Applications of Plant Biotechnology to Ornamental Crops

The ornamental plant industry is technically diverse and is characterised by the use of literally hundreds of different plant species. In many cases, numerous different cultivars of the same species may be available, and plants may be reproduced through both sexual and vegetative propagation methods. Due to the large amount of genetic diversity used by this industry, there are often very complex issues that arise during crop production and post-harvest handling throughout the wholesale and retail markets. For example, crop production practices for potted poinsettia and chrysanthemum plants require manipulation of the photoperiod to initiate flowering, and the application of growth regulators to keep the plants compact in habit. This type of production scheme is vastly different from the production methods used for producing bedding plant species such as petunia, geranium and impatiens, but a typical greenhouse grower may be producing all these plants simultaneously. At the post-harvest and shipping end of the industry, cut flower species grown in the tropical regions of Central and South America may have optimal shipping and storage conditions that are vastly different from the optimal conditions needed for cut flower species grown in Northern Europe. However, all the flowers may be exported to a common port such as Miami, then repackaged and shipped together in the same transport vehicle under the same conditions to retailers in virtually any part of the United States. This type of scenario often results in poor product performance when it reaches the florist or is displayed on retail shelves.

In addition to the demands of complex production and marketing systems, the demand from consumers for a constant supply of new and interesting flowering plants with unique characteristics continues to increase. In the past 5–10 years, there has been a dramatic increase in the number of flowering plant species available to consumers worldwide, and this trend is continuing. As a result, there is very little information available on appropriate production and post-harvest schemes for many of these crops. With such a diverse industry it is logical to assume that the application of biotechnology to ornamental crops is going to be very difficult. Applying biotechnology concepts is quite abstract in ornamental plants for several reasons. First, scientists attempting to improve these crops are trying to hit a moving target. Turnover of new cultivars is constant, and there are very few instances of consistent use of inbred lines in commercial breeding programmes. This means that by the time a researcher has transformed a new trait into a particular cultivar and proved that the trait is of commercial interest, the original cultivar may actually have been replaced by a new ‘improved’ cultivar. As a result, it will be of utmost importance to get commercially viable trangenes into breeding stocks, whether the goal is to produce a crop that will be reproduced by seeds or by vegetative propagules. Second, calculating the value added by a trait is very esoteric in ornamental crop species. Calculating increases in corn yield may be relatively straightforward, but determining how much a new colour of pansy is worth, or whether a more fragrant rose is something that someone will buy and actually pay more for, is a bit more cumbersome. Since it is well known that there is significant cost associated with developing an approach for improvement of any plant species using biotechnology, it becomes much harder to determine the potential economic value of a new ornamental cultivar until after it has proved to be successful. Much of the profit to be made in the ornamental plant industry through the use of biotechnology will be made by either the breeding companies who invest in technology or by retailers who have the ability to market the value of a novel trait. Value will also be added for growers if the introduced traits significantly reduce production costs, and this impact should be particularly significant because this segment of the industry currently has lower profit margins than any other. This scenario is very common in many types of plant-based agriculture, but with ornamental plant germplasm being spread amongst so many breeding companies, no one company can justify the investment in biotechnology that has been seen for crops of agronomic scale without making sure that the technology is a financial risk worth taking. It is likely that a vertically aligned strategy capturing as much value as possible from breeder to producer to retailer will be the best approach to making the benefits of biotechnology outweigh the high costs of technology development.

The last major issue that makes the concept of applying biotechnology approaches to ornamental crops more difficult is probably the most important—there is a significant bottleneck in the number of ornamental plant species that have been genetically transformed to date. Although there have been published reports describing the genetic transformation of several of the major floriculture crops such as chrysanthemum, rose, carnation and petunia, other important crops such as poinsettia, hibiscus and impatiens have still not been reliably transformed. Logically, most of the crops that have been the focus of transformation efforts to date have been those with the most value economically, or plants that have been discovered to be easier to transform or culture in vitro. Even in crops that have received a fair amount of research attention for genetic transformation, a significant amount of difficulty has been encountered with developing transformation protocols that can be used successfully on all cultivars of a given plant species. In many cases, transformation of different cultivars or breeding lines has required the development of several different transformation protocols. The more aggressive transformation efforts in ornamental crops are being undertaken by private corporations, with the remainder of the work being taken care of (one species or cultivar at a time) by smaller academic tissue culture laboratories.

