Haberlandt’s Dogmatic Dream and Its First Realisation

As vividly described in the review by Vasil (2008), the alleged friendship between the botanist Matthias Jakob Schleiden (1804–1881) and the animal physiologist Theodor Schwann (1810–1882) stimulated, among others, the formation of the “cell theory”. Schleiden (1838) was the first to formulate the hypothesis that all plant or animal structures are composed of cells (or their derivatives) that preserve the complete functional potential of the organism.

Cell Biology in Plant Propagation and Breeding

Self-repair of individual somatic cells is an almost universal property of multicellular organisms, both plants and animals. This ability is necessary to allow continuous replacement of cells lost through senescence or damaged by wounding. In both lower animals and most plants, the regeneration process can lead to the formation of new organs. In plants particularly, various regeneration strategies have culminated in mechanisms of vegetative propagation that either complement or even entirely substitute sexual propagation.

From Neˇmec and Haberlandt to Plant Molecular Biology

The high regenerative capacity of plants is a crucial feature of their life strategy. It is an essential part of the mechanisms that both allow these sessile organisms to repair injury caused by pathogens, herbivores and abiotic factors and to undergo rapid vegetative reproduction, so allowing them to dominate in particular environmental niches. Furthermore, various forms of natural regeneration contribute to techniques that are widely used in plant propagation and plant breeding. The biological nature of plant regeneration has been studied since the very beginnings of plant physiology as a science. Research on regeneration of intact plants in vivo was conducted by Bohumil Ne ˇmec, and early studies of in vitro regeneration in plant tissue cultures were carried out by Gottlieb Haberlandt. At this stage, however, suggestions that somatic plant cells possessed a regeneration “totipotency” were in practice often not acknowledged. Nevertheless, real experiments demonstrated that the regenerative ability of particular cells and tissues is clearly determined by the specific interplay of both genetic (or epigenetic) and physiological factors. This makes some systems “nonresponsive” to the standard regeneration procedures.

This regenerative recalcitrancy hampers both the routine vegetative propagation of various plant species and the construction of genetically modified crops. This chapter addresses the basic historical background of studies on plant regeneration and discusses both the results and ideas acquired by means of classical anatomical and morphological studies in the light of our current state of information obtained using modern molecular techniques. The present knowledge of plant regeneration is also viewed in the light of studies of structure and function of the “stem cell niches” of multicellular organisms, examining their role in the ontogenesis of intact plants and in the processes of embryogenesis and organogenesis in vitro. With reference to other chapters in this book, the role of genetics for the realisation of these processes as well as the role of various regulatory factors, of both exogenous and endogenous nature – especially phytohormones – is also examined. The importance to classify regenerative processes unambiguously using exact terminology (in the context of the allied field of regenerative medicine) as a prerequisite for the formation and validation of appropriate working hypotheses is discussed. Finally, this chapter summarises the main problems of current research on regenerative processes in plants and outlines possible directions for solving problems of recalcitrant materials in the context of their use for application.

Insertional Mutagenesis Using T-DNA Tagging

A soil bacterium, Agrobacterium, transfers the T-DNA containing genes that encode the proteins involved in biosynthesis of plant growth factors and bacterial nutrients. Because none of the genes carried by the T-DNA are required for the transfer, foreign DNA can be inserted into the plant chromosome by putting the sequences between two T-DNA borders and using the Agrobacterium transfer system. Utilising this naturally occurring property, efficient transformation protocols have been developed for Arabidopsis (Bechtold et al., 1993) and rice (Hiei et al., 1994). It is now feasible to rapidly generate the thousands of transformants necessary for investigating genome-wide mutagenesis (Clogh and Bent 1998; Jeon et al., 2000).

Plant Science Technologies to Improve Agriculture

Over the last 10,000 years, we have altered the genetic make-up of plants to produce food, fibres and other materials we need (1). For most of that time we have relied on fairly primitive selective breeding, with farmers selecting plants by the usefulness or desirability of their obvious traits, for example grain size, taste or colour. In the last 300 years, our expanding knowledge of plant science has provided more sophisticated technologies for improving crops. Here are some of the major ones:

Increase Tress Biomass Production

By modifying the gene expressions responsible for the branch growth during the first year of woody species, researchers of the Centre for Plant Biotechnology and Genomics (CBGP UPM-INIA), a joint centre of the Universidad Politécnica de Madrid (UPM) and the The National Institute for Agricultural Research and Experimentation (INIA), have found a way of increasing biomass production of a forest plantation without altering its growth, composition or the wood anatomy. These results have an important market value for the bioenergy sector, thus this study was protected by patent.

No Sign of Health or Nutrition Problems from GMO Livestock Feed

A new scientific review from the University of California, Davis, reports that the performance and health of food-producing animals consuming genetically engineered feed, first introduced 18 years ago, has been comparable to that of animals consuming non-GE feed. The review study also found that scientific studies have detected no differences in the nutritional makeup of the meat, milk or other food products derived from animals that ate genetically engineered feed.

How to Choice The Best Molecular Marker for Plant Breeding

The choice of the most appropriate molecular marker for genetic and plant breeding studies must be made on the basis of the ease of developing a useful technique coupled with the efficiency of data evaluation, interpretation, and analysis. The chosen marker must provide easy access and availability, rapid response and high reproducibility, and allow information exchange between laboratories and between populations and/or different species; it must also permit automation of data generation and subsequent analysis. Other desirable characteristics include a highly polymorphic nature, codominant inheritance (permitting the identification of homozygous and heterozygous individuals), frequent occurrence in the genome, and neutral selection (selection free from interference by management practices and environmental conditions). In addition to the characteristics of the marker, the goals of the project, the availability of financial, structural, and personal resources, convenience, and the availability of facilities for the development of the assay, as well as the genetic trait of the species under study, should all be considered.

Evolution of Genetics and Plant Breeding

Since the beginning of agriculture in approximately 10,000 BC, people have consciously or unconsciously selected plants with superior characteristics for the cultivation of future generations. However, there is controversy regarding the time when breeding became a science. Some believe that this occurred after Mendel’s findings, while others argue that it occurred even before the “era of genetics.”

