# Past and prospects of forage maize breeding in Europe.
## I. The grass cell wall as a basis of genetic variation and future improvements in feeding value

Yves Y. Barrière, D. Alber, O. Dolstra, Catherine Lapierre, M. Motto, A.

Ordas, J. van Waes, L. Vlasminkel, Claude Welcker, J.P. Monod

► To cite this version:

Yves Y. Barrière, D. Alber, O. Dolstra, Catherine Lapierre, M. Motto, et al.. Past and prospects of forage maize breeding in Europe. I. The grass cell wall as a basis of genetic variation and future improvements in feeding value. Maydica, 2005, 50 (3-4), pp.259-274. hal-02678747

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# PAST AND PROSPECTS OF FORAGE MAIZE BREEDING IN EUROPE.

## I. THE GRASS CELL WALL AS A BASIS OF GENETIC VARIATION AND FUTURE IMPROVEMENTS IN FEEDING VALUE

Y. Barrière<fnref n="1" />*, D. Alber<fnref n="2" />, O. Dolstra<fnref n="3" />, C. Lapierre<fnref n="4" />, M. Motto<fnref n="5" />, A. Ordas<fnref n="6" />, J. Van Waes<fnref n="7" />,
L. Vlasminkel<fnref n="8" />, C. Welcker<fnref n="9" />, J.P. Monod<fnref n="10" />

Received March 21, 2005

**ABSTRACT** - Although maize was early recognized as an excellent forage plant soon after its introduction in Europe, during a long time it was only bred for grain traits. Conversely to agronomic value, a steady decline in the average cell wall digestibility of hybrids was observed since the 1950s, despite a greater specialization of hybrids between forage and grain uses, except for the Netherlands where varietal registration has taken into account traits related to plant digestibility for longer times. Modern hybrids had on average a 5.5% lower *in vivo* cell wall digestibility than the old ones, leading to an organic matter digestibility reduction by 2%, despite a tendency to a slight, but significant, increase in grain content. When compared to the well-known early hybrid LG11, hybrids with a lower cell wall digestibility were 32, 63, 75, 84, and 93% in each of the registration periods before 1981, 1989, 1994, 1999, and in or after 1999, respectively. Alleles for highly friable and digestible cell walls were either eliminated during breeding for stalk standability and breakage resistance, or lost by genetic drift during breeding for grain yield. Breeding of highly digestible and ingestible forage maize may depend on the reevaluation and use of old genetic resources that are not currently used, and/or specific breeding of digestibility/ingestibility resource lines. Cell wall structure and organization, and lignified tissue patterning, are the key factor explaining silage maize variation both for cell wall digestibility and intake. In maize, as in other Poaceae, *p*-coumaric acid and ferulic acid, along with its array of dehydrodimers,

are ester and/or ether-linked to the cell wall polymers. Linkages of *pCA* esters to syringyl lignin units may markedly influence not only the binding mode of these lignin units, but also the spatial organization of the polymers and their interactions with polysaccharides. Similarly, ferulates cross-linking between guaiacyl residues and arabinoxylans is detrimental to cell wall digestibility, acting as a reinforcement of the wall structure, and decreasing wall component accessibility to microorganism enzymes. Moreover, diferulate cross-linking of arabinoxylan chains is also involved in cell wall digestibility decrease, and is commonly thought to play a role in stiffening cell walls. While first improvements of maize cell wall digestibility were (will still be) based on whole plant digestibility tests, understanding the biochemical and molecular basis of cell wall biogenesis, organization, and lignification, is now required to realize further progress in plant cell wall digestibility and intake.

**KEY WORDS:** Maize; Corn; Silage; Digestibility; Ingestibility; Lignin; *p*Coumaric acid; Ferulic acid; Germplasm.

## INTRODUCTION

Maize was early recognized as an excellent forage plant soon after its introduction in Europe, even if the first use of maize in Europe was for human feeding, as it was traditionally in America. However, maize was, for long time, considered as other common grasses were and mostly used as fresh green fodder for animal feeding during summer. The VIL-

<footnote n="1">¹ INRA, Unité de Génétique et d'Amélioration des Plantes, 86600 Lusignan, France</footnote>

<footnote n="2">² KWS SAAT AG, Grimsehlstrasse 31, 37555 Einbeck, Germany</footnote>

<footnote n="3">³ Plant Research International B.V., P.O. Box 16, 6700 AA Wageningen, The Netherlands</footnote>

<footnote n="4">⁴ INAPG-INRA, Chimie biologique, 78850 Thiverval-Grignon, France</footnote>

<footnote n="5">⁵ Istituto Sperimentale per la Cerealizzazione, 24126 Bergamo, Italy</footnote>

<footnote n="6">⁶ Mission Biologica de Galicia (CSIC), 36080 Pontevedra, Spain</footnote>

<footnote n="7">⁷ Agricultural Research Center, Ghent (CLO), 9820 Merelbeke, Belgium</footnote>

<footnote n="8">⁸ ADVANTA Seeds B.V., 4410 AA Rilland, The Netherlands</footnote>

<footnote n="9">⁹ INRA-ENSAM, UMR Ecophysiologie des Plantes sous stress, 34060 Montpellier, France</footnote>

<footnote n="10">¹⁰ PROMAIS, 39 Chemin Virebent, 31200 Toulouse, France</footnote>

<footnote>* For correspondence (e.mail: yves.barriere@lusignan.inra.fr).</footnote>
MORIN-ANDRIEUX catalogues thus recommended for forage use late genotypes since the first half of the 19th century. Silage maize development in Europe was low, and at the end of 1940s and early in the 1950s, ensiling of maize was still not common in France (CAUDERON et al., 1951) and in other European countries. Moreover, maize breeding was only focused on the improvements of grain traits over a long time. CAUDERON et al. (1951) considered that “both hybrids and landraces cropped for grain in the South-Western France were suitable for forage use” and that “choice of genotypes should be based on expected harvest date and water availability”. The second part of this view, related to water availability, has often been forgotten, and needs to be reconsidered. Conversely, the first part of the opinion, probably pertinent before intensive breeding of maize, and when maize were harvested with immature and little lignified tissues (low DM content), will have endured too many years with significant negative consequences. Genetic variation for digestibility in whole plant maize, and more especially genetic variation for maize cell wall digestibility, was indeed considered lately in France, even earlier in the Netherlands. This review describes the variation of forage maize feeding value observed during the past 50 years, and highlights the biochemical, genetic, and molecular traits of the grass cell wall which are (or could be) the basis of plant digestibility and intake. In addition an overview is given on genetic resources of interest in further breeding of high quality forage maize, whereas the evolution of maize germplasm used in (forage) maize varieties during the past 50 years is given in an additional review (BARRIÈRE et al., 2005).

