Breeding forage crops for increased nutritional value

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BREEDING FORAGE CROPS FOR INCREASED NUTRITIONAL VALUE M. D. Casler Department of Agronomy University of Wisconsin—Madison Madison, Wisconsin 53706-1597

I. Introduction II. Historical Background A. Natural Selection B. Unconscious Selection and Domestication C. Methodical (Artificial) Selection Prior to 1963 III. Methodical (Artificial) Selection Post-1963 A. Selection Criteria B. Selection Methodology IV. The Potential for Molecular Biology Contributions and Collaborations A. Molecular Markers B. Transgenic Plants V. Summary References

Plant breeding is an extremely cost-effective mechanism for increasing the nutritional value of forage crops. Genetic gains in in vitro dry-matter digestibility (IVDMD) have averaged 0.7–4.7% year−1 , similar to long-term gains for grain yield of many cereal crops. Relatively small increases in IVDMD typically result in measurable improvements in animal performance. Gains in IVDMD result from changes in chemical, anatomical, and/or morphological traits of plants, but rarely from genetic shifts in timing of reproductive maturity. These genetic gains are both genetically and environmentally stable and, for perennial forage crops, require only a one-time investment by growers. Selection for increased forage nutritional value is often associated with reductions in agricultural fitness traits, such as forage yield, disease and/or insect resistance, and stress tolerance. These characteristics can often be corrected by concomitant selection pressure in field-oriented plant-breeding programs. Transgenic plants represent a new mechanism for generating novel phenotypes with improved forage nutritional value. Many of these phenotypes appear to represent metabolic lesions that may also occur by natural mutations, but are more frequent within transgenic populations. Transgenic technology appears capable of 51 Advances in Agronomy, Volume 71 C 2001 by Academic Press. All rights of reproduction in any form reserved. Copyright ° 0065-2113/01 $35.00

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M. D. CASLER contributing novel phenotypes to improved forage cultivars, but only from collaboration between molecular biologists and plant breeders or agronomists with strong ° C 2001 Academic Press. field-oriented programs.

I. INTRODUCTION Forage crops, like most plant species, contain vast amounts of genetic variability. This variability causes differential phenotypic expression among plants within populations and among populations, providing the basis for selection pressure on desirable plant phenotypes. Selection pressure is the primary cause of phenotypic change in populations over time. Selection pressures fall into three general categories: natural selection, unconscious selection, and methodical selection (Darwin, 1859). Selection pressures act to favor reproduction by certain selected individuals, at the expense of less-desirable individuals, causing the genes contained in the selected individuals to increase in frequency within the population. Because of the various expenses associated with animal feeding and grazing trials, nearly all plant breeding and genetic research on forage nutritional value has been conducted using indirect laboratory measures of forage nutritional value. Some of these measures are plant traits per se, such as cell-wall concentration, lignin concentration, and etherified ferulic acid concentration. Others are not plant traits per se, but are laboratory-defined variables that are defined strictly by a physical operation (e.g., leaf shear strength) or an interaction between the plant sample and biological organisms (e.g., in vitro digestibility). Numerous studies have shown that many laboratory-defined variables are heritable and that they can be stably modified by selection and breeding, allowing them to be treated as plant traits per se in genetic studies, as they are in this review. Most forage nutritional value traits are relatively complex. Many traits are typically intercorrelated, such as most cell-wall constituents and the concentration of many mineral elements, and nearly all traits are regulated by numerous enzymes that are involved in their biosynthesis or metabolism. Thus, selection for one trait often affects other traits as well and typically acts to modify the frequency of numerous functional genes within the population of plants. Thus, most forage nutritional value traits are considered to be under oligogenic or polygenic control and have been treated as quantitative genetic traits. A very small number of functional genes have been discovered that have large and direct effects on forage nutritional value of plants, while gene cloning and plant transformation promise the potential for numerous additions to this library in the future.

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II. HISTORICAL BACKGROUND A. NATURAL SELECTION Natural selection is the process whereby those individuals best fitted to a particular environment, a range of fluctuating environments, or a human-imposed management system have the greatest survival rate or contribute the greatest numbers of viable progeny to succeeding generations. Historically, natural selection has had a tremendous influence on phenotypic expression of forage crops. Agronomic evaluations of large collections of forage crop germplasm routinely show large amounts of phenotypic variability, much of which can be attributed to specific environmental factors. For example, survival of perennial ryegrass (Lolium perenne L.) collections at freezing temperatures was closely related to the mean temperature of the coldest month at their site-of-origin (Tcacenco et al., 1989). Indeed, natural selection pressures for tolerance to a range of environmental and/or management conditions, including salinity, heavy metals, acidity, cold temperatures, and herbivory, have been documented in several forage crops (Casler, 1998; Casler et al., 1996). Forage grasses have most certainly coevolved with large mammalian herbivores (Stebbins, 1981) and have necessarily evolved traits that allow them to survive under grazing pressure. Mixtures of perennial ryegrass and Italian ryegrass (Lolium multiflorum Lam.) invariably show rapid changes toward the perennial ryegrass phenotype when subjected to grazing pressure, natural selective effects that grow stronger with increased grazing pressure (Brougham et al., 1960; Charles, 1964; Brougham and Harris, 1967). Natural selection for survival during 6 years of grazing led to a 7–10% increase in forage yield for survivors of “S23” and “S24” perennial ryegrasses (Charles, 1972). Perennial ryegrass plants adapted to grazing pressure are generally prostrate in growth habit, late in maturity, and have a high capacity for tillering (Breese, 1983). Perennial ryegrass appears to be specifically adapted to survive in association with grazers (Beddows, 1953; Breese, 1983) and is rarely found in natural ecosystems without large mammalian herbivores (Davies et al., 1973). Breese (1983) suggested that the evolution and migration of perennial ryegrass throughout Europe was linked to the evolution and development of ruminant livestock farming systems. Other grasses respond to grazing pressure in a similar manner but may require more long-term selection pressures to show the same degree of phenotypic change as observed for perennial ryegrass (Casler et al., 1996). Some grasses that have evolved in grasslands that have existed for millenia cannot survive without the presence of large mammalian herbivores (McNaughton, 1979). Despite the large knowledge base on the effects of natural selection pressures on adaptive traits of forage crops, little is known of the effects of natural selection

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pressures on traits related to forage nutritional value. All forage crops contain silica, lignin, and other phenolic compounds in quantities sufficient to limit intake by ruminants (Van Soest, 1994). These compounds may act as a general defense mechanism for plants to survive and/or reproduce under grazing pressure (McNaughton, 1979; Owen and Weigert, 1981). While it is well known that herbivores can select among species (Hodgson et al., 1994), little is known about the role of herbivore selectivity in regulating evolution and natural selection of a particular species within a sward. A high concentration of formononetin, which tastes bitter and causes infertility in ewes, was related to survival of subterranean clover (Trifolium subterraneum L.) plants under grazing (Cocks et al., 1979). Sheep preferentially grazed smooth bromegrass (Bromus inermis Leyss) plants with the lowest lignin and cell-wall concentrations, suggesting that these plants might be placed at a competitive disadvantage in heterogeneous populations (Falkner and Casler, 1998). An increased concentration of cell-wall compounds that serve to strengthen the cell wall may contribute to improved levels of adaptive traits such as seed production, lodging resistance, disease resistance, and insect resistance (Buxton and Casler, 1993; Vogel and Sleper, 1994). Long-term natural selection for these adaptive traits may favor plants with higher cell-wall concentration and/or stronger cell walls. Thus, favorable forage-quality traits, such as digestibility, rate of passage through the rumen, and ease of comminution of forage particles, may be negatively affected by natural selection. As Vogel and Pedersen (1993) pointed out, breeding for improved forage quality may be viewed as selection against fitness in natural environments, but in favor of fitness under agricultural conditions.

B. UNCONSCIOUS SELECTION AND DOMESTICATION Unconscious selection is the process by which humans save the phenotypically most valuable or desirable individuals, or their seed, and destroy or ignore the less valuable or desirable individuals. This process allows humans to facilitate genetic changes, insofar as allowed by genetic variation, without the need to define or premeditate specific selection criteria or potential correlated traits. This process has led to the domestication of numerous grain, fiber, fruit, and vegetable crops (Harlan, 1975; Heiser, 1990). Numerous crops fit Isaac’s (1970) strict definition of domesticated crops, those that are phenotypically distinct from their wild relatives. However, few traditional forage crops fit this definition, as cultivated forms are rarely phenotypically distinct from their wild or naturalized forms. With the exceptions of alfalfa (Medicago sativa L.) and perennial ryegrass, most perennial forage crops have undergone fewer than 10 generations of selection from their wild state. Furthermore, there are very few examples of traits in forage crops that are traditionally associated with the process of domestication. Shattering resistance has been discovered in a

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few perennial grass species and some morphological changes toward larger and more erect tillers with lower tiller density have occurred under long-term hay managements (Casler et al., 1996). Thus, there is little reason to believe, and no evidence to suggest, that cultivated forms of most forage crops differ in forage nutritional value from wild or naturalized forms. Nevertheless, there is evidence for unconscious selection against forage nutritional value in French maize (Zea mays L.) hybrids between 1958 and 1994 (Barri`ere and Argillier, 1998). In vivo digestibility of organic matter and crude fiber decreased in the population of French maize hybrids during this time. These authors attributed these changes to successful selection for whole-plant biomass and stalk standability. Interestingly, phenotypic variability for these two measures of digestibility increased dramatically from the 1950s to the 1980s, principally due to the development of numerous low-digestibility hybrids.

C. METHODICAL (ARTIFICIAL) SELECTION PRIOR TO 1963 Methodical selection comprises the forces that are applied by humans in their systematic attempts to create predetermined changes to populations. The technology exists today to apply selection pressures at the population or cultivar level, the individual plant or genome level, and at the individual gene level. Selection pressure at the individual gene level may be applied to existing genes with obvious phenotypic effects (e.g., brown-midrib genes) or to genes introduced from other species using the processes of gene cloning and plant transformation. Plant transformation has yet to contribute to the development of new forage cultivars with improved forage nutritional value. Nevertheless it offers considerable potential to complement breeding programs, contributing to the proven rate of gain that can be achieved by selection at the population and/or plant levels. The remainder of this chapter focuses on methodical selection applied at these three levels of organization. Although formal forage breeding did not begin until the 1880s in both North America and Europe, the concept of variability among strains and the ability to select superior strains from comparative trials dates back approximately to the late 17th century (Casler et al., 1996). Letters, notes, and papers describing some of the early trials and selection activities include references to selection of strains or genotypes with superior “quality” of forage (Beddows, 1953). While we cannot know the thoughts and intentions of these early selectionists, it is likely that they were attempting to identify genetic strains that had superior vigor, little or no disease symptoms, little or no senescence, and perhaps superior acceptability to livestock. Some of these traits tend to be positively associated with modern concepts of forage nutritional value and/or act to protect the nutritional value of the plant from degradation prior to feeding (Edwards et al., 1981; Lenssen et al.,

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1991). Thus, some of the initial conscious selection practiced on forage crops may have enhanced forage nutritional value as we know it or may have protected plants from degradation of nutritional value by pathogens. The concept of digestible nutrients as a basis of evaluating and discriminating among feeds was developed in the 1850s (Van Soest, 1982). While ruminant nutritionists and chemists worked throughout the remainder of the 19th century and early 20th century to develop techniques for quantifying the nutritional components of feeds, agronomic interest did not develop until the 1930s. During the mid-20th century agronomists worked closely with ruminant nutritionists to improve upon existing techniques and develop new methods of predicting feeding value of feeds using laboratory techniques. While agronomists applied many of these techniques to agronomic studies related to hay and pasture management, plant breeders paid little heed to the concept of forage nutritional value. They had greater concerns—development of breeding methodologies for forage crops, development of cultivars with improved adaptation and persistence, and development of a seed industry to market and deploy these cultivars. The discipline of forage breeding changed dramatically with the publication of the first in vitro digestibility analysis by Tilley and Terry (1963). For the first time, forage breeders had access to a laboratory analysis method that met their need for evaluating a large number of feed samples in a short time period and had direct and obvious relevance to animal performance. Indeed, the Tilley and Terry technique was probably developed purposely for use in forage breeding programs, as suggested in the first paragraph of their paper. Its first application actually predates publication of the technique itself (Cooper et al., 1962). J. P. Cooper of the Welsh Plant Breeding Station, with collaboration from J. M. A. Tilley and R. A. Terry, was the first to demonstrate that a laboratory measure of forage nutritional value could be treated as a heritable quantitative genetic trait. The Tilley and Terry procedure had nearly all the necessary characteristics for a reasonable selection criterion in a forage breeding program: rapid, repeatable, amenable to a relatively small sample size, heritable, and directly correlated with animal performance. Casler and Vogel (1999) described this procedure as the catalyst of the foragequality revolution that occured during the last third of the 20th century.

