site for main metabolic activities of cell. Main organelles which are part of almost every plant cell include nucleus, mitochondria, plastids, vacuoles, endoplasmic reticulum, Golgi apparatus and ribosomes. However, in addition to this, plant cell may also contain microbodies, tannosomes, anthocyanoplasts and oil bodies depending upon their physiological role (Figure 1.6).

1.2.1 A Cellulosic Cell Wall is Crucial for all Plant Cells

Angiosperms show diversity in chemical composition of their cell wall which is the outermost covering of every plant cell whether it is a cell of root, leaf, stem, flower, fruit or seed. Each cell of these organs possess their own cell wall which gives them rigidity, support and a definite shape along with cytoskeleton which is composed of a network of microtubules and actin filaments. Cytoskeleton is involved in orientation of cellulose

0002896515.INDD 7 1/12/2017 3:55:33 PM Table 1.1 A comparison of two major group of core angiosperms.

Characteristics Monocots Eudicots

Root Fibrous, vascular bundles are Taproot, xylem in centre collateral Stem Soft, herbaceous with scattered Soft in non‐woody herbs or woody but vascular bundles vascular bundles are compactly arranged

Wheat (Triticum aestivum) Coriander (Coriandrum sativum)

Leaf Parallel venation Reticulate venation

Flower Ear of wheat Umbel inflorescence of coriander

Fruit Wheat hull or husk Coriander fruit

Seed Wheat grains (monocots) Seeds showing two cotyledons (eudicots)

Pollination Mostly wind pollinated Pollination through insects and animals Pollen grains Monocolpate Tricolpate

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Figure 1.6 (a‐e) Plant cells show differences depending upon their role: (a) A typical plant cell, (b) Cells of mesophyll of leaves contain abundant chloroplasts due to their role in photosynthesis, (c) Seed cells of many plants store lipid in form of oil bodies or oleosomes, (d) Cells of petals of many eudicots contain large vacuoles for storing water soluble pigments in order to attract their pollinators, (e) Vacuoles of sepals and petals (perianth) of most monocots are not as conspicuous as in eudicots, as many of them are pollinated by wind. (See insert for color representation of the figure.) 1/12/2017 3:55:36PM 10 Flowering Plants: Structure and Industrial Products

microfibrils and organizes the plane of cell division. Cellulose exists in the form of crystals in the cell wall and forms microfibrils which are embedded within the cell wall. The cell wall is also crucial for cell growth and development. Structural and chemical differences in the cell wall may exist within a tissue or and even within a cell. The cell wall of roots epidermal cells may be different from the cell wall of epidermal cell of stem, leaf or flower depending upon its physiological role. Primary cell wall is characteristic of all plants cells and is composed largely of cellu­ lose microfibrils which are embedded in a matrix of pectin and cross‐linking glycans. The matrix of the cell wall is laid down in cell plate followed by synthesis of cellulose microfibrils after the plate has reached the side of cells. However, secondary cell wall is characteristic of xylem tracheary elements and fibers and involves deposition of lignin, a phenolic compound. Primary cell wall is composed of almost 35% of cellulose, 35% pectin and 25% hemi­ celluloses compounds. Cellulose is the main carbohydrate of cell wall which exists as unbranched polymer of D‐glucose molecules connected by ß‐1,4 glycosidic linkage. Major hemicelluloses (branched polymer) of the cell wall are xylans, glucomannans, xyloglucans and ß‐D‐glucans. The protein part of the cell wall includes cyclins and expansins which are important for growth and development of the cell wall. Pectin compounds are important constituents of the cell wall which are present in middle lamella which is the outer cementing layer of the cell wall. Galactouronic acids connected by α‐1,4‐D are basic units of pectins. Incorporation of methyl groups to carboxylic groups of these units make them esterified. Their linkage with Ca++ and Mg++ makes pectic compounds insoluble, thus, limiting cell wall application in food industry. Pectic compounds like galactouronic acids, rhamnogalactouronins (RGI and II) prevent the cell wall from dehydrating, give them shape and cause expansion. However, RGII in primary cell walls exists in form of dimer cross‐linked with borate. This dimer provides enough support to the cell wall for its growth. Secondary cell wall is different from primary cell wall in having more cellulose and due to the presence of lignin. Both are attached with each other by means of covalent bonding. In addition, secondary cell wall is composed of hemicellulose and lignin which is deposited between plasma membrane and primary wall and prevents enlargement of the cell. Precursors of secondary wall synthesis like monolignols are secreted into the cell wall space and become randomly cross‐linked depending upon reactive oxygen spe­ cies, generated by laccases and peroxidases which makes the cell wall resistant against pathogens and also gives structural support to the cell wall. Some alcohols like coniferyl, caumaryl and sinaply groups are also part of secondary cell wall along with deposition of lignin. Monocots are different from eudicots in many ways. Some differences also exist in the cell wall, that is, presence of different polymers type and their abundance in the cell wall, presence of SiO2 forming phytoliths, especially cell walls of commeliinids includ­ ing grasses contain relatively small amount of pectin and structural proteins. The cell walls of monocots are composed of upto 30% cellulose, 25% hemicelluloses, 30% pectin and up to 10% glycoproteins with an increased amount of ferulate in plants like wheat, maize, rice and sugarcane which are linked with glucurunoarabinoxylans (GAX) (Molinari et al., 2013). Furthermore, a unique feature of the cell walls of grasses includes accumulation of β‐glucan in addition to GAX during their elongation.

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Presence of gelatin‐like properties of the cell wall which is due to O‐acetylated of the cell wall polymers, increases its applications in food industry. However, their presence also limits their use in biofuel technology and modern research is focusing on reduction of O‐acetylation of plants’ cell wall (Gille & Pauley, 2012). In the cell walls of dicots, only side chains of galactosyl‐residue in xyloglucans are O‐acetylated, however, in mono­ cots‐like grasses glucosly‐residues of xyloglucans are O‐acetylated. Recent emerging trends in cell wall technology include determination, modification and isolation of cell wall polymers for use in biomaterial, pharmaceutical and food industries through techniques like NMR and mass spectrometry. Orientation of cellulose fiber in the cell wall can be determined through Raman micro spectroscopic methods in herbaceous plants (Sun et al., 2016). Recently, cotton fibre is considered to be a single‐cell model for cell wall and cellulose research. The cell wall not only provides protection to the cell but also provides passages for the entry and exit of molecules in the form of pores, known as plasmodesmata which are extensions of smooth endoplasmic reticulum and regulate transport of molecules within range of 900 daltons. A typical plant cell may have more than 10,000 plasmodes­ mata on all sides of the cell wall, the exception being the outer side of epidermal cells. Plasmodesmata provide connections among cells for the transport of molecules, however, transport through plasmodesmata is not specific like cell membrane. There may be up to 1,000 to 10,000 or even more number of plasmodesmata present within a single plant cell. Plasmodesmata are of two kinds, primary and secondary. Primary plasmodesmata are formed during cell division. They are extensions of smooth endo­ plasmic reticulum in the form of tube, that is, desmotubules which are lined with plasma membrane through filamentous proteins (Figure 1.7). Secondary plasmodesmata are branched which develop during cell expansion. Plasmodesmata play important role during floral development and also involved in intercellular signaling especially in ovules.

1.2.2 Plant Plasma Membrane Allows Molecules to Enter Only Through Their Respective Channels

Plasma membrane is the lipid and protein part of a plant cell next to the cell wall which regulates the transport of molecules. The lipid part of membrane makes a bilayer of phospholipids which provides a barrier for transport of molecules due to its hydropho­ bicity. Significance of bilayer is to give support to many protein channels which are embedded within plasma membrane, and also for better trapping of molecules inside the cytosol. Non‐lipid part of plasma membrane is composed of proteins which makes up to 75% of membrane and allows transport of molecules which cannot simply cross the membrane through diffusion. They include polar and large molecules like sugars or charged molecules like amino acids, nitrates, sodium, potassium, calcium, and so on. Concept of plasma membrane is just like a room, whereas proteins make the gates, where molecules will cross their respective gates or channels (Figure 1.8). Plasma mem­ brane also regulates the movement of water molecules through aquaporins channels. Water molecules pass in a single row through this pore. Although plasma membrane is the main membrane of cell, but organelles like mitochondria, chloroplasts, nucleus, vacuoles also have their own lipids bilayers which perform role of plasma membrane. Plant membranes also are composed of several

0002896515.INDD 11 1/12/2017 3:55:36 PM 12 Flowering Plants: Structure and Industrial Products

(a)

Middle lamella

Secondary Primary plasmodesmata plasmodesmata Plasma membrane

Cell wall

(b) (c)

Flimentous protein Middle lamella Flimentous protein Desmotubule Central rod on desmotubules Central rod Plasma membrane Cell wall Plasma membrane Smooth ER

(d)

Figure 1.7 (a‐d) (a) Plasmodesmata are extensions of smooth endoplasmic reticulum (SER) and main connections within the cell wall which allow transfer of water and other molecules from one cell to other, (b) Detailed structure of plasmodesmata showing desmotubule which extends from one cell to next through smooth ER, (c) Cross section of a desmotubule, (d) A view of plasmodesmata view within plant cell.

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Plasma membrane

Protein channels on plasma membrane Plasmodesmata (top view) Apoplast Cell wall

Figure 1.8 Plasma membrane of plant cells is enclosed in the cell wall which surrounds it from all sides (top view). Round circles on plasma membrane represent proteins which provide passage for regulation through plasma membrane. Gaps in the cell wall indicate plasmodesmata.

sterols, for example, sitosterols, stigmasterol being the most abundant one. However, membrane sterol composition is dependent upon ecophysiological conditions involved in growth and development.

1.2.3 Mitochondria Convert Energy of Glucose in ATP and in Reducing Powers

Mitochondria are the site of cellular respiration and synthesize energy in the form of ATP for various metabolic needs of cells. Mitochondria multiply by division and are maternally inherited. Mitochondria have two membranes, outer and inner which are made up of phospho­ lipid bilayers. However, outer membrane of mitochondria comprises mostly lipids, whereas the inner membrane contains almost equal amount of lipids as well as proteins. The inner membrane is folded to increase surface area for metabolic processes like the electron transport chain and these invaginations are known as cristae. Outer membrane of mitochondria have proteins, that is, porins through which they communicate with the cytosol of cell and allow transfer of molecule upto 10KD, such as glucose and many ions. However, inner membrane is more permeable to molecules like CO2, H2O having pores for transport of molecules like ATP, ADP and pyruvate. Many proteins which are required for metabolic activities of mitochondria are carried through binding with receptor in an energy‐dependent transport. Space between two membranes is inter membrane space and fluid part of mitochon­ drion within cristae is known as mitochondrial matrix which is site of many processes that are crucial for providing energy to the cell. Products from glucose breakdown are transported to mitochondria for further breakdown in order to release energy. Mitochondria have their own DNA which is present in from of loops similar to bacterial DNA. It is present in multiple copies in matrix along with ribosomes. However, mitochondrial genome encodes only a few proteins, most of them are encoded in the

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nucleus. Mitochondrial genome of plants is larger in plants as compared with humans, and its size varies among different plants as in Arabidopsis it is 20 times larger, whereas in melon it is 140 times higher than in humans. Mitochondria synthesize energy in the form of ATP (adenosine triphosphate), there­ fore, protein channels in membranes allow for transfer of ADP (adenosine diphosphate), Pi and ATP (also known as ATP and ADP translocators). ATP formation is immediately followed by its export through translocators to other organelles or cytosol of cell wherever energy is required within cell. ATP and ADP transport is important for cell as many processes, such as DNA replication, transcription, formation of larger molecules, translation, secondary metabolite formation and cell division are all dependent upon the availability of ATP. Therefore, mitochondria also accumulate near the site where energy demand is high in germinating pollen grain. Mitochondria also play role in cell adaptation to abiotic stress and regulate metabo­ lism of proteins which regulate homeostasis and are involved in programmed cell death. They are also source of providing energy to rapidly growing pollen tube cells.