*In vivo* digestibility variation in forage maize from the 1950s to the 2000s

It has long time been considered that the first digestion trial of maize silage in Europe was done in the Netherlands by DIJKSTRA and BECKER (1960), as quoted in DEINUM et al. (1984). However, WOODMAN and AMOS (1928) already conducted one in the 1920s. The objective of this study was to determine the feeding value of forage maize in comparisons with other forages usually fed to cattle. The experiment had a duration of 14 days and it was carried out with only two purebred Suffolk wether sheep. The ensiled variety of maize was “Jaune Gros du Domaine”. Silage maize fed to sheep had a 21% DM

content and the observed *in vivo* organic matter digestibility (OMD) was equal to 70.9%. Due also to the low DM content, crude fiber digestibility was high and equal to 70.3%, a value corresponding to about 65% in NDF *in vivo* digestibility (NDFD). To date, maize digestion trials with sheep that could have been done between 1928 and 1960 were not found again. In France, DEMARQUILLY (1969), and ANDRIEU and DEMARQUILLY (1974), established silage maize feeding values to underpin the framework of the INRA system of animal nutrition. OMD in maize silage was found to be on average equal to 71%, and could reach 74%, according to cropping conditions, maturity and/or grain content (ANDRIEU and DEMARQUILLY, 1988). Later studies dealt with the comparison of hybrids, and included investigations of cell wall digestibility, a trait nowadays considered to be the most relevant digestibility criterion. Comparisons between early maize genotypes showed that OMD of silage ranged from 70.6 to 73.6% in registered hybrids, and from 71.0 to 75.0% in experimental hybrids (GALLAIS et al., 1976). OMD of 25 maize silages was on average equal to 72.8%, and the average NDFD was equal to 52.7% with a range from 47.5 to 57.1% (DEINUM et al., 1984). The NDFD of hybrids tested in the study of ANDRIEU and DEMARQUILLY (1974, 1988) was later estimated to be 53.1%, and ranged from 49.8 to 54.8% (BARRIÈRE and ARGILLIER, 1997). From 50 maize silages cropped between 1971 and 1993, DE BOEVER et al. (1988, 1997) reported average OMD values of 74.7%. The average NDFD value of these hybrids, computed from crude fiber values, was 61.4%, and ranged from 56.1 and 68.8% (DE BOEVER unpublished data, quoted in BARRIÈRE et al., 2003a).

However, most of these original forage maize *in vivo* reference values were predominantly based on a limited number of early “old” genotypes, most of which having a good feeding value, such as Fronica, Circé, LG11, (but not Eta Ipho, related to Co125), in the Netherlands, and INRA258, Funk245, Dekalb204, LG11, in France, which are no longer representative for the present hybrids in cultivation. Hybrids developed in the following decades tended to have a lower *in vivo* digestibility value. Hybrids registered between 1989 and 1994 or 2002 appeared to be less digestible than hybrids registered before 1989. Based on data from the long term experiment of silage maize feeding value measurements with sheep at INRA Lusignan (BARRIÈRE et al., 2004a), agro-nomic and digestibility traits were re-investigated in registered hybrids (mostly in France, but also in
Germany, Belgium and the Netherlands) split in five groups representing five successive eras of breeding. The groups thus consisted of hybrids registered i) between 1958 and 1980, before the Iodent germplasm introduction in early maize breeding, ii) between 1981 and 1988, as year 1989 was previously proven as an important switch in digestibility values of hybrids, likely related to a greater frequency of BSSS germplasm in early hybrids, iii) between 1989 and 1993, as 1994 was the registration year of Anjou285 which illustrated the emergence of a new generation of hybrids iv) between 1994 and 1998, as 1999 was the first year in France with an *in vitro* digestibility value used as a registration trait, and v) between 1999 and 2002. A steady and regular trend towards a lower feeding value was observed in the most recent hybrids registered during the period from 1958 to 2002 (Table 1). Modern hybrids had on average a 5.5% lower cell wall digestibility (NDFD) than the old ones, leading to an OMD reduction by 2%, despite a tendency to a slight, but significant, increase in grain content. The decrease in whole plant digestibility was thus directly related to a decrease in cell wall digestibility. When compared to the well-known early hybrid LG11 (NDFD = 50.2%), hybrids with a lower cell wall digestibility (NDFD) were 32, 63, 75, 84, and 93% in each of the registration periods before 1981, 1989, 1994, 1999,

and in or after 1999, respectively. The use of an *in vitro* digestibility trait for hybrid registration in France since 1999 does not still appear efficient to modify this evolution. However, four years is probably too short a period to reverse such a long term trend. Forage maize breeding is still heavily depending on germplasm from grain breeding investigations. Moreover, registration control hybrids also originated from grain breeding and, for a significant part of all, are of mediocre digestibility. Nevertheless, some modern hybrids, of high agronomic value, had a similar cell wall digestibility and energy value as hybrids of the era before the 1980s, strengthening the interest and the possibility of further improvements.

Significant areas of silage maize are cropped on southern European lands and genetic variation for cell wall digestibility was similarly established in late maize late genotypes (FAO 400 to 700), even most of (all) hybrids were bred for grain production. Ranges of variation were larger for the different fiber component contents, *in vitro* organic matter digestibility, and for the UFL energy value, among the hybrids of the latest maturity class (FAO 700). DM matter yields were higher than 20 t/ha, with an average energy value equal 0.82 UFL/kg DM (BERARDO *et al.*, 1999), about 0.10 UFL lower than currently observed in earlier hybrids (FAO 250-

<table><caption>TABLE 1. Variance analysis for OMD, NDFD, NDF, grain and crude protein contents, and yield in silages of maize hybrids registered in five successive eras from 1958 to 2002 (OMD = in vivo organic matter digestibility, NDFD = in vivo NDF digestibility, NDF = neutral detergent fiber, and C Protein = crude protein).</caption><thead><tr><th>Early registered hybrids</th><th></th><th>OMD %</th><th>NDFD %</th><th>NDF %</th><th>Grain %</th><th>C Protein %</th><th>Yield t/ha</th></tr></thead><tbody><tr><td></td><td>df</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Registration period MS</td><td>4</td><td>107.0 **</td><td>349.3 **</td><td>67.5 **</td><td>37.7 *</td><td>3.2 **</td><td>165.9 **</td></tr><tr><td>Genotype / registration period MS</td><td>241</td><td>10.6 **</td><td>44.9 **</td><td>19.6 **</td><td>40.9 **</td><td>1.0 **</td><td>10.8 **</td></tr><tr><td>Residual MS</td><td>783</td><td>2.4</td><td>14.2</td><td>5.1</td><td>10.5</td><td>0.3</td><td>1.6</td></tr><tr><td></td><td>nb</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>mean registered 1958 - 1980</td><td>22</td><td>70.9</td><td>51.1</td><td>39.9</td><td>43.8</td><td>8.2</td><td>12.5</td></tr><tr><td>mean registered 1981 - 1988</td><td>43</td><td>70.7</td><td>49.9</td><td>40.3</td><td>42.9</td><td>8.1</td><td>14.4</td></tr><tr><td>mean registered 1989 - 1993</td><td>60</td><td>69.8</td><td>48.4</td><td>40.9</td><td>44.9</td><td>8.0</td><td>16.1</td></tr><tr><td>mean registered 1994 - 1999</td><td>77</td><td>69.7</td><td>47.6</td><td>40.3</td><td>44.5</td><td>7.9</td><td>16.4</td></tr><tr><td>mean registered 1999 - 2002</td><td>44</td><td>69.0</td><td>45.7</td><td>39.9</td><td>45.1</td><td>7.7</td><td>18.1</td></tr><tr><td>mean registered 1958 - 2002</td><td>246</td><td>69.9</td><td>48.2</td><td>40.3</td><td>44.4</td><td>8.0</td><td>15.9</td></tr></tbody></table>

df = degree of freedom; nb = number of hybrids; MS = mean-square.

** = significant at P < 0.001; * = significant at P < 0.01.
350). These results corroborated data of INRA Lusignan showing a range of variation from 32.9 to 58.1% for NDFD, and from 0.74 to 0.90 for UFL, in 53 late hybrids (FAO 500-650) registered in France between 1951 and 2001 (BARRIÈRE *et al.*, 2004a).