III. METHODICAL (ARTIFICIAL) SELECTION POST-1963 A. SELECTION CRITERIA The decision to devote resources toward forage nutritional value traits in a breeding program is usually made in response to a real or perceived need, typically

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in consultation or cooperation with agronomists and ruminant nutritionists. In some cases, such as with antiquality components of some species, the need for breeding and selection activity and the selection criteria to be used are obvious. In other cases, such as energy content, intake potential, and ease of comminution of forages, disagreements, confusion, scientific curiosity, and human ingenuity have resulted in a wide array of selection criteria, many of which are reviewed here. Many more potential selection criteria have been proposed than are reviewed here—this chapter focuses on those for which selection pressure has been applied and documented. In the first of two Delphi surveys, most forage-livestock researchers tended to agree that some measure of forage digestibility was the most important potential selection criterion for improving overall nutritional value of both grasses and legumes (Wheeler and Corbett, 1989). In the second survey, digestibility ranked first for grasses and third for legumes, having been surpassed in perceived importance by the need to eliminate antiquality components and create an optimum balance between rumen-degradable vs undegradable protein (Smith et al., 1997). Recent scientific advancements in the nutritional implications of secondary compounds of legumes and the fate of protein in the ruminant digestive tract may play a role in the evolution of these attitudes and perceptions. Finally, decision support systems for livestock production systems may play a role in future decision making within forage breeding programs. Current mathematical models and computer programs are sufficiently sophisticated to allow predictions of economic impact of new cultivars with a wide array of new characteristics, such as increased digestibility or reduced fiber concentration (Clark and Wilson, 1993; Donnelly et al., 1994; Undersander et al., 1993). Activities of plant breeders in this area, and quantitative reports of their progress, are sufficiently numerous that realistic estimates of long-term gains are possible for many forage nutritional value traits which are used as input criteria in decision support systems. Coupling simulation results of breeding objectives and hypothetical (or desired) breeding progress with the costs of plant-breeding programs can provide data upon which to base rational and objective decisions with regard to breeding objectives. 1. Measures of Digestibility Digestibility is one of the most important characteristics of a forage, in terms of its nutritional value. It is a measure of energy availability to the ruminant. It also influences rate of passage of forage particles from the rumen, as feed particles must be broken down sufficiently to pass from the rumen (Poppi et al., 1985; Waghorn et al., 1989). The rate at which forage particles are broken down and cleared from the rumen is a major constraint to voluntary intake by ruminants (Weston, 1985; Wilson and Kennedy, 1996). Thus, improved digestibility may lead to improved animal performance by improving energy availability, rate of passage, and intake.

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This almost universal benefit from improved digestibility is likely responsible for its overwhelming popularity among plant breeders as the most important selection and evaluation criterion for improving forage nutritional value. Modified Tilley and Terry procedures represent the most common selection criteria for improving the digestibility of forage crops. As of 1993, genetic variation for in vitro dry-matter digestibility (IVDMD) had been documented for 17 species (Buxton and Casler, 1993) and several more species that are described in more recent publications. Table 25-4 in Buxton and Casler (1993) clearly shows that relatively small differences among clones, families, or cultivars can be detected for IVDMD, with 18 of 32 studies capable of detecting genetic differences as small as 30% of the range among genotype means with 95% confidence. The wealth of genetic variation for measures of digestibility in most forage crops supports the previous conclusions that natural and unconscious selection have had little impact on forage nutritional value traits. Genetic progress for increased IVDMD, or some related measure of forage nutritional value, has been documented in several species, including legumes, warm-season grasses, and cool-season grasses (Table I). This species list is shorter than that for documentation of genetic variation per se because more effort and time is required to document genetic gains and because improved populations and cultivars are not always evaluated in direct comparison to their parents. To properly document genetic progress, populations that have undergone selection for IVDMD (as well as any other trait) should be compared to the original, unselected populations in replicated and randomized trials, preferably in multiple environments. Genetic gains documented in this manner have ranged from 1.0 to 4.7% year−1 , as a percentage of the original population mean (Table I). These values are all much higher than long-term rates of gain for forage yield (Casler, 1998), suggesting that improvements in IVDMD may be obtained more easily than improvements in forage yield. Dry matter disappearance from the in situ nylon-bag procedure (NBDMD) has been one of the principal selection criteria of the USDA-ARS bermudagrass [Cynodon dactylon (L.) Pers.] breeding program at Tifton, Georgia, U.S.A. since the early 1960s. This program was the first to document genetic gains in forage nutritional value (Burton et al., 1967). Genetic gains in NBDMD averaged 2 g kg−1 year−1 between 1963 and 1993 (Fig. 1). Relatively small improvements in NBDMD can provide large improvements in animal performance. A 3.8% superiority in NBDMD of “Grazer” bermudagrass over three check cultivars resulted in a 9.0% improvement in mean liveweight beef production over the same three check cultivars (Eichhorn et al., 1986). In vitro digestibility methods have been adapted to study the disappearance of several components of forage-plant dry matter. In smooth bromegrass, genotypic correlations among the in vitro digestibilities of neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose, and hemicellulose were all 0.87 ≥ r ≥ 0.99

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BREEDING FOR INCREASED FORAGE NUTRITIONAL VALUE Table I

Summary of Experiments Documenting Genetic Gains for Measures of Digestibility in Forage Crops Measured as the Difference between Selected and Unselected Populations Selection criteriona / forage species High IVDMD Orchardgrass

Smooth bromegrass

Timothy Perennial ryegrass Switchgrass

Digitaria milanjiana Alfalfa

High NBDMD Bermudagrass Low ADF and high CP Alfalfa Low lignin concentration Smooth bromegrass Alfalfa

Rate of gain for IVDMDc rb

C b g kg−1 cycle−1

% cycle−1

Reference

pb

Breese and Davies (1970) Rind and Carlson (1988) Saiga (1983) Carpenter and Casler (1990) Ehlke et al. (1986) Surprenant et al. (1990) Beerepoot et al. (1994) Godshalk et al. (1988b) Hopkins et al. (1993) Hacker (1986) Shenk and Elliot (1970) Jung et al. (1994)

NAd

3

3

20

3.0

0.07

2

1

15

2.2

0.01 0.12

1 1

1 1

14 8

2.1 1.3

0.03 0.03

1 2

1 1

26 8

4.0 2.4

0.09

1

1

7

1.0

0.04

1

2

10

1.6

0.19

1

3

13

2.6

<0.01 0.06

6 1

1 1

31 24

4.7 7.0

0.16

1

1

5

1.2

Hill et al. (1993)

NA

≥2

4

66

3.1

Vaughn et al. (1990)

NA

1

2

16

2.1

Carpenter and Casler (1990) Kephart et al. (1990)

0.12

1

1

10

1.6

0.10

1

1

14

2.7

a IVDMD, in vitro dry-matter digestibility; NBDMD, nylon-bag dry-matter digestibility; ADF, acid detergent fiber concentration; CP, crude protein concentration. b p, harmonic mean of the proportion selected in each cycle; r, number of replicates or repeated observations made prior to selection; C, number of cycles or generations. c All gains were measured by some form of Tilley and Terry (1963) or fungal cellulase fermentation (IVDMD). d Information not provided by authors or by the authors’ references.

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Figure 1 Progress in breeding bermudagrass for nylon-bag dry-matter digestibility (NBDMD) and forage yield as a function of year of cultivar registration. Cultivars were Coastal (1947), Tifton 44 (1978), Tifton 68 (1984), Tifton 78 (1988), and Tifton 85 (1993). Data were taken from Hill et al. (1993).

(Casler, 1987). Furthermore, the mean in vitro digestibilities of these components were also similar. Thus, limitations to digestion of the cell wall appear to apply uniformly to different components of the cell wall, as determined by the detergent system. In vitro digestibility of fiber components had lower genotypic correlations with IVDMD than among themselves (0.72 ≥ r ≥ 0.79), probably due to genetic variation for the concentration of cell solubles (Casler, 1987) or to genetic variation for the digestibility of cell solubles (Radojevic et al., 1994). In vitro digestibility of most cell-wall polysaccarides (xylose, arabinose, galactose, rhamnose, fucose, and uronic acids) showed similar responses to divergent selection for IVDMD or lignin concentration in alfalfa (Jung et al., 1994). In vitro digestibilities of glucose and mannose did not respond to selection, suggesting that degradation of these two polysaccarides is unrelated to lignification in this alfalfa population. Because a reliable source of rumen fluid may not always be available, the IVDMD procedure may not be practical for all breeding programs. Fungal cellulase solutions have been used in conjunction with neutral-detergent or acid-pepsin pretreatments as a substitute for rumen fluid in estimating IVDMD (Bughrara et al., 1989). Mean estimated digestibilities are similar for the two procedures and the genotypic correlation for the Tilley and Terry vs the fungal cellulase digestion is generally high, indicating that the two procedures could substitute for each other in a breeding program (Casler and Sleper, 1991; Lila et al., 1986), although there may be exceptions to this generalization (Ames et al., 1993). There is also recent interest and potential for adapting measures of gas production during in vitro fermentation for use as a forage breeding selection criterion (Van Loo et al., 1994). Most efforts to develop perennial ryegrass germplasm with improved forage nutritional value are based on high water-soluble carbohydrate (WSC) concentration

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as the principal selection criterion (Humphreys, 1989a, 1994). High WSC concentrations offer several nutritional advantages to a feed: buffering mid-summer declines in IVDMD due to reduced fiber digestibility (Radojevic et al., 1994), increasing animal preference for fresh forage or pasture (Jones and Roberts, 1991), providing adequate silage fermentation without additives (Haigh, 1990), promoting more rapid digestion in the rumen and particle passage from the rumen (Moseley and Antuna Manendez, 1989), and providing a regular supply of readily fermentable carbohydrates as an energy source for rumen microbial growth (Beever, 1993). There appears to be more genetic variation for WSC than for IVDMD in perennial ryegrass (Humphreys, 1989a, 1994; Radojevic et al., 1994). Selection for increased WSC has been effective in perennial ryegrass, although its inheritance appears to be more complex than for IVDMD, with an apparently large nonadditive component (Humphreys, 1989a). The concentration of WSC is not correlated with forage yield (Humphreys, 1989b), but is positively correlated with IVDMD (Humphreys, 1989b; Radojevic et al., 1994). The concentration of ADF has been used as an indirect predictor of forage digestibility (Undersander et al., 1993). The use of ADF offers an opportunity to avoid dependence on a biological system (rumen fluid or fungal cellulase) and the laboratory variability associated with enzymatic solutions derived from biological organisms. Two cycles of recurrent selection for combined low ADF and high crude protein (CP) concentration in alfalfa resulted in average reductions of −12.5 g kg−1 in ADF and −15.5 g kg−1 in NDF and increases of 12.0 g kg−1 in CP, 16.1 g kg−1 in IVDMD, and 16.4 g kg−1 in in vitro fiber digestibility, IVFD (Vaughn et al., 1990). It appears that genetic reductions in ADF (lignin + cellulose) may improve digestibility partly by reducing cell-wall concentration per se and partly by reducing cell-wall lignification (Coors et al., 1986). However, it is not clear from this study whether the gains in in vitro digestibilities were entirely due to the reduction in ADF concentration or were partly due to the increase in CP concentration, which might serve to enhance the efficiency of microbial degradation by providing additional soluble protein for rumen microorganisms. Successful selection for increased CP concentration in two grass species has also led to correlated responses for increased IVDMD (Clements, 1969; Surprenant et al., 1990), suggesting that microbial degradation may be enhanced by increased CP concentration. However, in the Surprenant et al. (1990) study, selection for increased IVDMD also led to increased CP concentration, suggesting that the genetic correlation between IVDMD and CP may arise developmentally within the plant rather than as a result of enhanced anaerobic microbial degradation. Measures of digestibility are defined only in terms of a plant–microbe interaction within an anaerobic environment, i.e., they are not genetic traits per se. While IVDMD and other measures of digestibility are under genetic control, this genetic regulation must be manifested through some plant trait(s) that are directly coded by the plant’s genes. Selection studies to date have conclusively demonstrated several

