Genetic variation and breeding strategies for improved cell wall digestibility in annual forage crops. A review
Genetic variation and breeding strategies for improved cell wall digestibility in annual forage crops. A review
s BARRIÈRE 1
h GOFFNER 0
0 UMR CNRS UPS Signaux et Messagers Cellulaires Végétaux , 31326 Castanet-Tolosan , France
1 Unité de Génétique et d'Amélioration des Plantes Fourragères, INRA , 86600 Lusignan , France
- Forage plants are the basis of ruminant nutrition, and cell wall digestibility is the limiting factor of their feeding value. Cell wall digestibility is therefore “the” target for improving the feeding value of forage crops. Among annual forages, maize cropped for silage making is the most widely used, and much research in genetics, physiology and molecular biology of annual forages is devoted to maize. Sorghum, immature small grain cereals and straws of small grain cereals are also given to cattle. Some dicotyledons are or were also used, such as forage beets, kales, canola in temperate areas and amaranths in tropical and subtropical areas. Large genetic variation for cell wall digestibility was proved from both in vivo and in vitro experiments in numerous species. Among the regular maize hybrids (excluding brown-midrib ones), the NDF in vivo digestibility nearly doubled from 32.9 to 60.1%. Correlations between in vivo and in vitro estimates of cell wall digestibility were often close to 0.75, but in vitro estimates of cell wall digestibility significantly reduced the range of variation between genotypes. Despite lignin content is well known as the major factor making cell wall undigestible, breeding for a higher digestibility of plant only from a lignin content trait appeared impossible. Correlations between lignin content and cell wall digestibility were indeed greatly variable according to the genetic background. Moreover, enzymatic solubilities were excessively dependent on lignin, and correlation between in vivo estimates of cell wall digestibility and lignin content were always lower than correlation between in vitro estimates of cell wall digestibility and lignin content. Among brown-midrib genes, the bm3 mutant in maize, and the bmr12 (and possibly bmr18) mutant in sorghum, which are both altered in the COMT activity, appeared as the most efficient in cell wall digestibility improvement. Moreover, a great genetic variation in the efficiency of the maize bm3 gene for cell wall digestibility improvement was observed according to the genetic background, with a lower efficiency when the normal germplasm was of better cell wall digestibility. Efficient breeding maize and others annual forage plants demands a renewing of genetic resources. Because resources of interest in cell wall digestibility improvement could be of poor agronomic value, the best is likely to
use a marker assisted selection, after identifying alleles of interest in these resources. Results
obtained on forage plants and model plants strengthened also the interest of genetic engineering in the
lignin pathway for improving the cell wall digestibility of forage plants.
cell wall digestibility / ingestibility / annual forage / maize / genetic variation / breeding /
Résumé — Amélioration de la digestibilité des parois des fourrages annuels. Les plantes fourragères
sont à la base de l’alimentation des ruminants, mais leur valeur nutritive est limitée par la digestibilité
variable des parois végétales, dont l’amélioration est alors la cible essentielle des programmes de
sélection. Parmi les fourrages annuels, le maïs est la plante la plus cultivée, et c’est sur cette espèce que
porte l’essentiel de la recherche en génétique, physiologie et biologie moléculaire. Toutefois, le
sorgho, les céréales à paille et des dicotylédones comme les betteraves, les choux, les colza en régions
tempérées et les amarantes en zones tropicales sont également utilisées comme plantes fourragères.
Une importante variabilité de la digestibilité des parois végétales a été mise en évidence pour de
nombreuses espèces, à la fois à partir de mesures in vivo ou in vitro. Ainsi, pour le maïs (sans plantes bm3),
la digestibilité in vivo du NDF varie du simple au double entre 32,9 et 60,1 %. Les corrélations entre
les estimations in vivo et in vitro de la digestibilité des parois sont en général de l’ordre de 0,75, mais
les estimations in vitro de la digestibilité réduisent significativement la gamme de variation entre
génotypes. Alors que la teneur en lignine est le facteur majeur d’ingestibilité, il n’est toutefois pas
possible de sélectionner pour une digestibilité plus élevée à partir des teneurs en lignine. Les corrélations
entre teneur en lignine et digestibilité des parois sont en effet très variables en fonction du fond
génétique. De plus, les solubilités enzymatiques apparaissent exagérément liées à la teneur en lignine,
avec des corrélations entre teneur en lignine et digestibilités in vitro bien supérieures à celles
observées avec les digestibilités in vivo. Parmi les mutants à nervures brunes, les mutants bm3 du maïs et
bmr12 du sorgho (et peut-être bmr18), avec des mutations liées à la COMT, sont apparemment les
plus efficaces en terme d’amélioration de la digestibilité. Il existe par ailleurs une variabilité de l’effet
du gène bm3 du maïs en fonction du fond génétique, avec une efficacité plus élevée quand le géniteur
de départ est de plus faible digestibilité. Un progrès génétique significatif pour la digestibilité des
parois des fourrages annuels nécessite un renouveau important des ressources génétiques
habituellement utilisées. La mise en évidence des allèles d’intérêt pour la digestibilité des parois et leur
introduction par sélection assistée par marqueurs évitera les pertes de valeur agronomique lié à
l’utilisation de ressources dépassées par ailleurs. Enfin, au vu des données actuellement disponibles sur
plantes modèles et plantes d’intérêt agronomique, il ne faudrait pas sous-estimer l’intérêt du génie
génétique pour l’amélioration de la digestibilité des parois végétales.
digestibilité des parois / ingestibilité / fourrage annuel / maïs / variabilité génétique /
amélioration génétique / lignine
Forage plants are the basis of ruminant
nutrition, but although forages contain
almost the same amount of gross energy as do
grains per unit of dry matter, the energy
value of forages is lower and much more
variable, ranging approximately from 80%
(leafy ray-grass) to 33% (wheat straw) of
maize grain value. This difference results
from the high content of cell wall in forage
plants, and to the limited digestion of this
fiber part by the micro-organisms of rumen
and, to a lesser degree, of large intestine of
animals. Lignins are the cell wall part
resistant to fungal and bacterial degradation.
Because of their quantitative importance in
the cell wall, of their variable structure, and
of the way in which they embed and bind to
hemicelluloses and cellulose, lignins have a
variable depressive effect on carbohydrate
degradation by micro-organisms.
Most spontaneous or cropped forages
are perennial plants, and are mainly
grasses. For annual forages, maize cropped
for silage making is the most widely used.
Because of the economic importance of the
“corn” crop worldwide, and of the
economic importance of forage maize in
Europe, much research in genetics,
physiology and molecular biology of
annual forages is devoted to maize, and there
are extensive data available. Sorghum and
immature small grain cereals (wheat,
barley, triticale…) are also given to cattle,
either as green plants, but often after ensiling.
Straws, including rice straws in tropical
areas, are also sometimes used for cattle
feeding after grain harvest. All these major
annual forage plants are monocotyledons.
Some dicotyledons such as forage beets
and kales were used in temperate areas and
given as fresh plant to cattle, but this is not
longer done due to the cost of harvesting
and difficulties in conservation. Rape-seed
or canola forages are also used in cattle
feeding, grazed, given fresh or ensiled.
Amaranths are widely grown as a leafy
vegetable for human feed in tropical and
subtropical areas, but some cultivars are
highly prized as forage crops, because of
their rapid growth rate, good yielding, and
high protein content. Annual grain legumes
of temperate areas, such as peas, are also,
but rarely, used as green forages of high
protein content. Despite its fiber crop
origin, kenaf has been viewed as an alternative
forage crop with a high drought tolerance.
A significant part of this review will deal
with investigations of cell digestibility
variation in cattle, and with relationships
between in vivo and in vitro results, because a
breeding strategy based on in vitro criteria
has to be first validated in vivo. Genetic
resources, including specialty plants, will be
investigated to highlight the most suitable
germplasm for breeding plants of higher
feeding value. A subsequent part of the
review will develop a tentative breeding
strategy based on the available data on cell wall
traits related to feeding value for ruminants,
and genetic advances or methodologies in
plant breeding. The focus of this review
will be on maize, as there are little data
available on cell wall digestibility
improvement in other annual forage crops.
However, whenever possible, information on
other annual forage crops will be reported.
2. CELL WALL DIGESTIBILITY IS
“THE” TARGET FOR THE
IMPROVEMENT OF FORAGE
The part of available energy in a forage
that is effectively used by an animal has
been proved strongly correlated to the
forage digestibility. The energy supplied by a
forage in a ruminant or herbivore animal
diet is thus related to the forage ingestibility
and digestibility. The digestibility of any
forage constituent (dry matter, organic
matter, or cell wall) is usually measured as the
percentage of each constituent that has
disappeared in the animal digestive tract. The
ingestibility of a forage is the quantity that
is taken by an animal when this forage is
offered ad libitum, as an unique meal.
Ingestibility is usually measured as kg DM
per animal and per day, but can also be
considered as g·kg–1 of metabolic weight (live
weight 0.75). When a plant is fed to an
animal, ingestibility and digestibility are plant
characteristics resulting of cell wall
development, and are subject to plant genetic
Assuming a normal efficiency of the
rumen micro-organisms, the variation in
energy feeding value of a forage plant is
related (i) to the variation in digestibility
of the stover part of the plant, and therefore
to the variation in cell wall digestibility,
(ii) to the variation in grain content, if
grains, that are a highly digestible part, are
present in the forage (maize, sorghum, …),
(iii) to the digestive interactions between
the forage and the concentrates present in
the diet and (iv) to the variation in
ingestibility, that is related to the transit
rate of particles out of the rumen, and then
to the intensity and rate of particles
Cell wall digestibility is “the” target for
improving the feeding value of forage
crops. This is obvious when the harvested
plant has no grain, particularly as soluble
carbohydrates are subject to extensive
environmental variation. But it is also true in
maize silage, for three independent
reasons. Firstly, in vitro digestibility of the
whole maize plant has most often been
proved to correlate better with stover cell
wall digestibility than with grain content.
From Deinum and Bakker [
and Struik [
], Wolf et al. [
] and data
reviewed by Cox et al. [
between maize whole plant digestibility and
cell wall (or stover) digestibility ranged
from 0.60 to 0.96, with average values close
to 0.80, whereas correlations between
whole plant digestibility and grain or ear
content were only nearly equal to 0.45.