From data of BARRIÈRE *et al.* (2004a), genotypic correlations between *in vivo* traits showed that energy value and OMD were related greatly to NDFD ($r^2$ = 0.59), but little to grain content especially for early registered hybrids ($r^2$ = 0.10). The correlation between NDF content and NDFD was close to zero, indicating that no significant relationship existed between the cell wall digestibility and the cell wall content when maize plants were harvested at a similar maturity stage. According to the diet usually given to cattle, hybrids with high cell wall digestibility, but more or less rich in starch should be proposed to farmers. For French conditions of rearing, with silage maize and energy and nitrogen rich concentrates, a starch content close to, but not higher than 30%, was shown to be sufficient. For German or Dutch conditions, with a mixed diet with maize and grass silages, maize starch content could be greater without risks of induced acidosis. The forage quality differences among early or late hybrids are of sufficient magnitude to be of significant interest for the silage maize producers. Published experiments with dairy cows also confirmed the lower feeding efficiency of maize with low NDFD, but also highlighted that hybrids with low cell wall digestibility were furthermore penalized by a lower correlative intake (BARRIÈRE *et al.*, 1995; VALENTIN *et al.*, 1999; BARRIÈRE *et al.*, 2003b). Very few hybrids of good cell wall digestibility additionally showed an unexpected and repeatedly extra intake by about 1.5 kg DM/cow.day (BARRIÈRE *et al.*, 2004b).

*In vitro* digestibility variation in forage maize from the 1950s to the 2000s

The first and in-depth thoughts on a forage maize ideotype were likely developed in the Netherlands and genetic variation for *in vitro* digestibility of the whole plant (IVDOM) has been next reported in numerous studies over the past 30 years using rumen fluid or enzymatic solutions. More recently, it was largely established that enzymatic solubility, but also quality traits such as starch, cell wall or lignin contents, could be accurately predicted by near infra red spectroscopy (NIRS) for routine analysis of bred genotypes. For breeding and molecular approaches,

it is completely relevant to take the starch content of the plant directly into account, and to use an *in vitro* criterion of cell wall digestibility, rather than a criterion of the resulting whole plant digestibility. STRUIK (1983), and DOLSTRA and MEDEMA (1990), proposed computing a cell wall digestibility index, assuming that the whole non cell wall part of the plant was completely digestible (IVNDFD). ARGILLIER *et al.* (1995) and BARRIÈRE *et al.* (2003a) proposed taking into consideration the non starch, non soluble carbohydrate, non crude protein part of the plant, assuming that these three constituents were completely digestible (DINAGZ). Genetic variation for IVNDFD or/and DINAGZ was proved in numerous investigations. Moreover, these cell wall digestibility traits also have a higher heritability than whole plant digestibility, mostly because genotype x environment interactions are much lower for cell wall digestibility than for starch and soluble carbohydrate contents.

In the Netherlands, during years of breeding without digestibility test for registration, a decrease in IVDOM was observed between hybrids registered between 1968 and 1984, close to 4% when based on 100% in LG11 (STRUIK, 1983; DEINUM and STRUIK, 1985). Leopard, registered in 1969 had an IVDOM equal to 101% of LG11 IVDOM, whereas Eta Ipho and Markant, registered in 1979 and 1983, respectively, had an IVDOM a little lower than 95% of LG11 IVDOM. In 1988, the feeding value came up as a registration criterion, and was mentioned as VEM/kg DM on the variety list. The effect on the development of new maize varieties was significant, because varieties with too low feeding values were kept away from the variety list. From 1999, the starch content of varieties estimated at 28, 32 and 36% DM is mentioned in the Dutch variety list as a supplementary quality criterion, but it is explicitly stated that the whole diet determines the optimal maize starch content. Finally, since 2003, the cell wall digestibility of the varieties is mentioned on the variety list. Since the start of varietal testing for IVDOM in 1988, the average feeding value of hybrids registered in the Dutch list increased by 8%, and the IVDOM yield was by 30%. The beneficial change in the feeding value of hybrids cropped in the Netherlands in the 1990s and 2000s is a consequence of breeding efforts that started in this country in the 1980s, directed to improve the forage quality, in particular NDFD.

Breeding for IVDOM and for a higher cell wall digestibility began significantly in France in the mid
<table><caption>TABLE 2 - Average values of best registered hybrids in the three maturity groups of the French varietal list between 1999 and 2004 for yield and UFL energy value, respectively.</caption><thead><tr><th rowspan="2">Hybrid registration</th><th colspan="2">Best hybrids for yield</th><th colspan="2">Best hybrids for UFL</th></tr><tr><th>Yield</th><th>UFL</th><th>Yield</th><th>UFL</th></tr></thead><tbody><tr><td>Very early</td><td>% Antarès</td><td>% Antarès</td><td>% Antarès</td><td>% Antarès</td></tr><tr><td>1999 - 2001</td><td>104.9</td><td>100.9</td><td>100.8</td><td>101.4</td></tr><tr><td>2002 - 2004</td><td>108.8</td><td>98.9</td><td>106.4</td><td>100.2</td></tr><tr><td>Highest UFL value in Baxxao (2000)</td><td>—</td><td>—</td><td>98.2</td><td>102.0</td></tr><tr><td>early</td><td>% Anjou285</td><td>% Anjou285</td><td>% Anjou285</td><td>% Anjou285</td></tr><tr><td>1999 - 2001</td><td>105.7</td><td>99.1</td><td>97.6</td><td>102.5</td></tr><tr><td>2002 - 2004</td><td>106.5</td><td>100.7</td><td>98.0</td><td>102.9</td></tr><tr><td>Highest UFL value in Colombus (2003)</td><td>—</td><td>—</td><td>98.9</td><td>104.4</td></tr><tr><td>Medium early</td><td>% Anjou37</td><td>% Anjou37</td><td>% Anjou37</td><td>% Anjou37</td></tr><tr><td>1999 - 2001</td><td>107.0</td><td>100.0</td><td>98.5</td><td>102.5</td></tr><tr><td>2002 - 2004</td><td>108.0</td><td>101.5</td><td>103.4</td><td>102.7</td></tr><tr><td>Highest UFL value in Phobos (2003)</td><td>—</td><td>—</td><td>104.3</td><td>103.6</td></tr></tbody></table>

of 1980s with the availability of enzymatic solubility (AUFRÈRE and MICHALET-DOREAU, 1983), the availability of accurate NIRS apparatus and the development of calibrations under the impulse of the Centre de Recherches Agronomiques de Gembloux (BISTON and DARDENNE, 1985) and Limagrain. However, whereas the importance of Dutch works and first works of INRA Lusignan (GALLAIS et al., 1976) were soon acknowledged by a small group of scientists and breeders, genetic variation of forage maize for NDFD was considered dubitatively, indeed even suspiciously, by several people involved in maize breeding and animal nutrition. The majority thought was that genetic effects, if present, were greatly negligible compared to environmental effects. Limagrain scientists then set up the network “Club digestibilité” (with CRA Gembloux, INRA Lusignan, INRA Theix, AGPM) which contributed to the knowledge of genetic variation for quality traits in forage maize, and led to the equations “Modèle 4” allowing DOM and energy value calculations from IV-DOM and CP contents (ANDRIEU et al., 1993; DARDENNE et al., 1993). Specific maize breeding for whole plant and cell wall digestibility thus began in France following these experiments, with a variable intensity depending on companies. The use of such a feeding value trait for maize registration was decided later in France, because animal nutritionist, breeder, trader and farmer opinions on forage specificity moved slowly, and also because of the

economic cost of managing two experimental networks, one for grain and one for forage.