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mechanisms by which IVDMD can be increased in forage plants: decreasing cellwall concentration (usually measured by NDF), decreasing lignin concentration within the cell wall, and reducing the extent of lignin–polysaccaride cross-linking. Genetic changes in plant anatomy and morphology also may bring about changes in whole-plant IVDMD, but it is not yet clear whether anatomical structure or chemical composition is the cause or the effect. This is discussed in a later section (III,B,4,c). Lignin concentration and composition appear to regulate much of the genetic variation in IVDMD within forage crops. In smooth bromegrass, a series of experiments showed that each 10-g kg−1 increase in IVDMD was apparently caused by a 1.3-g kg−1 decrease in acid detergent lignin (ADL) concentration (Carpenter and Casler, 1990; Casler, 1986, 1987; Ehlke et al., 1986). Genetic variation in ADL concentration accounted for over 80% of the genetic variation in IVDMD in each of the above studies. Genetic changes in IVDMD were also associated with cell-wall lignin concentration in switchgrass, Panicum virgatum L. (Hopkins et al., 1995), and alfalfa (Coors et al., 1986; Kephart et al., 1989). Furthermore, selection for low cell-wall lignification can be more effective for increasing in vitro digestibility of dry matter and cell-wall carbohydrates than selection for high IVDMD (Jung et al., 1994). Lignin concentration and composition do not appear to be independent traits in forage crops. Low-lignin plants tend to have increased molar ratios of esterified ferulic acid to esterified p-coumaric acid, EstFA:EstPCA (Jung and Casler, 1990), and lower levels of etherified ferulic acid (Argillier et al., 1996). Thus, high-IVDMD plants tend to have higher EstFA:EstPCA ratios, as observed in smooth bromegrass (Jung and Casler, 1990), switchgrass (Gabrielson et al., 1990), and silage maize (Barri`ere and Argillier, 1993). These results suggest that ferulic cross-linkages between arabinoxylans and lignin may be more important in regulating genetic variation for IVDMD than the concentration per se of these constituents (Jung and Deetz, 1993). Ferulic acid esterifies to arabinose subunits of arabinoxylan chains while p-coumaric acid esterifies to lignin during plant development (Jung, 1989). As plants mature, esterified ferulates become etherified to lignin, forming cross-linkages between lignin and cell-wall polysaccarides (Iiyama et al., 1990). Ferulate-polysaccaride esters act as initiation sites for lignification and may regulate the degree of lignin–polysaccaride cross-linking that occurs as plants develop (Ralph et al., 1995). Because the concentrations and relative ratios of lignin monomers (Jung and Casler, 1990) and polysaccaride monomers (de Ruiter et al., 1992; Godshalk et al., 1988a) appear to be under genetic control in forage crops, it is theoretically possible to create genotypes with an altered rate or extent of lignin–polysaccaride cross-linkage formation during development. The relatively consistent negative genetic correlation between EstFA:EstPCA and lignin concentration represents an obstacle to gaining a clearer understanding of the genetic regulation of IVDMD and IVFD. Casler and Jung (1999) used

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divergent selection to create groups of smooth bromegrass genotypes that differed in Klason lignin (KL) concentration but had uniform etherified ferulic acid (EthFA) concentration as well as groups with the converse characteristics (differences in EthFA, but uniform in KL). Their selection protocol allowed independent statistical tests of the genetic effects of EthFA or KL on IVFD. They found that the amount of ferulic acid etherified to lignin was not dependent on the amount of lignin per se, i.e., that the two traits could be independently adjusted. In vitro fiber digestibility was reduced by one of three mechanisms: (1) additional ferulate cross-linking, (2) additional biosynthesis of lignin per se, or (3) a combination of both processes. The concentration of EthFA appeared to have a slightly greater and more consistent effect on IVFD than the concentration of KL, but this may have been due to an apparent lower heritability for KL (Casler and Jung, 1999). These results clearly indicate that additional research on the molecular structure and cross-linking relationships of forage-plant cell walls would be valuable to forage breeders. Finally, a genetic reduction in NDF concentration typically results in increased IVDMD (Carpenter and Casler, 1990; Casler, 2000; Shenk and Elliot, 1970). However, the converse is not true—selection for increased IVDMD does not generally decrease NDF concentration (Carpenter and Casler, 1990; Gabrielson et al., 1990; Surprenant et al., 1990), probably because selection for high IVDMD per se acts on genes that control lignin concentration or composition as described above. Furthermore, genetic reductions in NDF concentration do not affect the digestibility of the NDF fraction. Thus, increased IVDMD associated with reduced NDF appears to represent a dilution effect in which the greater concentration of highly digestible cell solubles results in higher dry-matter digestibility (Casler, 2000). This asymmetrical relationship between IVDMD and NDF points out the potential danger in drawing inferences about correlated selection responses simply from correlation coefficients per se. Nearly all correlation coefficients reported in the literature suggest that IVDMD and NDF have a high negative correlation, an oversimplification of the above relationships. While it requires more labor than IVDMD, in vitro digestibility of the NDF fraction (IVFD) is a more meaningful selection criterion to improve digestibility of the cell wall per se. In vitro NDF digestibility is not genetically correlated with NDF concentration per se (Casler, 2000), so selection pressure for IVFD will apply directly to cell-wall structure and/or composition rather than to concentration. Despite a recent increase in the use of IVFD as a measurement tool, use of IVFD as a selection criterion has been reported in the literature only for silage maize (Barri`ere et al., 1997; Dolstra et al., 1993). Genetic increases in IVDMD have led to the development of several cultivars, some of which have been evaluated in grazing or feeding experiments with ruminants. In general, a 1% increase in IVDMD gave rise to a 3.2% increase in average daily gains (Casler and Vogel, 1996). In addition, concomitant increases

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in forage availability for most cultivars also led to increased animal production per hectare (Casler and Vogel, 1994). These authors also documented the importance of animal performance data and its use in extension, outreach, or marketing programs to successfully promote use of high-digestibility cultivars. 2. Measures of Intake Genetic improvements in voluntary intake potential of forage crops may be potentially more valuable than genetic improvements in digestibility, given the greater importance of intake to animal performance (Fahey and Hussein, 1999). For most forage diets, intake cannot be maximized due to limitations in feed quality (Van Soest, 1994). Fibrous bulk, the plant cell wall, is generally considered to be the factor most limiting to feed intake. Intake of fibrous bulk generally causes rumen fill and satiation before the ruminant has maximized its caloric intake, resulting in a reduced plane of nutrition (Van Soest, 1994). While increased fiber digestibility may partly ameliorate this limitation by increasing rate of particle size breakdown and passage, it does not necessarily contribute to increased intake because fiber concentration and digestibility are not necessarily correlated. The concentration of NDF in forage crops, which is generally accepted as an approximate measure of cell-wall concentration, appears to be under genetic control in a manner similar to that for IVDMD (Table II). Ranges of variation within populations, realized heritability, and genetic gains, although reported for fewer species, are similar to those for IVDMD. Genetic progress toward reduced NDF concentration has been reported for smooth bromegrass (Casler, 1999a); reed canarygrass, Phalaris arundinacea L. (Surprenant et al., 1988); and maize (Wolf et al., 1993). Genetic progress has ranged from 5 to 13 g kg−1 year−1 (0.8 to 2.0% year−1 ). An alternative strategy toward improving intake would be to develop forages that are degradaded into smaller particles at a more rapid rate, i.e., to reduce their breakdown resistance. Smaller particles have a greater surface-area-to-volume ratio and more sites for enzymatic degradation of carbohydrates. More rapid particle size breakdown and subsequent enzymatic degradation would increase rate of passage, reducing the time required before hunger is triggered again. Ruminants reduce the particle size of feeds largely through the processes of chewing during eating and rumination (McLeod and Minson, 1988; Wilson et al., 1989). Artificial measures of breakdown resistance that have shown high correlations with voluntary intake include a particle size index following artificial mastication (Troelson and Bigsby, 1964), the energy required to grind forage samples to pass a given mesh size (Laredo and Minson, 1973; Weston, 1985), and the energy required to shear leaf tissue (Henry et al., 1996; Mackinnon et al., 1988). The last of these is the only variable to have been used as a selection criterion in developing new germplasm. Divergent selection for leaf-blade shear energy in

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Table II Summary of Experiments Documenting Genetic Gains for Measures of Intake in Forage Crops Measured as the Difference between Selected and Unselected Populations

Selection criteriona / forage species Low NDF Smooth bromegrass

Reed canarygrass Maize stalks and leaf sheaths

Maize stover

Rate of gain for specified selection criterion Reference

pb

rb

Cb

g kg−1 cycle−1

% cycle−1

Carpenter and Casler (1990) Casler (2000) Surprenant et al. (1988) Buendgen et al. (1990) Ostrander and Coors (1997) Wolf et al. (1993)

0.12

1

1

−11

−1.4

0.10 0.02

1 1

3 1

−7 −21

−1.2 −3.5

0.06

2

1

−17

−2.5

0.10

2

2

−25

−4.0

0.08

4

1

−7

−1.0

High particle-size reduction Smooth bromegrass Culvenor and Casler (1999) Low leaf shear energy Perennial ryegrass Mackinnon et al. (1988)

0.01

1

1

24

% cycle−1 7.0

0.02

1

1

kg−1 cycle−1 1.0

% cycle−1 14.7

g

kg−1

cycle−1

a NDF,

neutral detergent fiber concentration. harmonic mean of the proportion selected in each cycle; r, number of replicates or repeated observations made prior to selection; C, number of cycles or generations. b p,

perennial ryegrass led to a low-shear breaking load (LS) population that required 59% as much energy to shear as the high-shear breaking load (HS) population (Inoue et al., 1994a). However, these two populations did not differ in intake, particle size reduction, chewing time or frequency, or live-weight gains (Inoue et al., 1994b). The latter authors attributed the lack of differences in animal performance to correlated responses toward longer leaves per unit of dry weight in the LS population, which they felt resulted in a greater masticatory load for leaves of the LS population. A ball-milling procedure was adapted to predict the breakdown resistance of smooth bromegrass leaves following a 30-s ball-milling period (Casler et al., 1996). The particle-size reduction index (PSRI) computed after seiving the ball-milled particles had realized heritabilities of 10–21% (Culvenor and Casler, 1999), which are similar to most selection studies for IVDMD and NDF. One cycle of divergent selection created an average divergence of 21% of the original mean (Low-PSRI = 29.3%, Original = 34.0%, High-PSRI = 36.4%).

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3. Protein Concentration and Composition Crude protein (CP) concentration has received relatively little attention. For grasses, this is likely because inexpensive nitrogen fertilizer is very effective at increasing CP concentration of forage. For legumes, this may be because of relatively high existing CP concentrations. In addition, protein ingested by ruminants is often used inefficiently. Most soluble protein is degraded to ammonia in the rumen, much of which is absorbed into the bloodstream and eventually excreted as urea. Nondegradable protein passes through the rumen where it is more efficiently utilized in the lower digestive tract. Thus, CP concentration per se is of questionable relevance to the nutrition of ruminants and generally not considered to be a useful selection criterion for improving nutritional value of forages (Smith et al., 1997). Genetic progress for increased CP (or N) concentration has been documented in several species (Table III). The progress achieved from selection in perennial ryegrass was measureable at N-fertilization rates of 0 to 100 kg N ha−1 ; N rates above this level negated the differences between selected and unselected lines (Arcioni et al., 1983). Conversely, alfalfa populations were selected for high reduced-N Table III Summary of Experiments Documenting Genetic Gains for Crude Protein (CP) or N Concentration in Forage Crops Measured as the Difference between Selected and Unselected Populations Rate of gain for specified selection criterion Selection criterion/ forage species High CP concentration Perennial ryegrass Timothy Alfalfa High N concentration Phalaris Alfalfa

Reference

pa

ra

Ca

g kg−1 cycle−1

% cycle−1

Arcioni et al. (1983) Surprenant et al. (1990) Vaughn et al. (1990)

0.11

6

1

17.0

6.5

0.04

1

1

7.4

7.7

NAb

1

2

6.0

3.2

0.09 0.03

1 1

3 2

1.0 0.7

3.5 2.3

0.05

1

2

0.7

2.6

Clements (1969) Demment et al. (1986) Teuber and Phillips (1988)

a p, harmonic mean of the proportion selected in each cycle; r, number of replicates or repeated observations made prior to selection; C, number of cycles or generations. b Information not provided by authors or by the authors’ references.

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concentration under conditions of N2 fixation and NH4 NO3 fertilization. Selection for high reduced-N concentration under both conditions resulted in improved populations that maintained superior reduced-N concentration under both conditions (Phillips et al., 1982) and regardless of the Rhizobium strain used for inoculation (Phillips et al., 1985). However, in another alfalfa population, genetic progress was partially negated by fertilization with NH4 NO3 , as observed in the perennial ryegrass study (Teuber and Phillips, 1988). Genetic variation for ruminal protein degradation parameters is present within alfalfa (Broderick and Buxton, 1991), suggesting the possibility that traditional breeding methods may be useful for improving protein quality. However, efficient and repeatable measurement of nondegradable protein for thousands of samples is a difficult and expensive proposition. It is more likely that gene cloning, combined with plant transformation, will contribute to genetic improvements in protein quality (see Section IV). 4. Antiquality Components Saponins are biological toxins that are found in alfalfa leaf tissue and cause reduced growth rates and production in monogastrics (Elliot et al., 1972). Selection for decreased saponin concentration has been highly successful but may decrease the resistance of alfalfa to insect pests (Pedersen et al., 1976; Small, 1996). Saponins were once thought to be important in regulating bloat of ruminants, but comparisons of high- and low-saponin alfalfa populations showed no differences in bloat characteristics (Majak et al., 1980). Tannins are thought to form insoluble complexes with proteins, reducing or eliminating bloat potential in some legumes. However, genetic variation for tannins has not been found within bloat-causing legumes such as alfalfa (Hill et al., 1988). Furthermore, somatic hybridization between alfalfa and bloat-safe sanfoin (Onobrychis viciifolia Scop.) failed to produce tannin-positive regenerants (Li et al., 1993). Estrogenic compounds of some legumes, such as formononetin, cause reduced fertility in grazing ruminants that can become progressive and permanent with long-term grazing of estrogenic legumes (McDonald, 1995). Selection for low formononetin has been successful in both red clover (Trifolium pratense L.) and subterranean clover (T. subterraneum L. var. yanninicum Zohary and Heller). Several cultivars have been developed with acceptably low formononetin levels and no detrimental effects on agronomic performance (e.g., Dear et al., 1997; Rumball et al., 1997). Low-formononetin red clover reduced infertility problems and breeding delays of ewes (Ovis aries) relative to a high-formononetin cultivar (McDonald et al., 1994). Numerous alkaloids are present in the herbage of reed canarygrass (Marten et al., 1976) and phalaris, Phalaris tuberosa L. (Oram et al., 1985), reducing intake and liveweight gains and, for phalaris, even causing death. In phalaris, five