Construction and Application of Genomic DNA Libraries in Plant

Crop failures, pathogenic outbreaks and famine are serious problems facing society. Understanding an organism's genome will help provide the genetic tools needed to solve these complex problems in a shorter time and with lesser effort. To efficiently study an organism's genome, it can be partitioned into a permanent and stable collection of DNA fragments, called a library. Such libraries provide convenient access to a genome for both laboratory and breeding applications. Genomic libraries can be used as substrates to physically map and sequence entire genomes, clone agriculturally important genes and to investigate gene expression patterns. Further, genomic libraries also provide powerful tools and resources for evaluating germplasm conservation stocks and biological diversity. Since the ongoing explosion of genetic data and molecular clone resources has opened new scientific possibilities with unfamiliar terms and acronyms to researchers venturing across all avenues of applied science

Serious consideration must be given to the various kinds of libraries that can be constructed with respect to different insert sizes, fragmentation methods, vectors, coverage, downstream operations, etc. Though downstream applications (see the Applications section of this chapter) differ for types of libraries, fragmentation size is the first consideration in genomic library construction. Genomic DNA libraries are classified as shotgun libraries, medium-size insert libraries and large insert libraries depending on insert sizes. A shotgun library, with a smaller insert size (generally 1.5–10 kbp), is made using high-copy plasmid vectors. It is the most common resource for complete sequencing of large genomic DNA clones (e.g. cosmid, PAC, BAC and YAC clones) and large DNA fragments (PCR products and restriction fragments). With the introduction of capillary sequencers for large-scale, high-throughput DNA sequencing, the shotgun library approach is considered a standard method for generating sequence-ready sublibraries for genome projects and positional cloning studies.

Cosmid and fosmid libraries contain medium-sized inserts (35–45 kbp). Cosmids are hybrids of λλ-phage and plasmids. They can replicate in the cell like a plasmid due to the plasmid replication origin and be packaged like a phage because of existing cos sites. Since most of the λλ-phage structure has been deleted, they can carry DNA inserts up to 45 kbp. This type of library is useful for cloning studies because of its simplicity and economic efficiency for construction in comparison with large insert library construction. The fosmid cloning system is similar in size to cosmids and the vector is derived from the endogenous E. coli F1 factor that maintains inserts in a single-copy state adding to the fidelity and stability of the DNA insert. The fosmid system is useful for easy generation of medium-sized insert (45 kbp) genomic libraries produced from small amounts of source DNA such as flow-sorted chromosomal DNA (Gingrich et al., 1996).

Large-insert genomic DNA libraries are essential for physical mapping, positional cloning and genome sequencing of complex genomes. There are two principal large-insert cloning systems: yeast and bacterial artificial chromosome systems (YACs and BACs). The YAC cloning system was first developed in 1987 (Burke et al., 1987), and uses Saccharomyces cerevisiae as the host and maintains large inserts (up to 1 Mb) as linear molecules with a pair of yeast telomeres and a centromere. Although used extensively in the late 1980s and early 1990s, this system has several disadvantages (Anderson, 1993; Zhang and Wing, 1997). The recombinant DNA in yeast can be unstable. DNA manipulation is difficult and inefficient. Most importantly, a high level of chimerism—cloning of two or more unlinked DNA fragments in a single molecule—is inherent within the YAC cloning system. These disadvantages impede the utility of YAC libraries and subsequently this system has been gradually replaced by the BAC cloning system introduced in 1992 (Shizuya et al., 1992).

The BAC system uses a derivative of the E. coli F-factor as a vector and E. coli as the host, making BAC cloning and subsequent down-stream procedures efficient and easy to perform. Recombinant DNA inserts of up to 200 kb can be efficiently cloned and stably maintained as single-copy plasmids in E. coli. BAC libraries have been developed for essentially all major crop plants, as well as model plant species, such as Arabidopsis thaliana and rice, and can be accessed from the laboratories that made the libraries or through various stock centres (e.g. AGI, CUGI and ABRC).

Insect-Resistant Crops

Insect-resistant crops were developed to contain genes from Bacillus thuringiensis (Bt) that encode for proteins toxic to insects. Bt is a soil bacterium, which produces crystalline proteins (referred to as Cry proteins) that are toxic, to select insect orders such as lepidoptera, diptera and coleopera (Swadener, 1994). When insects ingest the protein produced by Bt, the toxin binds to specific receptors on the mid gut epithelial cells. As a result, the cell membrane develops pores, which affects the insects' ability to regulate osmotic pressure. The function of insects' digestive system is thus disrupted, leading to starvation and eventual death.

The Cry proteins have been used as insecticides since 1961, mainly in organic crop production. The commercial Bt products are available in powder formulation, containing a mixture of dried spores and toxin crystals. They represent about 1% of the total agrochemical market across the world.

There are four insect-resistant Bt crops that have been approved for commercial production in the United States to date. They are field corn, cotton, potato and sweet corn. Field corn, cotton and potato were commercialised in 1996 while sweet corn was commercially introduced in 1998.

Insect-resistant field corn was developed to express Cry1A(b) and Cry1A(c) proteins that confer protection against European corn borer (ECB), south-western corn borer, fall armyworm, corn earworms and stalk borers. Insect-resistant Bt corn has no activity on other corn pests such as aphids, spider mites, cut worms and soil insects such as rootworms, wireworms and seed corn maggots. European corn borer is the primary target pest for Bt corn. Since its introduction, adoption of Bt corn steadily increased until 1999 (26%) but remained flat at 19% in 2000 (Table 61.2). Low corn prices and low pest pressure in the past two growing seasons were suspected to be the reasons for the slight decline in adoption rate in 2000
Insect-resistant cotton acreage steadily increased since its introduction and was planted on about 28% of the total cotton acreage in 2000 (Table 61.2). Bt cotton adoption rate is higher in certain states in the United States such as Alabama, where losses from bollworm/budworm infestations are huge. Target pests for Bt cotton are tobacco budworm, cotton bollworm and pink bollworm. While cotton bollworm and tobacco budworm infest south-east and mid-south production areas, pink bollworm is prevalent in western states of the United States.