Information on correlative improvement in yield and whole plant digestibility or energy value are available from official results given on the French registration list only for six years, between 1999 (first year with a digestibility traits for registration) and 2004. Average improvement was computed from the values of the best hybrid in yield and comparatively to the best hybrid in UFL (French Feed Unit for Energy, 1 UFL = 7.11 MJ) in each earliness group, and with splitting data in two three-year periods (Table 2). The control hybrids were those used in registration experiments. Antares has a cell wall digestibility and energy value close to LG11 or Dea, whereas Anjou285 and Anjou37 are both of similar and low cell wall digestibility and energy values. Among very early hybrids compared to Antares, no improvement was observed in UFL value, and higher yielding hybrids had a tendency to a lower UFL value. Among early and medium early hybrids compared to Anjou285 and Anjou37, there was a lower yield improvement in hybrids with the highest UFL values, especially in early hybrids, even if improvements in UFL value were rather limited. No improvement in feeding value was observed in hybrids having the highest yield. This was even more expected that control hybrids were of low feeding value. Based on average values of all hybrids registered between 1999 and 2003, LUCIANI
(2004) observed no improvement in UFL value of early and medium early hybrids, and a significant decrease equal to -0.01 UFL in very early hybrids, corroborating the results based on best hybrids in yield or UFL. UFL value used as registration trait in the French maize list did not allowed improvement of feeding value, but it efficiently discarded hybrids significantly lower than control hybrids. The value of control hybrids in each earliness group is therefore the relevant basis of further improvements. Hybrids with high UFL values and high cell wall digestibility should be suggested as the UFL controls for registration, in addition with hybrids with high agronomic values.

In Belgium, during the 1991-2001 period and on basis of the five best silage maize varieties for IV-DOM yield of the recommended list, total DM and IVDOM yields increased by 0.85% and 1.2% per year, respectively (VAN WAES, 2003), indicating an improvement on both yield and whole plant digestibility. However, because the grain yield of plants increased by 2.8% per year in the same period, although it was observed in another experimental network, this whole plant digestibility improvement occurred likely without improvement of cell wall digestibility. A significant increase in resistance to lodging and to stalk rotting was also observed in the same period.

## Breeding for higher cell wall digestibility and intake in forage maize from *in vitro* traits

The efficiency of breeding forage with improved cell wall digestibility is limited by the value of correlations between *in vivo* and *in vitro* traits. From a review in BARRIÈRE *et al.* (2003a), determination coefficients between OMD and IVDOM ranged from an r² = 0.67 (DE BOEVER *et al.*, 1997, 1999) and an r² = 0.32 (ANDRIEU *et al.*, 1993). *In vitro* estimates of whole plant digestibility thus explained only a part, nearing 50-60%, of the variation observed in cattle. Similarly, DINAGZ trait explained only a little more than 50% of NDFD (BARRIÈRE *et al.*, 2003a). Moreover, while average values were equal in OMD and IVDMD, or in NDFD and DINAGZ, respectively, the *in vitro* solubility heavily reduced the range of variation between genotypes. Among a set of 248 registered early hybrids, the minimum - maximum range was 19 percent points in NDFD, but only 9 percent points in DINAGZ. For breeding purpose, both NDFD and DINAGZ criteria lead efficiently and similarly

to the elimination of hybrids with low cell wall digestibility, or to the choice of the rare hybrids with very high cell wall digestibility. However, from a simulation of a hybrid choice performed in the INRA Lusignan database, the selection among numerous hybrids with intermediate cell wall digestibility could partly differ according to the NDFD or DINAGZ trait used, that did not cover the same part of digestibility variation found in plants fed to cattle. Correlations between lignin content in the cell wall and *in vitro* digestibilities were always higher than correlation between lignin content and *in vivo* digestibilities. Moreover, from unpublished data or information, enzymatic solubility of whole plants with high starch content appeared to be overestimated in comparisons between plants of similar cell wall digestibility, but lower starch content. With enzymatic treatment, cell wall degradability would be lower than potential, while starch solubilization would be complete. A NIRS calibration of the *in vivo* OMD and NDFD estimate of cell wall digestibility would then be of interest. BISTON *et al.* (1989) built up the first calibrations for OMD, with an r² value equal to 0.60. BARRIÈRE *et al.* (2004a) had then r² values equal to 0.68 and 0.63 for OMD and NDFD, respectively. The limitation is related to the capability in updating NIRS calibrations based on animal values, since their development and maintenance require an experimental sheep flock. The very small number of locations producing data could be compensated for by a great number of years of experimentation, and the low values of genotype x environment interactions for cell wall digestibility. Moreover, the number of reference measurements could decrease as the robustness of the calibration could increase after each year of experiments.

Voluntary intake, which is also a primary nutritional factor controlling animal production, was very often underestimated by plant breeders due to the lack of proper and relevant criterion for selection, to the correlative great difficulty for plant breeders to work with cattle. Animal productions of cattle fed maize hybrids with low cell wall digestibility are thus depressed by both the low intake of forage and its low energy value. Conversely, the most demonstrative results evidencing genetic variation for ingestibility in forage corn were obtained with the DK265 hybrid, which was registered in France in 1987. The voluntary intake of hybrid DK265, which is of good cell wall digestibility, was always found greater than that of other hybrids, in-
cluding hybrids with similar DM or cell wall di-
gestibility and grain content, by about 1.3 kg/day
more, when corn silage was given as about 75% of
the diet (BARRIÈRE et al., 1995, 2003b, 2004b). This
specific trait, related in the three-way hybrid DK265
to the flint and one dent lines (BARRIÈRE et al.,
2004b), seems very uncommon in germplasm used
in corn breeding, and was also only suspected in
the related hybrids Bahia or Briard (CIBA-SEMENCES,
1990, 1995). From simultaneous measurements of
intake and cell wall digestibility, the NDFD ex-
plained only 50% of the variation observed in intake
(estimated by the r² value, BARRIÈRE et al., 2003b),
and therefore about 50% of the specific intake varia-
tion has to be considered in traits different from cell
wall digestibility. Ingestibility of a forage is thus
considered as controlled by the time this forage is
retained in the rumen (reviews in MINSON and WIL-
SON 1994; JUNG and ALLEN, 1995; FERNANDEZ, 2003),
and particles have to be broken down to a size
close to 2 - 4 mm before they can escape from
the bovine rumen (ANDRIEUX and BAUMONT, 2000).
Chewing during eating and ruminating is responsi-
ble for most of the particle breakdown in chopped
forage (MINSON and WILSON, 1994). ULYAT (1983) and
INOUÉ et al. (1994), mentioned that food is chewed
during eating to a common end point, so that the
bolus may be easily swallowed. Chewing during in-
take thus led to similar particular size in bolus for
two corn hybrids having differences in silage parti-
cle size, but there were correlative differences in
eating time per unit of feed intake (FERNANDEZ, 2003;
FERNANDEZ et al., 2004). In tropical forages, MCLEOD
et al. (1990) concluded that "voluntary intake was
associated with the resistance of large particles to
breakdown by chewing during both eating and ru-
minating". This conclusion was in agreement with
works of INOUÉ et al. (1994) considering that both
shear breaking load of tissue and plant morphology
had to be considered for intake improvement in
Lolium. As a consequence, from a plant breeder
standpoint, the ingestibility (the filling capacity) of
maize forage depends on genetic traits of the maize
plant related to both its cell wall digestibility, and its
cell wall digestibility rate, by rumen microorgan-
isms, and its mechanical resistance to chewing by
cows. Cell wall digestibility and cell wall friability
(resistance to breakdown) are traits that should be
considered with (partly) independent biochemical
and genetic determinants. Up to now, no in vitro
traits are available for breeding for a higher intake
and/or friability, and one way would be to find bio-

chemical or mechanical traits (possibly) involved in
intake variation, and to have plant breeding directly
for these traits or their genetic markers.