1.2.4 Plant Vacuoles Store Water, Pigments and Compounds of Defensive Nature

Vacuole of plant cells serve as lytic and storage compartment, which maintains turgor and homeostasis under stress conditions. Storage vacuoles store water, pigments, nutrients, crystals, starch, protein bodies for the plant cell and also involved in growth of cell and also help in signaling. Molecules like calcium, sodium, potassium, magne­ sium, chloride, nitrate and water are stored in vacuoles and help increase in height and surface area. Calcium and potassium are required for many processes within plant cells, therefore, vacuoles serve to store them and facilitate their transport within the cell. Vacuoles have many enzymes which convert toxic products into non‐toxic forms. Therefore, they also detoxify harmful products like xenobiotics, as revealed by experi­ mental techniques involving isolation of vacuoles and through replacement of vacuolar content and mutants of vacuoles. Just like membranes of many other organelles, vacuolar membrane or tonoplast is also a lipid bilayer with protein transporters. Vacuoles of floral cells occupy more parts of cell as they store pigments for pollination. They also modulate turgor during growth. Vacuoles also digest old organelles due to hydrolytic enzymes which are no longer required by cells and are therefore known as lytic vacuoles. Mitochondria are transferred to vacuoles by endocytosis. Aquaporins were first discovered in the vacuolar membrane of seeds so named r‐TIP (tonoplast intrinsic proteins). Vacuoles of many pollens are elongated and tubular in shape. In the petals of many flowers, sometimes vacuoles of neighbouring cells store different shades of pigments within the same petal, which differentiates it from the rest of petal, thus making contrasting patterns known as a nectar guide which serve as a guide for pollinators. Vacuolar pH is important factor in determining the color of flowers as an increase in pH may give the blue shade in morning glory (Yagamuchi et al., 2001). This increase in pH is due to an active transport of Na+/K+ from cytosol into the vacuole with the help of Na+/K+ pump. Vacuoles of epidermal cells store pigments like anthocyanin which pro­ tect them against UV radiations. Tonoplast of vacuoles storing anthocyanins pigments require transporters like ATP binding cassettes (ABC). Vacuoles, like protein‐storing

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vacuoles, transfer nutrients to germinating seeds and main source of storing nutrients like Ca, Fe, Mg, P and Zn, thereby serving important role in nutrition. Vacuoles of all plants are composed of H + ‐ATPase, V‐PPase and TIP, such as aquaporins which may differ in their role depending upon their location. Plants vacuoles are also the site for storage of industrially important products like alkaloids and anthocyanins which are widely used in textile and food industries and are reported to have anti‐cancerous properties. Anthocyanins are known to accumulate in pigmented structures within vacuoles known as anthocyanoplasts in plants like red cabbage, which may also contain enzymes for anthocyanins biosynthesis. Channels like H+‐antiports, electrogenic uniport ion and ABC transporters facilitate transfer and accumulation of secondary metabolites in vacuoles. Two proton pumps on vacuolar membrane, that is, V‐ATPases and V‐PPases develop gradient across tono­ plast membrane. Vacuoles are crucial for storage of plants’ defensive compounds in order to prevent toxicity to the rest of the cell. They are also important for synthesis of saponarin which is inhibited without vacuoles (Marinova et al., 2007). New technolo­ gists have developed to express economically important proteins in plants which accumulate in vacuoles and can further be used for cultivation of plants so large num­ ber of proteins can be produced at low costs.

1.2.5 Golgi Apparatus

It is involved in the modification of carbohydrates and proteins, and also transports them within the cell wherever they are needed in the form of vesicles. Golgi apparatus also modifies many proteins which are involved in pollination and fertilization pro­ cesses within different floral cells. Within a plant cell, it constitutes a system of stacks of thin vesicles that are held together either in a flat or in a curved array. Individual stacks of cisternae that are filled with fluid with modification are present almost all over the cytosol within plant cell. Golgi complex coordinates with endoplasmic reticulum, and form vesicles, that fuse and form a wider thin vesicle cisternae. Vesicles formed by smooth ER form cisterna on cis face of Golgi apparatus, however, at the trans face, vesicles are released or carried to their destination. These vesicles may move to plasma membrane and discharge their contents. Golgi bodies are also involved in forming cell wall machinery through the same process.

1.2.6 Nucleus Encodes Genes Required for Enzymes Forming Products of Commercial Applications

Nucleus is an important organelle due to the presence of nucleic acids DNA (deoxyribo­ nucleic acid) and RNA (ribonucleic acid). DNA is main heredity material which is present on genes present on chromosomes which are thread‐like structures. RNA synthesizes proteins for plants growth and metabolism through a process which we call gene expression. All cells of plants possess their own nuclei. However, sieve cells of phloem loss their nuclei upon maturity and they are assisted by companion cells. Nucleus communicates with other organelles and cytosol by means of pores within nuclear membrane. Nuclear envelope is similar to plasma membrane and comprise of phospholipids as dominating lipids. Proteins which are part of nuclear membrane, facilitate transport of molecules and allow mRNA to leave the nucleus through nuclear pore. They also provide passage

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for transport of molecules like ATP from mitochondria within the nucleus to energize energy‐dependent reactions like DNA and RNA synthesis. Nucleotides which are build­ ing blocks of DNA and made up of repeating units of sugar, phosphoric acid and nitrog­ enous bases that are synthesized within the cytosol of the cell. Four nitrogenous bases, adenine (A), guanine (G), cytosine (C) and thymine (T) exist in a pair attached by means of hydrogen bonds. Adenine and guanine form double hydrogen bonds within the cen­ tre of double helical structure. However, guanine and thymine are attached by means of three hydrogen bonds. In RNA, instead of thymine, uracil is present. Nucleus encodes genes for formation of enzymes which are involved in pathways which leads to the for­ mation of products of industrial importance.

1.2.7 Plastids are Sites of Sugar and Fragrance Formation

Plastids are double membrane organelles derived from proplastids, which are undif­ ferentiated plastids in meristematic cells of root and stem. Detailed sequence analysis of genes have revealed that all plastids are evolved from a single ancestral source and thought to be originated by the engulfing of a photosynthetic organism by a non‐ photosynthetic organism. Like mitochondria, they multiply by division and have mater­ nal inheritance. Plastids possess their own circular genome known as ctDNA (circular DNA) or ptDNA (plastid DNA) which is 120‐160kbp in size and makes up to 0.1% of the size of a nuclear genome. Many enzymes like DNA polymerase, helicase and primase have been purified from them. Angiosperms plastids possess both plastid‐encoded and nuclear‐encoded RNA polymerases (Sato et al., 2003). During cell differentiation, they either differentiate into chloroplasts, chromoplasts or leucoplasts. Leucoplasts are colorless organelles which synthesize lipids and store starch in roots, tubers or seeds. Chloroplasts arise from differentiation of proplastids. They possess their own circu­ lar chromosome and enzymes for gene duplication, gene expression and protein syn­ thesis, however, majority of proteins are encoded in nucleus and later transported to chloroplasts. Chloroplasts are known to be evolved from cyanobacteria because their genome is similar to prokaryotic genome. Chloroplasts are one of the most abundant plastids which are present in leaves of green plants. Their presence in any organ of plant is indicative of their role in formation of sugar through photosynthesis. They contain a lipid bilayer similar to other mem­ branes, however, glycolipids are more common in a chloroplast membrane. Chloroplast lipids like monogalactosyl diacylgylcerols (MGDG) are in fact one of the most abundant lipids on our planet. The membrane of chloroplast is composed of pores which facilitate exchange of molecules and allow them to communicate with the cytosol of cell. Porins in outer membrane of chloroplasts are non‐specific and allow transfer of molecule upto 3 nm in size. However, the inner membrane is composed of specific protein transloca­ tors which allow transfer of molecules between the cytosol and stroma. Pigments which are involved in photosynthesis, that is, antenna pigments which are embedded in the lipid membranes of thylakoids which are disc shaped structures. A pile of thylakoids make grana which are interconnected through lamellae within chloroplasts. All angiosperms possess chlorophyll a as necessary pigments which are crucial for light reactions, however, other pigments, that is, chlorophyll ‘b’, carotenes and luteins, are also present on thylakoid membranes and transfer energy of

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sunlight to chlorophyll a. Fluid of chloroplast is known as stroma which contains starch grains, free ribosomes and enzymes for formation of carbohydrates during the Calvin cycle. Energy molecules like ATP and NADPH are synthesized through a series of photochemical reactions on thylakoids which diffuse in the stroma to provide energy for sugar synthesis. ATP and ADP translocators are present in the inner membrane of the chloroplast envelope. Plastoglobuli are oil containing bodies which are found within plastids of senescing leaves. Chromoplasts are usually present in the petals of flowers giving red, organ and yellow shades. They have an undulated system of membranes without grana. They contain pigments either on the membrane or in the form of plastoglobuli as droplets. During fruit ripening, many chloroplasts are converted into chromoplasts due to the formation of lipid pigments which is evident through a change of color in many fruits like tomato, organ, mango, and so on. They are green when unripened, and turn into chromoplasts when mature. Plastids without pigments and lipids are leucoplasts, they are colorless plastids like amyloplasts. The white color of petals of many flowers is due to leucoplasts. Carotenoids are lipid‐soluble pigments having 40 carbon atoms which accumulate in chromoplasts and give them that orange, yellow and red color to attract pollinators. However, the presence of carotenes in the chloroplast protects antenna pigments against high intensities of light and helps in the absorption of energy for photosynthesis.

1.2.8 Tannosomes are Chloroplast‐Derived Organelles Which Contain Polymers of Tannins

Plants synthesize many molecules in response to the defense against pathogens and herbivores. Proanthocyanidins are type of flavonoids which are also known as condensed tannins. They play a defensive role against pathogens and UV radiation. Although tannins inclusions are previously reported to be synthesized by endoplasmic reticulum, recent ultrastructure studies revealed that they are derived from chloroplasts (Brillouet et al., 2013). Figure 1.9 shows developmental stages in the differentiation of chloroplast into tannosomes (tannin‐containing organelles) which involves unstacking and inflation of thylakoids grana forming tannosomes, which are later encapsulated and transferred as shuttle from cytosol to vacuoles where they are stored in order to protect the rest of the cell from toxic effects of tannins.

1.2.9 Ribosomes

are the site for protein synthesis. They are membrane‐bounded structures and consist of one large and one smaller subunit. However, the number of ribosomes depends upon the function of cell where they are present. Plant cells contain fewer ribosomes than animal cells. However, the cells of seeds in legumes contain large numbers of ribosomes.

1.2.10 Endoplasmic Reticulum

(ER) forms part of the endomembrane system and communicates with the nuclear envelope. It is a system of flattened membrane, sacks and tubules. ER without ribo­ somes is smooth endoplasmic reticulum (SER), whereas ER with ribosomes is rough

0002896515.INDD 17 1/12/2017 3:55:37 PM 18 Flowering Plants: Structure and Industrial Products

Intrathylakoidal Grana lumen Stromal Inating lamella thylakoids

Tannins Pearling thylakoid

Stroma Outer Inner membrane membrane Tannosome Shuttle Stroma

Shuttle CYTOSOL membrane

Tonoplast Tonoplast Stroma

Cytosol Vacuole

Figure 1.9 Stages in formation of tannosomes from chloroplast. During formation of tannosomes, chloroplasts membranes are unstacked and inflated forming tannosomes which are encapsulated and transferred as a shuttle through cytosol to vacuoles where they are stored (Brillouet et al., 2013). Not to scale; (used with permission from Oxford University press).

endoplasmic reticulum (RER) which is involved in protein formation with the help of ribosomes; all the while, SER synthesizes lipids for cells. SER in plant cells also forms a system of transport of molecules in the form of plasmodesmata. It forms phospholipids and proteins for the plasma membrane. RER forms vesicles which transport large molecules in coordination with Golgi bodies.

1.2.11 Peroxisomes

are single membrane bounded organelles responsible for oxidative reactions due to catalases and for oxidation of fatty acids. They are involved in detoxification of many waste products. Glyoxysomes convert stored fats into sugars and important in germina­ tion of fat rich oily seeds. They contain enzymes for breakdown of fatty acids through beta‐oxidation. Peroxisomes are thought to be originated either through de novo synthesis from invagination of endoplasmic reticulum membrane or through division of preexisting peroxisomes.

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1.2.12 Oleosomes

or oil bodies are spherical organelles which contain triacylgylcerols surrounded by protein oleosins in their monolayer phospholipid membrane. They are found in seeds and pollens of many angiosperms. They act as storage sites of many triacylgylcerols. They are detected in tapetum of developing anthers of olive and Arabidopsis. Oleosins are low molecular weight proteins (<30 kDa) which give enough stability to oleosomes and also help in degradation of oils during germination. However, in addition to oleosins, oleosomes may also be composed of other proteins, that is, caleosins and steroleosin (Purkrtova et al., 2008). Caleosins bind to Ca2++ and increase stability of oil bodies while steroleosins are reported to bind with sterols. Oleosomes based emulsions are commonly used in many food industries for dressings and as sauces and they have applications in cosmetic and food industries. In order to understand their future role in industries, artificial oil bodies are also being produced.