The Colorado potato beetle-resistant potatoes developed to express Cry proteins (CryIIIA) from Bacillus thuringiensis var tenebrionis were marketed as NewLeafpotatoes in 1996. NewLeafPlus with resistance to the potato leafroll virus; and NewLeafY with additional resistance to potato virus Y were developed by stacking the virus-resistance traits with Bt and were introduced in 1999. Combined adoption of all three types of Bt potatoes has been limited and planted acreage never exceeded greater than 4% in the United States (Table 61.2). The low adoption is attributed to marketing concerns.

Insect-resistant Bt sweet corn [Cry1A(b)] tolerant to lepidopteran pests such as corn earworm and fall armyworm was commercialised in 1998. Adoption of Bt corn has been very low thus far due to the reluctance of fresh corn marketers' to purchase biotechnology-derived produce.

Use of insecticides has been the most commonly used method for insect control since 1930s in the United States. Insecticides have routinely been used in an integrated approach along with cultural practices such as crop rotation, tillage, and insect-resistant crop varieties. Major limitations to the use of insecticides are resurgence of primary and secondary pests and development of insect resistance to insecticides.

Resurgence occurs when insects normally killed by insecticides return in larger numbers. When insecticides remove target insects and their natural enemies, opportunity exists for the temporarily removed pests to reproduce before their natural enemies return. Spider mites, for example, caused havoc when DDT and other insecticides killed their predators.

The problem of insect resistance to conventional insecticides is already a serious issue, estimated to contribute about 25% of the pest control expenditure in the United States. Insect-resistance results in diminished utility of insecticides and places tremendous selection pressure on few products, which could further aggravate the problem. Resistance is the result of selection, where few insects in the population with genes of specific resistance mechanisms survive the insecticide sprays and multiply, thereby increasing the proportion of resistant insects in the population. The Colorado potato beetle is considered to be the most resistant pest in North America since it has developed resistance to every group of insecticides that growers have used against it. Other gaps in insect management using conventional tactics specific to each crop for target Bt pests are discussed below. Biotechnology-derived insect-resistant crops bridge the gaps in conventional insect management tactics with no need for chemical sprays to control target pests.

Corn

European corn borer damage results in poor ear development, broken stalks and broken ears, and ultimately yield losses due to larval feeding on kernels, leaves and conductive tissue. Its feeding on stalks and kernels increases the incidence of secondary infestations of stalk-rot fungi and mycotoxin-producing fungi, respectively. European corn borers also carry spores of secondary pathogens such as ear rot fungi from the leaves to the developing kernels and thereby increase the incidence of kernel rot and symptomless infections.

European corn borer is a difficult pest to control for two reasons. First, European corn borer levels are difficult to predict and vary greatly from year to year. As a result, growers are usually reluctant to incur costs on scouting to determine the feasibility and profitability of insecticide applications. Second, ECB control is complicated due to the feeding and survival behaviour of the insect. Corn borer larvae feed in leaf whorls after hatching and eventually move into the stalks to pupate inside the stem burrows thereby avoiding insecticide applications. Insecticides need to be applied during the two to three days period between egg hatching and their burrowing in the stems. Thus, carefully timed insecticide applications are the key for the successful control of ECB.

European corn borer control with the conventional insect-tolerant varieties and available insecticide options is only marginal to good. Consequent yield losses from ECB have been as high as 300 million bushels per year accounting to monetary losses of up to one billion dollars in the United States (Mason et al., 1996).

Cotton

Chemical control costs for cotton bollworm, tobacco budworm and pink bollworm amount to about 60% to 70% of the total pesticide costs to US cotton growers. Cotton insect management is very intensive; more than 90% of the entire cotton acreage is treated with insecticides and use of about ten insecticide applications was not uncommon in one season. More insecticides are applied to cotton than in any other crop in the United States (Gianessi and Marcelli, 1997). A limitation to cotton insect management using conventional insecticides has been the development of resistance in insects to pyrethroids, organophosphates and carbamates.

Potato

Roughly two-thirds of the total potato insecticides are targeted to control two insect pests, Colorado potato beetle (CPB) and green peach aphids. Colorado potato beetle is the most devastating and is particularly difficult to control as it has developed resistance to a broad range of insecticides such as arsenicals, organochlorines, organophosphates, carbamates and synthetic pyrethroids. Sprays of Bt are not widely used due to lack of effectiveness on early instars, lack of residual activity and stringent requirements on application timing (Whalon and Ferro, 1998).

The green peach aphid serves as a vector in transmitting potato leaf roll virus (PLRV). Potato leaf roll virus causes net necrosis in tubers, thereby lowering the marketable value of the crop. Since no chemical control options are available for virus control, the only way to limit virus infestations is to control insect vectors that transmit the virus.

Sweet Corn

Similar to ECB, the internal feeding habit of corn earworm and fall armyworm on sweet corn makes them only susceptible to pesticide applications during a narrow window of time when they migrate to newly developing ears. Once the larvae enter the ears, they are virtually impervious to chemical sprays. Most sweet corn growers consider 2% to be the maximum tolerable damage level for fall armyworm or corn earworm. As a result, it is not uncommon for sweet corn growers in states such as Florida to make up to 12 insecticide applications throughout silking stage to control these pests. Insect-resistant sweet corn varieties and biological methods have been used to no avail.

Herbicide Tolerant Crops

Herbicide-tolerant crops have been the most widely used application of agricultural biotechnology in the United States. Currently, crops have been modified to be tolerant to three herbicides: bromoxynil, glyphosate and glufosinate. Bromoxynil controls broadleaf weeds only while glyphosate and glufosinate are broad-spectrum with activity on both grass and broadleaf weeds.