In many cases, biotechnology applications are proving to be very difficult, but several advances have been made with engineering a wide variety of genetic traits in floriculture crops and turfgrass. Significant gains have been made in cloning important genes that are proving to be involved with biological processes that scientists hope to manipulate in ornamental crops in the future. The purpose of the rest of this chapter is to provide a status report on the progress that has been made in applying biotechnology to plants grown for their ornamental characteristics, and to project where research efforts will be focused over the next few years.

New Traits—Old Concepts

Flower Colour

Flower colour is a key component that influences consumer choice among crops grown commercially for their ornamental characteristics. Flower colour has been the subject of a large amount of biochemical and applied genetics research for many years (Mol et al., 1998). Flower breeders have been supplying the markets with new colours using traditional breeding techniques and selection, and to a lesser extent mutation breeding. The main biological function of flower colour pigments is to attract pollinators. Various pollinators are attracted to a flower by particular colours, and the patterning of flower coloration makes flowers easy for pollinators to distinguish as they move about.

Although many species have a wide variety of possible flower colours, no single species contains all possible flower colours. Flower colours are produced in plants biochemically as betalains, carotenoids and flavonoids. Betalains are synthesised almost exclusively in the Caryophyllales, and are yellow to red nitrogen-containing compounds derived from tyrosine (Stafford, 1994). Because of their limited presence in the plant kingdom, very little attention has been given to this class of compounds for the purpose of genetically engineering flower colour. Carotenoids are responsible for yellow and orange colours such as those observed in sunflower, marigold and tomato flowers, and over 600 different carotenoid structures have been identified (Straub, 1987). Carotenoids function mainly in photosynthesis by assisting with light harvesting and by quenching singlet oxygen and triplet chlorophyll species that are derived from excessive light energy (Demmig-Adams and Adams, 2000). Carotenoid synthesis and storage occurs in plastids, but all of the biosynthetic genes isolated to date are nuclear-encoded.

Although there are many plant species in which yellow flower colour is not produced and cannot be introgressed through traditional genetics, very little progress has been made in terms of genetically engineering yellow flower colour in plants. The general biochemistry of carotenoid synthesis has been studied for over thirty years, but the carotenoid biosynthetic pathway genes have only been identified and characterised since the 1990s (reviewed by Hirschberg, 2001). There are many different genes involved in carotenoid synthesis, and many of these genes are represented in plants in small multigene families (Zhu et al., 2002; Moehs et al., 2001). Since carotenoids are involved in such a range of diverse biological functions, most of the research on engineering carotenoid synthesis in transgenic plants has focused on the alteration of nutritional characteristics in food plants. Interestingly, rice engineered to over-express the daffodil phytoene synthase gene for increased pro-vitamin A content had a characteristic ‘golden’ colour (Burkhardt et al., 1997). Although these plants may be valuable for helping solve worldwide vitamin A deficiency, they also demonstrate the ability to drive the expression of yellow colour in plants. It is likely that progress in engineering yellow flower colour through the manipulation of carotenoid synthesis will be aided by observations on food crops with altered carotenoid content. It is also possible that attempts to genetically manipulate enzymes of the carotenoid biosynthesis pathway for yellow flower colour may affect unintended physiological processes in the plants leading to undesirable horticultural characteristics. Thus, the choice of appropriate transcriptional promoters to drive the expression of carotenoid biosynthetic genes in specific plant tissues will be imperative in determining the success of engineering yellow flower colour in ornamental plants.