1.3 Intracellular and Extracellular Communications are Crucial for Cells’ Metabolic Demands

Molecules like water, sucrose, signaling molecules, many defensive compounds, and phytohormones, and so on are transported in plants from one organ to another through vascular tissues. These molecules are required for many metabolic activities, that is, growth, development, reproduction, protection against herbivores, insects and micro­ bial pathogens. Although xylem and phloem serve as main tissues for transport of many such molecules in plants from one organ to next, however, cell‐to‐cell transport takes place through passages in the cell wall of plants, that is, plasmodesmata (Figure 1.10). They are channels in symplast which mediate trafficking of molecules from cell to cell. Such transport is vital because all cells are in need of water, nutrients and sucroses which can only enter in cells through PD. However, once molecules cross PD, they will have to cross next barrier, that is, cell membrane. Small RNA molecules (sRNA) are also known to move symplastically through PD. Primary PD develop during cytokinesis, whereas secondary PD develop de novo in existing cell walls during cell expansion. For intracellular communication plants have evolved a system of proteins, porins which are localized on all membranes within cells. Molecules within a cell are trans­ ported through these pores from one organelles to another. Water molecules cross plasma membrane through specific protein channels, that is, aquaporins which belong to intrinsic protein superfamily and exist in wide range of organisms including bacteria and eukaryotes. Almost 35 different aquaporins have been identified in Arabidopsis and maize. Aquaporins are found on plasma membrane as well as tonoplast as tonoplast intrinsic proteins (TIPs). Phosphorylation and dephosphorylation of aquaporins regu­ late activity of water transport in some cells. Transport across plasma membrane membrane takes place through channels, gates and pumps. Gas molecules like CO2, O2 and N2, ethanol can simply diffuse against the membrane. However, many molecules of relatively large size or molecules with electrical charges, that is, cations or anions cannot simply enter through diffusion; they depend upon channels and gates for entry or enter the cell through membrane proteins, that is, transporters. These transporters are transmembrane proteins which form a

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(a) (c)

(b)

(d)

Nutrients

Figure 1.10 (a‐d) Intercellular and intracellular communications in plants; (a & b) Organ to organ transport within plants takes place through vascular tissues, that is, xylem and phloem, (c) Plasmodesmata allow cell to cell communication within plants, (d) Intracellular communication (within cell) takes place through porins which are part of plasma membrane as well as other organelles.

channel comprising a passage or pore through which molecules pass through and provide hydrophilic environment for molecules to enter (Figure 1.11). Carriers are membrane proteins with high transport substrate specificity, whereas channels have low substrate specificity. Channels are passive transporters and direction of transfer of molecules is determined by solute concentration and electrochemical potential. Channels which allow molecules to pass most of the time are ungated, how­ ever, gated channels open only in response to a chemical or electric signal. Channels for transport of K+ and Cl− channels are ungated, however, Ca++ and Na+ channels are gated. In addition to this, cell membrane possesses many anion channels which may be dependent upon voltage, light activated channels and mechanical stress activated channels. Voltage activated channels may be depolarized‐activated channels which are either slow‐activating (S‐type) or rapid‐activating (R‐type) anion channels and are found mostly in plasma membranes of epidermal cells of root, guard cells and hypocotyls. R‐Type channels allow transport of chloride, nitrate, malate, sulfate, and citrate, how­ ever, S‐Type facilitate rapid transfer of chloride, citrate and phosphate. Mechanoactive channels are also present in stomatal cells and involved in osmoregulation. In addition to this, aluminum‐activated malate channels (ALMT) also exist in stomatal cells and in Arabidopsis, ALMTs are localized in membrane compartments and comprise a gene

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Plasma membrane

+ channels H Antiporters

Cd Anthocyanin + GSX pump + H Plasma membrane H AA AA Transporters

symporters + + Anions

H – H – Channels

+ + H H Anions Cations + + K H

Suc Suc + + + H H H pumps

Plasma memb ra Antiporters

Glu Fru Suc Cd Mg

pumps pumps

+ + + + H H H H Mg + + H H pumps ne Sucrose efux carrier

Figure 1.11 An overview of intracellular communication within a plant cell which is achieved through porins present on plasma membrane as well as on other organelles. Many channels, gates and pumps present within membranes of a plant cell allow transport of molecules from one organelles to other. Note some transporters within membranes allow only one molecule to pass through them, they are uniporters, whereas some allow two molecules to pass through them within same direction, they + are symporters. However, some are antiporters which allow counter‐exchange of molecules. NO3/H , 3− + + + + + PO4 /H , Suc/H are symporters on plasma membrane, whereas Na /H is an antiporter. Glu/H , Suc/H+, Ca2+/H+, Cd/H+, Na+/H+ and Mg+/H+ are antiporters in vacuoles. Pumps like H+, K+, Ca2+ and Cl− are present on plasma membrane. (See insert for color representation of the figure.)

family of 14 members (Kovermann et al., 2007). Temperature responsive channels open and close in response to low and high temperature, for example, capsaicin opens the channel in response to heat while menthol opens the channel regulated by low tem­ perature. However, coupled transport also takes place which involves binding of two molecules to a carrier proteins. If transport is in same direction it is known as cotransport, however, if it takes place in opposite direction, it is known as counter transport. In addition, plasma membrane comprises channels for K+ transport, high affinity K+ transporters, KUP permeases, Ca2+channels, cyclic nucleotide‐gated channels, GluR, annexins and mechanosensitive Ca2+channels.

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Active transport involves transfer of molecules in one direction at the expense of energy and is therefore dependent upon ATP hydrolysis. Many pumps like Na+/K+ pumps, H+/K+‐ATPases, ABC transporters are energy dependent. Three types of pumps are present mainly within plant cells, for example, ABC trans- porters, vacuolar (V‐type) H+ pumps and P‐type pumps which depend upon ATP as a fuel for energizing them. ABC transporters are generally found in the cell membrane as well as other endomembrane including vacuoles and allow efflux of molecules like organic acids, hormones, alkaloids, toxic products and xenobiotics (Verrier et al., 2008). P‐type pumps are cationic pumps which become phosphorylated during catalytic reac­ tion which are present in plasma membrane, vacuole and chloroplast membranes, ER and Golgi. Binding of molecule causes a conformational change in these pumps in order to facilitate transfer of substrate across the membrane. Other pumps which are present in plasma membrane may include P‐type pumps, + 2+ 2+ + ATPases, P3A‐type H ‐ATPase, P2B‐(Ca ) ATPase, P1B‐Zn ‐ATPase, H ‐Pyroph­ osphorylase, pin‐formed (PIN) transporter family, amino acid/auxin permease transporters, amino acid‐ polyamine‐choline transporters, amino acid transporter fam­ ily, amino acid permeases, AAAP transporters for auxin transport, lysine/histidine transporters, proline transporters, GABA transporters, aromatic and neutral amino acid transporters, peptide transporters, PTR/NRT1pepetide, sugar transport proteins, inositol, polyol, sucrose, purine, aquaporins, nitrate, ZIP, copper, SLC40, chelation‐based strategy of iron uptake in grasses like maize and rice, yellow stripe‐like transporters and phosphate transporters.

1.4 Future Perspectives

Angiosperms phylogenetic system (APG IV) is still characterizing and classifying plants in order to develop evidence‐based molecular systematics. Molecular cues are being explored that can form foundation of modern classification will be more reliable. Therefore, advancement in molecular phylogenetics can provide more accurate information for classification of plants. Molecular phylogenetics can also serve as a useful tool to provide adequate knowl­ edge about evolutionary relationships of angiosperms which will not only help in characterizing many products but can help implement data provided by phylogenetics that can be used for metabolomics to increase further applications. Tannosomes are relatively new structures reported in the plant cell. Many analytical and spectrometric techniques can further aid our knowledge of the plant cell and their metabolites (both primary and secondary) which will increase their use in industries. Tools in molecular biology, proteomics and metabolomics and further development of nano‐techniques can reveal different aspects of plant cells biology. Many cell membrane carrier proteins in flowers are still being explored. Reduction of O‐acetylation units in the cell wall will be helpful in biofuel technology. Determination of orientation of cellulose fiber in the cell wall through Raman micro­ spectroscopic methods and other spectroscopic methods in herbaceous plants can open directions for cell wall research in the future. In order to increase use of oleosomes in industries, artificial oil bodies are also being produced.

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References

Bremer, B., Bremer, K., Chase, M.W., Fay, M.F, Reveal, J.L, Soltis, D.E, Soltis, P.S., & Stevens, P.F. (2009). ‘THE ANGIOSPERM PHYLOGENY GROUP: An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III’, Botanical Journal of the Linnean Society, 161, pp. 105–121. doi:10.1111/j.1095‐8339.2009.00996.x. Brillouet, J.M., Romieu, C., Schoefs, B., Solymosi, K., Cheynier, V., Fulcrand, H., Verdeil, J., & Cone jé ro,́ G. (2013). ‘The tannosome is an organelle forming condensed tannins in the chlorophyllous organs of Tracheophyta’, Annals of Botany, 112, pp. 1003–1014. doi:10.1093/aob/mct168. Chase, M. (2003). ‘The Linnean Society of London’, Botanical Journal of the Linnean Society, 141, pp. 399–436. Gille, S. & Pauly, M. (2012). ‘O‐acteylation of plant cell wall polysaccharides’, Frontiers in Plant Science, 3, pp. 1–7. Kovermann, P., Meyer, S., Hortensteiner, S., Picc,o C., Scholz‐Starke, J., Ravea, S., Lee, Y., & Martinoia, E. (2007). ‘The Arabidopsis vacuolar malate channel is a member of the ALMT family’, Plant Journal, 52, pp. 1169–1180. Marinova, K., Kleinschmidt, K., Weissenbck, G., & Klein, M. (2007). ‘Flavonoid biosynthesis in barley primary leaves requires the presence of the vacuole and controls the activity of vacuolar flavonoid transport’. Plant Physiology, 144. pp. 432–444. Molinari, H.B., Pellney, T.K., Freeman, J., Shewry, P.R, & Mitchell, R.A. (2013). ‘Grass cell wall feruloylation: distribution of bound ferulate and candidate gene expression in Brachypodium distachyon’, Frontiers in Plant Science, 4, p. 50. doi: 10.3389/fpls.2013.00050. Purkrtova, Z., Le Bon, C., Kralova, B., Ropers, M.H., Anton, M., & Chardot, T. (2008). ‘Caleosin of Arabidopsis thaliana: Effect of calcium on functional and structural properties’, Journal of Agricultural and Food Chemistry, 56, pp. 11217–11224. Sato, N., Teraswa, K., Miyajima, K., & Kabeya, Y. (2003). ‘Organization, developmental dynamics and evolution of plastids nucleotides’, International review of Cytology, 232, pp. 217–262. Sun, L., Singh, S., Joo, M., Vega‐Sanchez, M., Ronald, P., Simmons, B.A., Adams, P., & Auer, M. (2016). ‘Non‐invasive imaging of cellulose microfibril orientation within plant cell walls by polarized Raman microspectroscopy’, Biotechnology & Bioengineering, 113, pp. 82–90. Verrier, P., Bird, D., Burla, B., Dassa, E., Forestier, C., Geisler, M., Klein, M., Kolukisaoglu, U., Lee, Y., Martinoia, E., Murphy, A., Rea, P., Samuels, L., Schulz, B., Spalding, E., Yazaki, K., & Theodoulou, F.L. (2008). ‘Plant ABC proteins – a unified nomenclature and updated inventory’, Trends in Plant Science, 13, pp. 151–159. Yamaguchi, T., Fakuda‐Tanaka, S., Inagaki, Y., Saito, N., Yonekura‐Sakakibara, K., Tanaka, Y., & Iida, S. (2001). ‘Genes encoding the Vacuolar Na+/H+ Exchanger and Flower Coloration’, Plant Cell Physiology, 42, pp. 451–461.

Further Reading

Bar‐Peled, M., Urbanowicz, B.R., & O’ Neill, M.A. (2012). ‘The synthesis and origin of pectic polysaccharide rhamnogalacturonan II‐insights from nucleotide sugar formation and diversity’, Frontier in Plant Science, 92, pp. 1–12.

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Blom, T.J.M., Sierra, M., van Vliet, T.B., Franke‐van Dijk, M.E.I., de Koning, P., van Iren, F., Verpoorte, R., & Libbenga, K.R. (1991). ‘Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don and its conversion into serpentine’, Planta, 183, pp. 170–177. Carpita, N.C., Defernez, M., Findlay, K., Wells, B., Shoue, D.A., Catchpole, G., Wilson, R.H., & McCann, M.C. (2001). ‘Cell wall architecture of the elongating maize coleoptile’, Plant Physiology, 127, pp. 551–565. Cosgrove, D.J. (2000). ‘Loosening of plant cell walls by expansins’, Nature, 407, pp. 321–326. De Oliviera, D.E., Franco, L.O., Simoens, C., Seirinck, J., Coppieters, J., Botterman, J., & Van Montagu, M. (1993). ‘Inflorescence‐specific genes from Arabidopsis thaliana encoding glycine‐rich proteins’, Plant Cell, 2, pp. 427–436. Gilley, S., de Souza, A., Xiong, G., Benz, M., Cheng, K., Schultink, A., Reca, I.B., & Pauly, M. (2011). ‘O‐acetylation of Arabidopsis hemicellulose xyloglucan requires AXY4 or AXY4L, protein with a TBL, and DUF231 domain’, Plant Cell, 23, pp. 4041–4053. Gaxiola, R.A., Fink, G.R., & Hirschi, K.D. (2002). ‘Genetic Manipulation of Vacuolar Proton Pumps and Transporters’, Plant Physiology,129, pp. 967–973. Haigler, C.H., Betancur, L., Stiff, M.R., & Tuttle, J.R. (2012). ‘Cotton fiber: a powerful single‐cell model for cell wall and cellulose research’, Frontier in Plant Science, 3, p. 104. Huang, L., Takahashi, R., Kobayashi, S., Kawase, T., & Nishinari, K. (2002). ‘Gelatin behavior of native and acetylated konjac glucomannan’, Biomacromolecules, 3, pp. 1296–1303. Jia, Z., Cash, M., Darvill, A., & York, W. (2005). ‘NMR characterization of endogenously O‐acetylated oligosaccharides isolated from tomatoes (Lycopersicon esculentum) xyloglucan’, Carbohydrates Research, 340, pp. 1818–1825. Lucas, W.J., Yoo, B.C., & Kragler, F. (2001). ‘RNA as a long‐distance information macromolecule in plants’, Nature Reviews Molecular Cell Biology, 2, pp. 849–857. Martinoia, E., Klein, M., Gessler, M., Sanchez‐Fernandez, R., & Rea, P.A. (2001). ‘Vacuolar transport of secondary metabolites and xenobiotics. In: Robinson, D., Rogers, J, (eds). Vacuolar Compartments. CRC Series. Sheffield Academic Press, Sheffield, UK, pp. 221–253. Maurel, C., Reizer, J., Schroeder, J.I., & Chrispeels, M.J. (1993). ‘The vacuolar membrane protein y‐TIP creates water specific channels in Xenopus oocytes’, Journal of European Molecular Biology Organization, 12, pp. 2241–2247. Otani, M., Shitan, N., Sakai, K., Martinoia, E., Sato, F., & Yazaki, K. (2005). ‘Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica’, Plant Physiology, 138, pp. 1939–1946. Otegui, M.S., Capp, R., & Staehelin, L.A. (2002). ‘Developing seeds of Arabidopsis store different minerals in two types of vacuoles and in the endoplasmic reticulum’, Plant Cell, 14, pp. 1311–1327. Ross, J.H.E. & Murphy, D.H. (1996). ‘Characterization of anthers‐expressed genes encoding a major class of extracellular oleosin‐like proteins in pollen coat of Brassicaceae’, Plant Journal, 9, pp. 625–637. Simpson, M.G. (2010). Plant Systematics, 2nd ed. Elsevier Academic Press. Shimaoka, T., Ohnishi, M., Sazuka, T., Mitsuhashi, N., Hara‐Nishimura, I., Shimazaki, K., Maeshima, M., Yokota, A., Tomizawa, K., & Mimura, T. (2004). ‘Isolation of intact vacuoles and proteomic analysis of tonoplast from susprension‐cultured cells of Arabidopsis thaliana’, Plant Cell Physiology, 45, pp. 672–683.