Herbicide-tolerance results from three mechanisms: metabolic detoxification, resistance at the site of action and prevention of the herbicide from reaching the site of action. Bromoxynil-tolerant crops were developed by introducing a gene that encodes for bromoxynil-specific nitrilase from a soil bacterium, Klebseilla ozaenae (Stalker et al., 1996). Crop tolerance to bromoxynil results from metabolic detoxification. While introduction of the glyphosate-insensitive EPSPS from Agrobacetrium sp. strain CP4 into crops by genetic modification techniques was successful in conferring glyphosate tolerance (Padgette et al., 1996), glufosinate tolerance was achieved through the use of bar gene isolated from another soil bacteria, Streptomyces hygroscopicus. The bar gene encodes for an enzyme, phosphinothricin acetyl transferase, which detoxifies the herbicide glufosinate (Vasil, 1996).

Commercialised herbicide-tolerant crops to date include bromoxynil-tolerant cotton, glyphosate-tolerant soybean, cotton, corn, sugarbeet and canola, and glufosinate-tolerant corn and canola (Table 61.1). Bromoxynil-tolerant cotton was introduced in 1995, while glyphosate-tolerant soybean, cotton, corn, sugarbeet and canola have been available in the United States since 1996, 1997, 1998, 1999 and 1999, respectively. Glufosinate-tolerant corn and canola were commercialised in 1997 and 1999, respectively.
The adoption of bromoxynil-tolerant cotton has been low in the United States (Table 61.1) due to several reasons. Although bromoxynil provides effective control of problem weeds such as morning glory and cocklebur, it is weak on sickle pod, which is a key weed species in several cotton growing states and has no activity on grass weeds. Marketability of bromoxynil-tolerant cotton is further limited, as the herbicide-tolerance trait has not been stacked with the insect-resistance trait.

On the other hand, the commercial adoption of glyphosate-tolerant soybean, cotton and canola has been the most rapid cases of technology diffusion in the history of agriculture. In 2001, glyphosate-tolerant soybean, cotton and canola were planted on approximately 68, 70 and 50% of the total planted acreage, respectively (Table 61.1). Herbicide-tolerant (glyphosate and glufosinate tolerant included) corn was planted on only 7% of the total acreage in 2001. Lack of approval for biotechnology-derived glyphosate-tolerant corn imports into the European Union and non-availability of herbicide-tolerance trait in popular varieties adapted for various corn growing regions have hindered the adoption of herbicide-tolerant corn thus far. Although glyphosate-tolerant sugarbeet has been available for commercial planting since 1999, adoption has been zero due to issues related to marketing.

A traditional weed control program in conventional crops involves the use of several herbicides, targeted to a specific weed or groups of weeds. Herbicides are typically applied either as preplant incorporated (PPI) treatments prior to planting, pre-emergence (PRE) applications at planting or before crop emergence, post-emergence (POST) applications after the crop has emerged or a combination of PRE followed by POST applications. Several constraints limit the success of PPI and PRE herbicide applications. Preplant incorporated and PRE treatments involve guesswork as herbicide applications are made anticipating the weed species that may emerge. The efficacy of soil-applied PRE herbicides is highly dependent on rainfall, with poor weed control under extremely low or high rainfall conditions. As a result, there is an increasing trend towards total POST herbicide programmes. Herbicide-tolerant crops, on the other hand, facilitate the use of POST herbicides, such as glyphosate and glufosinate, wherein herbicides are selected based on emerged weed species in the field within the limits of crop and weed growth stages.

Conventional herbicides pose carryover concerns resulting in planting restrictions as many of them have long soil persistence periods. For example, herbicide labels suggest that crops such as alfalfa, dry beans, cabbage, lima beans, muskmelon, onions, peas, peppers and pumpkins should not be planted for 18 months following the application of a premix of atrazine/rimsulfuron/nicosulfuron (trade name: Basis Gold) in corn. Similarly, 26 months should elapse after imazethapyr application when planting potato, and 40 months for tomato, watermelon, squash and pumpkin following imazethapyr application in soybean (Pest Management Recommendations for Field Crops, 2000). Herbicide-tolerant crops resolve this problem because herbicides used in biotechnology-derived crops, such as glyphosate and glufosinate, have no residual activity and thus no carryover potential.

Crop injury from herbicide applications is a major concern in crop production. The potential for crop injury is generally greater with certain conventional herbicides in crops such as cotton and soybean. For instance, herbicides, such as acifluorfen and 2,4-DB, can cause substantial injury to conventional soybean leading to yield losses (Kapusta et al., 1986; Wax et al., 1973). Weed control is compromised if herbicide rates are decreased to lessen crop injury. Herbicide-tolerant crops offer growers remarkable flexibility because crop injury is practically non-existent.

Herbicide-tolerant crops add flexibility to weed management as they offer programmes that are less restricted by crop growth stage, weed species, weed size, tank-mix partners and adjuvant type. Herbicides used in conjunction with herbicide-tolerant crops can be applied at later crop growth stages compared to conventional herbicides, and the maximum height at which they are effective on weed species is greater than that of currently used herbicides. For example, glyphosate can be used up to the 4-leaf stage on cotton, 6-leaf stage on canola and up to flowering on soybean. These application windows are much wider than those with conventional herbicides. Labels instruct that maximum height up to which glyphosate applications can be made for the control of foxtail and fall panicum, two problem weeds in corn, is 6 inches in contrast to 3 inches with the premix of conventional herbicides atrazine/rimsulfuron/nicosulfuron (Curran et al., 1999).

Herbicides used in herbicide-tolerant crops are broad-spectrum and non-selective in activity. As a result, control of both annual and perennial broadleaf and grass weeds can be obtained with one herbicide application alone and with no need for a tank-mix partner in most situations. This is in contrast to intense weed management programmes used in crops such as cotton, which on average receive three herbicide applications consisting of three active ingredients in combination with one to three cultivations. This simplicity in weed control coupled with no crop injury is the reason cited most often by growers for the adoption of herbicide-tolerant crops.