In vivo genetic correlations obtained at
INRA Lusignan (France) corroborated
these results. In vivo organic matter
digestibility (OMD) correlated most (rg = 0.77)
with in vivo NDF digestibility (NDFD),
whereas the correlation with starch content
was lower (rg = 0.54). Moreover, NDFD
and ear or starch content were found to be
independent traits (rg = 0.09), as also
observed by Deinum and Struik [
r = –0.01. NDFD and starch content both
explained at least 90% of the genetic
variation in OMD, while the remaining percent
points were probably related to the
variation in soluble carbohydrates content, and
to the imprecision of the estimates. The
second reason why cell wall digestibility is the
leading target to improve the feeding value
of forage crops is related to animal
digestive physiology. Due to rumen
micro-organism ecology and due to acidosis risks,
whatever the conditions of ruminant cattle
feeding, the optimal grain content in a cereal
silage has to be adjusted according to the
extra starch content of the diet, and
according to the proportion of by-pass starch. The
optimum starch content in maize was thus
proved to be close to 30% in European
conditions of dairy cattle rearing [
result, which was proved in maize, is very
likely true in other immature cereals.
Finally, a higher NDF digestibility should
result in higher energy intake by cattle, even
if the DM intake has not been affected
. An efficient genetic progress in
feeding value is then intrinsically related to
NDFD improvement, both for forages with
and without grain.
3. IN VIVO GENETIC VARIATION
FOR CELL WALL DIGESTIBILITY
IN ANNUAL FORAGE CROPS
3.1. In vivo genetic variation for cell wall digestibility in maize
The reference value of a forage
digestibility is established through measurements
with animals, and mostly often with sheep
(adult wethers) in digestibility crates. Most
often, work has been devoted to studies of
whole plant digestibility, and only few
studies have investigated cell wall digestibility.
The first digestion trials in Europe with
ensiled forage maize seem to be those of
Dijktra and Becker ([
], quoted in [
the Netherlands, and those of Demarquilly
], and Andrieu and Demarquilly , in
France. From 25 measurements reported by
Deinum et al. [
], OMD of silage maize
had an average value of 72.8%. The average
NDF in vivo digestibility (NDFD) of
hybrids was 52.7%, and ranged from 47.5 to
57.1% (NDFD of one bm3 hybrid was also
65.1%). Andrieu and Demarquilly 
reported an OMD in maize silage equal to
71% that could reach 74%, according to
cropping conditions, maturity and/or grain
content. The NDFD of hybrids involved in
this French study was later estimated to
53.1%, and ranged from 49.8 to 54.8% [
From 50 maize silages cropped between
1971 and 1993, De Boever et al. [
reported average OMD values equal to
74.7%. The average crude fiber
digestibility (CFD) of these hybrids was 66.3% and
ranged from 60.4 to 74.6% (de Boever,
personal communication), corresponding
approximately to NDFD values equal to 61.4,
56.1, and 68.8%, respectively. Most of
these forage maize in vivo reference values,
thus estimated more than 30 years ago,
were based on a limited number of early
genotypes with good feeding value (Fronica,
Circé, LG11, but not Eta Ipho, in the
Netherlands, and Inra258, Funk245, Dekalb204,
LG11 in France), which are no longer
representative of the presently available
Many measurements with sheep in
digestibility crates have been made on a much
wider genetic basis at INRA Lusignan (and
first reported in [
]). Today, data are
available from measurements of 2100 mini-silos
and 431 hybrids with an average 34% dry
matter content at harvest. Among these
hybrids, 167 were experimental hybrids and
264 were registered hybrids, representative
of all seed companies present on the French
and North European markets. Genetic
variation in OMD and NDFD of silage maize
were thus proved to be very large (Tab. I).
Among the regular maize hybrids
(excluding brown-midrib ones), the NDFD nearly
doubled from 32.9 to 60.1%, and it was
similar for the sub-sample of registered
hybrids for which the NDFD went from 32.9
to 58.4%. Among the 220 registered early
hybrids, the range in NDFD was 19 percent
points. Among the 41 registered late
studied varieties, the range in NDFD was
14.9 percent points. Late hybrids had an
average NDFD (46.1%) slightly lower than
early hybrids (48.0%), but a few late
hybrids had also a much lower cell wall
digestibility (the minimum value was 32.9%)
than early hybrids (the minimum value was
39.4%). Genetic correlations between
NDFD and other traits related to feeding
value are given in Table II. The correlation
between NDF content and NDFD was close
to zero. No significant relationship existed
between the cell wall digestibility and the
cell wall content when maize plants were
thus harvested at a similar maturity stage.
Consequently, for a given starch content,
OMD was only related to NDFD. The
genetic progress in feeding value was thus
directly related to NDFD improvement. The
narrow sense heritability of in vivo
digestibility traits was high (h2 > 0.70), and
assuming a selection intensity of 25%, the
genetic progress in NDFD was expected to
OMD: organic matter digestibility; NDF: neutral detergent fiber; NDFD: NDF digestibility.
be equal to 5.4 percent points per breeding
Based on European or Northern Amer
ica experiments, the genetic variation in the
NDFD value of maize silage was also
proved to have consequences on young bull
or dairy cow performances, even if maize
was not the only constituent of the diet, [
36, 53, 95, 118, 120
]. All other factors
being equal, when comparing hybrids with
poor or good cell wall digestibility in dairy
cows, fat corrected milk (FCM) yields
differed from 1 to 3 kg among hybrids. The
protein contents in milk were also equal or
higher in hybrids that allowed greater milk
yields. In a similar way, differences in
average daily gains of young bulls reached
100 g per day among hybrids. When it was
investigated, differences between hybrids
found in cows were often slightly lower
than expected according to their estimated
value with standard sheep, despite the fact
that Aerts et al.  showed no systematic
differences between sheep and cows in the
measurement of digestibility.
Genotype × environment interactions
affect both the extent of genetic progress in
plant breeding and the efficiency of variety
choice by farmers. From a specific
experiment in 14 locations [
], in vivo CFD
genotype × environment interaction was non
significant, whereas the location and
genotype main effects were highly significant.
Genotype × environment interactions could
be also investigated from a sub-sample of
the measurements in the 2100 mini-silos
and 431 hybrids fed to sheep. Data were
then considered for only 703 mini-silos,
corresponding to 35 hybrids studied over 5
or more years, with at least 2 replicates per
year (Tab. III). The genotype effect for
NDFD was highly significant (P < 0.001),
whereas the NDFD genotype × year
interaction was not significant. For OMD,
genotype × year interactions were significant,
but the mean-square was about 10 times
lower than the mean-square of the genotype
effect. This was related to high interactions
observed for starch content.
Genotype × year interactions were also
significant for NDF content, but this could only be
related to the variation in starch content, as
these two values were expressed in
percentage of the total dry matter. Therefore, maize
breeders are able to improve maize feeding
value from estimates of NDFD, after maize
cropping in a limited number of locations
and/or years, provided the locations are
well chosen, and provided a relevant
in vitro criterion is available.
3.2. In vivo genetic variation for cell wall digestibility in other annual forage crops
Cell wall digestibility was lower in sorghum silages than in maize silages, especially in sweet × grain sorghum hybrids, and OMD was also much lower (Tab. IV).
Sorghum silage appeared to have
maximum NDFD or OMD values that were
much lower than the highest values of
maize, despite the fact that some grain
sorghum silages had a higher grain content
than maize. Aydin et al. [
] also reported
that most studies that compared sorghum
with maize silage have shown that milk
production was consistently higher for cows
fed the maize silage than for those fed the
sorghum silage, which was of lower cell
wall digestibility. Few data were available
for genetic variation in cell wall
digestibility of small grain cereals. However, the
variation in feeding values of straws of
different varieties of cereal crops affected
the performance of lactating cows,
lactating sheep and steers [51, 147, 156 quoted in
52]. Garnsworthy and Stokes [
reported a modified ADF in vivo digestibility
of oat silage ranging from 46% (45 days
after emergence) to 41% (65 days after ear
emergence). From INRA Lusignan
unpublished data, average NDFD in triticale and
wheat were close to 49%, and close to 46%
in rye, but low or very low intakes were
observed with awned plants (especially all
triticale lines), which very likely led to
overestimated NDFD values. The cell wall
digestibility of rape-seed and canola, both
for green forage and silage, was closely
related to the stage of harvest. Rape-seed
CFD in sheep was very high in leafy stages,
nearing 90%, but swiftly decreased towards
50% as stems developed [
]. Winter type
genotypes were more suitable for cattle
feeding than spring types, as their leafy
stage lasted longer. No difference in
digestibility and/or growth performance of cattle
was observed between genotypes with high
or low glucosinolates content [
Demarquilly and Andrieu  gave an
average OMD of forage kale close to 83%,
related to the weak crude fiber content of this
forage, but also likely related to its good
cell wall digestibility. Compared to others
grasses and forage crops, the cell wall
digestibility of maize or sorghum varied little
during the period of ensiling compared to
great degrees in variation of cell wall
digestibility in other forage crops, due to the
rapid increase in stem content.
4. GENETIC IMPROVEMENT
OF CELL WALL DIGESTIBILITY
IN ANNUAL FORAGE CROPS
FROM IN VITRO TRAITS
4.1. Devising a breeding criterion for genetic improvement of cell wall digestibility
For obvious practical and financial rea
sons, digestibility assessments have to be
performed using in vitro tests of dry matter,
organic matter, or cell wall digestibility.
Moreover, this plant digestibility value
must be easily and accurately predicted
through NIRS. But this approach is
pertinent only if the in vitro method used is
proved to be a good predictor of animal
behavior. For maize breeders, in large trial
networks, a cheap and easy digestibility test
also has to be devised on whole plant
samples, without separating grain from stover,
or leaf from stalk. When using an
enzymatic solubility on whole plant samples, a
given digestibility value can be related to
large grain or soluble carbohydrate
contents, but with a low digestibility of cell
walls, or from a higher digestibility of the
stover, with lower starch or soluble
carbohydrate contents. Breeding forage plant for
feeding value makes it therefore necessary
to assess at the same time the digestibility
of the whole plant, and to have the
possibility of assessing or computing the
digestibility of the cell wall part.
The in vitro digestibility of plant was
first estimated through the Tilley and Terry
 method, using rumen fluid. This
method was later modified by Goering and
van Soest [
], Marten and Barnes [
Craig et al. [
] and recently by Lauer et al.