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cycles of selection reduced tryptamine concentration by 96%, lessening, but not eliminating, the toxicity of phalaris herbage (Oram et al., 1985). Selection has been successful at creating new cultivars of reed canarygrass with low levels of gramine and absense of tryptamines and ß-carbolines. These changes have led to reduced toxicity and increased liveweight gains of grazing ruminants (Marten, 1989). The successes in breeding low-gramine and tryptamine/b-carboline-free reed canarygrasses has had negative effects as well. The combination of breeding successes; scientific documentation of the problem and solution; and the efforts of extension, outreach, and marketing programs has virtually eliminated any North American demand for reed canarygrass cultivars that do not fit the above alkaloid profile. Seed of many older cultivars is no longer maintained or increased and some germplasm has undoubtedly been lost. In some cases, the only viable seed that represents these cultivars may be that held in governmental germplasm collections. Furthermore, the development of favorable alkaloid-profile cultivars was based on the rapid and intensive elimination of all genotypes that did not fit the desired alkaloid profile. This practice likely has narrowed the germplasm base of the current agriculturally desirable reed canarygrass phenotype, potentially leading to genetic vulnerability and limitations to long-term breeding progress. Current interest in reed canarygrass germplasm for use as a biofuel crop, for which animal preference and performance is unimportant, underscores the potential dangers of commodity- or market-driven germplasm management. 5. Mineral Element Concentrations and Ratios Hypomagnesemia is likely the most serious ruminant disease caused by mineral imbalance of forage crops. It is caused by a deficiency of Mg and/or an excess of K in the forage tissue. Two breeding programs have attacked this problem by selecting for increased Mg uptake. In Italian ryegrass, a 56% increase in Mg concentration of the forage led to a 13% increase in blood serum Mg and a 12% increase in dry matter intake of grazing ewes (Moseley and Baker, 1991). Ewe and lamb liveweight gains increased by 10%, while the proportion of clinical cases of hypomagnesemia was reduced by 88% with no fatalities among 120 ewes grazing the high-Mg population. In tall fescue (Festuca arundinacea Schreb.), selection for increased Mg and a reduction in the mineral ratio K/(Ca + Mg) (Reeder et al., 1986) led to a reduction in the mineral ratio of 18% (Mayland and Sleper, 1993). This resulted in a 12% increase in blood serum Mg levels of grazing cattle and an 8% reduction in the blood serum mineral ratio K/(Ca + Mg) (D. A. Sleper et al., 1997, personal communication). While other mineral imbalances exist in many forage crops, they can generally be corrected by mineral supplementation in the diet of ruminants or by corrective fertilization of the forage crop. These solutions are often sufficiently inexpensive that forage breeders can rarely justify efforts to correct mineral imbalances by

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breeding, unless they seriously threaten animal health, as with Mg deficiency. Lack of initiative by the seed industry and/or lack of interest among producers may provide an additional disincentive for such a breeding objective. The highMg tall fescue cultivar has failed to generate interest in the seed industry, despite the availability of animal performance data (D. A. Sleper, 1998, personal communication). Breeding for high Mg concentration ranked 12th of 13 possible selection criteria related to forage nutritional value of grasses, probably because most researchers felt that mineral supplementation is the simplest and most economical preventative for hypomagnesemia (Smith et al., 1997).

B. SELECTION METHODOLOGY Technological and methodological improvements and refinements during the past 40 years have greatly improved the efficiency of forage breeding for improved nutritional value. Many of these contributions derive from collaborations between plant breeders and ruminant nutritionists, such as the Tilley and Terry in vitro digestibility test. 1. Contributions of Near-Infrared Reflectance Spectroscopy (NIRS) Technology J. S. Shenk is the one person most responsible for adapting NIRS technology to predict forage nutritional value traits of forage samples. Indeed, his name has become synonymous with the use of NIRS in forage evaluation laboratories. He and his colleagues have made numerous methodological and technological contributions toward making NIRS technology repeatable, understandable, and affordable for most forage researchers. His successful efforts to develop NIRS into a routine and acceptable forage evaluation method probably rank as the second most important development in the history of breeding forage crops for improved nutritional value, behind the Tilley and Terry procedure. The improvements to NIRS technology have resulted in an ability to predict animal response directly from forage spectra with greater accuracy and precision than from standard laboratory reference methods (Shenk and Westerhaus, 1995). What history has not recorded is John Shenk’s original motivation for adapting NIRS to forage evaluation—to improve the efficiency of his forage grass breeding program at The Pennsylvania State University (Shenk, 1975, 1977). His successful applications quickly led him to conclude that he could serve a greater good by terminating the grass breeding program and devoting himself full-time to improving NIRS technology and methodology. While the forage breeding community lost another grass breeder, it gained an amazingly effective new technology that improved the efficiency of numerous forage-breeding programs around the world.

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The use of NIRS improves the efficiency of forage-breeding programs by three mechanisms. First, it reduces the cost associated with laboratory analysis. For example, in our laboratory, a typical selection experiment for NDF concentration may include 1000 forage samples. Amortizing the NIRS equipment and software over a 10-year period and assuming that it is in nearly constant use, total expenses for the NIRS analysis (labor and equipment) would be 19% of those for the wetlaboratory analysis (labor, supplies, and equipment). This figure will vary with the wet-laboratory cost of the variable (e.g., 12% for IVFD to 30% for IVDMD) and with the size of the experiment (larger experiments increase the cost advantage of NIRS). These figures are based on a calibration sample of approximately 8–10% of the total number of samples (M. D. Casler and K. A. Darling, 1999, unpublished data). These figures did not include the cost of harvesting, processing, and grinding forage samples, which is equal for the two analytical procedures. Second, NIRS is much faster that wet-laboratory analysis. In the above example of 1000 NDF samples, a trained half-time laboratory technician can deliver the data 4 weeks from receipt of samples using NIRS with a closed-population calibration. The closed-population calibration involves development of a new calibration equation specifically for the experiment in question, differing from the open-population calibration in which a general calibration sample set is maintained over time and updated with a relatively small number of samples from the experiment in question (Windham et al., 1989). Using an open-population calibration would reduce this time requirement from 4 to 3 weeks, although open-population calibrations tend to be less efficient (Aastveit and Marum, 1993). An exclusively wet-laboratory analysis of these 1000 NDF samples would require the same technician 28 weeks to deliver the data (M. D. Casler and K. A. Darling, 1999, unpublished data). With other competing demands on the technician’s time, such a time committment is nearly impossible to justify. Many forage crop breeders could not justify their efforts to improve forage nutritional value without the use of NIRS. Indeed, selection for improved forage nutritional value became a major objective of the U.S. alfalfa industry in the early 1990s only because of the existence of NIRS and the technological improvements that were made to both hardware and software in the 1970s and 1980s (Hill et al., 1988; Shenk and Westerhaus, 1995). Third, the above two factors (reductions in both monetary and time requirements) combine to allow the plant breeder to analyze many more genotypes and/or families than would be possible with wet-laboratory procedures. The clich´e, “plant breeding is a numbers game” is also one of the most important principles in plantbreeding theory. Larger populations of plants allow the plant breeder to maintain a larger effective population size, reducing the potential for inbreeding depression and allowing the breeder to use a greater selection intensity than would be possible with smaller populations (Comstock, 1996, Chapter 11). Furthermore, NIRS technology allows large selection pressures to be applied to many more populations than would be possible with wet-laboratory techniques (Casler, 1999a; Casler

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and Jung, 1999). Larger selection intensities and application of selection pressure to multiple populations greatly increase the likelihood of making measurable and economically viable genetic gains (Casler et al., 1996). The above three factors combine to create flexibility in a forage crop-breeding program that would not exist for a selection criterion based on a wet-laboratory procedure. Wet-laboratory procedures require such large amounts of time that forage samples cannot be processed to completion within the duration of the growing season. Therefore, a field-based breeding program would require a minimum of 3 years to complete one cycle of selection: Year 1 for establishment of the nursery, Year 2 for sample collection, and Year 3 for intercrossing selections. The use of NIRS could reduce this to 2 years if selections can be identified and transplanted into crossing blocks prior to anthesis during Year 2. Finally, the reduction in laboratory expense associated with NIRS is further enhanced if the forage nutritional value selection program is based on multiple traits, such as the Cornell University alfalfa-breeding program, which is based on low ADF combined with high CP (Vaughn et al., 1990). Because forage sample spectra can be used to predict a wide array of plant traits, the only additional expense associated with multiple-trait selection would be the expense of wet-laboratory analysis of the additional traits on the calibration subset of forage samples. The NIRS procedure is sufficiently versatile that it can efficiently predict a wide array of chemical traits (Barton, 1989), IVDMD (Barton, 1989; Shenk and Westerhaus, 1991), and mineral element concentrations (Clark et al., 1987). While NIRS technology is based on differences in bending and stretching of C-H, O-H, and N-H bonds under near-infrared light (Shenk and Westerhaus, 1995), the concentration of mineral elements can be predicted with reasonable accuracy and precision through their associations with these bonding patterns in forage samples. In smooth bromegrass, the genetic correlation between vegetative growth stages in early spring and the target growth stage for a late-spring hay crop is sufficiently high that selection at either growth stage should largely identify the same groups of genotypes (Reich and Casler, 1985). The NIRS procedure was required to generate all laboratory data within the 3-week window between the vegetative growth stage and anthesis. This hypothesis was validated in a selection experiment that showed selection at the vegetative growth stage (using NIRS) to be nearly as effective as selection at the heading growth stage (using wet-laboratory analysis) in terms of reducing NDF concentration at the heading growth stage (−2.6 vs −3.4 g kg−1 year−1 ; Casler, 1999a). The vegetative growth-stage procedure appeared to suffer from incomplete and nonrandom pollination in the in situ crossing block, a problem that could be solved by a more imaginative procedure to promote more complete intercrossing of selected plants (Burton, 1982). A direct comparison of NIRS vs wet-laboratory procedures, both conducted on forage samples collected at the heading growth stage, showed that the exclusive wet-laboratory procedure was more than three times more effective than the NIRS procedure

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(−2.5 vs −0.8 g kg−1 year−1 ), likely due to the extreme sensitivity of NIRS to uncontrolled sampling variation in plant-part composition at the heading growth stage (Casler, 1999a). This conclusion was supported by the observation that the NIRS procedure was three times more effective when applied to forage samples collected at the vegetative vs heading growth stage (−2.4 vs −0.8 g kg−1 year−1 ). 2. Recurrent Selection Nearly all genetic gains in forage nutritional value traits have been made with some form of recurrent selection. Recurrent selection consists of repeated cycles of selection in which each cycle involves the evaluation of a population of plants, identification of a subset of superior plants, and intercrossing of the selected plants (Comstock, 1996, p. 55). Recurrent selection can be conducted in either a closed fashion, in which several cycles of selection are conducted within a defined population (Comstock, 1996, Chapters 6–8), or in an open fashion, in which new and unrelated germplasm can be hybridized with selected plants from the recurrent selection program (Cramer and Kannenberg, 1992). The goal of recurrent selection, regardless of the specific procedure, is to increase the frequency of favorable alleles controlling the trait of interest, without unnecessarily restricting genetic variability for other traits (Comstock, 1996, pp. 69–72). While closed-population recurrent selection is the most common approach for improving forage nutritional value traits, open-population recurrent selection has been used in the longest running recurrent-selection program, the bermudagrass program at Tifton, Georgia (Fig. 1). Because bermudagrass cultivars are clonally propagated, the single best individual plant within each cycle can be clonally increased and released as a new cultivar. Because of this, all forms of genetic variation (additive, dominance, and epistasis) can be utilized in the selection process, much like in hybrid maize breeding. Each new cultivar was crossed to other, unrelated cultivars or to accessions collected from other regions of the world, allowing the breeder to select plants with unique and favorable dominance and epistatic gene combinations, the best of which represent the culmination of that selection cycle (Burton, 1989). Most other forage crops are seed-propagated perennials that are commercialized as synthetic cultivars, which are heterogeneous mixtures of highly heterozygous individuals. While open-population recurrent selection could also be used on seed-propagated species, closed-population methods seem to be the method of choice. Heritability is generally sufficiently high that progress can be achieved by phenotypic selection, avoiding the need for more complicated and lengthy family selection methods (i.e., selection of the best plants based on their performance per se rather than on the basis of their progenies’ performance). Realized heritability for forage nutritional value traits is generally 0.2–0.4 (Carpenter and Casler, 1990; Culvenor and Casler, 1999; Hopkins et al., 1993).