Flavonoids are a class of secondary metabolites that are responsible for pale yellow, red, purple and blue colours in flowers. The flavonoid biosynthetic pathway, in particular, the anthocyanin synthesis pathway, has been extensively reviewed (Heller and Forkman, 1994; Forkman, 1994; Holton and Cornish, 1995). Hundreds of anthocyanins have been purified and their chemical structures have been determined (Strack and Wray, 1994), and virtually all of the genes that encode anthocyanin biosynthetic enzymes have been isolated. Thus, the anthocyanin biosynthetic pathway has served as an excellent target for transgenic manipulation because of the extensive background studies of its chemistry and genetics.

There are six main anthocyanins in plant tissues: pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. These compounds are usually modified by glycosylation and acylation to produce a broader range of anthocyanins. In addition to the variety of anthocyanin chemical structures, colour variation produced by anthocyanins can be influenced by vacuolar pH, quantities of co-pigments and metal ions, and cell shape (Tanaka et al., 1998; Mol et al., 1998). Increased vacuolar pH is known to correlate with the ‘blueing’ of flowers of many plant species as they age and senesce (Yoshida, et al., 1995), and it is likely that both genetic and environmental factors influence the control of vacuolar pH. Genetic research on petunia has defined seven genetic loci that produce flower blueing when mutated (Chuck et al., 1993; van Houwelingen et al., 1998). Although these mutations are known to lead to measurable increases in the pH of petal extracts, but not an alteration of the actual anthocyanin composition, the actual mechanism of cellular pH control is still unknown. The range of the possible colours produced by anthocyanins can also be affected by the presence or absence of metal ions and co-pigments. In particular, the degree of blueness in anthocyanin pigments can be greatly influenced by metal ions and co-pigments which form stacked complexes with anthocyanins, and change their light absorption spectra (Kondo et al., 1992; Brouillard and Dangles, 1994). Perceived colour is also influenced by the shape of cells that accumulate anthocyanins. Petal epidermal cells more flattened in shape produce fainter colours, while conically shaped epidermal cells produce a sheen that gives the colours a more ‘velvety’ appearance (Noda et al., 1994). Since the underlying molecular mechanisms for changes in cell shape are poorly understood, it may be a few years before a complete understanding can be had of how pH changes and co-pigmentation and cell shape work together to produce an almost infinite array of flower colours through anthocyanins. However, it is well established that many genes involved in anthocyanin synthesis are regulated at the transcriptional level, which suggests that different flower colours and pigmentation patterns in flowers must be largely controlled by the expression patterns of regulatory genes (Holton and Cornish, 1995).

Over the past 10–15 years, many researchers have been able to successfully alter flower colour by manipulating the expression of anthocyanin biosynthetic genes in transgenic plants. Since no species has the ability to produce all possible flower colours, most of the research conducted to date has focused on the modification of already existing anthocyanin production systems in plants, or on the introduction of new biosynthetic enzymes that are not normally found in a particular species to make novel colours. For example, rose and carnation do not produce purple/blue delphinidin derivatives because they lack flavonoid 3′5′-hydroxylase (F3′5′H) activity (Mol et al., 1998). Also, plants such as petunia do not normally produce orange pelargonidin derivatives because the petunia dihydroflavonol reductase enzyme does not use the required dihydrokaempferol precursor as a substrate (Meyer, 1987).

Much of the research, focused on manipulating anthocyanin levels in flowers, has centred around the enzymes chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR). The CHS enzyme is the first enzyme committed to flavonoid production and catalyses the formation of chalcones, which are the intermediates used in the synthesis of all flavonoids. The DFR enzyme reduces dihydroflavonols to leucoanthocyanidins, another early rate-limiting step in anthocyanin synthesis. Many attempts at producing white flowers by suppression of flavonoid synthesis through the suppression of CHS and DFR activities have been successful. This approach is useful in a practical sense because in many species used as an ornamental crop, it has proved to be rather difficult to produce purely white flowering plants through traditional breeding methods.