]. NIRS calibrations for Tilley and
Terry modified tests with rumen fluid were
developed by Lauer et al. [
], and also in
labs in the Netherlands. Different whole
plant enzymatic solubilities (IVDMD)
were developed in Europe by Aufrère, [
slightly modified by Aufrère and
], Lila et al. [
], De Boever
], and Ronsin and Femenias [
Correlations between these different IVDMD
were very high (r > 0.90), but the mean
value of the Aufrère and Michalet-Doreau
IVDMD is about 5 percent lower than the
others, with an approximately 25% lower
residual error (Barrière et al., unpublished
data). NIRS calibrations of all these
IVDMD were computed and available at
CRA Libramont (Belgium). For maize
official registration, De Boever et al. [
Aufrère and Michalet-Doreau [
are used in Belgium, France, Germany, and
the United Kingdom, whereas in the
Netherlands and in Switzerland, digestibility of
maize for official registration is estimated
through a Tilley-Terry test. For all INRA
data reported here, IVDMD was assessed
with the Aufrère and Michalet-Doreau [
For plant breeding purpose, and from
results on maize, cell wall digestibility can be
cheaply computed using three different
estimates, based on a Tilley-Terry test or on
an enzymatic solubility of the whole plant
(both predicted through NIRS
calibrations). According to Struik  and
Dolstra and Medema [
], the in vitro NDF
digestibility (IVNDFD) is computed
assuming that the non-NDF part of plant
material is completely digestible. According
to Argillier et al. [
], the in vitro
digestibility of the “non starch and non soluble
carbohydrates” part (DINAG, or English
acronym IVDNSC) is computed assuming
that starch and soluble carbohydrates were
completely digestible. A modified DINAG
criterion, namely DINAGZ, was later
estimated in a similar way as DINAG, after
adding the crude protein to the completely
digestible constituents [
]. The formula
IVNDFD = 100 × (IVDMD – (100 – NDF))/
DINAG = 100 × (IVDMD – ST – SC)/
(100 – ST – SC)
DINAGZ = 100 × (IVDMD – ST – SC –
CP)/(100 – ST – SC – CP)
where ST, SC and CP are starch content,
soluble carbohydrate content and crude
protein content, respectively. These
estimates of cell wall digestibility have proved
their great relevance and efficiency in plant
11, 15, 24, 90, 133, 160
However, IVNDFD does not give the true NDF
digestibility value, and these three
estimates are not relevant in interspecific
comparisons. Moreover, the values of cell wall
digestibility obtained with any of these
estimates could be seriously biased if
anti-nutritional compounds such as tannins have
an impact on non-fiber digestibility.
The use of a Tilley-Terry estimate of
plant digestibility rather than an enzymatic
solubility could be questioned, both when
estimating whole plant IVDMD, and when
computing cell wall IVNDFD or DINAGZ.
Obviously, the enzymatic methods are
easier and cheaper, as they do not require the
maintenance of animals producing rumen
fluid. Only few data are seemingly
available giving correlations between
TilleyTerry and enzymatic IVDMD. From data of
Capper et al. [
] investigating 30 barley
genotypes, the correlation between a
pepsin-cellulase and the Tilley-Terry
digestibility was only 0.49. De Boever et al. [
reported a correlation r = 0.84 between a
Tilley-Terry and a pepsin-cellulase IVDMD,
from measurements in 50 maize silages.
From measurements on numerous maize
samples, Van Waes  reported
correlations between pepsin-cellulase and
Tilley-Terry digestibility equal to 0.60, 0.55
and 0.88 in each of the 3 years of
experiments, respectively. In the German network
for forage maize evaluation, the correlation
was close to 0.84, observed from about 100
samples both in 1998 and 1999 (Tillmann,
personal communication). Over a large
range of variation in digestibility values, a
European maize breeder (personal
communication) also observed a correlation close
to 0.80, but this correlation fell to 0.50
when only genotypes with average or good
cell wall digestibility were taken into
consideration. Differences between rumen
fluid digestibility and enzymatic solubility
also arose from the study of De Boever et al.
] on 50 maize silages, since correlations
were higher between enzymatic IVDMD
and ADL (r = –0.81) than between
TilleyTerry digestibility and ADL (r = –0.66).
4.2. Genetic variation in cell wall digestibility estimated from in vitro criteria
Much research has proved that there are
large genetic variations in the in vitro
digestibility of maize, either for whole plants
or cell wall parts, and both in inbred lines
and hybrids (reviews in [
considering the digestibility of whole
plants, as well as cell wall digestibility
(even if the latter was less frequently
studied), additive genetic effects for in vitro
values of digestibility were preponderant over
more complex genetic effects. Similarly,
genotype × environment interactions for
cell wall digestibility were very often small
compared to main effects. Heritabilities of
quality traits were high, ranging between
0.65 and 0.80, and at least equal to those of
10, 72, 160
]. The heritability of
DINAG and DINAGZ was always greater
than that of IVDMD, and equal or higher
than heritability of IVNDFD. Breeding for
higher in vitro cell wall digestibility value
should therefore be very efficient, and the
expected progress for the first selection
cycle of breeding for cell wall digestibility
could easily reach 3.0 percent points.
According to Garnsworthy and Stokes
], some comparisons have been made
on the effects of species, varieties and times
of harvest on the quality of small grain
cereals for silage. Notably, Tingle and Dawley
 reported differences in IVDMD
between varieties of barley and between
varieties of oats harvested at the soft-dough
stage. Large differences in IVDMD of
barley straw were also reported by Capper
et al. [
], but no data were available for cell
wall digestibility. Varietal differences in
IVDMD of rice straw have been reported
from many countries and have been first
summarized by Doyle et al. [
 observed later a whole plant IVDMD
ranging from 23.6 to 36.9 in rice straws,
when the IVDMD of stems ranged from
27.6 to 43.3%. Agbagla-Dohnani et al. 
observed an in sacco degradability of
organic matter in straws of 15 rice varieties
ranging from 23.6 to 35.6%. Genetic
variation in cell wall digestibility of rice straw
was first reported by Abou-el-Enin  from
53 varieties in 5 Oryza species. NDF
digested after a 48 h in sacco incubation
ranged from 21.2 to 31.1%, and varieties
with high or low IVNDFD were found
within each species group. It is doubtful if
the cell wall digestibility of straw could be
used directly as a breeding criterion in
small grain cereal improvement programs,
but identification of varieties with
consistently better straws is of interest to
determine the most economical use of straw for
cattle feeding, especially in areas where
straws represent an important part of the
Large morphological diversity exists
amongst kales and cabbages. Given that
four generations of half-sib family
breeding for a higher yield of IVDOM in an
initial population which included different
types of kales and cabbages, led to a
population of marrow-stem kale, Bradshaw and
] concluded that this was the
type of kale most suitable for the
simultaneous improvement of yield and feeding
value. From Kunelius et al. [
of a marrow kale variety was indeed very
high all along the season, close to 92% from
110 to 185 days after sowing, whereas
lignin content remained low and increased
only from 2.5 to 3.5%. These results were in
agreement with in vivo estimates of
Demarquilly and Andrieu [
]. No cell wall
digestibility of green forage brassicas was
seemingly published, but, computed from
data of Kunelius et al. [
], IVNDFD of
kale remained high and close to 71% during
the 110 to 185 day period after sowing. This
plant, that withstands low temperature
during autumn and early winter, is probably
underrated in cattle feeding, but this may be
related to the labor costs of harvesting and
The forage nutritive value of amaranths
was considered by Sleugh et al.  to be
equal to or better than commonly used
forage in IVDMD, with an average value
across genotype and harvest date equal to
71.1%. Amaranths have also a high crude
protein content (14.5%), which have high
digestibility and quality (amino acid
composition and content in bypass protein). For
7 species or ecotypes, harvested from 42 to
112 after planting, average NDF content
was 37.4%, but average IVNDFD was low,
nearing only 27.7%, and this could be
related to the C4 anatomy of amaranth plants.
Average IVNDFD remained close to 25%
from 42 to 84 days after planting, and
neared 20% from this date. The optimum
harvest date was probably close to 84 days
after planting, when the IVNDFD was still
close to 25%, and the nitrate and oxalate
concentrations were sufficiently low in
fresh forage to avoid toxicity risks in
animals. Ensiling the forage was considered as
an alternative for reducing the nitrate
concentration and then improving its
] quoted in ). Out of the
genotypes studied by Sleugh et al. , A
hybrid and A hybridus had the higher
IVNDFD at 84 days (28% on average), but
had the lowest protein content (11%). A
hypochondriacus (from Colorado), which
was also the species with the lowest ADL
content 84 days after planting, probably
gave a better compromise between
IVNDFD (23%), IVDMD and protein
content (14%). In any case, and according to
], quoted in ), A
hypochondriacus is an amaranth cultivated
solely for use as forage for cattle, and an
improvement in its cell wall digestibility
would very likely be possible. The search
for Amaranths adapted to temperate areas
could also be considered as an alternative
answer in countries facing difficulties in
crude protein supplies for cattle feeding.
Contradictory results were published on
kenaf as a forage plant. Xiccato et al. [
showed that digestibility was low in the
apical portion of full blooming kenaf. They
also established that kenaf silage was
unpalatable and often refused by ewes. But
Muir et al.  considered that the overall
plant digestibility of kenaf, harvested 60 to
120 days after planting, compared
favorably with traditional forages in semiarid
regions. The genotypic effect was significant
for in sacco NDF disappearance (ISNDFD),
with an average value close to 50% .
Low palatability to cattle, often cited as
limiting the use of kenaf as a forage plant, was
considered as more related to initial rather
than long term difficulties, as illustrated by
observation of favorable intake rates relative
to those of alfalfa ([
], quoted in ).
4.3. Relationships between estimates of in vitro cell wall digestibility in maize lines and hybrids
Both during elite hybrid breeding, for
QTL analysis, or for evaluation of genetic
resources, it is easier and cheaper to have
whole plant and cell wall digestibility
estimates in lines rather than after top-crossing.
Moreover, variance of traits is greater in
lines than in hybrids. Gurrath et al. [
reported correlation between maize inbreds
and hybrids stover digestibility, and ADL
content, at silage harvest equal to 0.75, and
0.81, respectively. Wolf et al. [
reported a correlation between S2 families
per se value and average top cross value
(two testors) for cell wall digestibility equal
to 0.62 in whole plant. Dolstra et al. [
reported a correlation between mid-parent
and hybrid stalk cell wall digestibility equal
to 0.70. Argillier et al. [
also good or very good relationships
between lines per se and top cross values in
two factorial designs. Correlations between
hybrid values and per se values ranged
between 0.76 and 0.94 for DINAG, and
between 0.63 and 0.87 for lignin content. On
the contrary, the correlation between hybrid
values and line values was very low for
starch content (r = 0.28). In a RIL progeny
study , correlations between cell wall
digestibility estimates in RILs per se and
top cross were high (r = 0.71 and 0.79 for
DINAGZ and IVNDFD, respectively), and
these correlations were, as expected, higher
than for IVDMD (r = 0.63). Correlation
between lignin content in RILs per se and top
cross was higher for ADL/NDF (r = 0.75)
than for KL/NDF (r = 0.62). Correlations
between RILs per se and top cross were also
high for other constituents of NDF, such as
Hemicellulose/NDF (r = 0.78) and
Cellulose/NDF (r = 0.81), but lower for NDF
content (r = 0.58). All these results proved
the feasibility of preliminary tests from
lines per se value in breeding for the
improvement of forage cell wall digestibility
in maize. This is very likely true in other
hybrid forage plants.