Perennial weeds are a major issue in crop production as they are difficult weeds to control. The difficulty arises due to their propagation behaviour that includes both vegetative and reproductive methods. Many of the currently available conventional herbicides are not effective on perennial weeds. Though herbicides such as clopyralid are effective, high cost limit their use. Herbicide-tolerant crops provide a viable choice for perennial weed management as herbicides such as glyphosate provide excellent perennial weed control in addition to control of other weeds and are cost-effective.

Benefits of Commercialised Biotechnology-Derived Crops in the United States

Agricultural biotechnology has been a significant step forward inpest management in the United States. The range of its applications has been extensive and is expanding rapidly. The principal commercialised applications thus far include herbicide tolerance, insect resistance and virus resistance.

Biotechnology-derived crops were first introduced for commercial production in the United States in the mid 1990s. In spite of the dichotomy of opinion regarding biotechnology-derived crops, adoption has been dramatically rapid in the United States since their introduction. The United States is the principal country that planted most of the biotechnology-derived crop acreage (68% of the global) followed by Argentina (22%), Canada (6%) and China (3%) in 2001 (James, 2001). In 2001, biotechnology-derived crops were planted on 88 million acres of US crop acreage, up by 18% compared to 2000 (James, 2001). James (2001) noted that adoption of biotechnology-derived crops has been the highest ever for new agricultural technologies and attributed this to grower satisfaction and significant benefits. Current trends suggest that in the next few years almost all acreage of the major crops grown in North America will be biotechnology-derived.

A conflicting aspect of agricultural biotechnology is the amount of public debate and furore it has generated. Opposing opinions regarding biotechnology-derived crops centre on different perceptions regarding their benefits, environmental and ecological safety, implications on human health and ethics. An understanding of the benefits of agricultural biotechnology for pest management is pivotal to judge the merit of the technology and to resolve the public discussion.

This chapter examines the importance of pest management in crop production and details the commercially available biotechnology-derived traits and their need in the context of available pest management options in conventional crops. The discussion is mainly focused on the actual and potential benefits of this innovation for crops commercialised so far in the United States. Economic advantage to growers is the ultimate key factor, which determines the adoption and success of biotechnology-derived crops. Economic benefits normally result from reduced input costs or increased yields or both.

Importance of Pests in Agriculture

Pest populations have been and will continue to be the major constraints to crop production in the United States. Based on a 1988–1990 estimate, the impact of weeds, insects and pathogens on the production value of eight major crops grown in North America was 11.4, 10.2 and 9.6%, respectively, amounting to a total of $23 billion (Oerke et al., 1994). A recent estimate suggests that impact of weeds alone on US economy exceeds $20 billion annually (Bridges, 1994).

Pest management in crops is a dynamic activity that evolves as new technologies are developed. Growers have relied on a variety of tactics such as manual methods, cultural practices, biological control, quarantine and pest-resistant cultivars to combat pests thus far. Use of chemicals replaced manual and cultural methods in the 1940s, after which crop productivity increased dramatically in the United States.

Weeds are a constant and major challenge to farmers worldwide. About 72% of the pesticides used in the United States are herbicides, 21% are insecticides and 7% are fungicides (Duke, 1998), which emphasises the importance of weeds as crop pests. Control of weeds is critical as they compete with crops for nutrients, water, sunlight and space resulting in significant yield and quality losses. Season-long weed infestations can result in severe yield losses depending on the competing weed species and their density. For example, corn yields were reduced 10% by giant foxtail, 11% by common lambsquarters, 18% by velvetleaf and 22% by common cocklebur at a density of two per foot of row (Beckett et al., 1988). Additionally, weeds increase the cost of agricultural production, reduce land use and human efficiency, and act as hosts for insects and pathogens thereby increasing their control costs. As a result, almost all of the acreage of major crops in the United States is treated with herbicides to avoid yield loss.

Insects infest crop plants for the most basic reasons: to obtain food or protection for overwintering and oviposition or to complete their life cycle. The direct impact of insects result from their feeding on plant parts, which leads to reduction in crop productivity and quality. Losses due to insects each year in the United States were estimated to be 13% or $33 billion (USBC, 1998). The concentrated large acreages of a single crop in successive years (monoculture) have led to a general increase in insect pest populations in the United States. Monocultures lead to unstable agroecosystems due to increased abundance of food supply, decreased competition, low diversity of insect pests and increased ease of locating food supply.

Pathogens that infect plants fall under diverse groups such as viruses, bacteria, fungi, algae, protozoans and nematodes. These pathogens cause harmful physiological and metabolic effects in crop plants thereby resulting in significant yield losses. For example, estimated crop losses due to diseases in the United States are over 10% (El-Zik and Frisbie, 1991). Annual crop losses to all plant pathogens total an approximate $33 billion in the United States (USBC, 1998).

Crop loss estimates due to various pests are often misleading as they represent average loss over a wide area of production. In reality, losses are usually much higher on individual farms. Thus, crop losses definitely justify research to explore new methods such as modern biotechnology to manage pest populations.

The following discussion centres on why growers have adopted biotechnology-derived crops. It outlines the shortcomings in conventional pest management and suggests how biotechnology-derived crops offer solutions to these problems. Finally, the benefits derived from the technology for specific crops are highlighted.

Application of Proven Agronomic Biotechnologies to Ornamental Plants

In 1996, interdisciplinary scientists ushered in a new age of collaboration between molecular biologists and plant breeders with the introduction of herbicide- and insect-tolerant transgenic plants on a commercial agronomic scale. In the past 6–7 years, herbicide and insect tolerance traits have been utilised in many different agronomic and vegetable crops, and now make up a significant percentage of the acreage planted yearly in the United States for corn, soybeans and cotton. Although there have been no published reports on the production of herbicide- or insect-tolerant ornamental crops, research is currently being conducted on turfgrass, and a select number of significant floriculture crops to engineer glyphosate resistance. The transfer of glyphosate resistance into creeping bentgrass is an obvious example of how proven agronomic traits can be used to make weed control in municipal and highly managed turf environments more efficient. It is very likely that any number of horticultural crops could be engineered with herbicide resistance, but the trait will probably only be commercially viable in crops grown in the field or planted in the ground in large public areas, such as vegetable crops, turfgrass, bedding plants and nursery crops. The use of herbicide resistance as a selectable marker in tissue culture, and subsequently as a ‘stacked’ trait on top of other introduced traits may influence the appearance of this technology in future crops grown for ornamental purposes as well.