Suppression of CHS via antisense or co-suppression has been demonstrated to suppress anthocyanin formation in a wide variety of plant species such as petunia (van der Krol, 1988; Napoli et al., 1990), chrysanthemum (Courtney-Gutterson et al., 1994), gerbera (Elomaa et al., 1993) lisianthus (Deroles et al., 1998) and torenia (Aida et al., 2000a). Similar to CHS, suppression of anthocyanin synthesis has also been achieved through suppression of DFR activity, leading to reduced anthocyanin synthesis in petunia (van der Krol, 1990) and torenia (Aida et al., 2000b). With the suppression of both CHS and DFR, there have also been reports of the generation of novel flower colour patterns in addition to the reduction in anthocyanin in petunia (van der Krol, 1988, 1990), torenia (Aida et al., 2000a, 2000b) and lisianthus (Deroles et al., 1998; Bradley et al., 2000). Interestingly, a consensus developed as a result of suppression of CHS and DFR is that the consistency of new flower phenotypes in these transgenic plants can vary within and between individual transgenic lines (van der Krol et al., 1990; Meyer et al., 1992; van Blokland et al., 1993; Elomaa and Holton, 1994; Deroles et al., 1998). Since colour patterns of some transgenic CHS lines of lisianthus have proved to be more stably inherited than others (Bradley et al., 2000), it is likely that any transgenic approach to the alteration of flower colour or colour patterning will require a significant amount of breeder selection over successive generations to stabilise the phenotype.

Controlled over-expression of anthocyanin biosynthetic genes has resulted in the production of novel flower colours in transgenic plants. One particularly dramatic example has been illustrated with petunia plants engineered for orange coloration. Normally, petunia does not produce orange colour because its intrinsic DFR protein does not accept dihydrokaempferol as a substrate, so pelargonidin-based pigments are usually absent. Maize DFR, which has a different substrate specificity than the petunia DFR, is able to produce pelargonidin derivatives if dihydrokaempferol is present and available. By expressing the maize DFR gene in petunia, Oud et al. (1995) were able to produce flowers with a brick red colour resulting from the accumulation of pelargonidin-derived pigments. Although this colour was not considered commercially acceptable, hybrids based on F4 genetic lines derived from commercial germplasm were obtained that had a unique orange flower colour. By introgressing the maize DFR gene into various breeding lines, DFR expression was stabilised, thus establishing new colour profiles that were not already present in petunia. Once stabilised through successive generations of breeding, the maize DFR gene behaved normally, and was used to successfully develop F1 petunia varieties with orange flower colour with no deleterious side effects (Oud et al., 1995).

Perhaps the best-known example of the production of novel flower colours in plants has been the transgenic ‘blue’ carnations produced by Florigene Ltd. and Suntory Ltd., which have been marketed in the United States, Australia and Japan under the name ‘Moonshadow’. These transgenic carnations contain the petunia flavonoid 3′5′-hydroxylase gene (F3′5′H), which encodes a cytochrome (cyt) P450 enzyme that catalyses the 3′5′ hydroxylation of dihydroflavonols, the precursors of purple anthocyanins. The F3′5′H enzyme activity is normally absent in carnation (and roses), but when the petunia F3′5′H gene is expressed in carnation, the plants produce and accumulate delphinidin, making the flowers a violet-bluish colour. It is likely that further improvements will be possible in producing blue colour using F3′5′H combined with other enhancers of anthocyanin pigmentation. It is known that the addition of cyt b5 activity enhances cyt P450 hydroxylation reactions, and is required for full activity of F3′5′H (de Vetten et al., 1999). Since the substitution pattern of anthocyanin pigments is a main determining factor in flower colour, it is thought that the stimulation of F3′5′H activity by the cyt b5 protein will be useful in enhancing blue flower colour (de Vetten et al., 1999).

Although much research has been conducted to date on anthocyanin biosynthesis and manipulation in plants, many issues related to the mechanisms of flower colouration are still poorly understood. To date, success has been achieved in the manipulation of flower colour through engineering anthocyanin biosynthetic genes and proteins. It is encouraging that most of the transgenic plants resulting from these efforts have been produced through driving transgene expression with constitutive promoters, and few deleterious side effects have been observed (Tanaka et al., 1998). However, the controlled manipulation of co-pigmentation and vacuolar pH have still not been addressed to a great extent. Once these factors are understood, the production of blue colour in flowers may become a reality.

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