The grass cell wall, towards target characterization in forage maize breeding

Variation in cell wall digestibility is the key fac-
tor explaining variation of energy content in forage
maize. Relationships between cell wall chemistry,
anatomy and cell wall degradability are not yet fully
established, but cell wall structure and organization,
and lignified tissue patterning, are undoubtedly in-
volved in both silage maize digestibility and intake.
While first improvements of maize cell wall di-
gestibility were based on whole plant digestibility
tests, understanding the biochemical and molecular
basis of cell wall biogenesis, organization, and ligni-
fication, is now required for further progress in
plant cell wall digestibility and intake. The lignified
grass cell wall is a composite material with cellulose
microfibrils and an amorphous matrix consisting
predominantly of hemicelluloses, mainly glucurono-
arabinoxylans (but very little pectins), and pheno-
lics. Phenolics comprise lignins and cell wall-linked
*p*-hydoxycinnamates, *p*-coumaric (*p*CA) and ferulic
(FA) derivatives, which are reported to be negative-
ly related with cell wall digestibility. The participa-
tion of *p*-hydroxycinnamates with lignins in the cell
wall composition and organization of the mature
vascular system is certainly the most remarkable
trait of grasses. Another characteristic of maize glu-
curono-arabinoxylans is the importance of acetyl
residues linked to xylan chains. Acetyl residues may
account for 5.5% of the cell wall. Acetyl groups es-
terified to the xylan backbone may hamper the ac-
tion of endoxylanases and thus restrict the degrad-
ability of the cell wall. While it is well established
that cellulose and hemicellulose components are
tightly associated through hydrogen bonds, the oc-
currence of covalent bonds between cellulose and
any other cell wall constituents has, thus far, not
been demonstrated.

The lignin polymer in grass cell walls

The first step of lignin biosynthesis occurred af-
ter the shikimate pathway with the deamination by
phenylalanine ammonia lyase (PAL) of L-phenylala-
nine to cinnamic acid. However, a tyrosine ammo-
nia lyase (TAL) activity catalyzing tyrosine deamina-
tion to *p*-coumaric acid has been observed mainly
in grasses (HIGUCHI et al., 1967; BAUCHER et al., 1998). RÖESLER et al. (1997) thus established that the maize PAL also had a TAL activity, suggesting that the same enzyme could perform the deamination reactions of both phenylalanine and tyrosine. Successive steps of hydroxylation, methylation, formation of hydroxycinnamoyl-CoA thioesters, reduction of hydroxycinnamoyl-CoA thioesters to hydroxycinnamaldehydes, and reduction of hydroxycinnamaldehydes leads to the three *p*-coumaryl, coniferyl, and sinapyl monolignols (BOUDET, 2000, 2003; BOERJAN et al., 2003). The three resulting *p*-hydroxycinnamyl alcohols are transported from the cytosol to the apoplast and there they undergo dehydrogenative polymerization via peroxidases, lacases and (poly)phenol oxidases to form lignins (BOERJAN et al., 2003; BOUDET, 2003). Whereas the lignin pathway was considered for a long time to be a metabolic grid, the lignin biosynthesis pathway in maize appears likely to be based on a C3H-CCoAOMT hub (BARRIÈRE et al., 2004c). Feruloyl-CoA is thus the common precursor of ferulate derivatives, and of guaiacyl and syringyl lignin units in the maize cell wall.

The lowering of lignin in the cell wall was established numerous times as increasing cell wall digestibility. The brown-midrib-3 (bm3) mutant is a convincing demonstration of the higher cell wall digestibility in lower lignified plants, even if lignin structure and organization are also affected in bm3 mutant plants. Out of different published results, the *in vivo* average NDFD was equal to 49.4% in a set of 31 normal hybrids, and reached 58.1% in their isogenic bm3 counterparts (BARRIÈRE et al., 2004a), with a lignin content reduced by about one third. Negative consequences on plant standability, on water and sap transport, and correlatively on yield and earliness, prevented breeders from registering early bm3 hybrids, while few late hybrids are available on the US market (OBA and ALLEN, 2000; BALLELARD et al., 2001; SCHWAB et al., 2002). However, if highly digestible lines such as F4 have low lignin content, lines with an even lower lignin content such as F7 are of lower cell wall digestibility than F4, and lines such as F7012 with a significantly higher lignin content than F4 or F7 have a high cell wall digestibility (FONTAINE et al., 2003). From a large review of maize data, correlations between lignin content and cell wall digestibility ranged from -0.3 to -0.9, depending on the germplasm involved, and depending on the methods used to estimate lignin content or cell wall digestibility (BARRIÈRE et

al., 2003a). The correlation between lignin content and cell wall digestibility appeared to be even greater given that the genetic basis of compared genotypes was more homogeneous, or when cell wall digestibility was based on *in vitro* traits. The part of cell wall digestibility variation, often higher than 50%, which is not explained by lignin content, could be due to variation in lignin structure. In particular, ferulate-mediated cross-linking by phenolics is also greatly involved in cell wall digestibility variation.

Maize lignins mostly comprise of guaiacyl (G) units, derived from coniferyl alcohol, and syringyl (S) units, derived from sinapyl alcohol, whereas *p*-hydroxyphenyl units (H), derived from *p*-coumaryl alcohol, occur as a minor component. The average proportions of H, G, and S units has been reported close to 4, 35, and 61% in mature maize stalk lignins (LAPIERRE, 1993). The S/G molar ratio of thioacidolysis monomers released from the mature maize stalk lignins of 13 normal inbred lines ranged between 1.0 and 1.8 (MÉCHIN et al., 2000). The low amount of H units, which can be detected in maize cell walls, is a specific trait of grass lignins and it contrasts with the trace amount recovered from constitutive dicotyledon lignins. The impact of these minor *p*-hydroxyphenyl units on the properties of maize cell walls should not be underestimated as these units increase the frequency of resistant interunit bonds (CABANE et al., 2004). Together with a higher lignin content, an increased proportion of H units has been proposed to contribute to the hardening of maize endodermis cell walls (DEGENHARDT and GIMMLER, 2000). Precise information on the frequency of H units in maize lignins is scarce due to analytical limitations. The H, G and S units of maize lignins are interconnected through labile β-O-4 ether bonds, and through a series of resistant carbon-carbon and biphenyl ether linkages, referred to as the condensed bonds. The native lignins of maize stalk yield thioacidolysis monomers ranges in values between 600 and 800 µmoles per gram lignin. These values are three times lower than what was observed in hardwood lignins, despite of a frequency of S units that is similar in maize and hardwood lignins. Attempts to predict the impact of lignin structure, commonly described with the S/G ratio, on the cell wall susceptibility to enzymatic hydrolysis led to conflicting results, especially when including data of mutant, over-expressed or down-regulated genes in plants. Model studies with artificially lignified maize walls suggested that the fre-
quency of H, G and S units in these artificial lignins, which may substantially differ from native lignins, does not influence cell wall degradability (GRABBER *et al.*, 1997). In the case of maize inbred lines, the S/G ratio appeared to be positively correlated to the stem IVNDFD (MÉCHIN *et al.*, 2000). Consistent with the low thioacidolysis yield diagnostic for a high condensation degree, maize lignins are typified by a high content in free phenolic groups, a structural trait that is common to grass lignins (LAPIERRE, 1993). Up to 60% of native maize lignins can be solubilized in alkali at room temperature. This massive solubilization of lignins is precluded by a mild CH<sub>2</sub>N<sub>2</sub>-methylation of free phenolic groups. This unusual behaviour suggests that maize lignins are distributed in the cell wall as small and alkali-leachable domains. These domains have a high frequency in condensed bonds that constitute branching points and increase the abundance of terminal units with free phenolic groups. A high lignin condensation degree is not necessarily detrimental to cell wall digestibility provided that lignin domains are small enough to allow access of the polysaccharides accessible to microorganism enzymes.