4.4. Relationships between in vitro and in vivo cell wall digestibility traits in maize
Much research has focused on the
relationships between whole plant Tilley-Terry
or enzymatic IVDMD, and whole plant
in vivo OMD, in order to elaborate practical
rules for cattle feeding. Zimmer et al.
], quoted in [
]) reported in maize a
quadratic regression between OMD and
Tilley-Terry IVDMD with a correlation
r = 0.80. Givens et al. [
] obtained a
correlation r = 0.80 from 4 sets of maize silage
originating from the UK, the Netherlands
and Belgium (and a total of 106 silages)
between OMD and an enzymatic neutral
detergent cellulase OMD. De Boever et al.
] reported a correlation r = 0.82 from
50 maize silages between OMD and
enzymatic IVDMD. Andrieu et al.  observed
a correlation only equal to 0.57 between
OMD and IVDMD, but, despite 234
measurements were achieved in sheep, only
15 hybrids were used and harvested about
7 times from early milky stage to a grain
near maturity stage. These results
highlighted that in vitro estimates of whole plant
digestibility explained only a part, nearing
60%, of the variation observed in cattle.
But, while OMD and IVDMD have been
significantly investigated, very few papers
have reported data on intra-specific
relationships between in vitro and in vivo cell
wall digestibility estimates. Argillier et al.
, in an experiment with 58 maize
hybrids studied for both in vivo and in vitro
digestibility values, gave a correlation equal
to 0.55 between CFD in sheep and DINAG.
From a study with 36 maize silages, De
Boever (personal communication)
obtained a correlation r = 0.67 between CFD
and Tilley-Terry IVDNFD, and a
correlation r = 0.55 between CFD and an
IVNDFDr (estimate obtained from
incubation of a NDF residue with a cellulase), in a
very good agreement with the observations
of Argillier et al. [
However, correlations between in vivo
and in vitro values for whole plant and cell
wall digestibility were also recently studied
on a much larger genetic basis in an Inra –
ProMaïs network [
]. For 4 years, 165
maize hybrids were cropped in a balanced,
but incomplete, design with 2 replicates per
year and per genotype in Lusignan,
allowing the making of 560 mini-silos (one
mini-silo per hybrid and replicate). Feeding
values of these hybrids were estimated
through sheep experiments. Enzymatic
solubility and biochemical constituents were
measured on green forage, in samples taken
during harvesting. Genotype effects were
highly significant for in vivo traits OMD,
NDFD, for in vitro IVDMD or DINAGZ,
and for ADL/NDF content, even when bm3
hybrids were not taken into consideration in
the variance analysis (Tab. V). The
correlation between OMD and IVDMD was r =
0.74, in agreement with values observed by
Givens et al. [
] and De Boever et al.
]. The correlation was r = 0.75 between
NDFD and DINAGZ, in good agreement
with correlations observed by Argillier et al.
] or De Boever (personal
communication). Average values were equal in OMD
and IVDMD, or NDFD and DINAGZ,
respectively, but the in vitro solubility heavily
reduced the range of variation between
hybrids (Tab. V). Among normal hybrids, the
minimum – maximum range was 27
percent points in NDFD, but only 12 percent
points in DINAGZ. However, the greater
range of variation in cell wall digestibility
observed in vivo was partly balanced by the
higher error observed for NDFD than that
for DINAGZ. In vivo measurements of cell
wall digestibility were less precise than
in vitro estimates, as it was previously
reported by Deinum et al. [
]. But, greater
errors in NDFD than in DINAGZ were
probably also related to the necessity of
measuring, both in silage and in feces, the
NDF content, which had a slightly high
In vitro methods are screening tools for
determining the relative differences among
forages, and the real concern is that in vivo
and in vitro methods rank forages in a
similar order. As it was observed for whole plant
digestibility, in vitro estimates of cell wall
digestibility only explained 60% of the
variation observed in cattle. For breeding
purpose, both NDFD and DINAGZ criteria
lead efficiently and similarly to the
elimination of hybrids with poor cell wall
digestibility, or to the choice of good hybrids such
as the bm3 ones, or normal hybrids close to
the bm3 ones. However, from a simulation
of a hybrid choice performed in the INRA
Lusignan database, the selection of hybrid
with intermediate cell wall digestibility
could partly differ according to the NDFD or
DINAGZ trait used. The ranking of hybrids,
within a reduced variation range of cell wall
bm3 hybrids included
MS genotype × year
For abbreviations, see Table I; IVDMD: whole plant enzymatic solubilities; DINAGZ: in vitro digestibility of
non starch, non carbohydrate and non crude protein parts; ADL: acid detergent lignin.
digestibility, was nonetheless partly
different with DINAGZ and NDFD, as these two
traits did not cover the same part of
digestibility variation found in plant fed to cattle.
Similar results were, as expected, obtained
when simulated choices were based on
OMD or IVDMD, respectively, because
NDFD was the major determinant factor of
These results raised the question of the
interest in having a NIRS calibration of the
in vivo NDFD estimate of cell wall
digestibility. From the data set quoted above,
preliminary results [
] proved the feasibility
of such a cell wall calibration, with a r2
value equal to 0.63 for NDFD, with a
standard error of cross validation (SECV) equal
to 3.4. No other attempt to calibrate NDFD
seems to have been reported. However, a
first experimental calibration was built up
by Biston et al. [
] for silage maize OMD,
with a standard error of prediction equal to
1.6 and a r2 value equal to 0.60. A slightly
better result was obtained for OMD from
our data set, with a SECV equal to 1.4 and a
r2 value equal 0.68. An alternative strategy
in cell wall digestibility computing through
the IVNDFD criterion could then be
proposed, as suggested by Dardenne (personal
communication), using an OMD value
calibrated in NIRS rather than an IVDMD
value. The limit of procedures based on
animal values is the necessity to have an
experimental sheep flock, in order to get annual
reference data requested for NIRS
calibration maintenance. However, the number of
reference measurements could decrease as
the robustness of the calibration could
increase after each year of experiments.
4.5. Breeding from cell wall digestibility traits or from lignification traits
Lignin content is well known as the ma
jor factor making cell wall undigestible.
The interest of working simply with a
lignification trait, rather than with a more
complex cell wall digestibility trait, could thus
be questioned in plant breeding for
improved digestibility traits. However, for
such an objective, it is absolutely essential
to evaluate lignin content as a part of the
cell wall, NDF for example, and not as a
part of the whole plant (DM or OM). If not,
the lignin content is largely biased due to
the variation in soluble carbohydrates or
starch content. Moreover, the choice
between a cell wall digestibility trait or a
lignification trait has to be considered from
in vivo and in vitro values of digestibility,
because relationships between digestibility
and lignification could be different in
in vivo or in vitro traits.
Correlations between NDFD and lignin
content were also available from the
experiment based on 560 mini-silos and
165 maize hybrids [
between NDFD and ADL/NDF or LK/NDF
were r = –0.75 and r = –0.65, respectively,
with bm3 genotypes, and r = –0.45 and r =
–0.25, respectively, without bm3
genotypes. But, in the same set of data, the
correlations between DINAGZ and ADL/NDF
or KL/NDF were much higher, with r =
–0.94 and r = –0.75, respectively when bm3
hybrids were included, and r = –0.88 and r =
–0.45, respectively, without bm3 hybrids.
From a sub-sample of 19 maize silages
among the 50 studied, De Boever (personal
communication) observed similarly a
correlation r = –0.39 between NDFD and
Wolf et al. [
] reported correlations
between maize stover IVNDFD and
permanganate lignin content equal to –0.86,
–0.37 and –0.64 in a set of 24 S2 family and
their top cross by 2 inbred lines,
respectively. In Lundvall et al. [
], and from a
study of 45 maize lines, the correlation
between ADL/NDF and IVNDFD was equal
to –0.62. Méchin et al. [
] observed a
genetic correlation only equal to –0.51
between ADL/NDF and IVNDFDr in basal
stalks of a set of 18 normal maize lines
whose IVNDFDr ranged between 25.7 and
38.0%. The correlation was r = –0.80 when
4 bm3 hybrids were added. From a 3 year
experiment with 125 early and medium
early inbred lines harvested at silage
maturity stage, whose DINAG value ranged
from 53.0 to 64.5 (68.7 with bm3), and
whose ADL/NDF value ranged from 1.29
to 4.69 (0.56 with bm3) [
correlations between DINAG and ADL/NDF were
r = –0.79, and r = –0.72, with and without 6
bm3 lines, respectively. From a study of cell
wall digestibility in RIL progenies ,
the correlation between DINAGZ and
ADL/NDF or KL/NDF in lines per se were
r = –0.93 and –0.63, respectively, and were
in top cross r = –0.86 and –0.34,
respectively. Unpublished results at INRA
Lusignan showed a correlation r = –0.33
between ADL/NDF and DINAGZ in stover of
a set of 23 normal maize lines, whose
DINAGZ values ranged from 43.1 to
60.9%. This correlation increased up to
–0.47 when 2 bm3 lines were added.
All these results highlighted a great vari
ation in correlations between lignin content
and cell wall digestibility depending on the
method used in lignin content estimation,
and depending on the germplasm involved.
The correlation between ADL/NDF and a
cell wall digestibility trait appeared to be
even greater given that the genetic basis of
compared genotypes was more
homogeneous, as within a RIL progeny or a set of
registered or elite hybrids. It was also the
case when bm3 genotypes were added,
mainly increasing the range of variation
and then the linkage between the two traits.
The lignin content could explain only 20 to
50% of the in vivo variation in cell wall
digestibility, but often more than 50% of the
in vitro variation in cell wall digestibility.
The ADL/NDF value also appeared as a
measure of the part of lignin mainly
involved in the indigestibility of the cell wall,
whereas the KL/NDF value measured a
whole lignin content of the plant cell wall.
However, when breeding normal lines or
hybrids, a large part of the DINAGZ variation
remained unexplained by the ADL/ NDF
value, and the genetic unexplained part
could reach 75% among normal lines of
diverse origins. This unexplained part
corresponded to variation in lignin structure and
cell wall phenolics involved in cell wall
digestibility. Moreover, because the
correlation between cell wall digestibility and
lignin content of the cell wall was much
higher for in vitro data than for in vivo,
in vitro solubilities has probably led to an
overestimated effect of lignin content on
cell wall digestibility. These results have
highlighted the main disadvantage of
enzymatic solubilities, which are excessively
dependent on lignin content. The variation
in lignin content and in lignin structure
may have induced non-proportional
mechanical effects, and/or non proportional
effects on the rate of degradation, that
could not be approached through an in vitro
test, or at least, through their actual in vitro
estimates. Breeding for cell wall
digestibility improvement cannot be based
only on a lignin content trait, and must
also involve a cell wall digestibility trait.