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Recurrent selection for forage nutritional value traits is typically conducted on the basis of unreplicated plants which are transplanted as seedlings into spacedplant nurseries. The spaced planting allows samples to be clipped and data to be collected rather easily from each individual plant. While replication of the individual plants, using clonal propagation, could be practiced for nearly any forage crop, it is rarely done, mostly likely due to the time and labor expense of clonal propagation and the added record-keeping complexity that would be required. Nevertheless, replication of selection units could increase heritability and selection gains (Aung et al., 1994) and has been recommended for traits, such as Klason lignin concentration, that appear to be highly sensitive to genotype × environment (GE) interactions (Casler and Jung, 1999). However, selection on replicated units will be advantageous over unreplicated units only when individualplant heritability is low, as can happen with extreme GE interactions (England, 1977). Furthermore, if replication of selection units causes the breeder to reduce selection intensity, due to cost constraints on the number of samples that can be processed, then replicated selection will be less efficient than unreplicated selection (England, 1977). Instead of spatial or clonal replication, many researchers practice replicationin-time, using repeated measures on unreplicated spaced plants to improve the precision of their selection criterion. This system is typically manifested as twostage selection, in which mild selection pressure is applied to the first harvest, followed by more intensive selection pressure on subsequent harvests (Casler and Jung, 1999; Godshalk et al., 1988b; Vogel et al., 1981). Selection in the second or later stages is often based on the mean over all available harvests. Multistage selection for IVDMD, N concentration, and forage yield in a switchgrass population was predicted to increase selection gain by 6–7% for each additional selection stage beyond the first (Godshalk et al., 1988b). Conversely, there are many examples of selection gains that have been achieved on the basis of a single observation per plant, unreplicated in either space or time (e.g., Casler, 1999a; Coors et al., 1986; Wolf et al., 1993). In a long-term selection program, a 6–7% improvement in gain per cycle is insufficient to offset the increased time required to conduct multistage selection, which may increase cycle time from 2 to 3 or 4 years. Traits that are highly sensitive to GE interactions are likely to show the greatest benefit from multistage selection (Casler and Jung, 1999). Recurrent selection for high or low CP concentration and high or low oil concentration of maize grain has shown nearly continual progress for 90 cycles of selection (Dudley and Lambert, 1992). The long-term nature of these selection gains indicate that a relatively large number of genes are segregating for these two traits in this population and that many cycles of selection are required before these genes become fixed in the homozygous state. Similar results have been demonstrated from recurrent selection for a wide array of traits in a wide array of plant and animal species (Falconer and Mackey, 1996). For nutritional value

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Figure 2 Responses to selection: (A) three cycles of positive selection and one cycle of negative selection for in vitro dry-matter digestibility (IVDMD) in switchgrass and (B) three cycles of negative selection and one cycle of positive selection for neutral detergent fiber (NDF) concentration in smooth bromegrass. Switchgrass data taken from Hopkins et al. (1993). Smooth bromegrass data taken from Casler (2000).

traits of forage crops, the longest term recurrent selection programs and their selection criteria are NBDMD of bermudagrass, four cycles (Fig. 1); IVDMD of switchgrass, three cycles (Fig. 2A); and NDF of smooth bromegrass, three cycles (Fig. 2B). In the bermudagrass program, gains in NBDMD were erratic due to simultaneous selection pressure for forage yield and other agronomic traits. In the switchgrass and smooth bromegrass programs, selection was largely for a single trait, and progress was linear through three cycles of selection for increased nutritional value (high IVDMD or low NDF) and one cycle of selection in the opposite direction (Fig. 2). Finally, recurrent selection can be a powerful tool for developing inferences about populations. Estimating genetic variances and the potential for making gains within populations is a time-consuming and expensive proposition, and reliability of results is typically limited by low degrees of freedom. Instead, many forage breeders have practiced selection for increased forage nutritional value traits in multiple populations. Because gain from selection is directly proportional to additive genetic variance for the selection criterion within the population, indirect

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estimates of additive genetic variance can be obtained from selection experiments. Instead of relying on huge numbers of families to build up degrees of freedom, the reliability of estimates based on realized gains depends on the experimental design and degree of replication, factors that are simple and relatively inexpensive to manipulate. 3. Genotype × Environment and Genotype × Management Interactions Documentation of genetic gains from recurrent selection for a forage nutritional value trait is a two-step process. In the first step, one or more cycles of selection are conducted, with the breeder saving seeds produced from intercrossing the selected plants in each cycle. In the second step, a sample of remnant seed from the original population and all selection cycles are placed in replicated and randomized trials, often at multiple locations. Because the exact environments in the original selection nurseries cannot be repeated (for field-based selection), the presense of a significant genetic gain in the second-step evaluation is, in itself, evidence that GE interactions are relatively unimportant for the selection criterion (Casler and Vogel, 1999). Furthermore, because selection is typically conducted under spaced-planted conditions, evaluations that are conducted as sward plots represent particularly robust tests of GE interaction. If GE interactions are important for the selection criterion, then the inability of forage breeders to reproduce the selection environments would lead to a high frequency of studies in which genetic gains would be highly variable among test environments and/or generally nonsignificant. As discussed above, this has not been the case. While it is possible that studies with such an undesirable result may be unpublished due to either the perception or reality that inconsistent and/or unrepeatable results are also uninteresting, the literature suggests GE interactions have been historically unimportant for forage nutritional value traits. Furthermore, a survey of 113 forage researchers indicates that this seems to be a generally accepted truism (Cherney and Volenec, 1992). Smooth bromegrass has received the greatest attention with respect to GE interactions of forage nutritional value traits. Eight experiments conducted in Wisconsin, South Dakota, and Ontario over a 20-year period show a general pattern of consistent genotype rankings across locations, years, and harvests (Carpenter and Casler, 1990; Casler et al., 1990; Christie, 1977; Collins and Drolsom, 1982; Ehlke et al., 1986; Reich and Casler, 1985; Ross et al., 1970; Sleper et al., 1973). Genetic gains were generally consistent across multiple locations and years and when measured in sward plots (Carpenter and Casler, 1990; Casler, 2000; Ehlke et al., 1986). Similar results have been reported for perennial ryegrass (Humphreys, 1989b; Wilkins, 1997), switchgrass (Hopkins et al., 1993; Vogel et al., 1981),

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bermudagrass (Hill et al., 1993), alfalfa (Coors et al., 1986; Vaughn et al., 1990), and silage maize (Argillier et al., 1997). For alfalfa and perennial ryegrass, the lack of GE interaction extended to multiple harvest managements (Vaughn et al., 1990; Wilkins, 1997). In some cases, significant GE interactions consist almost exclusively of unexplainable changes in magnitude of genotype differences, leading to relatively few or no changes in genotype ranking. Some examples of this include reed canarygrass, Phalaris arundinacea L. (Hovin et al., 1976); tall fescue, Festuca arundinacea Schreb. (Nguyen et al., 1982); quackgrass, Elytrigia repens (L.) Nevski (Greub et al., 1986); and maize (Wolf et al., 1993). However, there are a growing number of examples of significant and biologically meaningful GE interactions that are becoming increasingly important to forage breeders. Coors et al. (1986) attributed strong genotype × harvest and genotype × year interactions for forage nutritional value traits in alfalfa to genetic variation in growth habit, regrowth vigor, and fall dormancy within their populations. In smooth bromegrass, three cycles of selection were practiced for low NDF, independently at the vegetative and heading growth stages, after which all selected populations were tested at both vegetative and heading growth stages (Casler, 1999a). The most effective selection method evaluated at the vegetative growth stage was based on selection at the vegetative growth stage; results were similar for the heading growth stage. Similarly, selection for low NDF concentration in four diverse smooth bromegrass populations was evaluated throughout the growing season from early June to early October (Casler et al., 2000). Selection responses were significant only in mid-summer in both years, suggesting a photoperiod or temperature dependence of the NDF selection responses, which may be due to the fact that most selection pressure was based on one mid-summer harvest. A strong genotype × temperature interaction was also observed for IVDMD of timothy, Phleum pratense L. (Ames et al., 1993). Strong genotype × harvest and genotype × year interactions for lignin concentration of smooth bromegrass prevented detection of significant differences between divergent-lignin selections (Casler and Jung, 1999). While early results from selection experiments tended to suggest GE interactions were relatively unimportant (Buxton and Casler, 1993; Casler and Vogel, 1999), the growing database of selection experiments allows breeders to identify potentials for GE interactions and, more importantly, their biological explanations. These recent experiments indicate that GE interactions cannot be taken for granted but must continue to be evaluated in any experiment designed to estimate genetic gain from selection. In some cases, extreme GE interactions, such as those observed by Casler et al. (2000) and Casler and Jung (1999) may necessitate the use of multistage selection to ensure that gains made for the selection criterion are stable across the range of necessary environmental conditions. While the authors in both of these studies used two-stage selection, selection pressure in the second stage (50% in both cases) was likely insufficient to be useful.

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4. Potentially Undesirable Genetic Correlations with Agronomic Traits A long-time and highly successful forage breeder, R. R. Kalton, was often very critical of recurrent-selection programs that focused on improving forage nutritional value traits. He was concerned that large selection pressures were being applied to populations of plants, often based on a single trait such as IVDMD or NDF concentration. His concern was justifiable; in an applied forage-breeding program with the single goal of producing new cultivars, this could result in loss of important agronomic traits such as forage and seed yield, lodging resistance, disease and insect resistance, and stress tolerances. During the 1960s and 1970s, when few selection experiments for forage nutritional value traits were underway, many other forage researchers were skeptical of the Tilley and Terry procedure and the Van Soest detergent fiber analyses (Van Soest, 1994) as forage-breeding selection criteria. Many breeders were even more fearful than Dr. Kalton, suggesting that forage nutritional value traits could not be improved without causing losses in agronomic traits (van Bogaert, 1977). Nevertheless, single-trait recurrent selection experiments have served a valuable purpose in contributing to an increased understanding of the inheritance of forage nutritional value traits. A large proportion of these selection experiments have had dual purposes: (1) to generate germplasm that can be used to study the genetic nature of a forage nutritional value trait and (2) to produce improved germplasm or new cultivars. Many experiments have proven that predictions of correlated selection responses simply from correlation coefficients can be grossly inaccurate, leading to erroneous conclusions regarding true relationships between forage nutritional value traits and other agronomic traits. a. Reproductive Maturity Numerous changes occur as forage plants mature, most notably that all positive measures of forage nutritional value decline (digestibility and protein), while negative measures of forage nutritional value increase (NDF, lignin, and silica). Stage of reproductive maturity is probably the single most important factor, including both genetic and environmental factors, that controls the nutritional value of a feed. Indeed, ruminant nutritionists and agronomists traditionally use differential harvest dates to generate feeds of differing nutritional value. Of course, feeds generated in such a manner differ in many confounded factors. Unfortunately, some of the initial selection experiments related to forage nutritional value further confused the issue of maturity when it was observed that genetic changes in IVDMD were almost completely explained by genetic changes in maturity. For a fixed sampling date, IVDMD is negatively correlated with earliness (Frandsen, 1986; Lentz and Buxton, 1991, 1992), so that selection for increased IVDMD, ignoring maturity stage, will lead to a later heading date. In an attempt

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to correct this perceived problem, some researchers sampled plants at a defined stage of reproductive maturity, ignoring calendar date. In this scenario, IVDMD was positively correlated with earliness (Christie and Mowat, 1968; Davies, 1976), i.e., the plants with the highest IVDMD were those that had been sampled at the earliest calendar dates, resulting in the shortest time for cell-wall development to occur. Selection for high IVDMD in this scenario leads to an earlier heading date. Based partly on the these relationships and partly on cultivar evaluations that have shown similar associations between forage nutritional value and maturity (e.g., Gately, 1994), Van Wijk et al. (1993) erroneously concluded that “The progress we have witnessed in breeding for improved nutritional value largely consists of chance findings as well as of early-maturing selections from within a population” and that “Once the gross variation due to maturity classes has been taken care of by maturity-grouping there is little net variation left to be measured accurately within these maturity groups.” Recurrent selection for increased IVDMD rarely results in correlated responses for timing of reproductive maturity (Buxton and Casler, 1993; Casler, 1998; Casler and Vogel, 1999; Vogel and Sleper, 1994). In populations that are highly variable for maturity, potential correlations with maturity can be ameliorated by selecting only within maturity classes or by statistically adjusting forage nutritional value data to a constant maturity stage (Hopkins et al., 1995). There is considerable genetic variation in many species for forage nutritional value traits and a large proportion of this is independent or only weakly dependent on maturity. In smooth bromegrass, the wealth of genetic variability that exists for forage nutritional value traits is entirely independent of maturity—little genetic variation exists for timing of reproductive maturity due to extreme photoperiod sensitivity (Casler and Carlson, 1995). b. Forage Yield, Seed Yield, and Lodging Resistance Potential losses in forage yield, seed yield, and lodging resistance have been a major concern of forage breeders and agronomists for the past 40 years. Early literature on genetic variation for forage nutritional value traits showed a high frequency of negative genetic or phenotypic correlations between forage yield and forage nutritional value (Marten, 1989). Hacker (1982) was one of the first authors to point out that most of these negative correlations were sufficiently small that they could either be ignored or broken by simultaneous selection for increased forage yield and nutritional value. His prediction has largely been realized, with bermudagrass acting as the most prominent example (Fig. 1). The first cultivar in this series had increased NBDMD, but reduced forage yield. If the future of the program had depended on improving forage nutritional value without any loss in forage yield, the program might not have survived this first cycle of selection for NBDMD. Fortunately, the breeder, G. W. Burton, knew the value of patience, perseverence, and several cycles of crossing and recombination to break up undesirable genetic linkage blocks.