From a broad perspective, it appears that the future for application of biotechnology to ornamental crops is promising. Many technical advancements have been made that have provided ‘proof of concept’ of their commercial utility in ornamental crops, and this work will likely supply the next generation of flower breeders with many novel traits. As progress is made in sequencing new plant genomes and as functional genomic tools become more widely used in crops of lesser economic value, scientists working to apply biotechnology to ornamental crops will likely find themselves with more novel traits to work on than they have people in their labs. There is no question that the main technical limitations that exist for applying biotechnology to ornamental crops lie mainly in the area of development of transformation systems for the large number of plant species used in the industry. For this reason, it will be imperative for breeders to introgress commercially viable traits into breeding stocks, whether their goal is to reproduce their crops by sexual or asexual means.

The real potential of biotechnology in ornamental crops is probably not going to be determined by any major difficulties encountered in the technical realm—it appears that there will be many genes and promoters that will have commercial viability in the ornamental plant industry. The factors that will determine how the ornamental plant industry accepts and utilises biotechnology will be more influenced by economic and regulatory issues. It is obvious that in terms of regulatory hurdles, ornamental crops have a particular advantage over food crops because they are non-edible. Since consumers do not physically ingest these products, it is likely that much public relations benefit can be gained in terms of consumer perception of genetically engineered crops by providing them an ornamental plant that is extremely novel and desirable. The introduction of new and novel plants has been the basis of progress in the ornamental plant industry since its inception, so it should be no surprise if that happens.

It is also logical to think that since there may be fewer regulatory hurdles for ornamental plants, the cost of introducing a new biotechnological ornamental crop would be reduced. However, the current regulatory restrictions that have been developed around the introduction of genetically engineered food crops will have to be revisited when regulatory packages for new ornamental crops are submitted to federal agencies. While it may be feasible to submit regulatory packages for each individual cultivar of genetically engineered corn or soybean, it will be cost prohibitive for any company to acquire all of the required regulatory data necessary for the hundreds of individual engineered cultivars that could be developed and potentially released by any particular breeding company. Since an individual ornamental crop has much less commercial value than any individual agronomic crop and a much shorter market life expectancy, the costs of gaining regulatory approval from federal agencies will have far more impact on the economic decisions made by those parties interested in engineering these crops. With the current royalty structures and profit margins in the ornamental plant industry, combined with a comparatively low market volume, it would take a company several years to recur the initial cost of their biotechnology investment, even when they capture large percentages of a given market share. In addition, many cut flowers and vegetative propagules used in the ornamental plant industry are produced in Africa, Central and South America and Europe, and are imported into the United States and all throughout Europe and Asia. With the current global status of genetically engineered plants in most of these regions being more restrictive than in the United States, any segment of the industry depending on import or export of products outside the United States will have far more complications in the physical movement of genetically engineered ornamental plants. These types of scenarios ultimately restrict the application of plant biotechnology to ornamental crops far more than any limitations at the technical end.

Phytohormone Synthesis and Perception

Flower colour and scent of ornamental crops are important candidate target traits for genetic manipulation with biotechnology tools because they are the obvious reasons that these crops are grown. However, there has been much research attention given to the manipulation of phytohormone synthesis and perception in ornamental crops due to the wide range of physiological processes in which they are involved. Currently, several issues in crop production and post-harvest handling in the ornamental horticulture industry reflect a need to be able to understand physiological processes that are normally controlled by plant hormones. For example, significant effort and money is spent applying chemicals to plants to control the synthesis of gibberellic acid (GA) and subsequent plant height during crop production. In many potted flowering crops and most bedding plant crops, as many as three to five applications of growth-regulating compounds may be used during a crop cycle. This adds a significant amount of production cost for chemicals and labour to apply and handle them, while being a negative factor environmentally. In addition, many of the newly introduced ornamental species that have not been in commercial production for a long period of time are receiving particular attention in conventional breeding and selection for dwarf plants because their natural habit does not fit into the mass-market operation scheme requiring compact plants that fit into transport vessels. It is apparent that the manipulation of endogenous GA concentration in plants through genetic engineering has the potential to produce ornamental and flowering plants with a diverse array of reduced-height phenotypes.

Considerable research over the past two decades has been conducted to isolate GA biosynthesis mutants (for review, see Hedden and Proebsting, 1999) and to elucidate the GA biosynthetic pathway (for review, see Hedden and Kamiya, 1997; Hedden and Phillips, 2000). The initial cloning and characterisation of gibberellin 20-oxidase genes from Arabidopsis (Phillips et al., 1995) and spinach (Wu et al., 1996) provided some of the first practical molecular tools needed to develop plants with altered endogenous GA levels. In Arabidopsis thaliana, GA 20-oxidase catalyses consecutive steps late in GA biosynthesis, and is encoded by three genes with differential patterns of expression (Phillips et al., 1995; Coles et al., 1999). Over-expression of GA 20-oxidase in Arabidopsis leads to increased production of active GA (GA4) and subsequent stem elongation, while expression of antisense GA 20-oxidase leads to reduced levels of active GAs and reduced rates of stem elongation (Coles et al., 1999). Heterologous over-expression of a pumpkin GA 20-oxidase gene driven by constitutive promoters in both transgenic Solanum dulcamara (Curtis et al., 2000) and lettuce (Lactuca sativa ‘Vanguard’) (Niki et al., 2001) also resulted in the production of dwarf plants. In both cases, endogenous concentrations of active forms of GA, such as GA1 and GA4 were reduced in transgenic dwarf plants, while concentrations of various inactive forms of GA such as GA25 were increased, indicating a diversion of the normal pathway of GA biosynthesis towards the production of inactive forms of GA.