Another great disadvantage of breeding
with ADL/NDF rather than with a
DINAGZ trait is the weaker relationship
observed between lines per se value and
top cross value for lignin content,
compared to the relationship observed between
lines and hybrids for cell wall digestibility
5 . IN VIVO GENETIC VARIATION
FOR INGESTIBILITY IN ANNUAL
5.1. Plant traits related to variation for intake in ruminant cattle
Voluntary intake is a primary nutritional
factor controlling animal production.
Ruminants consuming diets high in cell wall
content often are unable to eat sufficient
quantities of forage to meet their energy
demands. Dry matter content of the silage is
an important factor of intake variation, and
optimum water contents have been
established, allowing a good conservation, a
good palatability and a good intake of every
forage (32 to 37% dry matter content in
maize silage). For a given dry-matter
content, ingestibility is the plant trait, subject to
genetic variation, estimated in animals as
intake. But, due mostly to the great
impossibility for plant breeders to work with
cattle, and in agreement with Minson and
], there was “a failure of most
scientists to recognize the importance of
voluntary intake, that has led to an
unnecessary and undesirable gulf between the
science and the practice”. Moreover, intake
responses are not totally similar in sheep
and in cows, and intake response by an
animal depends also on its energy needs [
84, 92, 143
]. The regulation of an animal’s
appetite is above all a physical regulation.
The ingestibility of a given forage is
controlled by the time this forage is retained in
the rumen (reviews in [
have to be broken down a size close to 1 mm
before they can go out of the rumen through
the digestive tract. Chewing during eating
and ruminating is responsible for most of this
breakdown of particle in chopped forage
]. As a consequence, the filling
capacity of a forage, and hence its ingestibility,
depends on (i) the rate of particle size
reduction while animal is eating and
ruminating; (ii) the rate and extent of ruminal
degradation of the cell wall constituents;
and (iii) the rate of passage of small
particles out of the rumen through the
reticulo-omasal orifice, which also depends
on the functional specific gravity of the
5.2. Variation in ingestibility and relationships with genetic variation in cell wall digestibility
When fed to cattle, intake of maize
hybrids of significantly lower cell wall
digestibility was lower than the intake of hybrids
of rather good cell digestibility [
35, 36, 53,
]. Although it has been reported in very
few experiments, some hybrids have indeed
a higher intake in dairy cows. A better
ingestibility was shown by Ciba-Semences
] in the kindred hybrids Briard and
Bahia, close to 0.5 and 1.0 kg, respectively,
compared to a commonly used hybrid. The
voluntary intake of hybrid DK265, which is
of good cell wall digestibility, was proved
to be greater than that of other hybrids [
]. When maize silage was given as about
80% of the diet, dairy cows fed a DK265
silage had an average intake reaching nearly
1.5 kg·day–1 more than hybrids with the
same dry matter and grain contents, and, in
two comparing experiments, with the same
cell wall digestibility. Intake of DK265
appeared indeed much closer to that of bm3
hybrids than to that of normal hybrids.
The effect of cell wall digestibility on
intake was also proved in inter-specific
comparisons. Cummins and McCullough [
and Aydin et al. [
] reported that most
studies that compared sorghum with maize
silage have shown that DM intake was
consistently higher for cows fed the maize
silage than for those fed the sorghum silage,
with lower cell wall digestibility. The
average dry matter intake of sorghum silage was
81% that of maize, when fed to heifers in
the Cummins and McCullough [
experiment. In a diet including 35% of sorghum or
maize silage, respectively fed to dairy
cows, the average dry matter intake of the
sorghum silage diet was 85% that of the
maize silage diet, whereas the IVNDFD of
sorghum silage was 75% that of maize [
5.3. Devising a breeding criterion for genetic improvement of ingestibility
The composite structure of many
thick-walled and lignified cells in vascular,
sclerenchyma strands and parenchyma
cells between bundles, in both leaves,
shanks and stems makes fiber particles
physically strong and difficult to reduce in
]. As a consequence, it is
obviously difficult to create a direct prediction
tool for estimating hybrid ingestibility.
Minson and Wilson [
] reported old
studies on the mechanical resistance of
tissue to grinding as correlated with intake
]. They also reported the
development of a mechanical masticator by
Troelsen and Bigsby , giving
encouraging results, but this technique was not
used later because “it was so laborious”.
Simpler methods were later developed for
breeding higher ingestible forages [
seemingly without significant further use.
According to Minson and Wilson [
Blaxter et al. [
] first reported that
voluntary intake was positively correlated with
digestibility, and Hawkins et al. [
reported that voluntary intake was negatively
correlated with lignin content. The rate of
NDF degradation, measured in situ in
fistulated animals, varied very significantly
among genotypes [
]. Such a trait
should be considered as being possibly
related to a genotype’s rumen-filling
capacity, and as a consequence, to genotype
ingestibility. However, from later results
, the ranking of hybrids for parameters
of the degradation kinetics was not
sufficiently related to the ranking of hybrids for
their ingestibility, and these parameters did
not appear to be useful for the improvement
of maize ingestibility.
From preliminary results , the best
multiple regression for the prediction of
intake by cows included as first regressor the
NDFD measured in sheep, and then the
energy content of the silage (also from sheep
measurements), with a r2 value slightly
higher than 0.6 (for a given DM content).
Even if the rate of breakdown is a main
factor of voluntary intake regulation, the cell
wall digestibility improvement is probably
a main target for ingestibility improvement,
all the more because it could be assumed
that the sheep NDFD included a component
related to particle friability. These results
are an a posteriori justification of the
priority given to studies on digestibility. The
improvement of cell wall digestibility in
maize (and very likely in other forage
plants) will also bring about an
improvement in ingestibility. However, some
specific and unknown characteristics in
hybrids such as DK265 have to be
elucidated. Today, mapping QTL traits related to
lignification and cell wall digestibility is
probably the best way to highlight the
important traits involved in maize ingestibility.
This could be considered more specifically
in related RIL progenies including, or not, a
parental line of hybrids such as DK265.
6. BROWN MIDRIB PLANTS
AND INGESTIBILITY IN ANNUAL
6.1. Brown-midrib plant discovery and main traits
The brown midrib plants exhibit a reddish
brown pigmentation of the leaf midrib and
stalk pith, associated with lignified tissues,
since the plants have about five expanded
leaves. Until now, and according to
Cherney et al. [
], brown midrib phenotypes
were only seen in maize, sorghum and millet,
which are all diploid monocotyledons
belonging to the Panicoideae subfamily. As
reported by Jorgenson [
], the first brown
midrib maize plant appeared in a
self-pollinated line of a northwestern dent maize in
1924. The gene was subsequently named
bm1 and three other genes inducing the
brown midrib phenotype were described
later, as bm2 by Burnham and Brink [
bm3 by Emerson [
], and bm4 by
]. Each bm1, bm2, bm3, or
bm4 gene originates from natural
mutations and segregates as a simple mendelian
recessive character. The effect of maize
brown midrib mutations on lignin content
was first evidenced by Kuc and Nelson
]. A few years later, the effect of these
mutants on forage feeding value
(digestibility or ingestibility) was first evidenced by
Barnes et al. [
] from in vitro studies. In
sorghum, 19 independently occurring
brown midrib mutants were identified in
segregating progenies of chemically treated
seeds of two lines by Porter et al. .
Some of the mutant lines had a significantly
reduced lignin content, and/or a
significantly higher in vitro digestibility of cell
wall constituents. Brown midrib mutants in
pearl millet also originated from
chemically induced mutations [
]. Many studies
were then made on brown midrib plants,
used as models in digestibility and
lignification studies (reviews in [
26, 63, 123
6.2. Genetic improvement of cell wall digestibility and ingestibility in brown-midrib crops
Most experiments evaluating the
improvement in performances of cattle fed
brown midrib plants were based on the
maize bm3 mutant. The effect of the bm3
mutation on forage maize feeding value
(digestibility or ingestibility) was first
demonstrated in vivo by Colenbrander et al. [
], through a comparison of intake and
growth of heifers fed on normal maize and
on bm3 maize. Different experiments with
lactating cows have been reported since this
work (Tab. VI), but it would seem that no
experiment of cattle rearing with bm3
genotypes were done between 1987 and 1998.
The intake of bm3 silage by dairy cows was
always higher than the intake of normal
silage, even if the difference was not always
significant (and for dairy cattle, the
apparent benefit of the bm3 mutation is from
increased silage intake). This characteristic
was recently observed with an
experimental medium early bm3 hybrid that was
ingested 2.1 kg·day–1 more than the average
of 10 control hybrids, and 0.6 kg more than
the highly ingestible normal hybrid DK265
]. Higher milk yield of cows fed bm3
hybrids were reported in eleven out of
fifteen experiments, ranging from 0.5 to
3.3 kg·day–1 (Tab. VI). Milk yields were not
significantly lower in four experiments.
Moreover, every time this trait was
recorded, increase of body weight was
observed in cattle fed bm3 silage. Hybrids with
very good digestibility and ingestibility,
such as bm3 hybrids, could indeed appear to
be no more efficient than normal hybrids in
dairy cows, when maize silage is too small
an ingredient in the diet, or when supplying
Ballard et al. [
] 2001 31 31 0.0 (3) 10.9 (4) 0.5 2.5o –0.01 –
(1) Cows fed bm3 silage were given 0.4 kg·day–1 soybean meal less and 0.4 kg·day–1 ground maize more than
cows fed isogenic normal hybrid; (2) Cows fed bm3 hybrids were given 0.1 kg·day–1 soybean meal less and
0.1 kg·day–1 high moisture maize more than cows fed isogenic normal hybrid; (3) Cows fed bm3 hybrids were
given 1.3 kg·day–1 alfalfa silage more; (4) Apparent digestibility measured in lactating cows.
the usual quantity of concentrates. An
excess in available energy is then partly
converted by cows into weight gain (meat or fat
tissue). Experiments of Hoden et al. [
Bal et al. [
], and Oba and Allen 
clearly supported the hypothesis that the
higher potential of such hybrids can only be
fully expressed when the supply of energy
concentrates is lower, taking into account
the extra intake of silage, and taking into
account the higher energy value of bm3
Comparisons involving the other differ
ent maize brown-midrib genes with meat or
dairy cattle are very rare. The bm1 and bm3
mutation in an isogenic background were
compared for the feeding of young bull
]. Compared to bulls fed with the normal
Inra260 hybrid, the average daily carcass
gain was 39 g·day–1 higher in bulls fed
Inra260bm1, but was 137 g·day–1 higher in
bulls fed Inra260bm3. This result,
corroborating measurements of cell wall
digestibility in sheep, clearly established the much
higher efficiency of the bm3 mutant for
cattle feeding. The digestibility and the
interest in cattle feeding of bm4, and on a lower
scale, of bm2 hybrids, remain unclear.