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Tanaka, Y., Tsuda, S., & Kusumi, T. (1998). ‘Metabolic engineering to modify flower color’, Plant Cell Physiology, 39, pp. 1119–1126. Vanholme, R., Demedts, B., Morreel, K., Ralph, J., & Boerjan, W. (2010). ‘Lignin Biosynthesis and Structure’, Plant Physiology, 153. pp. 895–905. Van Norman, J.M., Breakfield, N.W., & Benfey, P.N. (2011). ‘Intercellular communication during plant development’, Plant Cell, 23, pp. 855–864. Vitale, A., & Hinz, G. (2005). ‘Sorting of proteins to storage vacuoles: how many mechanisms?’ Trends in Plant Science, 10, pp. 316–323. Vogel, J. (2008). ‘Unique aspects of the grass cell wall’, Current Opinion in Plant Biology, 11, pp. 301–307. Werner, D., Gerlitz, N., & Satdler, R. (2011). ‘A dual switch in phloem unloading during ovule development in Arabidopsis’, Protoplasma, 248, pp. 225–235. Wu, X., Weigel, D., & Wigge, P.A. (2002). ‘Signaling in plants by intercellular RNA and protein movement’ Genes and Development, 16, pp. 151–158. Yoshida, K., Kawachi, M., Mori, M., Maeshima, M., Kondo, M., Nishimura, M., & Kondo, T. (2005). ‘The Involvement of Tonoplast Proton Pumps and Na (K+)/H+ Exchangers in the Change of Petal Color During Flower Opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue’, Plant Cell Physiology, 46, pp. 407–415. Zhen, R.G., Kim, E.J., & Rea, P.A. (1997). ‘The molecular and biochemical basis of pyrophosphate‐energized proton translocation at the vacuolar membrane’, Advances in Botanical Research, 25, pp. 297–337.

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Problems Chapter 1

1 Select the best answer from options given below.

i. One of the following is an important distinguishing criteria in classification of asterids and rosids. A Presence of tepals B Arrangements of petals C Presence of endosperm D Presence of tricolpate pollens

ii. Inflorescence of monocots like grasses is different from eudicots and known as A catkin B umbel C spikelet D cyathium

iii. Parallel venation is a characteristic of: A Zea mays B Coriandrum sativum C Mentha piperita D Rosa indica

iv. Cell wall is outermost boundary of plant cells which gives a definite shape to plant cell and is made up of carbohydrates, proteins and acids. However, one of the following carbohydrate is not constituent of cell walls. A Sucrose B Cellulose C Hemicellulose D Arabinose

v. Plasma membranes are lipid bilayer which serves as a barrier to many molecules and allow them to enter only through their specific channels which are made up of ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐. A lipids B proteins C carbohydrates D nucleic acids

vi. In symplast pathway for water transport, water and dissolved solutes will enter from one cell to another through plasmodesmatal connections which are extensions of ‐‐‐‐‐‐‐‐‐‐‐‐‐. A smooth endoplasmic reticulum B mitochondrial cristae C thylakoids of chloroplast D vacuoles

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vii. If you find a flower which is small, inconspicuous, and not scented, its most likely pollinator would be: A bees B birds C humans D wind

2 If anaerobes represent the common ancestors of modern living plants, eudicots and monocots have common origin, but they are evolved at different rate, eudicots faster than monocots, and monocots represent the highly evolved plants. How will you construct a Tree of Life based on the information given above?

3 Evolution is never‐ending process, responsible for modern plant architecture. Based upon this fact, predict ONE change in plant architecture after a million years.

4 Herb is a general term which is used to describe soft and herbaceous plant which never becomes woody throughout its life cycle and may be a monocot or a eud­ icot. However, many morphological and anatomical differences exist among monocot and eudicot herbs. Taking grasses like rice as an example of monocot herb and mint as a eudicot herb, fill the information in the table below showing main differences in the roots, stem, leaves and flowers of these economically important herbs.

Characteristic Rice Mint

Stem Leaves Flower Fruit Seed

5 Compare and contrast major anatomical traits of monocots and eudicots with the help of cladogram. How vascular tissues of monocots differ from eudicots? Explain with the help of diagram.

6 Suppose you find plant A which is non woody, have parallel venation and small inconspicuous flower. However plant B is woody, with reticulate venation and with large showy flower. How can you compare internal organization of plant A with plant B in concluding that plant A is possibly a monocot while plant B is an eudicot?

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7 Classify the following plants as asterids and rosids.

(a) (b) (c)

8 What evidences will you give to support that plant in picture (a) is a monocot and (b) is an eudicot?

(a) (b)

9 A team of Russian scientists reported (published in the New York Times February 2012) that a narrow‐leafed campion, living plant, which died 32,000 years ago, have been generated from the fruit of a little arctic flower. The fruit was stored by an arctic ground squirrel in its burrow on the tundra of northeastern Siberia and lay permanently frozen until excavated by scientists a few years ago. If the ancient campions are the ancestors of the living plants, this family relationship should be evident in their DNA. Based upon above information, briefly explain the advan­ tages of molecular phylogeny and which techniques helped scientists for drawing their conclusions?

10 Do you think that grasses are considered to be one of the highly evolved clade of flowering plants? If yes, give reasons to support your answer.

11 Give two main differences in cell walls of monocots and eudicots.

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12 Lipids and proteins make important part of plasma membranes. Hydrophobic nature of lipids acts a barrier for the transport of molecules and directs them toward their protein gates, many of which are molecule specific. Lipid‐protein organization is almost the same for the membrane of organelles as well. Keeping this mind, explain with the help of diagram that how organelles communicate within a cell for transport of molecules?

13 Vacuoles are organelles which perform multiple functions in plants. They are the site of storage of water molecules and minerals; they degrade harmful products and also digest old organelles. They also accumulate water‐soluble pigments within cells of flowers especially in cells of petals which are attractive for many pollinators. It indicates that vacuoles which are present in floral cells are different from vacuoles in cells of vegetative organs. Using this information, make two sepa­ rate diagrams of plant cells (i) a stem cell (ii) a petal cell (preferably eudicot as they form conspicuous petals) to show how vacuoles of different plant organs may differ in their functions.

14 Define oleosomes. Why many plants prefer to store triacylgylcerols within their seeds?

15 With the help of diagram, differentiate between (i) carriers (ii) channels (iii) pumps (iv) antiports (v) symporters and (vi) uniporters (vii) primary and secondary plasmodesmata

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Recent Advances in Sexual Propagation and Breeding of Garlic

Einat Shemesh‐Mayer and Rina Kamenetsky Goldstein Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Beit Dagan, Israel

ABSTRACT

The restoration of flowering ability, sexual hybridization, and seed production in garlic (Allium sativum L.) has resulted in an increase in genetic variability available to agriculture and has opened new avenues for the breeding of this important crop. In this review, the current status of flower development, fertil- ity, hybridization, sexual propagation, and seed production in garlic is dis- cussed. We summarize the main stages in the life cycle of garlic from true seeds to flowering and bulb formation, and recent advances in our understanding of floro‐ and gametogenesis. Flowering and fertility of garlic are tightly regulated by environmental conditions, and therefore the seed production cycles in vari- ous climatic zones are complex and challenging. Recent establishment of mod- ern molecular tools and the creation of large transcriptome catalogs provide a better understanding of the molecular and genetic mechanisms of flowering and fertility processes, and accelerate the breeding process by using molecular markers for desirable traits. KEYWORDS: Allium sativum, environmental regulation, fertility, genetic regu- lation, hybridization, male sterility, seed production

I. INTRODUCTION II. HORTICULTURAL DIVERSITY AND GENETIC RESOURCES III. LIFE CYCLECOPYRIGHTED AND THE FLOWERING PROCESS MATERIAL A. Seed and Seedling Development B. Annual Life Cycle and Florogenesis C. Environmental and Genetic Control of Flowering

Horticultural Reviews, Volume 46, First Edition. Edited by Ian Warrington. © 2019 John Wiley & Sons, Inc. Published 2019 by John Wiley & Sons, Inc.

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IV. FERTILITY BARRIERS A. Morphology and Anatomy of the Individual Flower 1. The Male Gametophyte 2. The Female Gametophyte B. Environmental and Genetic Control of Male Sterility V. UNLOCKING VARIABILITY BY SEXUAL REPRODUCTION A. Morphological Variability in Seedling Populations B. Environmental Regulation of Seedling Development C. Molecular Markers in Variable Garlic Populations VI. CONCLUDING REMARKS LITERATURE CITED

I. INTRODUCTION

Garlic (Allium sativum L.) is one of the most popular vegetable crops, being cultivated in different continents for flavor, nutrition, and medici- nal purposes. The wild ancestor of cultivated garlic probably originated in Central Asia, and was gathered by seminomadic tribes about 10 000 years ago. Later, traders introduced plants to the Mediterranean Basin, India, and China, and from there garlic spread across various regions of the world (Engeland 1991; Etoh and Simon 2002). Widespread geographical distribution of cultivated garlic resulted in its adaptation to different climatic conditions and in the development of many local types and varieties with specific morphological and physiological traits. Taxonomically, A. sativum belongs to the section Allium of the genus Allium. Among 114 species in this section, about 25 are closely related to the cultivated plant, such as A. tuncelianum (Kollman) Özhatay, Mathew, Şiraneci from Turkey and A. moschatum L. from the Caucasus (Mathew 1996). A. sativum is not found in native populations, but most garlic relatives grow wild in regions characterized by relatively cold winters and hot and dry summers, have garlic‐like taste and smell, and are used by local populations as food and nutraceuticals. Similar to many wild Allium species, the ancestors of garlic from Central Asia probably produced flowers, seeds, and relatively small bulbs. However, since the development and growth of flowering scapes consume energy at the expense of storage organs, it is likely that human selection for early maturation of large garlic bulbs deprived the develop- ing scapes of nutritional supplies. Cultivated garlic has lost its flowering potential and fertility, and today commercial production is based exclu- sively on vegetative propagation (Etoh 1985; Etoh and Simon 2002). Consequently, garlic breeding has been limited to selection from estab- lished genetic variation, and breeding was attempted only via mutation and in vitro techniques (Takagi 1990). In recent years, flowering ability

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was restored in several garlic genotypes, and an increase in garlic vari- ability was achieved via sexual hybridization and seed production (Etoh 1983b; Etoh et al. 1988; Pooler and Simon 1994; Inaba et al. 1995; Jenderek 1998, 2004; Jenderek and Hannan 2000; Jenderek and Zewdie 2005; Kamenetsky et al. 2005; Kamenetsky 2007). Fertility restoration and seed production have opened a new stage of genetic research into garlic. Similar to many other perennial monocots, Allium species possess a large genome size (7–32 Gb) (Ricroch et al. 2005). Despite its domestication, garlic has maintained its ploidy level (2n = 2x = 16), and the diploid garlic nuclear genome is estimated at 15.9 Gbp, 32 times larger than the genome of rice (Arumuganathan and Earle 1991; Fritsch and Friesen 2002; Kik 2002). Therefore, full sequenc- ing of the garlic genome is still a challenging task, but transcriptome assembly using next‐generation sequencing (NGS) might be efficiently employed for the generation of functional genomic data. At the same time, an enormous amount of genetic and molecular data, collected in model plants over recent decades, can be translated to commercial crops by using various experimental tools such as candidate genes, library screening, expressed sequenced tags (ESTs), and genomic, transcrip- tomic, proteomic, and metabolomic databases (Leeggangers et al. 2013). New genetic variability obtained by sexual hybridization, in combi- nation with research results, has provided solid ground for a new phase in garlic breeding (Pooler and Simon, 1994; Jenderek and Hannan 2004; Kamenetsky et al. 2004a, 2015; Jenderek and Zewdie 2005; Shemesh et al. 2008; Shemesh‐Mayer et al. 2015a). Generation of garlic S1 fami- lies provided the first source of variability for genetic studies for breed- ing purposes (Hong and Etoh 1996; Jenderek 2004; Jenderek and Zewdie 2005). In Israel, a breeding program was established 10 years ago and is currently focused on sexual hybridization and selection of superior gar- lic plants, the introduction of new useful traits that are uncommon in commercial clones, and the development of new cultivars for different climatic zones. In this review, the current status of sexual propagation, hybridization, and seed production in garlic is discussed.