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Simultaneous selection for increased forage nutritional value and yield has also been practiced in switchgrass (Fig. 2A), alfalfa (Coors et al., 1986), and reed canarygrass (Surprenant et al., 1988). In alfalfa, mild selection pressure for forage vigor and few leaf-disease symptoms was likely responsible for the lack of change in forage yield. In switchgrass, selection pressure for forage yield was often as large as that for IVDMD, resulting in increased forage yield for both the high-IVDMD and low-IVDMD directions (Fig. 2A). The huge loss in forage yield resulting from Cycle-3 selection can be explained by loss of cold tolerance, as is discussed in a later section. In reed canarygrass, simultaneous selection for high forage yield and low NDF concentration resulted in significant reductions in NDF with little or no change in forage yield. While Surprenant et al. (1988) included a negative control (selection for low forage yield), no authors have used the proper control to test this selection method, i.e., the comparison of selection for high nutritional value with and without concomitant selection for high forage yield. Large genetic correlation coefficients of forage yield with IVDMD (r = −0.88) and CP concentration (r = −0.97) were observed in a population of timothy halfsib families (Marum et al., 1994). Analysis of covariance adjustment, using heading date as a covariate, reduced the magnitude of these coefficients by 34% for IVDMD and 53% for CP. Thus from one-third to one-half of the observed genetic correlation between forage yield and nutritional value in this population was due to genetic variation for heading date and its secondary effect on IVDMD and CP. Single-trait selection for increased IVDMD and/or CP in such a population would most likely lead to undesirable correlated responses for both heading date and forage yield. Selection for increased IVDMD in smooth bromegrass, which was accomplished via reductions in cell-wall lignin concentration, did not affect forage yield (Carpenter and Casler, 1990; Casler and Ehlke, 1986) or seed yield (Carpenter, 1988). However, forage yield of smooth bromegrass was reduced by selection for either high or low NDF concentration (Fig. 2B). Buxton and Casler (1993) argued that reductions in NDF or cell-wall concentration may necessarily be associated with losses in forage yield due to the importance of cell walls in providing the structural framework of forage plants as well as typically greater than 50% of their dry matter. If this correlation is biologically necessary, then why was forage yield also reduced by selection for high NDF concentration (Fig. 2B)? The answer lies in a basic principle of plant breeding—recurrent selection necessarily involves a restriction on population size by the fact that relatively few parents are selected to produce progeny for the next generation. Restrictions on population size result in inbreeding in direct proportion to the number of parents chosen (Falconer and Mackey, 1996). Divergent selection for a single forage nutritional value trait can be used to separate the effects of inbreeding from selection per se because they are computed in an orthogonal manner (Fig. 3). For NDF concentration, the selection criterion, the effect of selection was 6.1 times greater than the effect of inbreeding, which

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Figure 3 Effect of one cycle of divergent selection for high (+1) or low (−1) NDF (neutral detergent fiber) concentration on NDF and forage yield. Selection response is partitioned into two single-degree-of-freedom components: b, selection per se (solid lines) and I, inbreeding (dashed lines). Data taken from Casler (2000).

was not significant (P > 0.05). Furthermore, the linear selection response, due to change in allele frequency, accounted for 99% of the variation among the three populations. Conversely, for forage yield, the effect of inbreeding was 1.6 times greater than the effect of selection per se and both were significant (P < 0.01). The effect of inbreeding accounted for 47% of the variation among the three populations. The theoretical inbreeding coefficient of the selected populations was F = 0.05 (5%) compared to an average inbreeding depression of 4.6% for forage yield. This nearly 1:1 ratio of inbreeding depression to theoretical inbreeding is similar to the average inbreeding depression rates of Wilsie et al. (1952) and the most severely affected clones of Hawk and Wilsie (1952) for smooth bromegrass. Casler (2000) proposed three solutions to this problem. First would be to increase the effective population size. However, maintaining relatively high selection pressures for NDF concentration would make this an expensive proposition. Second would be to practice simultaneous selection for low NDF and high forage yield, which was partially successful in reed canarygrass (Surprenant et al., 1988). Third would be to practice selection for low NDF in multiple unrelated populations, followed by strain crossing to produce chance hybrids between the improved strains (Fig. 4). If the A and B strains are unrelated to each other, there should be a forage yield improvement in the hybrid strain due to complementation of dominant genes in repulsion-phase linkage (Bingham, 1998). This effect will be even greater for polyploids, which includes most perennial forage crops (Bingham et al., 1994). Similarly, if selection within different populations acts to increase the frequency of different dominant genes conditioning low NDF, then the hybrid population may also show “heterosis” for low NDF, further reducing NDF of the hybrid. However, if low NDF is conditioned by recessive genes, the hybrid strain will, at best, have the average NDF of strains A and B but could show an increase in NDF due to complementation of dominant genes conferring high NDF (i.e., “heterosis” for high

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Figure 4 Schematic diagram of a breeding procedure designed to restore forage yield potential lost during recurrent selection for reduced neutral detergent fiber (NDF) concentration. Populations A and B are unrelated to each other, improving the potential for a heterotic yield response in the A×B hybrid.

NDF). The populations to test these hypotheses are currently under development. Heterotic responses observed in perennial ryegrass lend support to this proposed breeding method. Despite 6% average heterosis for forage yield among 25 twoclone hybrids, there were no differences between F1 (hybrid) and S1 (selfed; 50% inbred) family means for CP concentration or IVDMD (Posselt, 1994). There is little data on lodging or seed yield of forage crops as related to genetic improvements in nutritional value. Despite the numerous experiments that have measured genetic gains for forage nutritional value traits, most authors do not mention lodging or seed production, either as a measured trait or a visual observation. Most likely this is because very little lodging has been observed in these experiments or, if observed, was sufficiently uniform as to discourage any attempts to develop a post facto rating or measurement system. Furthermore, it is also likely that the researchers who have conducted these experiments have not purposely challenged these populations with conditions conducive to lodging, such as high N, wind, excessive precipitation, or hail. While it may be prudent to cautiously conclude that genetic increases in forage nutritional value do not bring about increases in lodging susceptibility in most field environments, lack of hard data prevents any inference regarding this relationship under conditions which promote lodging. No conclusions can be drawn with regard to seed yield because significant reductions in seed production could occur without any visual clues to the plant breeder or agronomist.

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Two barley (Hordeum vulgare L.) and two wheat (Triticum aestivum L.) cultivars that differed in straw strength in numerous field trials were evaluated for differences in stem anatomy and chemical composition (Travis et al., 1996). The weak-strawed wheat cultivar was characterized by thinner cell walls and smaller cells than the strong-strawed cultivar. The strong-strawed barley cultivar had a greater degree of ferulate–polysaccaride cross-linking, which the authors suggested gave it greater flexibility, allowing its stems to bend under load, compared to the weak-strawed cultivar. Resistance to bending stress of rice (Oryza sativa L.) cultivars was also positively associated with lignin concentration of stems (Ookawa and Ishihara, 1993). Similarly, recurrent selection for increased stalk-lodging resistance in two maize populations was associated with decreased cell-wall concentration and cellwall lignification (Fig. 5). Divergent selection for stalk-crushing strength in two other maize populations, which created a 45-fold difference in stalk-lodging percentage, resulted in no changes to lignin or fiber concentrations of the stalks (Undersander et al., 1977). These results uniformly suggest that increased forage nutritional value should not cause greater lodging potential and may actually enhance lodging resistance in some forage crops. Plant breeders and agronomists should undertake an effort to quantify lodging resistance in germplasm that has

Figure 5 Responses to selection for increased mechanical stalk strength (MS) or increased stalk rot resistance (SR) in BS1 maize. Data taken from Albrecht et al. (1986).

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been selected for increased nutritional value, partly to ensure that detrimental changes have not occurred, but also to document potential positive changes, as suggested by the results of the above studies. c. Plant Anatomy and Morphology Genetic changes in forage nutritional value may not necessarily be associated with changes in cell-wall development or biosynthesis of cell-wall constituents. Different types of cells range from rapidly and almost completely degradable, e.g., mesophyll cells, which are relatively nonlignified, to slowly or almost completely nondegradable, e.g., schlerenchyma cells, which are highly lignified (Akin, 1989). If plants differ in their ability to signal initiation and/or termination of development of specific cell types, changes in forage nutritional value (of either whole plants or specific organs) could result from changes in the relative proportion of cell types with differential degradability. Furthermore, such a change may not affect cell-wall development per se of any specific type of cell, despite our ability to detect genetic differences in cell-wall components using ground tissue samples (see Section III,A,1). In smooth bromegrass, high-IVDMD genotypes had more slowly and nondegradable tissue types in both stem and leaf cross-sections than lowIVDMD genotypes (Ehlke and Casler, 1985). Conversely, high- vs low-IVDMD genotypes of Digitaria milanjiana did not differ in leaf cross-sectional anatomy (Masaoka et al., 1991). More data is required on plant anatomy of genetic lines with differential forage nutritional value before any conclusion can be drawn regarding this hypothesis. This hypothesis also applies to the next higher level of organization: plant organs. For species such as legumes, in which leaves are often considerably higher in nutritional value than stems, selection for increased whole-plant forage nutritional value may arise simply from genetic changes in leaf:stem ratios. Divergent selection for lignin concentration in alfalfa showed this response, with higher leaf:stem ratios in all low-lignin lines, regardless of the evaluation environment (Kephart et al., 1989). The low-lignin alfalfa lines also had lower lignin concentration in stems (Kephart et al., 1990), indicating that at least two mechanisms were jointly responsible for the reduction in whole-plant lignification of cell walls: changes in leaf:stem ratio combined with chemical and/or anatomical changes of stems. Conversely, selection for increased whole-plant N concentration in alfalfa did not cause changes in leaf:stem ratios, despite two- to threefold higher N in leaves vs stems (Demment et al., 1986). In smooth bromegrass, divergent selection for whole-plant IVDMD did not affect leaf:stem ratios, probably because leaf blades and stem+leaf sheath fractions were similar in IVDMD (Casler and Carpenter, 1989). However, because of differential NDF concentration among smooth bromegrass plant parts (stems: 700 g kg−1 ; leaf sheaths: 664 g kg−1 ; leaf blades: 585 g kg−1 ; and panicles: 435 g kg−1 ), selection for reduced whole-plant NDF resulted in changes in plant

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structure (Casler, 1999b). Reduced whole-plant NDF concentration was due to two changes: (1) reduced stem concentration, which was compensated by increases in leaf blade, leaf sheath, and panicle concentrations; and (2) reduced NDF concentration per se of stems and leaf sheaths, the two components with the highest NDF concentration. Selection for reduced NDF concentration based entirely on smooth bromegrass leaves had no effect on plant structure at the heading growth stage (Casler, 1999b). Because of the genetic variability for plant structure at the heading growth stage, and the disruptive effects it may have on genetic gains for NDF concentration of individual plant parts, realized heritability was substantially lower for selection at the heading growth stage compared to the vegetative growth stage (Casler, 2000). Orchardgrass (Dactylis glomerata L.) clones that differed in leaf blade width by 2.5 mm (5.9 vs 8.3 mm) had an average difference in IVDMD of 31 g kg−1 (619 vs 650 g kg−1 ) in spring, but no difference in summer (Lentz and Buxton, 1991). Conversely, smooth bromegrass clones and populations differing in IVDMD showed either no difference in leaf blade width or an opposite difference (Casler and Carpenter, 1989). For smooth bromegrass, high-IVDMD plants tended to have fewer leaves, but larger leaves, as measured by leaf blade length, leaf blade thickness, and specific leaf weight (Casler and Carpenter, 1989). Conversely, in D. milanjiana, high-IVDMD clones tended to have more leaves, but smaller leaves (Masaoka et al., 1991). These results point out either extreme species specificity in the relationships between forage nutritional value and plant morphological traits or lack of repeatability for some of these measurements. Unfortunately, the number of relevant experiments is insufficient to choose between these two explanations. d. Disease and Insect Resistance The relationship between host–plant forage nutritional value and reaction to infection by pathogenic organisms, largely fungi, has been studied from two different perspectives. First, infection by fungi increases lignification and cell-wall development in host plants (Nicholson and Hammerschmidt, 1992; Ride, 1978; Vance et al., 1980), reducing forage digestibility (Vogel and Sleper, 1994). Resistance to fungal diseases can protect host plants from these losses in forage nutritional value, as observed for Colletotrichum trifolii Bain of alfalfa (Lennsen et al., 1991) and several fungal leafspots of intermediate wheatgrass [Thinopyrum intermedium (Host) Nevski] (Karn et al., 1989). An orchardgrass cultivar selected for increased resistance to stem rust (Puccinia graminis Pers. f. sp. dactylidis Guyot and Massenot) produced an average of 17% greater liveweight gains for grazing steers (Bos taurus) compared to three rust-susceptible cultivars (D. A. Sleper, 1995, personal communication), possibly resulting from this nutritional value protection mechanism. The second perspective derives from potential mechanisms for host resistance to pathogens. Because of the widespread host-cell lignification response to fungal