Lusk et al. [
], Grant et al. [
Aydin et al. [
] reported different
experiments with brown midrib sorghum in the
diets of dairy cows. DM intake and milk
yield were always lower in standard
sorghum diets than in brown midrib sorghum
diets. Brown midrib sorghum resulted
indeed in milk production similar to normal
maize silage. In a palatability trial with a
4 week regrowth, grazing lambs displayed a
marked preference for the brown-midrib
pearl millet, compared to normal [
When normal or brown-midrib pearl millet
were fed to wethers, average NDFD over
two cutting were 67.7 and 71.0% in normal
and brown midrib plants, respectively [
Whatever the involved species (maize,
sorghum or pearl millet), and whatever the
in vitro test used (Tilley-Terry or enzymatic
solubility methods), the higher digestibility
of brown midrib plants was also always
found. Results were doubtless similar for
lignin content. Using three chemically
induced mutants, selected by Porter et al.
 for the further experiments, Fritz
et al. [
] showed an improvement in stem
IVDMD ranging between 8 and 12% in
brown midrib sorghum, compared to
normal sorghum. From their results, the
ADL/NDF was reduced by 1.3, 2.2 and
2.5 percent point in bmr6 (BC2), bmr12 and
bmr18 (BC1) sorghum, compared to
normal, respectively. But IVNDFD was only
improved by 3.1, 5.2 points in bmr6 and
bmr12, respectively, and, surprisingly
1.6 percent in bmr18, due to a lower NDF
content in brown midrib mutants. Watanabe
and Kasuga  observed a higher
digestible structural matter (DSM) in bmr12 and
bmr18 sorghum (close to 49.6%), than in
bmr6 (42.4%), and they considered that
these brown midrib sorghum were 10–15%
higher in DSM than in comparable normal
sorghum. From Akin et al. , IVNDFD of
two pearl millet stems was on average
13.2 percent points higher than that of the
isogenic line. Many similar results were
also published for maize brown midrib
mutants, and most of them were reported in the
reviews already mentioned. However,
compared to other maize brown midrib mutants,
the maize bm3 mutant appeared to be
especially improved in cell wall digestibility.
Grand et al. [
] established the
quasilack of COMT (caffeic acid O-methyl
transferase) activity in bm3 plants, and
Vignols et al.  established that the bm3
mutation corresponded to a deletion or a
large insertion of a retrotransposon element
in the exon 2 of the COMT gene. Sorghum
bmr12, and perhaps bmr18, mutants likely
brought similar improvement in cell wall
digestibility to the maize bm3 mutant, and
appeared also altered in their COMT
]. Similarly, an Arabidopsis
mutant with a knocked-out COMT gene
had also a highly improved cell wall
digestibility of floral stems . Conversely to
COMT mutants, sorghum bmr6 and pearl
millet bmr mutants probably had an
efficiency similar to that of the maize bm1
mutant, which is altered in the CAD
(cinnamylic alcohol deshydrogenase)
]. New mutants of interest for
feeding value (brown midrib or not) should
be found through lignin or IVNDFD
measurements when using a transposon tagging
method, in search of genes involved in
lignification and cell wall biogenesis,
whatever the (diploid) species used as a model.
6.3. Genetic variation for cell wall digestibility among bm3 maize genotypes
Genetic variation was found among bm3
hybrids, as backcrossing the bm3 gene in
different genetic backgrounds did not lead
to similar improvement in cell wall
digestibility. Gentinetta et al. [
21 bm3 hybrids of a 7 × 7 semi-diallel
mating of late maize lines, and their normal
isogenic counterparts. Variation for lignin
content was significant in bm3 hybrids, and
ranged from 3.1 to 5.0%, whereas lignin
content varied significantly from 5.3 to
6.8% in normal hybrids. Moreover, lignin
content in bm3 hybrids ranged from 50.8 to
74.8% of the content observed in the
isogenic hybrid, with a weak correlation
between lignin content in normal hybrids
and in their bm3 isogenics (r = 0.48). In a set
of 12 experimental hybrids made from
crossing early and medium late inbred lines
], the average NDFD improvement
between normal and bm3 hybrids was 8.5
percent points, but the NDFD improvement
ranged from 4.4 to 17.9 percent points.
Similarly, from a comparison of 14
experimental and formerly or more recently
registered hybrids (Barrière et al., unpublished
data), the range of NDFD went from 43.4 to
55.8% (12.4 percent points) in normal
hybrids, and from 52.0 to 63.4% (11.4 percent
points) in bm3 isogenic hybrids. The
NDFD improvement brought by the bm3
gene ranged from to 5.3 to 12.3 percent
points, with an average value of 8.4 percent
points. The correlation between NDFD
values in normal and bm3 hybrids was only
0.65. Moreover, the correlation between the
NDFD in normal hybrids and the NDFD
improvement given by the bm3 gene in
each hybrid was –0.40. All these results
proved an effect of the genetic background
on the NDFD improvement obtained with
the bm3 gene, with a tendency to a lower
efficiency of the mutant gene when normal
hybrids were of higher cell digestibility.
Similar results were observed in vitro in
]. The range of DINAG in 7 early
flint and dent bm3 maize lines was only
2.4 percent points, lower than the 7.4
percent point range observed between the
isogenic normal lines. But, as it was
observed in hybrids, there was also a great
variation in improvement between lines as
the DINAG value was increased by 10.7
percent points in F271bm3, but only by 5.2
percent points in F113bm3 or W117bm3,
with a similar tendency to a lower
efficiency of the bm3 gene when normal lines
were of higher DINAG. Specific effects of
the genetic background should be
considered when the bm3 gene was used for
improving the cell wall digestibility. But this
variation never cancelled the interest of the
bm3 mutation for the cell wall digestibility
improvement, especially as the efficiency
of the mutation seemed greater when the
feeding value of the normal genotype was
6.4. Towards an improvement of agronomic value in brown midrib forage crops
The higher efficiency of bm3 maize for
cattle feeding was indeed clearly
established, as soon as in the 1980’s, but for a
long time, breeders were disappointed by
the lower yield, irregular earliness, and
susceptibility to lodging of bm3 hybrids. The
recent and renewed interest in bm3 hybrids
for dairy cattle feeding (references in
Tab. VI), or for growing steer feeding ,
especially in the USA, became possible
because of the great improvement in
agronomic value of normal hybrids in the last 25
years. This renewed interest may also be
related to the low feeding value of the
parental lines used in modern medium late and
late hybrids. With normal hybrids of good
standability, whose potential yield ranges
between 17 and 20 t·ha–1, it is conceivable
to breed isogenic or close bm3 hybrids
whose yield will be reduced by about 3
t·ha–1, but whose NDFD will be increased
by about 8 percent points. Ballard et al. [
and Cox and Cherney [
] reported a yield
reduced by 2 to 3 t·ha–1, with an IVNDFD
improved by at least 10% and sufficient to
increase the FCM yield, in non isogenic
commercial hybrids. The availability of
bm3 hybrids on the seed market in the USA
has proved the feasibility of the bm3
process for cell wall digestibility improvement
of commercial hybrids, at least for late or
medium late hybrids. But the higher seed
costs of bm3 commercial hybrids in the
USA, their lower yield, and their higher
intake in cattle, makes the economic interest
of such hybrids still unclear. In Europe, the
results obtained until now with early or
medium early bm3 hybrids have not made it
possible to draw any definite conclusion.
The reputation of bm3 genotypes is poor,
and they are still suspected of great
susceptibility to lodging, on top of their lower
yields. Nevertheless in INRA Lusignan, an
experimental medium early bm3 hybrid
was bred with a yield close to 14 t·ha–1, a
NDFD close to 59%, and an acceptable
standability, when normal hybrids of
similar earliness yielding about 18 t·ha–1 had a
NDFD equal or lower than 47%. However it
may be, the breeding effort of bm3 lines
was until now very low. The choice of using
lower yielding hybrids of higher feeding
value, such as bm3 hybrids, is a matter of
strategy which has yet to be agreed on, and
especially so in more friendly
environmental conditions of plant cropping and cattle
]. The search of COMT alleles,
with less drastic effects than that of the
bm3, is also very likely an efficient way of
improving cell wall digestibility in maize
(or in any forage plant).
7. GENETIC RESOURCES AND
STRATEGIES FOR CELL WALL
7.1. Improvement of cell wall digestibility by the use of specialty maize
Different types of specialty maize hy
brids (excluding here brown midrib
hybrids) were investigated, mostly in North
America, and different results were
reviewed by Coors et al. [
between multi-tillering and non tillering
maize for feeding value traits were mostly
investigated in Canada, but no clear
difference in nutritive value was found [
Dwarf genotypes were also considered for
cattle feeding many years ago, when the
role of grain in silage maize feeding value
was overestimated, because dwarf
genotypes potentially increased the grain to
stover ratio ([
] cited in [
and Jackson  compared normal,
“NutriDense” and waxy maize hybrids
given to cows as silage and grain. DM
intake, milk and FCM yields were higher for
cows fed the waxy diet. But, because
hybrids were not isogenic, it was not possible
to conclude on the greater efficiency of one
particular maize. In an IE/ARPEB [
experiment, a slightly higher FCM yield
was observed in animals fed the waxy
hybrids, compared to their isogenics. The
lower milk yield was assumed to be related
to a higher ruminal acidity in animals fed
normal hybrids, that probably led to a
decrease of cell wall digestion. Kuehn et al.
], Bal et al. [
], Thomas et al. ,
and Clark et al. [
] compared grain or dual
type hybrids, and leafy type hybrids. In the
two first papers, DM intake, milk yield and
milk components did not differ for cows fed
the grain or the leafy hybrid. In the two last
papers, cows fed the diet with the leafy
maize silage produced higher yields of
milk, FCM, and milk protein, than cows
that were fed the diet with the normal maize
silage. The leafy hybrid was also more
digestible in vitro. However, in these two
experiments, normal and leafy hybrids were
not isogenic, and the higher milk yield
cannot be surely related to the leafy trait. Bal et
] did not find any difference in NDF
in situ disappearance between a grain and a
leafy hybrid. In the measurements
performed in Lusignan [
], two leafy hybrids
had NDFD similar to values observed in
hybrids of rather low cell wall digestibility.