II. HORTICULTURAL DIVERSITY AND GENETIC RESOURCES

During a long cultivation history, garlic plants were grown in diverse climatic and biogeographic regions. They exhibit wide variations in bulb size, shape and color, number and size of cloves, peeling ability, maturity date, flavor and pungency, bolting capacity, and numbers and sizes of topsets and flowers in the inflorescence (Figure 1.1) (McCollum

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Figure 1.1 Morphological variation in garlic cultivars, propagated vegetatively in vari- ous climatic areas (Source: C. Aaron and R. Adams, poster, 2017, with permission.)

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1976; Astley et al. 1982; Astley 1990; Hong and Etoh 1996; Lallemand et al. 1997; IPGRI, ECP/GR, AVRDC 2001; Kamenetsky et al. 2005; Meredith 2008). A strong interaction between genotype and environ- ment has led to a variety of phenotypic expressions (Lallemand et al. 1997; Portela 2001; Kamenetsky et al. 2004b; Meredith 2008). Depending on the ability to develop a flower stem, garlic producers distinguish between softneck and hardneck varieties (Engeland 1991; Meredith 2008). However, from a physiological point of view, the termi- nology bolters and nonbolters is more accurate. Depending on the traits of scape elongation and inflorescence development, garlic varieties were classified by Takagi (1990) as: (i) nonbolters, which normally do not form a flower stalk or produce cloves inside an incomplete scape; (ii) incom- plete bolters, which produce a thin, short flower stalk, bear only a few large topsets, and usually form no flowers; and (iii) complete bolters, which produce a long, thick flower stalk, with many topsets and flowers. It was observed that these traits might be altered by different environmen- tal conditions, but the mechanisms of their regulation are still unknown. Based on morphological and physiological phenotype, worldwide garlic cultivars were classified into several horticultural groups, reflect- ing the broad diversity of the crop. The group named Purple Stripe, which includes bolting hardneck cultivars, is considered to be geneti- cally closest to the origin of garlic. The other groups include the Artichoke, Asiatic, Creole, Glazed Purple Stripe, Marbled Purple Stripe, Middle Eastern, Porcelain, Rocambole, Silverskin, and Turban types (Meredith 2008). These groups vary in bolting ability and bulb struc- ture. Moreover, plant performance is affected by environment, and therefore phenotypes of the same variety change dramatically under different climatic conditions. Amplified fragment‐length polymor- phism (AFLP) analysis of 211 genotypes indicated duplications of 41–64% of the garlic accessions in the National Plant Germplasm System (NPGS) and commercial collections in the USA (Volk et al. 2004). Therefore, accurate discrimination between different cultivars and groups requires further application of modern molecular tools (Meredith 2008; Volk and Stern 2009; Kamenetsky et al. 2015). Central Asia, the center of origin for many Allium species, is a valu- able source of garlic diversity (Hanelt 1990; Simon 2005). In the early 1980s, Japanese expeditions to Central Asia collected a number of gar- lic accessions in Uzbekistan, Tajikistan, Kirgizstan, and Kazakhstan (Etoh et al. 1988). Later, fertile garlic plants were also found in Armenia, Georgia, and Xinjiang. The garlic plants collected in these regions were grown at Kagoshima, Japan, and, following topset excision, some clones developed fertile flowers and viable seeds with germination up to 40%

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(Etoh 1983b, 1986; Etoh et al. 1988, 1991). Pooler and Simon (1994) improved floral production and seed set, but seed germination still remained low and ranged between 10 and 12%. Screening of several garlic collections identified larger variability of highly fertile clones, producing over 400 seeds per umbel, with seed germination of 67–93% (Etoh 1986, 1997; Inaba et al. 1995; Hong and Etoh 1996; Jenderek 1998; Jenderek and Hannan 2000, 2004). In 1995–2001, international collect- ing missions to Central Asia gathered over 300 garlic landraces and plants from natural populations (Baitulin et al. 2000; Kamenetsky et al. 2004b). The collected material was evaluated in Israel, and 30 acces- sions showed high ability for flowering and seed production, with ger- mination rates around 90%, and normal seedling development (Kamenetsky et al. 2005). These collections laid the groundwork for large scientific projects and the initiation of garlic hybridization and breeding programs in Israel and other locations.

III. LIFE CYCLE AND THE FLOWERING PROCESS

A. Seed and Seedling Development The seed shape, color, and seedling morphology of garlic are typical of the subgenus Allium (De Mason 1990; Druselmann 1992; Kruse 1992; Shemesh et al. 2008). The weight of 1000 fresh garlic seeds reaches 1.5–2 g, approximately half the weight of bulb onion and leek seeds. The germination process can take several weeks to several months (Etoh and Simon 2002; Shemesh et al. 2008). Scarification, stratifica- tion, and chilling promote germination, while phytohormone treat- ments have only little effect (Etoh and Simon 2002). The germination rate ranged between 20–40% (Etoh 1983b; Etoh et al. 1988; Shemesh et al. 2008) and 90% of viable seeds (Kamenetsky et al. 2004b). The germination of garlic seeds begins with the appearance of a loop‐shaped cotyledon, followed by the initiation of new leaves, and elongation and production of a cylindrical stem‐like structure termed the “false” stem (Shemesh et al. 2008). Limitations to seedling development include weak performance, abnormal morphology, low survivability, and dying at the stage of 2–3 leaves (Etoh 1983b; Pooler and Simon 1994).

B. Annual Life Cycle and Florogenesis The mature bulb of an adult garlic plant is a cluster of lateral cloves, which arise in the axils of foliage leaves (Mann 1952; De Mason 1990; Messiaen et al. 1993). At the end of the growth period, the aboveground

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Low temperatures Short day

Winter

Autumn Spring

Long day

Figure 1.2 Schematic presentation of the annual cycle of vegetatively propagated bolt- ing garlic. Low temperatures support spring elongation of foliage leaves and the flower stem. A long photoperiod enhances bolting and bulb development.

organs dry up, and following bulb maturation, the cloves enter a sum- mer dormancy period (Figure 1.2). After dormancy release and planting in the fall, adventitious roots arise from the base of the clove, and leaf primordia in the apical bud become active, producing characteristic flat leaves. Under suitable envi- ronmental conditions, some varieties bolt and develop inflorescences with flower buds and small bulblets (i.e. topsets) (Mann and Lewis 1956; Takagi 1990; Brewster 1994; Simon et al. 2003; Kamenetsky 2007). In bolting and flowering garlic genotypes, florogenesis consists of four main phases: meristem transition from the vegetative to reproduc- tive stage, scape elongation, inflorescence differentiation, and comple- tion of floral development to anthesis (Etoh 1985; Kamenetsky et al. 2004b) (Figure 1.3). The transition of the apical meristem from a vegeta- tive to a reproductive state occurs during the active growth stage (Kamenetsky and Rabinowitch 2001). An initial elongation of the flower stalk precedes spathe (prophyll) formation and the swelling of the reproductive meristem. This meristem subdivides to form several clusters, each of which gives rise to a number of individual flower pri- mordia (Figure 1.3b and 1.3c). Similar to other Allium species, floral primordia within each cluster (cyme) develop unevenly in a helical order (Qu et al. 1994; Kamenetsky and Rabinowitch 2001; Rotem et al.

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(a) (b) (c) sp (d)

fp vm

lp t fp sp

(e) (f) (g) (h)

Figure 1.3 Stages of reproductive development in flowering garlic (adapted from Rotem et al. 2007). (a) Vegetative meristem (vm) produces leaf primordia (lp), six weeks after planting. Scanning electron microscopy (SEM) image. Bar = 0.5 mm. (b) Inflorescence meristem, nine weeks after planting. Differentiation of first flower primordia (fp) is visible. Spathe (sp) removed. SEM image. Bar = 0.5 mm. (c) Inflorescence meristem pro- duces flower primordia (fp), 12 weeks after planting. Spathe (sp) removed. SEM image. Bar = 1 mm. (d) Differentiation of topsets (arrows) following flower differentiation. Bar = 1 mm. (e) Topset formation in the inflorescence. Flowers are squeezed and eventually aborted. Bar = 2 cm. (f) Fully differentiated inflorescence after spathe opening. Bar = 2 cm. (g) Seed setting in garlic hybrid with full capacity of seed production. Bar = 2 cm. (h) Garlic seed. SEM image. Bar = 0.5 mm.

2007). In most garlic genotypes, elongation of the floral pedicels is accompanied by quick differentiation of new meristematic vegetative domes, developing into small inflorescence bulbils (topsets) (Figure 1.3d) (Etoh 1985; Qu et al. 1994; Kamenetsky and Rabinowitch 2001; Rotem et al. 2007). The topsets are interspersed with young flowers and physi- cally squeeze the developing floral buds, thus causing their degenera- tion (Figure 1.3e). Therefore, in some garlic clones, perpetual removal of the developing topsets resulted in the development of a number of normal flowers, some of which produced viable pollen and seeds (Konvicka 1984; Etoh et al. 1988; Pooler and Simon 1994; Jenderek 1998; Jenderek and Hannan 2000; Kamenetsky and Rabinowitch 2002; Simon and Jenderek 2004). In flowering genotypes, a fully developed inflorescence consists of about 100 acropetal cymes, each made up of five to six flower buds and/ or open flowers (Figure 1.3f). Further seed development and ripening (Figure 1.3g) are associated with the location of the flower within the cyme and with the location of the cyme in the inflorescence. In different

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genotypes, the development of an individual flower from color break to senescence takes 15–25 days, while seed maturation occurs about one month after fertilization (Qu et al. 1994; Shemesh‐Mayer et al. 2013).

C. Environmental and Genetic Control of Flowering As in many other Alliums (Kamenetsky and Rabinowitch 2002, 2006), the environment plays a major role in garlic development. In bolting garlic clones, florogenesis is differentially regulated by photoperiod and temperature. Therefore, information on the interactions between genotype and environment might enable fertility restoration as well as effective seed production in different genotypes (Kamenetsky et al. 2004a; Mathew et al. 2011). Low temperature (vernalization) is the main factor affecting flower- ing in garlic. In general, vernalization is the induction of a flowering process by exposure to the prolonged cold of winter, or by an artificial cold treatment. Many plant species require vernalization in order to acquire the ability to flower. In the major cultivated Allium crops, including bulb onion, shallot (A. cepa L.) (Rabinowitch 1985, 1990; Krontal et al. 2000), chives (A. schoenoprasum L.) (Poulsen 1990), and Japanese bunching onion (A. fistulosum L.) (Inden and Asahira 1990), vernalization is required for floral induction. Similarly, in bolting gar- lic, cold storage of cloves prior to planting promotes the transition of the apical meristem from the vegetative to the reproductive stage with subsequent leaf and scape elongation and spathe breaking (Takagi 1990; Kamenetsky et al. 2004a; Rotem et al. 2007; Wu et al. 2015, 2016). Bolting garlic genotypes vary in cold requirements and in the number of days from planting to meristem transition and to elongation of flower stalks (Mathew et al. 2011). However, in the semibolting Israeli cultivar ‘Shani’, adapted to warm Mediterranean conditions, low storage tem- peratures inhibited meristem transition to flowering and promoted fast bulbing after planting, indicating the considerable genotype variation in plant response to environmental factors (Rohkin‐Shalom et al. 2015). Following meristem transition, flower differentiation and further development of the inflorescence are affected by growth temperatures (Shemesh‐Mayer et al. 2015a) (Figure 1.4). Study of the development of a fertile genotype under controlled conditions indicated that plant exposure to a sequence of moderate (22/16 °C day/night) and then warm (28/22 °C day/night) temperatures enhanced the differentiation of many intact flowers and viable anthers, while continuous exposure to moder- ate or relatively low temperatures during the entire growth period

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Many flowers with degenerated anthers 34/28°C

Many degenerated 28/22°C anthers

22/16°C

16/10°C 16/10°C

Flower initiation Bolting Spathe break Flowering Intact flowers

22/16°C 22/16°C 22/16°C 22/16°C Massive flower abortions

28/22°C Intact flowers 28/22°C

34/28°C 34/28°C Many flowers, Flower abortions degenerated anthers degenerated anthers