### The p-coumarate derivatives in grass cell walls

Depending on the tissue and its stage of development, cell walls of maize can contain up to 3% *p*-coumarate (GRABBER *et al.*, 2004). *p*-Coumarate is mainly esterified to the γ-position of phenylpropanoid side chains of S units in lignin (RALPH *et al.*, 1994; GRABBER *et al.*, 1996; LU and RALPH, 1999). Although a small amount of *p*CA is esterified to hemicelluloses in immature tissues, most *p*-coumarate accretion occurs in tandem with lignification, making *p*-coumarate accumulation a convenient indicator of lignin deposition, particularly of S units (GRABBER *et al.*, 2004). Syringyl units are enzymatically pre-acylated with *p*CA acid prior to their incorporation into lignin, so sinapyl *p*-coumarate is the logical precursor incorporated into lignin (LU and RALPH, 1999). Considering that most *p*CA esters are linked to S lignin units, 25 to 50% of S units may be acylated by *p*CA. This figure may markedly influence not only the binding mode of S lignin units, but also the spatial organization of the polymers and their interactions with polysaccharides. The possibility that *p*CA substituents on S lignin units could hinder the depolymerization process has been ruled out by the fact that a mild alkaline hydrolysis of *p*CA esters, performed without any lignin loss, does not improve the recovery of thioacidoly-

sis monomers (LAPIERRE *et al.*, unpublished results). The systematic low thioacidolysis yield is most likely indicative of a high content in condensed bonds of maize lignins. While sinapyl alcohol has a pronounced tendency to be involved in β-O-4 end wise type coupling upon peroxidasic polymerization in plant cell walls, its *p*-coumaroylation at Cγ might change the course of the reaction towards the formation of condensed bonds, such as the β-β ones. *p*CA content is negatively correlated with cell wall digestibility and it can explain an equal or higher statistical part of cell wall digestibility variation than lignin content (MÉCHIN *et al.*, 2000; FONTAINE *et al.*, 2003). Even if *p*CA does not likely have as such a direct effect on cell wall digestibility, it is very probably indirectly involved in cell wall digestibility variation of grasses. Moreover, its higher content indicates a higher plant cell wall maturity, and/or a higher proportion of secondary wall in tissue and, as a consequence, a lower cell wall digestibility. The use of *p*CA acid content as an additional trait in maize breeding appears possible because unpublished results at INRA Lusignan showed it was conveniently predicted in NIRS (r² = 0.90). This calibration was however built on plants derived of their ear the day of harvest, as reference analysis of *p*CA are more relevantly done in absence of starch and grain cell walls.

### The ferulate derivatives in grass cell walls

In maize, as in other Poaceae and Commelinoid monocotyledons, ferulic acid, along with its array of dehydrodimers, is ester and/or ether-linked to the cell wall polymers. Ferulic acid is the major hydroxycinnamic derivative in young cell walls and its content increases and then plateaus as the walls come to maturity (MORRISON *et al.*, 1998). Depending on the tissue and its stage of development, cell walls of maize can contain up to 5% ferulates (monomers plus dimmers, GRABBER *et al.*, 2004). At least 50 to 70% of alkali-labile ferulate deposition occurs during secondary wall formation and lignification (MACADAM and GRABBER, 2002; JUNG, 2003). Ferulic units are primarily esterified to non-cellulosic polysaccharides, such as glucurono-arabinoxylans. Through their peroxidase- and/or laccase-driven oxidative coupling, ferulates and diferulates provide points of growth for the lignin polymer, via ether bonds that anchor lignins to cell wall polysaccharides, and thereby direct cell wall cross linking (RALPH *et al.*, 1992, 1995; JACQUET *et al.*, 1995). JACQUET *et al.* (1995) established that ferulic acid
was ether-linked to the β-position of coniferyl alcohol (guaiacyl unit), conversely to the ester linkage of p-coumaric acid to syringyl units. Lignins can also be bound directly to non-cellulosic polysaccharides via direct ether or ester bonds resulting from opportunistic addition reactions between quinone methides and various nucleophiles. The presence of ferulates linked to arabinosyl sides-chains of arabinoxylans also provides a convenient and reliable way of cross-linking these polysaccharide chains. Feruloylated arabinoxylans may be primarily responsible for the scattered distribution of lignin domains in grass cell walls. Moreover, the cell wall organization of lignins in the stems of CCR-deficient tobacco lines is reminiscent of that of grass cell walls. The unusually high solubility of these tobacco lignins in alkali and at room temperature was linked to their enrichment in free phenolic groups, which is diagnostic for the occurrence of small and alkali-leachable lignin domains. This dramatic disorganization of the lignin network co-existed with the appearance of cell wall linked ferulic acid (CHABANNE et al., 2001). In addition to their impact on the organisation of the lignin network, ferulates cross-linking between guaiacyl residues and arabinoxylans is detrimental to cell wall digestibility, acting as a reinforcement of the wall structure, and decreasing wall component accessibility to microorganism enzymes. In a study based on cell suspensions, a reduction of ferulate-lignin cross-linking thus increased the extent of cell wall hydrolysis (GRABBER et al., 1998a). Monosaccharide analysis showed that this reduced cross-linking enhanced the hydrolysis of xylans, and to a lesser extent of cellulose. In a set of 25 lines at silage harvest, the best fitting regression explaining stover cell wall digestibility was based on esterified pCA and etherified FA (r² = 0.57), and the effect of etherified FA appeared more detrimental when lignin content was higher. However, progress in developing plants with lower levels of cross-linking is presently hindered by the non-availability of analytical methods of investigation (GRABBER et al., 2004). Only etherified ferulates are quantified by solvolytic methods, and only 40% of the ferulate which cross-links lignin to arabinoxylans is in ether linkage. Other couplings of ferulate or diferulate to lignin are not determined and such analyses may account for as little as 15% of total cross-linking. How etherified ferulates reflect total ferulate and diferulate cross-linking is actually not known (GRABBER et al., 2004). However, breeding for low level of ferulate ether-

linked to lignin has improved bromegrass cell wall degradation by rumen microorganisms (CASLER and JUNG, 1999), with a reasonable genetic independence between lignin and etherified FA contents. GRABBER et al. (2004) moreover suggested that, “at a given lignin concentration, a low p-coumarate to ferulate ratio might indicate that lignin deposition is restricted to a relatively low proportion of tissues, whereas a high ratio would suggest that lignin is more evenly distributed among tissues”, and that “this ratio may also indicate differences in the proportions of non-lignified and lignified tissues in grasses”.