Lax leaf inbreds had lignin concentration
nearly equal to those of the low lignin bm3
mutant, and they should be of interest for
cattle feeding. But no functional
relationships appeared between the lax leaf
phenotype and the digestibility or the lignin
]. The occurrence of the lax leaf
phenotype and the low lignin concentration
in the same early-generation inbred family
was considered as a “fortuitous random
combination of traits”. No specialty maize
has so far appeared of great interest for
silage use, except brown midrib types
7.2. Drift of cell wall digestibility in maize, and necessity of renewing genetic resources
Tremendous improvements in maize
yield, yield regularity, stalk standability,
stalk rot resistance and stay-green have
been achieved in the last five decades in
], and in the last century in the
USA [161, 175, 176]. In forage maize ,
the genetic progress was found to be close
to 0.17 t·ha–1·year–1 for hybrids registered
from whole plant experiments in France
between 1986 (the first year with registration
after forage maize official trials) and 2000.
In the period before 1986, whole plant
genetic progress was less important in hybrids
bred for grain yield, as Inra258 registered in
1958 yielded about 12 t·ha–1, LG11
registered in 1970 13 t·ha–1, Dea registered in
1980 15 t·ha–1, but Anjou285 and Nexxos
registered in 1994 and 2000 yielded 18 and
20 t·ha–1, respectively [
feeding value was not considered for forage
maize registration until 1998, and a
significant drift of hybrids towards lower in vivo
digestibility values was observed in the last
two or three decades (Tab. VII; and [
]). European hybrids of the 1960–70 era
had a higher OMD and NDFD than hybrids
registered since than. Statistical
computation proved that the most important switch
occurred in 1989 [
]. Hybrids with a low
cell wall digestibility were more numerous
after 1989 than hybrids previously
registered. When compared to the well-known
early hybrid Fanion (NDFD = 51.0%), 40%
of early hybrids registered before 1989 had
a higher cell wall digestibility than Fanion,
but this was only 15% of the hybrids
registered in and after 1989. The cell wall
digestibility upper values of hybrids registered in
the past decade were also lower than those
of hybrids registered before 1989, even if a
few modern hybrids had a good cell wall
digestibility. In the USA, Lauer et al. [
highlighted an annual rate of forage yield
increase of 0.13 to 0.16 t·ha–1 since 1930.
But they did not find any change of the
in vitro digestibility of the whole plant, nor
of the cell wall digestibility, whereas major
improvement in stalk standability, and in
stalk rot resistance, were achieved during
the same period. The discrepancy between
European and US results could be due to a
different evolution of hybrid germplasm in
Europe and in the USA. Dent lines in
modern European hybrids are now more related
to Iodent and Reid origins than were old
early dent lines used in Europe, with higher
cell wall digestibility. Old European flint
lines of high cell wall digestibility such as
F7 are not involved in the modern flint
germplasm, due to their low combining
ability values for yield, stalk rot or lodging
resistance. Some modern early European
hybrids are also dent or quasi-dent hybrids.
It seems likely that the maize improvement
in the US was carried without major
germplasm changes, and continuously
based on the Reid and Lancaster groups.
Moreover, in Lundvall et al. [
], no trend
towards higher or lower values has been
evidenced in stem IVNDFD, or in stem
ADL/NDF content, between lines of early
cycles of breeding in BSSS such as B14 or
B37, and lines of advanced cycles such as
B89 or B94. No difference was shown
between lines B73 (BS13C5) and B84
(BS13C7). The observed drift of maize
towards lower feeding values in Europe, and
similarly the necessity to improve feeding
value of late hybrids, highlighted the need
to investigate renewed and specific genetic
resources. Significant improvement of
maize cell wall digestibility in the USA or
in Europe will be based on targeted
introduction of original germplasm in currently
used elite germplasm.
7.3. Investigating genetic resources for cell wall digestibility improvement
Today, among maize hybrids registered
in France, hybrids with the highest NDFD
values are 6 percent points higher than the
average value of hybrids of similar
earliness, but they are 10 percent points lower
than the best bm3 hybrid. Most parental
lines currently used in commercial hybrids
are of medium or weak cell wall
digestibility. In Argillier et al. [
], a great range of
cell wall digestibility was found in a set of
125 normal maize lines of various origins
(mostly early or medium-early, but
including some medium-late). The DINAG value
ranged between 53.0 and 64.5%, and 68.7%
including bm3 lines. As also established by
Méchin et al. [
], few normal lines
such as F4 had a DINAG equal to the
DINAG of some bm3 lines. But, most of
early and medium early hybrids are crosses
between Iodent or Reid related dent lines
and early flint lines related to F2, both with
poor DINAG ranging between 56.6 and
58.2%. Lines of superior feeding value,
which are indeed available, are not used as
parents in elite varieties, partly because
they lead to poor-yielding hybrids (lower
additive value, and lower heterotic pattern)
and partly because their hybrid progenies
are susceptible to lodging. But there is no
evidence of an absolute or definite linkage
between these poor agronomic traits and
the feeding value. The correlation between
yield and NDFD was only –0.38 (without
bm3 hybrids, INRA Lusignan unpublished
data). Moreover, Argillier  proved the
independence between lodging
susceptibility and DINAG.
Breeding for higher cell wall
digestibility indeed demands to have a large
investigation in maize genetic resources, either
lines or ecotypes (and similarly in any other
forage crop). The germplasm investigated
until now for cell wall digestibility
measurements only represents a small part of
the available genetic resources in maize,
and is mostly based on lines currently or
quite recently used in grain maize breeding.
The old lines bred from the early selfing in
the different maize populations, and their
progenies obtained during the early cycles
of breeding, had to be investigated
systematically for cell wall digestibility traits. The
objective is to discover lines that were
considered not suitable for grain breeding, and
then forgotten for silage maize breeding.
Similarly, cell wall digestibility has also to
be investigated in ecotypes from which no
lines were developed. Because there is
obviously a great gap in agronomic value
between these old lines and elite modern
lines, specific strategies of introgressing
feeding value traits in elite germplasm have
to be considered.
7.4. Investigating QTL and allelic variation for cell wall digestibility improvement
Once lines of different feeding values
and different genetic background are
identified, different RIL progenies should be
developed in order to determine the
genomic location involved in feeding value
traits. In two RIL progenies, Méchin et al.
] and Roussel et al.  found three
major clusters for cell wall digestibility and
lignification traits, located in descending
order for both LOD values and percentage
of explained phenotypic variation in bins
6.06, 3.05/06, and 9.02. The bin 6.06,
which gathered together 11 individual
QTL, was also greatly involved in cell wall
carbohydrate composition because QTL
for hemicellulose and cellulose contents (as
percentage of NDF) were also found at this
location. Three other clusters were also
involved in cell wall digestibility and
lignification traits, located in bins 2.08, 4.08, and
6.01. The identification of the underlying
genes will be achieve in diverse ways
including colocalization with known
candidate genes, cDNA or EST mapping, and
BAC sequencing. Once genes are
identified, the choice of favorable alleles could be
carried out through allele sequencing in a
collection of lines, followed by the study of
the linkage disequilibrium between the
nucleotide polymorphism (SNP, single
nucleotide polymorphism, or INDEL, insertion –
deletion polymorphism) in alleles, and the
cell wall digestibility in lines. This strategy
also allowed a heavily targeted possibility
of marker assisted selection (MAS), with a
very small modification of the backcrossed
The different structural or regulatory
genes of the lignin pathway could also be
subjected to a SNP analysis, whether or not
QTL have been found close to these genes,
with a correlative study of the cell wall
digestibility in the different lines. Such
investigations are in progress in maize within the
Génoplante network. Allelic variation in
CCoAOMT1 and CCoAOMT2 genes
(caffeoyl CoA O-methyl transferase, both
sequenced by Civardi et al. [
COMT gene (sequenced by Collazo et al.
]) was analyzed in a set of 28 maize lines
chosen for their variable levels of
digestibility and the large diversity of their
genetic origins. As observed in other known
CCoAOMT genes, the sequence of the
CCoAOMT1 gene contained four introns.
However, the fourth intron was missing in
the CCoAOMT2 gene. The CCoAOMT1
gene was well-conserved among lines, and
its polymorphism was not associated with
DINAGZ digestibility values. At present,
this gene did not appear as a priority target
in cell wall digestibility improvement.
Polymorphism of CCoAOMT2 genomic
sequences was essentially located in
introns. However, one SNP, located in the
first intron, explained 30% of the observed
DINAGZ variation (P = 0.0026), and three
other SNP also appeared to be significantly
related to DINAGZ. Moreover, using the
RIL progeny described in Roussel et al.
, the CCoAOMT2 gene was mapped
on chromosome 9, in bin 9.02, and thereby
colocalizing with a QTL involved in the
DINAGZ, ADL/NDF and KL/NDF
variation. The first exon of CCoAOMT2 of one
early flint European line, which had a high
digestibility and a high S/G ratio, appeared
also deeply modified, differing greatly at
the N terminal region. Among
CCoAOMT1, CCoAOMT2, and COMT
genes, the COMT gene was the most
variable, not only with many SNP and INDEL
in its unique intron, but also several
variations in exons leading to several amino acid
changes. Association studies between these
allelic modifications and the cell wall
digestibility have shown that one INDEL,
located in the intron, explained 32% of the
observed DINAGZ variation (P = 0.0017).
When considering these results, results of
Guillet et al. [
], and unpublished
Génoplante data on maize genes, a high
degree of nucleotide polymorphism seemed
present in maize genes of the lignin
pathway. Feeding value of elite maize lines
should be greatly improved by studying
allelic variation through high throughput
SNP genotyping, in conjunction with the
measurement of cell wall digestibility in
very large collection of lines, including old
lines and ecotypes, followed by a MAS
targeted introgression of alleles or genomic
areas linked to the favorable SNP.
7.5. Genetic engineering of resources for cell wall digestibility improvement
Another relevant way in breeding forage
crops of higher digestibility is to devise
specific genetic resources through genetic
engineering in the lignin pathway. Boudet
], Dixon et al. [
], and Chen et al. [
for woody plants, have recently published
extensive reviews of genetic engineering of
the lignin pathway, and the resulting
consequences on lignin content and structure of
altered transgenic plants. Highly variable
results have often been observed regardless
of the transformation method. However,
these inconsistencies have highlighted the
necessity “to re-evaluate how we have
come to arrive at the current metabolic grid
model for the monolignol biosynthesis”
]. Data related to cell wall digestibility
were only provided in a part of the papers
reviewed by Dixon et al. [
] or in later
published works [
40–42, 104, 110, 150,
]. Very few results have been
published on maize and in general on
monocotyledons . However, whatever the plant
species, and whatever the enzyme
downregulated in the lignin pathway, an
increase in cell wall digestibility ranging
from 9 to 90% was observed in all
experiments, except inconsistencies between
glasshouse and field results in Baucher
et al. [
], with heterogeneous effects on
cell wall phenolics. Nevertheless, these
results clearly established the efficiency of
antisense or silencing strategies in
increasing the cell wall digestibility of plants.