More recently, additional tools for use in engineering dwarf plants have been developed as a result of the cloning and characterisation of genes involved in the regulation of GA degradation. The first steps in the degradation of biologically active GA appear to involve hydroxylation reactions that are similar to the final steps of GA synthesis. Similarly to the GA3-oxidases, which hydroxylate the 3-position of GA precursors to form active GAs (Chiang et al., 1995; Talon et al., 1990), GA-2 oxidases hydroxylate the 2-position of active C19-GAs as a first step to render them inactive (Sakamoto et al., 2001; Martin et al., 1999; Thomas et al., 1999). Two additional GA2-oxidases (AtGAox7 and AtGAox8) have recently been isolated from Arabidopsis that hydroxylate C20-GA precursors, but not active C19GAs (Schomburg et al., 2003in press). When over-expressed in transgenic Arabidopsis and tobacco, these genes result in plants with decreased levels of active GAs and a range of corresponding dwarf phenotypes. Transgenic CaMV35S::AtGAox7 and AtGAox8 petunias have a range of dwarf phenotypes, suggesting that the control of endogenous GA levels may be applicable to important floriculture crops as well (Figure 45.1). It is interesting to note that the dwarf phenotype can be reversed by treatment of plants with exogenous GA (Figure 45.2). This is very important for the application of dwarfing technology to the ornamental plant industry, because producers of vegetative propagules may actually want to temporarily increase shoot elongation at various points during cutting production, then have the plants resume a dwarf phenotype after propagation.Breeder selection for the appropriate plant height among transgenic lines will be critical for applying these technologies to any given crop, and breeders should be able to select any particular plant height they desire. Traditional horticultural experiments similar to those used in any breeding programme with a focus directed towards plant height control should be relatively straightforward, and are well established in the industry.

It is likely that dwarfing technologies will find utility in many areas of ornamental plant production. There should be ample room in the industry for potted plants that fit in transit vessels easily, annual bedding plants that do not grow out of the confines of a small garden spot, and shrubs and trees that require less pruning. One extremely important application for dwarfing technology will likely be observed in plant species used in the turfgrass industry. Slower growing lawn grasses that require significantly less labour input for maintenance have been a dream of suburban homeowners and turfgrass maintenance experts for many years. This concept is being developed by commercial scientists at the Scotts Company, with a great deal of progress being made in producing dwarf creeping bentgrass (Agrostis sp.). In high-management areas, these ‘low mow’ grasses have a great deal of potential for saving money on labour costs and equipment maintenance. In addition, slow-growing grasses could help reduce lawn mower pollution and use of fossil fuels, and may use less water and fertilisers. It is likely that these ‘low-mow’ turfgrasses will be important for homeowners and municipal or roadside situations where there is a low amount of foot traffic. It is not likely that these grasses will be used to any great extent on golf courses or sports turf facilities. Due to the high amount of wear and tear that is experienced under these conditions,they may not grow fast enough to cover the damage inflicted by constant use.

Another aspect of hormonal control of plant morphology currently being pursued in ornamental crops concerns the engineering of plants with delayed leaf senescence. Senescence is the final developmental process in the lifecycle of a leaf through which macromolecules (e.g. chlorophyll, proteins, nucleic acids) of leaf cells are metabolised to basic components and transported to the growing shoot and reproductive organs of the plant (Matile et al., 1996; Noodén et al., 1997). Natural leaf senescence in many plants is characterised by lower leaf yellowing or chlorosis as nutrients and other components of the cells are degraded (especially chlorophyll). In horticultural terms, leaf chlorosis can cause a decreased aesthetic appearance of ornamental plants and thus a decrease in the salability of those plants.

One way to prevent leaf senescence is through the manipulation of cytokinins. Cytokinins are an important class of phytohormones that influence numerous aspects of plant growth and development, and have been shown to delay and, in some cases, reverse the leaf senescence process (Gan and Amasino, 1996). The limitation of the use of cytokinins in the prevention of senescence has most often been related to difficulties of controlling temporal and spatial delivery of cytokinins. Exogenously applied cytokinins may not enter the cells, nor be transported to the area needed, nor quickly become conjugated or metabolised to non-active forms (Klee and Lanahan, 1995; Gan and Amasino, 1996; Kaminek et al., 1997). Researchers have recently turned to the manipulation and engineering of endogenous cytokinin biosynthesis in order to solve these problems.

After many years of research on cytokinins, the mechanisms of biosynthesis and perception are just now being elucidated. One enzyme, isopentenyl transferase (IPT), has received much research attention because of its known involvement in cytokinin synthesis. IPT enzyme activity has been isolated from many organisms including plants (Nishinari and Syono, 1980; Blackwell and Horgan, 1994), Dictyostelium discoideum (Taya et al., 1978; Ihara et al., 1984), and Rhodococcus fasciens (Crespi et al., 1992). Although it is well known that there is IPT activity in plants, the genes that encode IPT have only recently been isolated and cloned from plants (Kakimoto, 2001; Takei et al., 2001). As a result, experiments focused on the manipulation of endogenous cytokinin synthesis in transgenic plants with these genes have been lacking. In contrast, a gene from Agrobacterium tumefaciens that encodes the IPT enzyme has been available for use in transgenic plant research for a number of years. IPT is encoded on the Ti (tumour inducing) plasmid of Agrobacterium tumefaciens (Akiyoshi et al., 1984; Barry et al., 1984). It catalyses the condensation of dimethylallylpyrophosphate (DMAPP) and 5′AMP to form isopentenyladenosine 5′-phostphate ([9R-5′P]iP), which is then quickly converted to different cytokinins. This is presumed to be the rate-limiting step in cytokinin biosynthesis since expression of this one gene can cause an over-production of cytokinins (Medford et al., 1989; Klee and Lanahan, 1995; Gan and Amasino, 1996). Attempts have been made to use this gene under the control of various promoters, for example, modified CaMV 35S (Faiss et al., 1997), Cu2+-inducible (McKenzie et al., 1998), or heat shock inducible (Smart et al., 1991) to stop leaf senescence. However, the results of such experiments were often complicated by abnormal growth patterns, or the possibility that the induction treatment (i.e. high temperature or CuSO4 treatment) was causing the observed phenotype (Smart et al., 1991; Buchanan-Wollaston, 1997).