Figure 1.4 Effect of temperature regime on development of the reproductive organs in garlic. Plants were exposed to different growing temperatures at the stage of bolting or at the stage of spathe breaking. Alterations in temperature regime resulted in a var- ied response with respect to the inflorescence structure and number of viable flowers. Combinations of the controlled day/night temperatures are: low (16/10 °C), intermediate (22/16 °C), warm (28/22 °C), and hot (34/28 °C). (Source: Adapted from Shemesh‐Mayer et al. 2015a.)

resulted in the development of topsets in the inflorescence, massive flower degeneration, anther abortion, and reduced pollen production. Dense and viable inflorescences were promoted by outdoor growth conditions during winter and spring in Israel. It was proposed that since natural habitats at the center of the origin of garlic, in Central Asia, are characterized by a gradual warming during the growing sea- son (Hanelt 1990; World Weather Online 2014), such conditions are optimal for floral and pollen development (Shemesh‐Mayer et al. 2015a). However, bolted plants exposed to a sudden increase in tem- perature responded by a reduction in time to spathe opening and anthe- sis. Similar to other plant species (Erwin 2006), high‐temperature stress considerably shortened the period assigned for microsporogenesis, fer- tilization, and seed set in garlic (Shemesh‐Mayer et al. 2015a; Figure 1.4). Another important environmental factor is photoperiod. Photoperiodic signals are translated in plants into internal signals and to changes in the

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hormone profile. A long photoperiod (LP) often enhances endogenous gibberellin levels, with subsequent transition to florogenesis (King et al. 2006). Allium crops, including Chinese chives (A. tuberosum L.) (Saito 1990), leek (A. ampeloprasum L.) (Van der Meer and Hanelt 1990; De Clercq and Van Bockstaele 2002), and rakkyo (A. chinense G. Don) (Toyama and Wakamiya 1990), require LPs for inflorescence initiation and differentiation. In garlic, an LP promotes the elongation of the scape, but an extended LP also promotes topset development in the inflores- cence (Kamenetsky et al. 2004a). Garlic genotypes of different biomorphological groups are differen- tially affected by environment in regard to florogenesis and bulbing, suggesting that competition for resources by the bulb, topsets, and flowers varies among genotypes (Mathew et al. 2011; Figure 1.5). A combination of low storage and growth temperatures with LPs can pro- mote elongation of the flower stalk, while warm temperature combined with LPs led to the degeneration of the developing inflorescence and early bulbing. A short photoperiod (SP), interrupted with a one week LP, enhanced scape elongation and flower differentiation, supporting the concept of environmental manipulation as a tool for fertility resto- ration (Kamenetsky et al. 2004a; Mathew et al. 2011). To understand the genetic mechanisms of garlic development, two main strategies of knowledge transfer from model to nonmodel plants might be employed: (i) creating large‐scale transcriptome profiling and correlating the phenotype to the expression pattern and to specific genes, and (ii) searching for conserved candidate genes of known molecular pathways and their functions in the nonmodel crops (Leeggangers et al. 2013). Both strategies have been used in garlic. An initial search for the specific genes involved in the control of flowering in garlic resulted in the identification of gaLFY – a homolog to the key genes in flower development: LFY from Arabidopsis and FLO from Antirrhinum majus L. (Coen et al. 1990; Weigel et al. 1992; Rotem et al. 2007). In Arabidopsis, LFY is expressed during the development of flo- ral meristems and activates a group of floral‐organ identity genes within the flower (Krizek and Fletcher 2005; Moyroud et al. 2010). Similarly, an expression of LFY/FLO homologs during flower initiation and dif- ferentiation has been shown in many different plant species (Mouradov et al. 1998; Shu et al. 2000; Shitsukawa et al. 2006). In some plants, such as Eucalyptus (Southerton et al. 1998), apple (Wada et al. 2002), and maize (Bomblies et al. 2003), the presence of two differentially expressed LFY homologs was reported. In garlic, gaLFY was identified as a single‐copy gene with the two transcripts generated by alternative splicing (Rotem et al. 2007).

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0

1 1 0

1 1 0 0

11

1

1 1

Figure 1.5 Effect of photoperiod on inflorescence development in eight bolting geno- types. Three photoperiod treatments were applied: (1) a natural photoperiod in Israel, (2) interruption by a long photoperiod of 16 hours for 10 days, and (3) interruption by a long photoperiod of 16 hours for 30 days. Note the complete absence of an inflorescence in accessions #2509, #3026, and #2085 under the long photoperiod applied for 30 days. (Source: Mathew et al. 2011, with permission.)

The accumulation of gaLFY is associated with reproductive organs; it increases during florogenesis and gametogenesis in garlic, while it is downregulated in vegetative meristems and in topsets (Rotem et al. 2011). The transcripts of the gene are differentially expressed during inflorescence development and florogenesis, suggesting the involve- ment of gaLFY in different stages of sexual reproduction, similar to the

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Flower transition Flower differentiation Gametogenesis

Vegetative buds Individual Vegetative Reproductive Initiation of Differentiation differentiate in flower meristem meristem flower primordia of flowers inflorescence

Phase I Phase II Phase III

Figure 1.6 Ups and downs in flowering key gene gaLFY expression during florogenesis of fertile garlic. Red color marks gaLFY expression. Phase I: mRNA is detected during meristem transition from the vegetative to the reproductive phase. Phase II: Following initiation of the individual flower primordia,gaLFY is downregulated in the inflores- cence meristem and then expressed again during organ differentiation in the individual flowers. Phase III: The third peak is detected in the anthers and ovules of the fully differ- entiated mature flowers. Source:( Rotem et al. 2011, with permission.)

LFY homolog NFL in Narcissus tazetta (Noy‐Porat et al. 2010; Rotem et al. 2011). Thus, a specific increase in gaLFY expression was docu- mented during meristem transition to the reproductive phase, during the differentiation of individual flowers, and in the matured anthers and ovules (Rotem et al. 2011; Figure 1.6). Another group of genes, strongly associated with the reproductive process, belongs to the FLOWERING LOCUS T (FT) family, found in numerous model and crop plants. In onion, the induction of flowering and bulbing are tightly connected, and both processes are regulated by the genes of the FT family (Lee et al. 2013). Flowering promotion by vernalization in A. cepa is associated with upregulation of AcFT2, whereas bulb formation is regulated by the interaction of two antago- nistic FT‐like genes, AcFT1 and AcFT4. LP promotes the upregulation of AcFT1 and the downregulation of AcFT4. Four isoforms of FT, iden- tified in the garlic transcriptome, demonstrate high homology to the onion FT‐like genes and might similarly regulate developmental pro- cesses in garlic. FT‐like genes exhibited different expression patterns in garlic inflorescences, flowers, roots, basal plate, and cloves, suggesting the strong link between flowering and bulbing processes (Kamenetsky et al. 2015). Although FT genes are conserved in plant species, their expression and function in bulb formation, leaf elongation, and floral

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transition of the apical meristem can vary in species and even in cultivars that have adapted to different environmental conditions. Thus, in the short‐day Mediterranean garlic cultivar ‘Shani’, the induction of AsFT1 in the internal bud occurred under cold storage conditions, while the antagonistic AsFT4 was induced by warm‐ temperature storage. At the same time, the expression of AsFT2 was higher at cold versus warm storage temperatures in the internal bud and storage leaf (Rohkin‐Shalom et al. 2015). Transcriptome catalogs have been initially generated from garlic renewal buds (Sun et al. 2012). Later, deep transcriptome sequencing of roots, stems, leaves, and bulbs resulted in de novo assembly of 135 000 unigenes, with more than 50 000 unigenes being annotated. The authors were able to develop over 2000 simple sequence repeats (SSRs) that can be used for genetic studies, mapping, and fingerprinting (Liu et al. 2015). A comprehensive transcriptome catalog of fertile garlic was pro- duced by using multiplexed gene libraries based on RNA collected from several plant organs, including inflorescences and flowers (Kamenetsky et al. 2015). More than 32 million 250‐bp (base pairs) paired‐end reads were assembled into a broad transcriptome, contain- ing 240 000 contigs. Further analysis allowed the production of an edited transcriptome of 102 000 highly expressed contigs. This tran- scriptome catalog was annotated and analyzed for gene ontology and metabolic pathways. Organ‐specific analysis displayed significant vari- ation in gene expression between different plant organs, while the highest number of specific reads was found in the inflorescences and flowers (Figure 1.7). The de novo transcriptome catalogs of various gar- lic genotypes provide a valuable resource for research and breeding of this important crop, as well as for the development of effective molecu- lar markers for useful traits, including fertility and seed production, resistance to pests, and nutraceutical characteristics. Transcriptome analysis showed that the floral‐induction pathways in garlic are similar to those of model plants (Tremblay and Colasanti 2006; Tsuji et al. 2011). Orthologs of some of the key flowering genes [CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY), APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), PISTILLATA (PI), SEPALLATA1 (SEP1), SEPALLATA3 (SEP3), and AGAMOUS (AG)] were differentially expressed in reproductive tissues, leaves, and bulbs, proposing their role in both flower signal transduction and the bulbing process of garlic (Kamenetsky et al. 2015). In addition, the flowering genes of Arabidopsis, GI (GIGANTEA), FKF1 (FLAVIN‐BINDING), and ZTL (ZEITLUPE), asso- ciated with photoperiodic requirements, were found to be conserved in

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Roots 9080 Clove 21,862 2971 5578 11,638 Complete garlic Rice 10638 2164 3093 2280 2448 transcriptome database 2669 10591 1545 1137 2968 47,500 1533 2476 1946 125,436 23805 6991 849 5222 54,542 Reference garlic 5569 3909 1511 1460 transcriptome 4631 17201 2939 3837 68101 6439 6612 Reproductive Basal plate organs (c)

Color key

–5 05 Value

1

2

3

4 s s ots ve ve wers Ro Clo Lea Flo lorescence Basal plate Inf

Figure 1.7 De novo generation of transcriptome of fertile garlic. The sequencing data were deposited in the NCBI Sequence Read Archive (SRA) database as bioproject PRJ- NA243415 (Source: Kamenetsky et al. 2015). (a) Venn diagram of the distribution and similarity of sequences in extensive and abundant transcriptome catalogs of garlic in comparison with a rice protein database (www.phytozome.org). (b) Common and specific contigs found in the extensive transcriptome catalog of the various organs of fertile garlic. Note the high number of specific contigs in the reproductive tissues. (c) Hierarchical cluster analysis of gene expression patterns in six vegetative and reproductive organs of garlic. The heat map shows the relative expression levels of each contig (rows) in each sample (columns). Four identified gene clusters (shown in the left tree) are differentially expressed in one or more organs. Organs are clustered to reproductive and vegetative, with closer proximity between the roots and basal plates (upper tree).

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onion (Taylor et al. 2010). These genes are involved in both flowering and daylength‐dependent bulb initiation, and might also regulate similar processes in garlic.

I V. FERTILITY BARRIERS

In bolting garlic genotypes, the inflorescence can produce numerous flowers, usually mixed with topsets. However, even if an individual flower exhibits normal morphological development, it still might be sterile and incapable of seed production. Flower sterility can be caused by various factors, from anatomical malformations to genotype × envi- ronment interactions. This section summarizes research on mecha- nisms of floral sterility in garlic.

A. Morphology and Anatomy of the Individual Flower In the garlic flower, each perianth lobe and the subtended stamen arise simultaneously from a single primordium, as in bulb onion (Jones and Emsweller 1936; Esau 1965; De Mason 1990), shallot (Krontal et al. 1998), and other Allium species (Kamenetsky and Rabinowitch 2002). The flower has six perianth lobes and six stamens, and the carpels are initiated, within the inner whorl, when the outer perianth lobes over- arch the stamens (Etoh 1985; Kamenetsky and Rabinowitch 2001). In fertile clones, after spathe opening, the anthers develop purple color, the filaments elongate, the anthers open, and the pollen is released (Jenderek 2004; Shemesh‐Mayer et al. 2013). Most fertile garlic clones develop purple anthers at anthesis, although in some genotypes, the anthers lack the purple pigmentation (Hong and Etoh 1996; Etoh and Simon 2002; Jenderek 2004; E. Shemesh‐Mayer, personal observations). The ovary, composed of six ovules, turns from green to dark purple at anthesis. The style elongates beyond the anthers, and the stigma becomes receptive (Shemesh‐Mayer et al. 2013, 2015a). The garlic flower is characterized by protandry, where the stigma’s receptivity increases only when the anthers are withered (Shemesh‐Mayer et al. 2013). The protandry mechanism is known in other Alliums, including onion, Japanese bunching onion, leek, and chives (Trofimec 1940; Currah 1990). Although the gradual development of flowers within the same inflorescence enables pollination between flowers in the same Allium plant (Currah and Ockendon 1978), protandry encourages outcrossing, thus limiting inbreeding depression. It is known that inbreeding depression reduces seed production and germination rate,

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and promotes morphological defects and low plant vigor in the field. These observations were reported in A. cepa (Jones and Davis 1944; Pike 1986), A. porrum (Berninger and Buret 1967; Gray and Steckel 1986), A. fistulosum (Moue and Uehara 1985), and garlic (Hong and Etoh 1996; Jenderek 2004).