The formation of ferulate dimers is all that is required to covalently couple the two polysaccharides (HATFIELD et al., 1999). Over 50% of wall ferulates can thus undergo dehydrodimerization, and arabinoxylans become extensively cross-linked by ferulate dimerization (GRABBER et al., 2004). Diferulate cross-linking of arabinoxylan chains is also involved in cell wall digestibility. From experiments with non-lignified cell suspensions of maize, GRABBER et al. (1998b) established that changing the degree of cell wall feruloylation had little effect on their degradability, while increasing the proportion of ferulate dimers reduced carbohydrate release after enzymatic hydrolysis. Xylans with a high degree of diferulate cross-linking accumulated in the indigestible residue, but no effects were observed on the extent of cellulose degradation. Moreover, diferulate cross-linking of arabinoxylan chains is commonly thought to play a role in stiffening cell walls and decelerating growth (KAMISAKA et al., 1990; IYAMA et al., 1994; MACADAM and GRABBER, 2002). Whether ferulate and diferulate cross-linking is involved in tissue friability, and then in maize indigestibility, is very probable, but not definitively established for maize stems, in contrast to the envelopes of the maize grain. The pericarp layer, provided with the highest amount of diferulates, displayed the highest stiffness. In these bran layers, the intermediate strips, which were more plastic, were also found to be the more lignified (ANTOINE et al., 2003). The lignin fractions of low-molecular weight have been evidenced to act as plasticizers in artificial composites (BAUMBERGER et al., 1998). Moreover, lignin distribution has been reported to affect the position of fracture within various cell wall layers (DONALDSON, 1995). Together with ferulate derivatives, the lignification pattern of maize tissues could therefore affect the mechanical behavior of the stems, including friability and flexibility. Variation in
tissue patterning, in xylem and lignified tissue repartition, in lignin structure most likely have effects on mechanical qualities of stalks and leaves, such as their easiness to be torn up during chewing by cattle, but also their resistance to lodging and stalk breakage. Moreover, plant cell wall digestibility, and more likely plant mechanical properties may also arise from genes involved in cell wall organization and carbohydrate biosynthesis. Rice BRITTLE CULM1 (BC1) mutant plants, which are affected in the mechanical properties of culms and leaves, have a cellulose content reduced to 70% of the wild type, and have a lignin content in culm cell wall increased by 30% (Li *et al.*, 2003). Rice BC1 gene encodes a COBRA-like protein (Li *et al.*, 2003) having a suggested role in cellulose deposition or crystallization and oriented cell expansion (SCHINDELMAN *et al.*, 2001). The Arabidopsis FRAGILE FIBER1 (FRA1) gene causes a very important reduction in fiber mechanical strength, but without apparent alteration in cell wall composition (ZHONG *et al.*, 2002). The FRA1 gene encodes a kinesin-like protein that likely mediates the activity of cortical microtubules in orienting the cellulose microfibrils during differentiation of xylem cells (ZHONG *et al.*, 2002; NIEMINEN *et al.*, 2004). Other modification of lignified tissues in Arabidopsis, such as collapsed xylem cells, are in fact related to deficiency in cellulose-synthase genes, and correlatively in secondary cell wall cellulose deposition (TAYLOR *et al.*, 1999). MONTIES (1993) also noticed that anatomical variation such as “the interspersion of sclerenchyma bundles with lignified material” might significantly influence the brittle character of grass leaves. A lower sclerenchyma / vascular bundle ratio was found in fescue normal leaves than in brittle leaves, but both plant types had similar lignin content (MONTIES and CALET, 1992). Molecular (and biochemical) determinants of physical friability and disintegration during eating and chewing, and of cell wall digestibility, probably have to be investigated far beyond the lignification phenomena, including cell wall carbohydrate characteristics and organization, and tissue patterning.

## Genetic resources and alleles of interest in cell wall digestibility improvement

Most of (even all) modern forage maize hybrids are based on a germplasm previously bred and/or also used in grain maize hybrids. Moreover, in parallel of the decline in plant digestibility, was shown

an evolution of germplasm for both dent and flint parental lines. It is then very probable that alleles allowing a good cell wall digestibility and a good friability of plants were either eliminated during breeding for stalk standability and breakage resistance, or lost by genetic drift during breeding for grain yield. The future availability of highly digestible and ingestible maize will require a new investigation of old genetic resources that are not currently used, or that were never used in maize breeding. However, most of improvements progressively piled up in modern (grain) lines and hybrids of maize should be preserved in the future breeding of genotypes with high feeding value, but likely not all.

The (re-)investigation in forgotten genetic resources is all the more a necessity that cell wall digestibility and friability are polygenic traits, in agreement with numerous characteristics of the cell wall possibly involved in its degradability and stiffness. From a synthesis of data in three RIL progenies (RALPH *et al.*, 2004), QTL involved in cell wall digestibility were described in 11 maize bins. Complementary experiments in one of this progeny, and investigation in a new RIL progeny (F7025 x F4, GÉNOPLANTE, unpublished data) allowed finding extra QTL locations involving 13 new bins. All these QTL were not equally distributed on the maize chromosomes, with 3, 4, 3, 3 bins supporting QTL on chromosomes 1, 2, 7, 8, and 1, 2, 2, 2, 2 bins on chromosomes 3, 4, 5, 6, 9, 10, respectively. Based on highest values in percentage of both IVNDFD and DINAGZ genetic variation explained by individual QTL, in *per se* value experiments, maize genetic locations most involved in cell wall digestibility were bin 4.05 (r² = 9.7, RIL F838 x F286), bin 4.08 (r² = 8.3, RIL F7025 x F4), bin 6.06 (r² = 33.1, RIL F288 x F271), and bin 7.02/03 (r² = 13.3, RIL Io x F2). Moreover, a location in bin 1.02/03 was found involved in both cell wall digestibility traits in the four investigated RILs with r² values equal to 9.9, 8.9, 7.6, 6.0, in RILs Io x F2, F288 x F271, F7025 x F4 and F838 x F286, respectively (MÉCHIN *et al.*, 2001; ROUSSEL *et al.*, 2002; INRA Lusignan - PROMAIS and GÉNOPLANTE, unpublished data). According to publicly available data, different genes of the lignin pathway are mapped in these bins (CaldOMT, CCR2, C4H, Pal3, ZmPox3, ...). Other colocalisations probably exist as private data. However, unknown genes, or genes with unknown functions, but involved in i) cross-linking of components, ii) modes and duration of cell wall growth,
lignification, and stiffening, iii) tissue patterning, and iv) variation in regulation of these traits are also likely determinant factors of cell wall digestibility variation, instead of variation in efficiency of structural genes of the lignin pathway.