In an attempt to overcome the problems associated with past work on transgenic plants overproducing cytokinins, Gan and Amasino (1995) developed a genetic construct that utilised the highly senescence-specific promoter from SAG 12 (PSAG-12) to drive IPT expression. This construct had three important features due to PSAG-12 specificity: temporal regulation, spatial regulation and quantitative regulation (Gan and Amasino, 1995, 1996). When leaf senescence is triggered, the transcription of IPT is activated by PSAG-12, leading to the production of functional IPT enzyme. The enzyme then catalyses cytokinin production, which in turn delays senescence. Without senescence signals, the PSAG-12 promoter attenuates IPT transcription and subsequent enzyme production, thus providing autoregulatory control of cytokinin synthesis. The PSAG-12-IPT construct was transformed into tobacco. The transformed plants displayed a normal growth habit except that senescence was inhibited and there was a significant increase in flower number, an increased biomass due to the presence of lower leaves, and increased seed yield (Gan and Amasino, 1995). Since then, transgenic PSAG-12-IPT lettuce plants (McCabe et al., 2001) Nicotiana alata (Schroeder et al., 2001) and petunias (Clark et al., in press) with similar delayed leaf senescence phenotypes have been produced (Figure 45.3). The PSAG-12 promoter has also been used to drive the expression of the KNOTTED-1 mutant gene from maize in transgenic tobacco plants to confer a delayed leaf senescence phenotype (Ori et al., 1999), thus extending the utility of delayed leaf senescence technologies. Transgenic PSAG-12:KNOTTED-1 and PSAG-13-IPT petunias both have delayed leaf senescence after nutrition stress induced at the onset of flowering. It is likely that horticultural performance studies will show that breeder selection of transgenic plants under a wide range of selection criteria will be essential for providing the growers with the methods required to produce these plants effectively on a commercial scale.
One hormone of particular interest in the ornamental plant industry is ethylene gas. Ethylene is involved in many physiological processes in plants including fruit ripening, petal senescence, abscission and seed germination. Floral senescence is of particular interest because quality loss of many important floriculture crops is known to occur due to ethylene gas in the post-harvest environment. Treatment of ethylene-sensitive flowers with chemical ethylene biosynthesis or perception inhibitors delays visual symptoms of corolla senescence (Jones and Woodson, 1997; Whitehead et al., 1984; Serek et al., 1995). Many economically important crops are grown for their floral display; therefore the control of ethylene synthesis and perception is thought to be the key to increasing display life and enhancing visual quality. Since ethylene is such a significant problem in both potted flowering crops and cut flowers there have been several attempts to produce chemical control methods for both ethylene synthesis and sensitivity. Effective control of ethylene synthesis using a chemical approach has been much less effective across the industry than the manipulation of ethylene sensitivity. Ethylene sensitivity has long been managed in floriculture crops through the use of silver thiosulfate (STS), which makes plant tissue insensitive to ethylene, but also has environmental downsides that appear to restrict its commercial use. Another chemical approach gaining popularity within the industry is the use of 1-MCP (1-methylcyclopropene), which is a compound that blocks the ethylene receptor protein and makes plant tissue insensitive to ethylene. Typically applied as a gas, 1-MCP provides a means by which large amounts of tissue can be treated for a short time. Although this compound has proved to be effective in some crops, there is some difficulty with the use of 1-MCP in crops that continue to produce new ethylene receptor proteins through development during post-harvest transit, thus limiting the residual effect of the compound (Cameron and Reid, 2001).

A great deal of research has been conducted to produce floriculture crops that synthesise less ethylene, or are insensitive to ethylene. Transformation with an antisense ACC oxidase has been shown to be effective in producing carnation (Savin et al., 1995) and torenia plants (Aida et al., 1998) that produce significantly less ethylene, and have delayed flower senescence. As has been the case with chemical approaches to control the effects of ethylene in ornamental crops, the method of control receiving the most research attention in terms of genetic engineering has been at the level of ethylene perception. Wilkinson et al. (1997) transformed petunia with a dominant mutant Arabidopsis ethylene receptor, etr1-1, under the control of a constitutive cauliflower mosaic virus 35S (CaMV35S) promoter to produce ethylene insensitivity throughout the whole plant (Figure 45.4). These petunias had an increase in natural and pollination-induced flower longevity compared to wild-type plants, but had physiological side effects that limit their commercial use (Wilkinson et al., 1997; Gubrium et al., 2000). Ethylene-insensitive petunias showed a significant reduction in adventitious root formation, and even exogenous treatments with auxin did not increase adventitious root formation to untreated wild-type controls (Clark et al., 1999). Since many horticultural species are often propagated through vegetative cuttings this characteristic would severely limit the commercial utility of ethylene-insensitive plants propagated by vegetative cuttings. After much experimental effort, it is clear that the key to manipulation of ethylene sensitivity will be the promoters being used to drive transcription of these proven transgenes. Effective temporal and spatial control of transgene expression will ultimately lead to plants that have longer lasting flowers with no negative side effects that would limit commercial production. Bovy et al. (1999) have supported this idea by producing transgenic ethylene-insensitive carnations by driving the etr1-1 transgene with the flower-specific transcriptional promoter fbp1 from petunia. Although they were able to produce plants with a delayed flower senescence phenotype, they did not report heritability of the trait or extensive investigation of other horticultural performance characteristics that would limit commercialisation of these plants. Future experiments directed towards discovering the appropriate promoter to drive transgenes conferring ethylene insensitivity will likely be the key to success for the development of long-lasting flowers in the future. It will be imperative to conduct a significant amount of field and greenhouse trailing of these plants in order to completely eliminate the possibility of negative side effects.