1. The Male Gametophyte. The developed anther of garlic comprises the epidermis, the endothecium, a middle layer, and a secretory tape- tum. The microspore mother cells (MMCs) undergo meiosis, followed by the release of the microspores out of the callose wall into the locular space of the pollen sac. Later, following mitotic divisions and pollen maturation, the tapetum disintegrates and the endothecium cells stretch prior to stomium opening, dehiscence, and pollen release (Etoh 1983a,b, 1985, 1986; Hong and Etoh 1996; Shemesh‐Mayer et al. 2013, 2015a). A similar pattern of pollen development was also reported for other Alliums, including A. cepa (Holford et al. 1991a), A. triquetrum (Garcia et al. 2006), A. schoenoprasum (Engelke et al. 2002), A. mongolicum Regel (Wang et al. 2010), and A. senescens L. (Liu et al. 2008). In general, phenotypic expression of male sterility varies, from the entire absence of male organs, interference in the meiotic process, pollen abortion, and lack of anther dehiscence to the failure of viable pollen to germinate on the stigma. Usually, in genetically male‐sterile plants, the female functions remain intact (Budar and Pelletier 2001). Three types of inherited male sterility are known in plants: genetic male sterility (GMS), cytoplasmic male sterility (CMS), and cytoplasmic‐genetic male sterility (CGMS). GMS is controlled by two dominant genes (Ms and Rf): Ms is sterile, while Rf is fertile and has dominant epistasis over Ms (Shilling et al. 1990). Maternally inherited CMS is controlled by extra- nuclear genetic control, frequently associated with unusual open read- ing frames (ORFs) in the mitochondrial genome (Hanson 1991; Schnable and Wise 1998). An interaction between CMS and GMS (CGMS) was identified in bulb onion in 1925 (Jones and Emsweller 1937; Jones and Clarke 1943; Havey 1993, 1995) and subsequently in the bunching onion (Nishimura and Shibano 1972; Moue and Uehara 1985) and in chives (Tatlioglu 1982). Since male sterility is important in breeding and seed production, this trait was studied in various Allium crops: onion (Havey 2000, 2002; Kik 2002; Engelke et al. 2003), chives (A. schoenoprasum) (Engelke and Tatlioglu 2000), leek (A. ampeloprasum) (Havey and Lopes Leite 1999), and bunching onion (A. fistulosum) (Yamashita et al. 2010). In onion, two sources of CMS were defined: S cytoplasm (Jones and Clarke 1943), which is stable under wide envi- ronmental conditions and where female fertility is retained, and thus is

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used in most hybrid‐onion cultivar production (Havey 1995); and T cytoplasm (Berninger 1965; Schweisguth 1973). Abnormalities in onion plants containing S cytoplasm include irregular tapetum development prior to microspore abortion, while T cytoplasm plants exhibit irregu- lar development during meiosis (Monosmith 1928; Tatebe 1952; Peterson and Foskett 1953; Yen 1959; Dyki 1973; Patil et al. 1973). Further studies (De Courcel et al. 1989; Holford et al. 1991b; Havey 1993, 1995; Satoh et al. 1993; Sato 1998) classified normal (N) male‐fer- tile and S cytoplasm in the organellar genome of onion. Since emascu- lation of Allium flowers is practically impossible, male sterility is extremely important in seed production of hybrid varieties. The main types of garlic male sterility are characterized by disruption of the male organs and gametes at different developmental stages (Jenderek 2004; Shemesh‐Mayer et al. 2013) (Figure 1.8). In the com- pletely sterile garlic (Figure 1.8, Type 1), microsporogenesis ceases after the stage of meiosis, when the callose walls of the tetrads degrade due to a change in the activity of the callase (b‐1,3‐D‐glucanase) (Winiarczyk et al. 2012). This type is characterized by metabolic disturbances in the callose wall (Tchórzewska et al. 2017), the absence of a normal cortical cytoskeleton, and the dramatically progressive degeneration of cyto- plasm in the pollen mother cells (Tchórzewska et al. 2015). The abor- tion of the microspores in Type 1 is also associated with high levels of protease and acid phosphatase activity and a lower level of ester- ase activity in anther locules (Winiarczyk and Gębura 2016). It was

Complete sterility Male sterility Male sterility Female Fertility Type 1 Type 2 Type 3 sterility

Filament impediment Anther Pollen Non-receptive stigma Anther degeneration degeneration sterility Ovule abortion Pollen degeneration Ovule abortion Stigma non-receptive

Figure 1.8 Fertility barriers in individual garlic flowers. Fertility was impaired in differ- ent flower organs (e.g. non‐elongated filaments, anther degeneration, sterile pollen, and nonreceptive ovule and/or stigma). (Source: Adapted from Shemesh‐Mayer et al. 2013.)

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suggested that male sterility might be caused by irregular chromosome pairing, multivalents, and chromosomal deletions (Takenaka 1931; Katayama 1936; Etoh 1979, 1980, 1985; Pooler and Simon 1994). In the male‐sterile genotypes of Type 2 (Figure 1.8) (Shemesh‐Mayer et al. 2013), following meiosis, the microspores release from the tetrads, but then degeneration of the anthers occurs. The anthers exhibit enlarged tapetal cells, and pollen differentiation is arrested when postmeiotic microspores release from the callose wall (Novak 1972; Etoh 1979, 1980; Gori and Ferri 1982) (Figure 1.9). Abnormal tapetal development,

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 1.9 Comparative developmental anatomy of anthers in the fertile (a–d) and male‐sterile (e–h) garlic genotypes during microgametogenesis. Comparisons were made between early, mid, and late stages of flower development Source:( Shemesh‐Mayer et al. 2015b). (a) Cross section of pollen sac at the tetrad stage. Bar = 40 μm. (b) Longitudinal section of pollen sac after microspore (arrow) release from the callose. Endothecium (et) and tapetum (t) are visible. Bar = 60 μm. (c) Longitudinal section of an anther with mature microspores (arrow) that contain vegetative and generative cells. Tapetum (t) is degener- ated, and only remains are visible. Bar = 30 μm. (d) Mature flower of fertile genotype. Long filaments, dehisced anthers (a), and long style are visible. Bar = 1.5 mm. (e) Longitudinal section of pollen sac at the tetrad (arrow) stage. Typical tapetum (t) cells are visible. Bar = 30 μm. (f) Cross section of an anther, with microspores released from the callose. Note hypertrophy of the tapetum (t) cells. Bar = 45 μm. (g) Considerable enlargement to the tapetum (t) cells and degenerated microspores (arrow) in a male‐sterile genotype. Bar = 45 μm. (h) Mature male‐sterile flower. Degenerated yellow anthers (a) are visible, and the style is elongated. Bar = 1.5 mm.

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including hypertrophy and vacuolation, along with microspore degeneration prior to the first mitosis, was documented in the male‐ sterile bulb onion ‘Italian Red’ (Monosmith 1928), the bunching onion (Yamashita et al. 2010), the F1 hybrids of onion and A. pskemense B. Fedtsch (Saini and Davis 1969), as well as Arabidopsis (Chaudhury et al. 1994), rice (Nishiyama 1976), and other plant species. In the male‐sterile genotypes of Type 3, the androecium and the gynoecium exhibit normal development, but the visually normal pollen grains are sterile and do not germinate on the stigma or on an artificial medium (Shemesh‐Mayer et al. 2013) (Figure 1.8).

2. The Female Gametophyte. In flowering garlic genotypes, the mature ovule consists of tissues from both generations of the plant life cycle, the diploid sporophyte and the haploid gametophyte. The female organs usually exhibit normal development and have visu- ally vital ovules, a receptive stigma, and normal setting of seed (Shemesh‐Mayer et al. 2013, 2015a,b). However, in completely degenerated flowers, the abortion of female organs or abnormal for- mation of embryo sacs was observed at the early stages of develop- ment. Asymmetric development of integuments or incapacity of a micropylar channel to facilitate the entrance of the pollen tube might lead to ovule abortion (Etoh 1985; Winiarczyk and Kosmala 2009; Shemesh‐Mayer et al. 2013).

B. Environmental and Genetic Control of Male Sterility In general, male flower organs are more sensitive to environmental stress than female ones (Hedhly 2011). Microgametogenesis, pollen production and dehiscence, and fruit or seed setting can be damaged by temperature stress in different plant species, including Arabidopsis (Kim et al. 2001), rice (Oryza sativa L.) (Matsui and Omasa 2002), tomato (Solanum lycopersicum L.) (Peet et al. 1998), and wheat (Triticum aestivum L.) (Saini et al. 1983, 1984). In most Allium crops, including onion (Jones and Clarke 1943; Van Der Meer and Van Bennekom 1969; Ockendon and Gates 1976) and bunching onion (Yamashita et al. 2010), fertility may be negatively affected by nutrition, diseases, mutations, and an inappropriate growth environment. In fer- tile plants of garlic, intact flowers develop viable anthers under favora- ble temperatures, for example under a sequence of moderate (22/16 °C day/night) and warm (28/22 °C day/night) temperatures. However, a sharp transition to high temperatures (34/28 °C day/night), especially

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Tapetum Unfavorable hypertrophy Empty factors pollen grains

Pollen mother cell Dyad Tetrad Released Uni-cellular Bi-cellular microspore pollen grain pollen grain

Callose Meiosis I Meiosis II Mitosis I

Flower bud Closed flower Closed flower, Pre-anthesis 2.5–3mm green tepals, 4mm pink tepals, 4mm flower, purple tepaIs

Figure 1.10 Microsporogenesis course and pollen abortion resulting from genetic and/or environmental effects. Microsporogenesis stages correspond with flower bud development. Meiotic division of the pollen mother cell is followed by tetrad release and mitotic division, resulting in the bicellular pollen grain. The most vulnerable phase in garlic microsporogenesis is the unicellular microspore stage, whereas the early stages of pollen differentiation are more tolerant to unfavorable conditions. (Source: Adapted from Shemesh‐Mayer et al. 2015a.)

after spathe opening, induced rapid anther senescence, tapetal malfor- mation, and pollen abortion. Pollen degeneration may be induced by low temperatures during the pre‐anthesis stage or by high temperatures during anthesis. Unfavorable conditions cause tapetum hypertrophy and degeneration of the microspores prior to mitotic division, resulting in empty aborted pollen grains (Shemesh‐Mayer et al. 2015a). It was shown that the most vulnerable phase in garlic microsporogen- esis is the unicellular microspore stage, whereas the early stages of pollen differentiation are more tolerant to unfavorable conditions (Figure 1.10). However, in the male‐sterile genotype, pollen production cannot be restored by any favorable growth regime, suggesting that at least some types of male sterility are controlled by genetic mechanisms (Shemesh‐Mayer et al. 2015a). Different molecular techniques have been used for marker‐assisted selection of fertile or male‐sterile garlic genotypes. Random amplified polymorphic DNA (RAPD) analysis indicated polymorphism between pollen of fertile and sterile garlic clones (Etoh and Hong 2001).

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Furthermore, combined analysis of single‐nucleotide polymorphism (SNP), SSR, and RAPD generated 37 markers from a segregated popula- tion (Zewdie et al. 2005). These markers were used to construct a genetic linkage map, composed of nine linkage groups, one of which consisted of the major locus affecting male fertility (mf). Transcriptome analyses of fertile and male‐sterile garlic genotypes allowed the identi- fication of genes and biological processes involved in male gametogen- esis. More than 16 000 genes were differentially expressed between the flowers of fertile and male‐sterile genotypes (Shemesh‐Mayer et al. 2015b). In the fertile genotype, characterized by viable pollen develop- ment, the activity of genes was associated with the development of reproductive tissues (e.g. regulation of meristem structural organiza- tion, floral organ development, regulation of cellular component organ- ization, and sugar metabolism) (Figure 1.11). Real‐time polymerase chain reaction (PCR) validation for the expression analysis of four fer- tility‐related genes (homologs of Arabidopsis APETALA3 (AP3), MALE MEIOCYTE DEATH1‐LIKE (MMD1), MALE STERILITY2 (MS2), and GLYCEROL‐3‐PHOSPHATE ACYLTRANSFERASE2 (GPAT2)) confirmed their higher expression in fertile garlic genotypes in comparison with sterile genotypes. It was proposed that the selected genes are conserved and involved in male fertility regulation (Shemesh‐Mayer et al. 2015b). The MADS‐box transcription factor AP3 controls flower differentiation (Honma and Goto 2001). MMD1 is involved in the formation of pollen exine and cytosolic components, as well as in tapetum development and male meiosis (Ito and Shinozaki 2002; Yang et al. 2003; Ito et al. 2007; Li et al. 2011). MS2 is expressed during exine formation (Aarts et al. 1997; de Azevedo Souza et al. 2009), while members of the GPAT family are involved in pollen development and tapetum viability in Arabidopsis (Zheng et al. 2003). In contrast to fertile genotypes, in the male‐sterile plant the activity of genes was associated with modifications in the mitochondrial genome that affect the functions of anthers, pollen, or male gametes (Figure 1.11). Significantly higher expression of 23 garlic genes with high similarity to known mitochondrial genes (Figure 1.12) suggests that, similar to other higher plants, male sterility in garlic might be caused by the generation of chimeric ORFs in these genes, leading to the interruption of mitochondrial functions, respiratory restrictions, and nonregulated programmed cell death of the tapetum leading to energy deficiency and pollen abortion (Woodson and Chory 2008; Shaya et al. 2012; Chen and Liu 2014; Islam et al. 2014; Shemesh‐Mayer et al. 2015b). This hypothesis was supported by differential expression of three specific mitochondrial genes in the flowers of male‐sterile and