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infection, lignin and phenolic compounds are thought to act as mechanisms for resistance to many fungal diseases (Nicholson and Hammerschmidt, 1992; Ride, 1978; Vance et al., 1980). Indeed, inhibition or suppression of enzymes in the phenylpropanoid pathway decreases host resistance to fungal diseases (Carver et al., 1994a, 1994b). Thus, plant-breeding efforts to increase nutritional value by reducing lignin concentration and/or altering cell-wall phenolic composition may compromise existing fungal disease-resistance mechanisms of host plants. To date, selection for reduced lignin concentration in forage crops has not resulted in large losses of disease resistance. Several measures of leaf rust (Uromyces striatus J. Schr¨ot.) resistance of alfalfa were unaffected by divergent selection for lignin concentration (Webb et al., 1996). Similarly, reaction to brown leafspot [Pyrenophora bromi (Died.) Drechs.] or spot blotch [Cochliobolus sativus (Ito and Kuribayashi) Drechs.] did not differ consistently among smooth bromegrass clones with divergent lignin or etherified ferulic acid concentrations (N. J. Delgado, M. D. Casler, and C. R. Grau, 1998, unpublished data). A very thorough evaluation of five fungal diseases of alfalfa showed no evidence of genetic correlation between disease resistance and forage nutritional value (Fonseca et al., 1999). Selection for reduced NDF concentration in smooth bromegrass did not result in changes in resistance to spot blotch (L. X. Han, M. D. Casler, and C. R. Grau, 1998, unpublished data). Smooth bromegrass clones with reduced cell-wall lignification tended to be more susceptible to crown rust, Puccinia coronata Corda (N. J. Delgado, M. D. Casler, and C. R. Grau, 1998, unpublished data). High-IVDMD pearl millet [Pennisetum glaucum (L.) R. Br. K. Schum.] lines were more susceptible to rust (P. substriata Ellis and Barth. var. indica Ramachar and Cummins) than lowIVDMD lines (Wilson et al., 1991). Similarly, a 37% increase in WSC concentration due to selection resulted in a 128% increase in crown-rust infection of perennial ryegrass (Breese and Davies, 1970). However, in the latter study this apparent relationship was probably due to linkage disequilibrium (chance associations of genes) rather than to pleiotropy (common causal effects of genes) because it was broken rather easily by subsequent selection and recombination (M. O. Humphreys, 1997, personal communication). Genetic changes in forage nutritional value that can be brought about by relatively few cycles of recurrent selection do not appear to have affected forage crops’ inherent resistances to fungal diseases. Observed associations to date appear to be due to chance linkage relationships that can be broken by simultaneous selection for high nutritional value and disease resistance, the latter often based on field observations from natural inoculations rather than intensive evaluations following artificial inoculation. It remains to be seen what effect larger changes in forage nutritional value, such as the accumulated effects of five or more cycles of recurrent selection or the sudden and dramatic effects of plant transformation using cloned genes, will have on host-plant resistances. The brown midrib trait of maize, which

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Figure 6 Responses to selection for European corn-borer resistance in BS9 maize: (A) measured as number of cavities and (B) measured as neutral detergent fiber (NDF), lignin, or ash concentration. Lignin and ash were computed as a proportion of NDF. Data taken from Buendgen et al. (1990).

has a relatively large effect on cell-wall lignification, was associated with a decrease in resistance to ear rot of maize, Fusarium moniliforme (Nicholson et al., 1976). Cell-wall composition and concentration appear to have a direct causal relationship with European corn-borer (Ostrinia nubilalis H¨ubner) resistance in maize. Lignification and silica deposition are associated with reduced feeding by corn borers, possibly by increasing bulk density of the corn-borer diet, leading to greater energy expenditure to meet larval nutritional and water requirements (Ostrander and Coors, 1997). Indeed, recurrent selection for reduced feeding by corn borers was successful in maize and appeared to be due to indirect selection for increased NDF, lignin, and ash concentrations (Fig. 6). Lignin and ash, which is primarily made up of silica, had the largest proportional responses to selection, suggesting their importance as a mechanism of corn-borer resistance in maize. Leaf toughness, measured as the force required to puncture a leaf blade with an instron, is positively correlated with corn-borer resistance (Bergvinson et al., 1994). As expected, selection for reduced NDF and cell-wall lignification led to increased feeding by second-generation corn borers, suggesting a direct role of lignification as a defense mechanism to this insect (Ostrander and Coors, 1997). e. Abiotic Stress Tolerances Little attention has been given to the study of how abiotic stress tolerances are affected by selection for increased forage nutritional value. As with lodging

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resistance, this may be due to lack of severe stress in most field experiments. A lowlignin alfalfa population had 34% survival after 2 years in the field near Ames, Iowa compared to 64% survival in the high-lignin counterpart (Buxton and Casler, 1993). Surviving plants of each population were intercrossed in isolation and the next generation progenies were tested in Iowa, Nebraska, and Wisconsin. The high- and low-lignin populations did not differ in survival after 4 years in the three-location test, despite a mean survival of less than 10% at all three locations (M. D. Casler, D. R. Buxton, and K. P. Vogel, 1999, unpublished data). Approximately 81% of the alfalfa-plant mortality occurred during winter months. It appears that selection of the surviving plants from the original experiment in Iowa provided sufficient selection pressure in the low-lignin population to restore the two populations to similar levels of winter-stress tolerance. Divergent-IVDMD selections of orchardgrass (Rind and Carlson, 1988) and switchgrass (Hopkins et al., 1993) were included in the above three-location test. Over 4 years, mean mortality was 8% for orchardgrass and 47% for switchgrass, nearly all of which occurred during winter months. Both species showed a negative response of survival to selection for high IVDMD as early as 12 months after planting, an effect that increased progressively throughout the duration of the experiment. After 48 months, each cycle of selection for increased IVDMD led to an average mortality of 5.6 and 3.2 percentage units for switchgrass and orchardgrass, respectively. Because nearly all mortality occurred during winter, it appears likely that selection for high IVDMD (low lignin) led to reduced cold tolerance. In general, cold-hardy plants contain more lignin and phenolics than nonhardy plants, suggesting a possible causal relationship (Chalker-Scott et al., 1989; Chalker-Scott and Fichigami, 1989). Selection for increased particle size breakdown during ball-milling led to increased mortality of smooth bromegrass, but the source of stress could not be identified (Culvenor and Casler, 1999). Conversely, divergent-IVDMD populations of smooth bromegrass did not differ in survival in the above three-location test. 5. Major Genes for Increased Forage Nutritional Value Considering the activity and interest in breeding for increased forage nutritional value, relatively few genes have been identified to have large and unequivocal effects on forage nutritional value. The brown midrib genes of maize, sorghum, and pearl millet are the most well known and studied of these genes. Four distinct bmr loci are known to exist in maize, one bmr locus exists in pearl millet, and several bmr loci may exist in sorghum and sudangrass [Sorghum bicolor (L.) Moench.] (Cherney et al., 1991). All known bmr loci are inherited as Mendelian recessives. Brown midrib loci increase in vitro digestibility by decreasing lignin concentration by as much as 51% in stems and 25% in leaves (Porter et al., 1978) and by decreasing NDF concentration by as much as 13% (Fritz et al., 1981).

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These effects vary widely for individual bmr loci backcrossed into different genetic backgrounds, suggesting that bmr loci interact with other loci to produce observed phenotypes. These other loci are most likely involved in regulation of quantitative genetic variation for cell-wall development, providing the basis for genetic gains observed in recurrent selection programs, as reviewed above. A dwarf gene of pearl millet, when present as a homozygous recessive, causes shortened internodes, resulting in increased leafiness compared to normal millet, an effect that increases progressively with time (Burton et al., 1969). The dwarf phenotype had 21% greater dry-matter intake, 49% greater average daily gains in a feeding trial, and 20% greater average daily gains in a grazing trial. Interestingly, the increased forage nutritional value of the dwarf phenotype appears to be a result of two changes—an 11% increase in leaf concentration and a 17–21% increase in IVDMD of stems per se. Thus, it is very likely that stems of the dwarf and normal phenotypes differ in cell-wall development and/or concentration. Dwarf phenotypes of elephantgrass (P. purpureum Schumach.) were also characterized by increased leafiness and shorter internodes, although their genetic control is unknown (Williams and Hanna, 1995). However, dwarf, semidwarf, and normal elephantgrass clones did not differ in either IVDMD or CP concentration. Without exception, the major genes described above cause a dramatic reduction in forage yield. In maize, grain and biomass yields are generally reduced by 10 to 20% by backcrossing the bmr3 gene into a range of inbred lines (Barri`ere and Argillier, 1993). Furthermore, recurrent selection for increased yield in a maize bmr3 population failed to break this association (Barri`ere et al., 1988). The dwarf gene of pearl millet reduced forage yield by 30% and carrying capacity of pastures by 15%, but the trade-off between forage nutritional value and forage yield resulted in equal animal production per hectare for dwarf and normal phenotypes (Burton et al., 1969). Burton et al. (1969) argued that the dwarf phenotype would be easier to manage and require less energy for ensiling or dehydration. Additional selection for forage yield and disease resistance has resulted in improvements of the dwarf pearl millet phenotype (Hanna et al., 1988). Brown-midrib genes generally confer increased intake, digestibility, and rate of passage of feeds, although the literature does not contain economic analyses of the trade-off between increased forage nutritional value and decreased yield (Barri`ere and Argillier, 1993; Cherney et al., 1991). Genes that regulate leaf surfaces and epidermal features may also affect forage nutritional value traits. The trichomeless gene of pearl millet, a recessive, results in a smooth waxy surface without trichomes (Burton et al., 1977). The trichomeless (waxy) phenotype has higher palatability to livestock, but lower digestibility of intact leaves. The bloomless gene of sorghum, also a recessive, removes the bloom (a waxy surface) of sorghum leaves (Cummins and Dobson, 1972). The bloomless phenotype (nonwaxy) has higher digestibility of intact leaves. Because waxy surfaces may be important in preventing infection by some pathogens (Carver et al.,

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1990), the trichome phenotype of pearl millet and the bloomless phenotype of sorghum may be more disease susceptible. Stay-green mutations have been discovered in meadow fescue (Festuca pratensis Huds.) and perennial ryegrass. The stay-green phenotype of meadow fescue is controlled by a single Mendelian gene (Sid ) in which the homozygous-recessive condition prevents yellowing, which normally accompanies leaf senescence (Thomas, 1987). The stay-green phenotype is due to a disabling of the third step in chlorophyll breakdown, the oxygenolytic cleavage of the chlorin-macrocycle, regulated by phaeophorbide dioxygenase (Hauck et al., 1997; Vicentini et al., 1995). The stay-green phenotype has a pleiotropic effect on protein concentration, reducing the decline in protein concentration as leaves age (Humphreys, 1994; Thomas, 1987).

IV. THE POTENTIAL FOR MOLECULAR BIOLOGY CONTRIBUTIONS AND COLLABORATIONS Recent advances in molecular genetic knowledge and technology have radically changed scientific perceptions about the manner and degree to which plants can be genetically modified to improve their nutritional value. Whereas traditional methods of laboratory evaluation and recurrent selection are severely limited by time and labor, molecular methods appear to be limited largely by human imagination. Recent literature documenting numerous mechanisms for modifying cell wall structure and composition is so voluminous and varied that it warrants dedicated literature reviews (Boudet et al., 1995; Boudet and Grima-Pettenati, 1996; Campbell and Sederoff, 1996). While molecular biologists are making rapid advancements in human knowledge of cell-wall structure as it relates to degradability, few of these programs are currently partnering with forage-plant breeding programs. The documented potentials for detrimental effects of reduced or modified cell-wall composition on plant phenotype, such as reduced forage yield, decreased disease and/or insect resistance, and decreased stress tolerance, warrant collaborations with strong field-oriented plant breeding programs. Greenhouse and growth-chamber evaluations of transgenic plants are insufficiently predictive of plant phenotypes under field conditions (Baucher et al., 1999).