According to opinions of Halpin et al.
] and Casler and Kaeppler [
alteration of early steps in phenylpropanoid
metabolism (PAL, phenylalanine
ammonia-lyase; C4H, cinnamate 4-hydroxylase),
which are clearly involved in other
important processes in plants, could lead to too
many adverse pleiotropic effects to be
useful for cell wall digestibility improvement
of plants. CCR (cinnamoyl CoA reductase)
and CAD (cinnamyl alcohol dehydrogenase),
that “function after all possible branch
points in the pathway”, were then
considered as potentially suitable targets [
Downregulated CAD plants had no or
slight changes in lignin content, but some of
these plants had a higher cell wall
]. The recent discovery of a
SAD (sinapyl alcohol dehydrogenase) in
aspen  also opens new possibilities in
cell wall engineering. But, according to
literature data, the maize bm1 mutant proved
to be not very efficient in cattle feeding.
Therefore CAD (and perhaps) SAD are
probably better targets for paper pulping in
dicotyledons than for forage digestibility
improvement in monocotyledons.
Similarly, before concluding on the relevance of
CCR engineering for forage improvement,
it is necessary to further elucidate further
the possible specificity of different CCR,
and the independence of pathways leading
to guaiacyl and syringyl units. Moreover,
plants could show important growth defects
when CCR activity is very low .
Based on current knowledge of the
lignin pathway [
], CCoAOMT is
(are) probably a major hub in controlling
lignification and digestibility, and therefore
a preferential target for digestibility
improvement. Up to now, only the work of
Guo et al.  has illustrated the
efficiency of a CCoAOMT down-regulation in
digestibility improvement. Alfalfa plants
with a 5% residual CCoAOMT activity had
an increased cell wall digestibility of 34%.
No changes were observed in ADL content,
whereas KL content was reduced by 25%.
The bm3 maize is thus far the best model
available for improving digestibility
through endogenous enzyme silencing, and
COMT (caffeic acid O-methyltransferase)
and F5H (ferulate 5-hydroxylase) are also
probably key targets in forage digestibility
improvement. This hypothesis is also
strengthened by converging results
indicating that COMT, in maize and in vivo, is very
likely to be a 5-hydroxyconiferaldehyde
O-methyltransferase (AldOMT or CaldOMT)
rather than a caffeic acid O-methyltransferase
]. Among data published on COMT
down-regulation (review in [
greatest improvement in cell wall
digestibility was observed in tobacco in which
both a decrease in lignin content and an
unexpected increase in S/G ratio were
observed . Piquemal et al.  reported
only recently one COMT down-regulation
in maize, despite the fact the bm3 mutant
and the COMT gene were known for years.
In plants with 30% COMT residual activity,
they observed a 9 percent point increase in
maize cell wall digestibility . This
increase in cell wall digestibility was similar
to those observed in the bm3 isogenic lines.
The drawback of COMT down-regulation
or silencing is the correlative S/G decrease,
because a higher S/G ratio could impact
positively the cell wall digestibility in
]. CCoAOMT could be
considered a priori as an even better target than
COMT, because CCoAOMT
down-regulation in plants would logically result in a
lower lignin content without a decrease in
S/G ratio [
]. At present, data are not
yet available concerning the respective
roles or tissue specificity of the different
CCoAOMT genes in maize and other
forage crops. In poplar, CCoAOMT genes
exhibited precise cell-specific expression
The polymerisation reactions may also
be considered as good targets, even though
laccases and peroxydases are encoded by
multigene families. Ros Barcelo (
quoted in [
]) postulated that
peroxydases were the sole enzymes
involved in the ultimate step of lignin
biosynthesis, but most recent reports
considered the involvement of both laccases
and peroxydases in the formation of
phenoxy radicals [
]. Up to now,
mechanisms of phenoxy radical coupling
of monolignols are still unknown, and no
clear-cut results have been provided to
determine if lignin results from random
free-radical coupling or from dirigent
protein orchestrated polymerization .
In grasses, enzymes involved in (i) pcoumaric and ferulic acid biosynthesis; (ii) their transport to the cell wall; (iii) their coupling to carbohydrates and cell wall
polymers, could very likely be relevant
targets for plant engineering. Ranocha et al.
] established that the down-regulation
of one laccase in poplar led to plants
exhibiting no visible phenotype, but with highly
altered xylem fiber cell walls and
mechanical properties of the wood. These plants
were not modified in lignin content, nor
in S/G ratio, but accumulated soluble
phenolics. The authors speculated that such
a laccase was involved in the formation of
certain types of phenoxy radicals leading to
cross-linking of xylem fibers. It could also
be hypothesized that such plants could be
easier to break down by ruminating cattle,
and then of higher ingestibility. Laccase
down-regulated plants could therefore be
considered as resources of reduced
crosslinked fibers, and should be considered as
potential targets in forage digestibility and
Regulatory genes of lignification could
be hypothesized as potential targets for cell
wall digestibility improvement in plants.
Myb-related transcription factors are
involved in regulating phenylpropanoid
metabolism, and Tamagone et al.  proved
that lignin content was heavily reduced in
mature parts of tobacco plants
overexpressing an Antirrhinum Myb factor.
No measurements of digestibility were
given in this paper. Moreover, Myb genes in
maize belong to a very large family of
expressed regulatory proteins .
Although identification of Myb factors
specifically involved in the lignin pathway
is necessary before using them for cell wall
digestibility improvement, the
modification of such regulatory genes could allow
the control of the entire, or at least a part, of
the pathway in given tissues [
overall rate of lignin deposition in the cell
wall is regulated by monolignol synthesis,
but also by the transport of their precursors
to the cell wall. Transport forms have not
been clarified so far, but
4-O-β-Dglucosides, presumably synthesized by
UDPG-utilizing glucosyltransferases and
subsequently hydrolyzed by monolignols
specific β-glucosides, are the most
probable candidates [
], and are indeed targets
for plant engineering in cell wall
The simultaneous down-regulation of
two (or more) genes could be more efficient
than the down-regulation of only individual
genes. Tobacco hybrids resulting from the
crossing of transgenic lines down-regulated
for CCR and CAD, CCR and COMT, or
COMT and CAD, had a reduced lignin
content, but with no adverse impact on the
growth of the plants [
1, 56, 148
from results of Abbott et al. , chimeric
silencing constructs could also be more
efficient than achieving multiple suppressions
by crossing independent events. This
strategy should allow a synergetic enzyme
reduction that regulates the flux of
metabolites through the lignin pathway. Moreover,
the optimal construct could be designed to
reduce the expression of one gene while
increasing the expression of another. For
example, the simultaneous down-regulation
of CCoAOMT and over-expression of
F5H could be hypothesized as a first step in
a forage ideotype building, assuming that
this ideotype should logically have a
reduced lignin content with an increased S/G
Up until now, data on cell wall
composition and digestibility in transformants and
in naturally occurring mutants, were often
difficult to predict. In maize, more data will
be necessary concerning both the lignin
pathway and suitable promoters to drive
transgene expression. Comparison of
antisense data with data obtained in
knocked-out mutants will also be of
interest, especially for genes belonging to
multigenic families. Moreover, little data
are currently available on the stability of
transgene expression throughout
generation of selfing, crossing, and successive
backcrossing in elite germplasm. Feeding
value tests in cattle (with sheep in
digestibility crates and/or with milking cows) will
then be necessary to reinforce the relevance
of this new technology in cattle feeding.
Moreover, a complete knowledge of the
consequences of the genetic engineering
appeared all the more important that
unexpected increase in lignin content was
observed in Bt11 and Mon810 maize tolerant
to the European corn borer .
In the search for a forage ideotype, the
breeding effort to be placed respectively on
either biomass yield or biomass
digestibility is open to debate. A high digestibility
should allow farmers to provide less
concentrates to cattle, and is a necessity for
good forage intake. For a given quantity of
inputs (nitrogen fertilization, …), and
water availability, a forage ideotype
resembling bm3 maize would maximize the
production of energy having great
ingestibility and digestibility in cattle, and
it could increase the profit in cattle
Cell wall digestibility is undoubtedly
one of the major targets for the
improvement of forage feeding value. A higher cell
wall digestibility would lead to both a higher
overall digestibility and ingestibility.
Because lignin content is not the only trait
involved in cell wall digestibility, breeders
should use a trait directly related to cell wall
digestibility, such as IVNDFD or DINAGZ.
A large scale investigation of genetic
resources, including germplasm forgotten
after decades of breeding for agronomic
value and/or grain yield, is required.
Comprehensive knowledge of the lignin
pathway and cell wall biogenesis will allow
plant breeders to choose the best targets for
the improvement of plant digestibility. That
said, most of the lignification research has
been done on dicotyledons and woody
plants, and grass breeders should keep in
mind the specificity of grass cell walls.
The improvement of forage cell wall
digestibility may be envisaged both through
genetic engineering or marker assisted
introgression of favorable alleles. It may be
possible to improve similarly cell wall
digestibility with a targeted use of natural
genetic resources and genetic engineering.
However, with a transgene strategy, only
one parental line needs to be backcrossed,
because of the dominant behavior of
engineered genes, but the stability of transgene
expression over generations is still
unknown. Favorable alleles will probably
have partially recessive behavior, but
different favorable alleles could be
introgressed in different heterotic groups,
allowing the breeding of hybrids with the
best combination of alleles and epistatic
interactions between these alleles and others
genes related to lignification or
Maize may be considered as a model
plant for lignification and digestibility
studies in monocotyledons. At present,
similar research efforts are not being made
on other annual or perennial grass forage
plants. Because of the synteny between rice
and maize , the availability of the rice
genome will bring very valuable
complementary information, even if the lignin
pathway is seemingly not investigated in
rice. Moreover, gene mining and genetic
engineering in model plant and systems
(Arabidopsis, Zinnia, …) are also
complementary approaches for improvement of
cell wall digestibility in monocotyledon
forage crops. Finally, results obtained in
maize could also be exploited for
dicotyledons forage improvement, despite the
specificity of monocotyledon lignification.
Special thanks to J. De Boever (Gent,
Belgium) and P. Tillmann (Kassel, Germany) for
giving their valuable unpublished information
on the relationships between traits related to
silage maize feeding value. Thanks also to
J. Ralph (Madison, USA) who suggested us to
write this synthesis, even if the foreseen related
project was cancelled. Thanks to the maize
breeding companies involved in the ProMaïs –
INRA network “DINAG” that contributes to the
funding of the forage maize research in
Lusignan. Thanks to an anonymous reviewer for
his greatly fruitful criticisms.
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