0003960591.INDD 22 9/5/2018 2:27:59 PM 0003960591.INDD 23 stress. (Source: Shemesh‐Mayer et al.2015b.) ile genotype: Main patternsarerelatedto energy‐consumingactivities and/orresponseto genesis andcell‐division processes, and specific fertility-related processes. (b)Male‐ster are relatedtothegeneraldevelopment ofreproductivetissues,metabolism,microsporo - cluster, whilecolorindicatessemantic similarities.(a)Fertilegenotype:Mainpatterns VIGO algorithms.Circlesize isproportionaltotheabundanceofGOterm inthe by transcriptomeanalysis. GOtermdistributionwasperformedusingBlast2GO andRE- Figure 1.11 (b (a) o O rganization responses Stress rganelles ) regulation C ell cy Semantic space Y Semantic space Y –6 –5 –4 –3 –2 –1 Biological processes in fertile and male‐sterile garlic genotypes, as revealed –6 –4 –2 0 1 2 3 4 5 cle 0 2 4 6 modifi Cell wall Semantic space X Semantic space X stress response to Cellular 6– 4– 2–1 –2 –3 –4 –5 –6 cation activity Ca organismal process of multicellula Negative regulation –5–6 talytic To arrangement structure Anatomical xic responses –4 storage Lipid response hypersensitive Plant-type organization Actin filament 3– –1 –2 –3 reticulum stress endoplasmic Response to metabolism Methy r recombination Mito lglyox tic metabolism Disaccharide metabolism Starch Aging al 06 0123456 change conformation DNA senescence Or ga GTP catabolism n transport transmembrane Amino acid transport coupled proton AT biosynthesis Oligosaccharide P hy metabolism Oxylipin Cell divisions to protein localization Es drol membrane metabolism Suga tablishment of ys 54321 is r transpo Active metabolism Amino-acid initiation replication DNA metabolis Cy Glutamine, Seri steine ne, rt s m 9/5/2018 2:28:00PM - 24 E. SHEMESH-MAYER AND R. KAMENETSKY GOLDSTEIN

Color key

–1 0 1 Value 12 3 Late_96

Late_87

Early_96

Mid_96

Early_87

Mid_87 COX2 18S rRNA 18S rRNA 18S rRNA 26S rRNA 26S rRNA Nad7 Nad7 NADH-uo Nad1 Nad5 Nad4 Nad5 Nad3 Nad2 Nad5 Nad7 Nad1 CcmC Nad6 COX2 NADH-uo Nad5 comp54534_c0_seq5 comp51053_c1_seq1 comp51053_c0_seq1 comp53140_c0_seq2 comp14507_c0_seq1 comp15448_c0_seq1 comp18339_c0_seq1 comp15640_c0_seq1 comp58822_c0_seq10 comp32406_c0_seq1 comp34916_c0_seq1 comp56401_c0_seq28 comp58822_c0_seq6 comp56960_c0_seq10 comp53958_c0_seq3 comp58822_c0_seq15 comp53958_c1_seq5 comp46638_c0_seq3 comp66781_c0_seq1 comp36780_c0_seq2 comp56960_c0_seq12 comp16875_c0_seq1 comp58822_c0_seq16

Figure 1.12 Hierarchical cluster analysis of the expression patterns of 23 genes with high similarity to the published sequences of plant mitochondrial genes at three stages of flower development for fertile (#87) and male‐sterile (#96) garlic genotypes. The relative expression levels of each gene (column) in each sample (row) are shown. (Source: Shemesh‐ Mayer et al. 2015b.)

fertile garlic genotypes. The association between mitochondrial func- tions and male sterility in garlic is still awaiting further investigation.

V. UNLOCKING VARIABILITY BY SEXUAL REPRODUCTION

A. Morphological Variability in Seedling Populations Open pollination of fertile garlic genotypes resulted in large popula- tions of garlic seedlings. These populations demonstrated large varia- tion in many vegetative and reproductive traits, including germination

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(a) (b)

(c)

Figure 1.13 Variability in garlic bulbs obtained after the first growing season of a garlic seedling population. Bar = 1 cm. (a) Single‐clove bulbs; (b) multicloved bulbs; (c) bulbing ability varies significantly within the seedling population. Source:( Adapted from Shem- esh et al. 2008.)

and growth rates; number of foliage leaves prior to transition to the reproductive phase; ability for secondary growth (production of sec- ondary axillary branches); flowering, development of the inflorescence, pollen viability, and seed production; and bulbing ability, bulb matu- rity, earliness, and lateness. During the first growing season, seedlings develop 2–12 leaves (Etoh et al. 1988; Pooler and Simon 1994; Jenderek 1998; Kamenetsky et al. 2004b; Shemesh et al. 2008), but only 15% of the population produce reproductive organs, usually exhibiting weak inflorescence performance and flower degeneration (Shemesh et al. 2008). Under natural conditions in Israel, the first year of seedling development ends with the formation of 0.5–2 cm diameter single or cluster bulbs, varied in skin color (white, purple, gray, and brown), bulbing ability, and ripening date (Kamenetsky et al. 2004b; Shemesh et al. 2008) (Figure 1.13). The second year of development from bulbs originating from seeds is similar to that described for vegetatively propagated garlic plants (Abdalla and Mann 1963; Brewster 1987, 1994; De Mason 1990; Kamenetsky and Rabinowitch 2001; Shemesh et al. 2008). Plants

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(a) (b) (c)

(d) (e) (f)

Figure 1.14 Variability in inflorescence performance in garlic population originated from seeds. Bar = 1 cm. (a) Many green sprouting leaves and a few topsets; (b) many small topsets and a few developing flowers, later aborted; (c) many large topsets, with flowers aborted at early stages of differentiation; (d) only two–three large topsets, with no flowers; (e) normal flowers are mixed with topsets; and (f) numerous normal flowers and a few small topsets. (Source: Adapted from Shemesh et al. 2008.)

develop 9–14 foliage leaves, and 80% of them are able to produce flower stalks and vary largely in inflorescence performance (Etoh 1997; Shemesh et al. 2008) (Figure 1.14). Under natural conditions in Israel, 20% of the bolting plants within a seedling population developed fer- tile flowers (Shemesh et al. 2008). The newly formed bulbs differ in size, weight, color, shape, clove number, sulfur compound concentration, dry matter content, and response to environmental conditions (Etoh 1997; Jenderek 2004; Kamenetsky et al. 2004b; Jenderek and Zewdie 2005; Shemesh et al. 2008). In addition, seedling populations possess important traits, such as disease resistance (e.g. tolerance to rust; Puccinia allii) (Jenderek and Hannan 2004). This biological variability of seedling populations is comparable to the global variability within clonally propagated garlic (Kamenetsky et al. 2004a; Shemesh et al. 2008). New variation within seedling populations is now available for breeding, and will allow the

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development of new and better cultivars adapted to a variety of climates and to different production conditions (Etoh and Simon 2002; Jenderek and Hannan 2004; Kamenetsky 2007).

B. Environmental Regulation of Seedling Development Seedling populations are highly variable, and, therefore, individual plants within those populations differ significantly in their reactions to environmental factors. In general, cold storage prior to planting of one‐ year‐old bulbs that originated from seeds promoted the transition of the growing plants to reproductive development (Shemesh et al. 2008). Moreover, in some genotypes, pre‐planting cold storage induced bul- bing, secondary sprouting of axillary buds, and earlier leaf senescence. In contrast, pre‐planting storage in warm temperatures inhibited flower stem elongation and caused inflorescence abortion at an early develop- mental stage (Shemesh et al. 2008). Similar to bulb onion (Sinnadurai 1970a,b; Rabinowitch 1990), garlic genotypes vary in their cold require- ments. Thus, several genotypes were able to flower without cold treat- ment, but others did not bolt even in response to cold storage for eight weeks, possibly due to suboptimal induction (Shemesh et al. 2008). In the future, the effects of pre‐planting and growth temperature, as well as photoperiod, on garlic performance should be considered by breed- ers for tropical, subtropical, and temperate zones, where environmental conditions differ significantly (Shemesh et al. 2008).

C. Molecular Markers in Variable Garlic Populations In the past, the genetic heterogeneity of clonal garlic collections was estimated using isozyme analysis, RAPD and AFLP (Maaß and Klaas 1995; Garcia Lampasona et al. 2003, 2012; Ipek et al. 2003), and SSRs (Ma et al. 2009; Cunha et al. 2012; Liu et al. 2015). Assessment of garlic diversity with isozymes and RAPD markers generally agrees with mor- phological observations, but fails to discriminate clones. The introduc- tion of AFLP techniques facilitated evaluation of the genetic diversity in garlic collections and gene banks. For example, AFLP techniques were used for the genetic analysis of 211 garlic accessions available through the US Department of Agriculture’s (USDA) National Plant Germplasm System (NPGS) and from commercial growers (Volk et al. 2004). In spite of extensive duplications within the surveyed acces- sions, AFLP analyses revealed substantial diversity that is largely con- sistent with major phenotypic classes. SSR markers benefit from their

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high abundance and polymorphism, as well as their co‐dominant heredity and ease of use. In comparison with AFLP or RAPD, SSRs are very variable and thus can be used in assessing genetic variability within clonal collections. Initial studies produced 8 and 16 SSRs in collections of 90 and 394 garlic accessions, respectively (Ma et al. 2009; Cunha et al. 2012). Furthermore, 1506 SSR markers were produced out of 135 360 Expressed Sequence Tags (ESTs), thus providing the basis for effective genetic analysis, mapping, identification of quantitative trait loci (QTL), and fingerprinting (Liu et al. 2015). Recent studies indicate the possible use of microRNAs (miRNAs) as molecular markers for disease resistance (Chand et al. 2016, 2017). In general, small RNAs (sRNAs) are involved in the mechanisms of plant defense to biotic stresses by negatively regulating gene expression via silencing processes (Yang and Huang 2014). In plants, most of the sRNAs are short interfering RNAs (siRNAs) and miRNAs. Both are 20–24 nucleotides in size and have different structures, biological pathways, and activity. Plant miRNAs regulate the nucleotide bind- ing site leucine‐rich repeat (NBS‐LRR) proteins, which can recognize the presence of pathogen effectors, trigger cellular changes that cause rapid cell death, and eventually restrict the growth of the path- ogens (Yang and Huang 2014). It was found that miRNAs are involved in the response of garlic to Fusarium oxysporum f. sp. cepae (FOC), which causes fusarium basal rot, a severe disease resulting in 60% loss of garlic yield (Chand et al. 2016, 2017). A total of 28 NBS sequences were isolated in an FOC‐resistant garlic genotype, based on the NBS conserved motif of NBS‐LRR resistance proteins (Rout et al. 2014). This pioneering research promotes new approaches toward the use of molecular markers for identifying disease resist- ance in garlic. Modern molecular tools, such as RAPD, AFLP, SSR, NBS‐profiling marker technology, SNP, and insertion–deletion (indel), have already been applied to the detection of molecular markers in garlic popula- tions generated from seeds. This approach has yielded markers for alii- nase, chitinase, sucrose 1‐fructosyltransferase (SST1), and chalcone synthase (CHS) (Ipek et al. 2005); was used for the construction of a genetic linkage map, including the major locus affecting male fertility (mf) (Zewdie et al. 2005); and was used in evaluations of genetic vari- ability, including identification of identical lines in garlic collections (Havey and Ahn 2016). Once the fertility barriers of garlic were eliminated and variability was unlocked, novel tools for marker development became available. Current goals include the search for fertile parents with useful quality

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traits and disease resistance. These parents will be introduced into hybridization processes for the generation of segregating populations and for further phenotyping and genotyping of the progeny.

VI. CONCLUDING REMARKS

Thousands of years of active selection for larger bulbs resulted in the loss of garlic fertility, and therefore modern garlic cultivars are com- pletely sterile and are propagated only vegetatively. The discovery of fertile garlic genotypes in Central Asia in the 1980s, restoration of fertility in bolting plants, acquisitions of new variations in useful traits, and establishment of transcriptome catalogs have opened new ways for in‐depth physiological, genetic, and molecular research in garlic and have provided the ground for modern breeding programs in this crop. The main objectives of garlic breeding and selection include smooth, round bulbs; an even distribution of clove size within the bulb; an even skin and flesh color; and genetic resistance to viruses and diseases (Messiaen et al. 1993). Massive seed propagation of garlic is already exploited in plant breeding for improvement of yield, seed germina- tion, seedling vigor, tolerance to biotic and abiotic stresses, disease resistance, and quality traits. These efforts will be combined with the utilization of new technologies for gene transfer, which are expected to facilitate the integration of useful agronomic and quality traits into new garlic varieties.

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