A. MOLECULAR MARKERS Because so few major genes are known to regulate forage nutritional value traits, forage breeders must largely rely on quantitative trait loci (QTL) for genetic improvement of forage crops. Nearly all the genetic variation described in this chapter is based on QTL which, to the best of our knowledge, are most likely numerous and

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have small individual effects on any given trait, such as NDF or IVDMD. Molecular marker technology, which allows the development of genomewide linkage maps, presents the possibility of identifying and selecting on the basis of individual QTL for forage traits. Marker-assisted selection (MAS) consists of (1) identifying putative QTL by correlating phenotypic data to markers within a linkage map, (2) determining which putative QTL explain the largest amount of phenotypic variance and have the desired effect (positive or negative) on the phenotypic trait, and (3) selecting plants on the basis of the molecular marker(s) linked to the putative QTL (Dudley, 1993). While the concept of MAS dates back to 1967 (Smith, 1967), it has received very little attention in forage crops. Because so many forage crops are complex polyploids with genomes that derive from multiple progenitors or with polysomic inheritance, development of reliable linkage maps in most forage crops is more complicated than in simple diploids. In two maize crosses, a total of 11 putative QTL for IVDMD and 13 putative QTL for CP were identified (L¨ubberstedt et al., 1997). The maximum allelic substitution effect for an individual QTL was 5.6 g kg−1 for IVDMD and 2.34 g kg−1 for CP, which accounted for 13.0 and 7.6% of the total phenotypic variance for each trait, respectively. In two pearl millet × elephantgrass crosses, a total of 15 putative QTL were identified for in vitro organic matter digestibility, NDF, and CP, six of which were associated with more than one trait (Smith et al., 1993), suggesting either pleiotropic effects or close linkage of genes controlling two different forage nutritional value traits. In both studies, there was little or no similarity among populations for QTL associated with forage nutritional value traits (L¨ubberstedt et al., 1998; Smith et al., 1993), and the degree of association decreased as population pedigrees diverged (L¨ubberstedt et al., 1998). These results are fairly typical of marker–QTL associations and indicate a fundamental inability to use generalized MAS selection criteria across multiple populations or crosses. In contrast to the above studies, an association between isozymes of phosphoglucose isomerase (PGI-2) and WSC concentration has proven relatively consistent in perennial ryegrass. The b allele of the Pgi-2 locus has been associated with high WSC concentration in populations undergoing natural selection (Hayward et al., 1994), in a range of cultivars that differed in Pgi-2 allele frequency and WSC concentration (Smith et al., 1998), and marker-selected progenies of a cross between parents homozygous for different isozymes of PGI-2 (Humphreys, 1992). In the latter study, 69 to 98% of the range of variation in WSC between the two parents was recovered in the marker-selected homozygotes of various F2 populations. This enzyme catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate, an essential step preceding carbohydrate metabolism in plants. While the association between high WSC concentration and the b allele of the Pgi-2 locus may be due to linkage disequilibrium between different genes (Humphreys, 1992), there is a metabolic explanation for the Pgi-2 locus to act as a QTL for WSC concentration (i.e., pleiotropic effects).

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Recent efforts to develop MAS protocols and selection criteria in alfalfa have grown rapidly (Bouton and Bauchan, 1998). Nevertheless, most of these efforts focus on forage yield and/or various adaptation traits. For alfalfa, and proably most legumes, yield and adaptation traits remain more important selection criteria than forage nutritional value traits, most likely because persistence is often the most important factor limiting legume production (Marten et al., 1989).

B. TRANSGENIC PLANTS 1. Modification of Lignin Concentration and/or Composition Lignin results from the oxidative coupling of three monolignols: coumaryl, coniferyl, and sinapyl alcohol. Lignin biosynthesis develops through a series of reactions involving (1) the shikimate pathway which provides phenylalanine as a substrate, (2) the phenylpropanoid pathway which results in several cinnamoyl CoAs that act as precursors for a wide array of phenolic compounds, and (3) the ligninspecific pathway which converts cinnamoyl CoAs into monolignols and lignin (Fig. 7). While there are still some unknowns in the biosynthetic pathway of monolignols, most of the enzymes and reactions are known to exist in many plant species (Boudet and Grima-Pettenati, 1996). For most of these enzymes, cDNA sequences are available from one or more sources, allowing production of transgenic plants that are either down-regulated from antisense RNA sequences or overexpressed from sense RNA sequences. Most of this research has been conducted on model systems such as Arabidopsis thaliana L. and tobaccco (Nicotiana tabacum L.). Transgenics down-regulated with antisense RNA for phenylalanine ammonialyase (PAL) have reduced lignin concentration but also possess numerous potentially detrimental pleiotropic traits, such as reduced vigor, reduced pollen viability, altered leaf shape, and altered flower morphology and phenology (Elkind et al., 1990). These results suggest that down-regulation of steps early in the lignin biosynthesis pathway (Fig. 7) may be unsuitable for specific modification of lignin concentration or composition because of the wide array of other secondary metabolites (and plant processes) that may be altered in transgenic plants (Boudet and Grima-Pettenati, 1996). These authors suggested that ligninspecific isoforms of enzymes such as PAL, cinnamate-4-hydroxylase (C4H), and 4-coumarate-3-hydroxylase (C3H) are not currently known to exist. Nevertheless, down-regulated PAL transgenics of tobacco showed a range of lignin concentration and NDF digestibility as well as a high negative correlation between lignin concentration and NDF digestibility (Sewalt et al., 1997), all strikingly similar to results from nontransgenic plants (Casler and Jung, 1999). Unlike PAL transgenics, plants down-regulated or overexpressed for downstream enzymes in the phenylpropanoid or monolignol pathways (Fig. 7) typically

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Figure 7 Monolignol biosynthesis pathway. The enzymes are PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; C3H, 4-coumarate-3-hydroxylase; COMT, caffeic acid O-methyltransferase; F5H, ferulate-5-hydroxylase; 4CL, hydroxycinnamate:CoA ligase; CC3H, coumaroyl CoA hydroxylase; CCOMT, caffeoyl CoA 3-O-methyltransferase; CCR, cinnamoyl CoA reductase; and CAD, cinnamyl alcohol dehydrogenase. Dotted arrows indicate reactions that have not been experimentally verified. Adapted from Sewalt et al. (1997) and Zhong et al. (1998).

have modified lignin composition but may not have modified lignin concentration. An A. thaliana mutant in which ferulate-5-hydroxylase (F5H) is nonfunctional was morphologically normal, but unable to produce sinapyl alcohol, containing only guaiacyl lignin (Chapple et al., 1992). Down-regulated caffeic acid O-methyltransferase (COMT) transgenics share many features of the brown-midrib plants of maize and sorghum, including a decrease in the syringyl:guaiacyl (S:G) monomer ratio; a reduction in COMT activity; and frequent occurrence of red, brown, or orange plant coloration

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(Boudet and Grima-Pettenati, 1996; Bernard Vailh´e et al., 1996; Tsai et al., 1998; Zhong et al., 1998). Indeed, the maize bmr3 mutation represents a structural change in the COMT gene (Vignols et al., 1995). Cosuppression or overexpression of COMT from sense constructs led to a greater reduction in the S:G ratio than observed from down-regulation (Bernard Vailh´e et al., 1996). Neither method of reducing the S:G ratio had an effect on lignin concentration of tobacco (Bernard Vailh´e et al., 1996). Conversely, down-regulation of COMT in alfalfa transgenics led to reduced lignin concentration (Sewalt et al., 1997). Both the reduction in the S:G ratio of tobacco and the reduction in lignin concentration of alfalfa resulted in increased cell-wall degradability. This parallels the results of Casler and Jung (1999) that both lignin concentration and composition regulate cell-wall degradability in nontransgenic plants (see Section III,A,1). Taken together, these results suggest that the mechanisms by which cell walls are modified in lignin-regulated transgenics differ little from naturally occurring mechanisms. In many studies of transgenic plants, the ranges of variation in lignin concentration, cell-wall degradability, and S:G ratio are similar to that observed within populations of nontransgenics. Thus, the basic effect of up- or down-regulating many enzymes in the phenylpropanoid and monolignol pathways may be similar to that of native genes coding for increased or decreased activity of these enzymes. Multiple isoforms of several enzymes in the phenylpropanoid and monolignol pathways are known to exist, providing a basis for natural genetic variation in lignin development (Campbell and Sederoff, 1996). Except for a few highly unusual transgenics, the biggest difference between plant transformation and natural variation may be that novel lignin phenotypes occur at higher frequency within transgenic lines (Boudet and Grima-Pettenati, 1996), making them easier to identify than novel lignin phenotypes that occur relatively infrequently in natural populations (Casler and Jung, 1999). Development of more extreme novel lignin phenotypes may require simultaneous down-regulation of multiple enzymes (Zhong et al., 1998) or single enzymes that have specific roles in the phenylpropanoid pathway, such as F5H (Chapple et al., 1992; Meyer et al., 1998). Some of the most unusual novel lignin phenotypes have resulted from downregulation of cinnamyl alcohol dehydrogenase (CAD), the last enzyme in the monolignol biosynthesis pathway (Fig. 7). Transgenic plants with reduced CAD activity typically have increased incorporation of cinnamaldehyde moieties in their lignin (Boudet and Grima-Pettenati, 1996) and reduced S:G ratios (Baucher et al., 1999; Bernard Vailh´e et al., 1998; Yahiaoui et al., 1998). A naturally occurring loblolly pine (Pinus taeda L.) mutant, with reduced CAD activity, increased the incorporation of unusual alcohols and aldehydes into its novel lignin compared to a normal lignin phenotype (Ralph et al., 1997). Furthermore, two enzymes (COMT and caffeoyl CoA O-methyltransferase, CCOMT) function as methylating agents in the phenylpropanoid pathway (Zhong et al., 1998), resulting in a metabolic grid which provides alternate routes for monolignol synthesis (Fig. 7; Chen et al.,

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1999; Inoue et al., 1998). This adaptability of phenolic metabolism and the largely normal physical appearance of many novel lignin plants suggests that lignins are highly plastic and that much of this plasticity is not disruptive of essential metabolic processes in plants. Field testing for vigor and stress tolerances will be required before the agricultural fitness of these transgenics can be judged. Indeed, the bmr1 maize mutant, which is associated with reduced biomass and grain yield (Barri`ere and Argillier, 1993), appears to be a mutation of the CAD gene, sharing many properties of down-regulated CAD transgenics (Halpin et al., 1998). Reduced vigor and abnormal growth of novel lignin transgenics is limited to cases where transgenes cause large reductions in lignin concentration (Jung and Ni, 1998), paralleling results from all known bmr mutants. Woody species may avoid these vigor reductions by compensatory mechanisms such as increased cellulose deposition in reducedlignin transgenics (Hu et al., 1999), but such mechanisms are not known to exist in herbaceous plants. 2. Modification of Protein Quality While forage crops are not specifically deficient in sulfur-containing amino acids, dietary supplementation with methionine or cysteine may increase liveweight gain, milk production, and wool growth (Tabe et al., 1993). In addition to nitrogen losses during rumen fermentation, ruminant production may suffer from a proportional loss of sulfur during the conversion of ingested plant protein to microbial protein, limiting sulfur utilization in the abomasum and small intestine (Kennedy and Milligan, 1979). Sunflower seed albumin (SSA), rich in both methionine and cysteine, is highly resistant to degradation during rumen fermentation (Tabe et al., 1993). Both alfalfa and subterranean clover were succcessfully transformed with a chimeric SSA gene that, in the case of subterranean clover, was stably expressed through one generation of sexual reproduction (Khan et al., 1996; Tabe et al., 1995). Transgenic plants with the highest levels of SSA expression had 0.1 and 0.75% of soluble leaf protein in the form SSA in alfalfa and subterranean clover, respectively (Khan et al., 1996; Tabe et al., 1995). For subterranean clover, this level meets the lower end of the range at which a wool-growth response would be expected, based on dietary supplementation research (Khan et al., 1996). Plant transformation appears to be the most promising genetic approach to creating and identifying novel protein phenotypes of forage plants. Furthermore, because each transgenic event is inherited as a simple Mendelian dominant, it can be backcrossed relatively easily into other germplasms and different transgenic events can be combined to potentially increase the phenotypic response. While natural genetic variability is present for rumen-degradable protein composition of forage crops (Broderick and Buxton, 1991), the current process of evaluation and selection is expensive, time-consuming, and tedious. Unless the evaluation

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process can be improved, genetic efforts to improve ruminant amino acid utilization from high-forage diets should focus on transgenic strategies. These efforts must incorporate a strong field-oriented plant breeding component to identify potential pleiotropic effects on agronomic performance, ensuring a commercially viable product.

V. SUMMARY There is a wealth of genetic variation for plant traits related to forage nutritional value. Most of these traits can be modified relatively easily by traditional laboratory evaluation and selection methods. Several cultivars with improved in vitro digestibility represent documented improvements in animal performance. Most commercial cultivars with improved forage nutritional value are agronomically acceptable largely because of concomitant selection for important agronomic traits. Selection for increased forage nutritional value, ignoring agronomic traits, may result in detrimental correlated responses, including reduced forage yield, disease resistance, insect resistance, or stress tolerance. A few major genes have large effects on forage nutritional value traits, but many of these genes carry severe reductions in agronomic performance. Transgenic technology has the potential to create novel plant phenotypes unlike any found in nature. Potential achievements from application of this technology appear to be limited only by human imagination. While transgenic technology may simplify the initial screening of novel plant phenotypes, it will not shorten plant breeding cycles or reduce the time required to develop new cultivars. Transgenic plants are subject to the same environmental effects and genotype × environment interactions that affect all crop plants. In addition, their utility and value will depend on transgene stability and expression in future sexual generations. While transgenic plants are currently providing valuable information about plant metabolic processes, their value for improving forage nutritional value will remain in doubt until their agronomic performance and the stability of transgene expression has been adequately documented. Plant breeders and plant molecular biologists should strive to forge mutually beneficial partnerships aimed at commercialization of transgenic products.

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