According to the study of early maize germplasm evolution in hybrids released between 1950 and 2004, flint lines, or flint origin in lines having a “flint” combining ability, are mostly related to two origins, Lacaune and EP1 for (Caribbean) flint originally introduced through Spain, Portugal or Italy, and Gelber Badischer Landmais (Bade yellow) for German flint and Northern Flint origins. To a lesser extent, introductions of landraces or lines from central Europe (Hungary, Danube valley) and from southern or central America have also to be considered. Among Lacaune lines, F2 germplasm is significantly more present in modern lines than F7 germplasm, despite the fact that F7 had a significantly higher cell wall digestibility. According to GUILLET-CLAUDE *et al.* (2004), the ZmPox3 peroxidase gene is disrupted in F7 by a MITE insertion in the second exon. A part of the interest of F7 towards cell wall digestibility appeared related to this insertion, even if other traits inducing a high digestibility also exist in F7. Another flint line of high cell wall digestibility is F4 (MÉCHIN *et al.*, 2000, FONTAINE *et al.*, 2003), with DINAGZ or NDFD values higher than in F2bm3. The particularities of F4 are not yet understood, but from histological observations, the lignification of its parenchyma was very discreet, and the lignification of its vascular bundles was also less pronounced than in other flint lines such as F2 (MÉCHIN, unpublished data). Other INRA flint lines not used in grain or modern maize breeding could be used as genetic resources for cell wall digestibility improvement, such as F66 (Sost landrace), and more recently released F286 (F7, F64 related), F324 (F7, F64, Du101, EP1 related), and F7012 (F7, EP1, Du101 related). These three later lines probably gathered favorable alleles (or gene clusters) originating from F7, but also from Argentina (F64), or Bade Yellow (Du101) and Spanish (EP1) germplasm.

Evolution of dent germplasm in early hybrids during the period between 1950 and 2004 is more complex than for flint parents (BARRIÈRE *et al.*, 2005). However, early dent lines appeared more and more related to Iodent, Reid and BSSS germplasm, with a disappearance of the Minnesota13 germplasm. Conversely, from INRA Lusignan *per se* value experiments, early dent lines of high cell wall digestibility

such as F113, F292, F7019, W33, and W117 are all related to the Minnesota13 origin. Medium early lines (W94129, W95115) bred in the Wisconsin Quality Synthetic (WQS; FREY *et al.*, 2004) also appeared of high cell wall digestibility in European (Lusignan) conditions, with a lignin content significantly lower than lines of similar earliness. Re-investigation of cell wall digestibility could be devoted to old dent or flint x dent lines whose digestibility value is unknown, especially first cycle lines of every maize breeding countries, and about simultaneously to landraces that were of importance in forage (grain) maize before hybrid arrival, but without any available progenies.

Enough genetic resources are likely available for an efficient short and medium term cell wall digestibility improvement of flint and dent lines, even if most of modern elite dent lines appeared of low cell wall digestibility. Conversely, investigations have to be continued for discovering genetic and biochemical basis of cell wall digestibility and intake, prerequisites of medium and long term forage maize improvements in feeding value.

## CONCLUSION

DOLSTRA and DE JONG (1984) soon considered that in forage maize improvement “a breeder must make a lot of choice, sometimes between conflicting wishes”. A forage maize ideotype is probably not a plant maximizing DM or digestible DM yields. The best forage maize allows maximizing the energy ingested and digested by cattle. In short-term breeding, the priority is to re-increase the cell wall digestibility of maize hybrids towards value similar to the ones observed in INRA258 or INRA240 era hybrids, with a slight decrease of DM yield in comparison with today hybrids. In long-term breeding, the most limiting factor is certainly the intake, especially because this trait is neither easily visible nor quantifiable by breeders. Breeding for plant ingestibility and cell wall digestibility improvement cannot be based only on a single cell wall digestibility trait, but it must also involve a comprehensive genetic dissection of wall digestibility and friability in its underlying determinants, such as lignin content, S and G content in the different tissues, hydroxycinnamic acid contents and cross-linking between cell wall components, and cell wall carbohydrate composition and deposition (the importance of the latter is probably underestimated). This dissection should be
favored as it allows piling complementary traits favorable to digestibility in elite lines through marker assisted selection. It is also all the more relevant as lignin content is also involved in plant standability, and water and sap conduction. However, the understanding of the respective importance of all traits involved in cell wall biogenesis will still require numerous further and heavy investigations. The use of model system lines bred for a high or low specific component (or genetically engineered), the use of quasi-isogenic lines in RIL progenies, and the use of model system plant (rice or Arabidopsis) are likely major tools for forage maize quality improvements.

Considering a more fundamental approach, maize is probably the best plant allowing the study of cell wall deposition and lignification in monocotyledons. As it clearly appears in the review of MONTIES (2003), there is a great specificity of lignification in monocotyledons, which was little investigated because paper pulp is only produced from dicotyledons. The monocotyledons vascular system, without bifacial cambium, without secondary growth, but with lignification that proceeds from an intercalary meristem in each internode, and with vascular bundles scattered, penetrating radially and present in medulla and in cortex, has no homology with the dicotyledons vascular system, which could not be considered as ancestral (TOMLISSON, 1995). The understanding of the specific organization of the grass cell wall, of the specific vessel organization and lignification patterning in grasses will also provide valuable information on plant emergence, evolution, and phylogeny.

ACKNOWLEDGEMENTS - PROMais head staff and members are greatly thanked for their interest in this review on forage maize, and for their efficient help in information gathering. VILMORIN Seeds Company is especially thanked for allowing old VILMORIN-ANDRIEUX catalogs to be consulted.

In addition, thanks are given for valuable and original information to: B. AIZAC (GEVES, F-17700 Surgères), C. BAULAND (INRA, F-80200 Péronne), J. BEIGBEDER, (SYNGENTA, F-31790 Saint-Sauveur), L. VAN BEUNINGEN (ADVANTA Seeds B.V., NL-4410 AA Rilland), P. CARRÉ (PROMAIS, F-31200 Toulouse), F. CASAÑAS (Universidad Politécnica de Cataluña, Barcelone, Spain), B. CHABBERT (INRA, F-51686 Reims), M. CHAMPION (LIMAGRAIN Genetics, F-63720 Chappes), A. CHARCOSSET (INRA, F-91190 Gif-sur Yvette), J.G. COORS (Wisconsin University, Madison, USA), T. CROSBIE (MONSANTO Company, IA-50021 Ankeny, USA), J. DALLARD (INRA, F-34130 Mauguio), J. DE BOEVER (Agricultural Research Center, B-9090 Melle-Gontrode), P. DESBONS (PIONEER Génétique F-41290 Oucques), T. GOCKEN (MONSANTO Company, MO-63167 St Louis, USA), A. LACOMBE (ADVANTA France, F-35520 La Mézière), J. LALLEMAND (GEVES, F-17700 Surgères), A. LAMBERT (LIMAGRAIN, F-49160 Longué), B. LEFEVRE (RAGT-R2, F-12510 Olemps), R. LEIPERT (KWS SAAT AG, D-37555 Einbeck), J. LORGEOU (Arvalis, F-91720

Boigneville), T. LUEBBERSTEDT (Danish Institute of Agricultural Science, DK-4200 Slagelse), M.J. MILLAR (USDA PI center, Iowa, USA), T. MOLNAR (PIONEER, F-35740 Pacé), B. MONTIES (INAPG-INRA, F-78850 Thiverval-Grignon), J.P. MULLER (SYNGENTA, F-50170 Les Pas), P. NOURRISSAT (SYNGENTA breeder, retired), M. OUZUNOVA (KWS SAAT AG, D-37555 Einbeck), M. PENOT (VILMORIN seeds, F-49250 La Ménitré), J.P. SAMPOUX (INRA, F-86600 Lusignan), L. SAULNIER (INRA, F-44316 Nantes), P. SEHABIAGUE (MONSANTO Company, F-40305 Peyrehorade).

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