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Published online 27 October 2005
Published in Crop Sci 45:2425-2429 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP BREEDING, GENETICS & CYTOLOGY

Leaf and Stem Characteristics of Timothy Plants Divergently Selected for the Ratio of Lignin to Cellulose

Annie Claessensa, Réal Michaudb,*, Gilles Bélangerb and Diane E. Matherc

a Dep. of Plant Science, McGill University, Sainte-Anne-de-Bellevue, QC, Canada H9X 3V9
b Agriculture and Agri-Food Canada, Soils and Crops Research and Development Centre, 2560 Hochelaga Blvd., Sainte-Foy, QC, Canada G1V 2J3
c Molecular Plant Breeding Cooperative Research Centre and School of Agriculture and Wine, Univ. of Adelaide, PMB 1, Glen Osmond SA 5064, Australia. Contribution no. 779 Agriculture and Agri-Food Canada

* Corresponding author (michaudr{at}agr.gc.ca)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Improved dry matter (DM) digestibility of herbage can be due to changes in fiber composition of leaves and/or stems, and/or to changes in the proportion of leaves and stems in the herbage. Previously, we reported that divergent selection for the ratio of acid detergent lignin (ADL) to cellulose (ADL/CEL) led to differences in timothy (Phleum pratense L.) DM digestibility. Here, we examined plant morphology (leaf-to-stem ratio, plant height and area, and stem diameter), leaf and stem fiber composition, and leaf and stem digestibility in timothy genotypes selected for high or low values of ADL/CEL and in divergent progeny populations derived from these selections. Parental genotypes and their progeny populations were field grown for 2 yr, and measurements were taken at the first harvest of each year. Selection for low values of ADL/CEL reduced ADL and neutral detergent fiber (NDF) concentrations in stems and increased the in vitro true digestibility (IVTD) of stems but had no consistent direct effects on plant morphology or leaf characteristics. Averaged over 2 yr, stems of the low-ADL/CEL group of genotypes had lower ADL and NDF concentrations (by 3.4 and 12 g kg–1 DM, respectively) and a higher IVTD value (by 22 g kg–1 DM) than those of the high ADL/CEL group of genotypes. In addition, stems of the low-ADL/CEL progeny population had lower ADL and NDF concentrations (by 4.2 and 26 g kg–1 DM, respectively) and a higher IVTD value (by 22 g kg–1 DM) than those of the high ADL/CEL progeny population. Thus, the observed changes in overall herbage DM digestibility due to selection for this ratio can be attributed mainly to modification of stem digestibility.

Abbreviations: ADF, acid detergent fiber • ADL, acid detergent lignin • CEL, cellulose • DM, dry matter • HEM, hemicellulose • IVTD, in vitro true digestibility • NDF, neutral detergent fiber


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
IN FORAGE CROPS, selection for improved digestibility and/or for related traits can result in marked changes in digestibility itself, in the fiber composition of leaves and stems, and in plant morphology. In alfalfa (Medicago sativa L.), Kephart et al. (1989) observed that lines selected for low acid detergent lignin (ADL) concentration had lower ADL concentrations in stems and higher leaf-to-stem ratios than lines selected for high ADL concentration. In smooth bromegrass (Bromus inermis Leyss.), the selection for reduced neutral detergent fiber (NDF) resulted in plants with a reduced proportion of stems and a reduced NDF concentration in those stems (Casler, 1999).

For most cool-season grasses, stems have a greater concentration of fibers than leaves, and the fibers of stems usually have a higher lignin concentration than those of leaves (Buxton, 1990). As a result, stems are usually less digestible than leaves. In many studies, the leaf-to-stem ratio was positively correlated with digestibility (Buxton et al., 1987; Bélanger and McQueen, 1996, 1998).

Other morphological traits have been found to be associated with digestibility of forage grasses, with negative correlations for plant height in timothy (Durand and Surprenant, 1993), smooth bromegrass (Ross et al., 1970; Sleper and Drolsom, 1974; Tan et al., 1978) and reed canarygrass (Phalaris arundinacea L.) (Christensen et al., 1984), positive correlations for plant area in spaced plant trials of reed canarygrass (Marum et al., 1979), and positive correlations for stem diameter in smooth bromegrass (Sleper and Drolsom, 1974; Ehlke and Casler, 1985), bermudagrass (Cynodon dactylon L.) (Hanna et al., 1973), and limpograss (Hemarthria altissima [Poir.] Stapf and C.E. Hubb) (Schank et al., 1973). Van Soest (1994) hypothesized that large stems may be more digestible than small stems because their lignified tissue may be more thinly distributed.

We have previously reported on fiber traits, forage digestibility, and plant biomass of timothy after divergent selection for various criteria related to fiber concentration (Claessens et al., 2004, 2005). We concluded that the ADL/cellulose (ADL/CEL) ratio is an effective and stable selection criterion for improving timothy in vitro true digestibility (IVTD) and in vitro NDF digestibility while maintaining plant biomass. Given that timothy has a wide range of variation in morphological traits, including leaf-to-stem ratio, plant height, plant area, and stem diameter (Durand and Surprenant, 1993), we hypothesized that selection based on the ADL/CEL ratio could affect plant morphology and/or could differentially affect the digestibility and composition of the leaf and stem fractions.

The objectives of this study were (i) to assess the effect of the divergent selection based on ADL/CEL on plant morphology and on the digestibility and fiber composition of the leaf and stem fractions and (ii) to explain differences in forage digestibility that we had observed between materials selected for high and low ADL/CEL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The genetic material for which results are reported here consisted of two sets of eight timothy genotypes divergently selected respectively for high or low ADL/CEL ratios and the two first-cycle progeny populations derived from these selections. The development of this material was described in detail by Claessens et al. (2004)( 2005). Plants of the 16 parental genotypes and the two progeny populations were started in April 1998 in a greenhouse and transplanted in May 1998 to a field at Lévis, QC, Canada (46°9'15'' N, 71°12'00'' W; altitude {approx} 45 m) along with similar materials selected for other criteria. The parental genotypes were arranged in a randomized complete block design with four blocks, with each plot consisting of two plants per genotype. The progeny populations were arranged in a randomized complete block design with eight blocks, with each plot consisting of five plants. Plants were spaced at 90 cm between and within rows.

At head emergence in June 1999 and 2000, plant height and plant area were measured. Plant height was measured from ground to tip of the highest panicle. Plant area was estimated using the equation 3.1416 x R1 x R2, where R1 = the longest radius at ground level and R2 = the radius perpendicular to R1. Each plant was harvested at a 10-cm stubble height with a hand sickle. For each parental plant or each plot of the progeny populations, a representative forage sample was separated into a leaf fraction consisting only of leaf blades and a stem fraction consisting of stems and leaf sheaths. Within each stem fraction sample, stem diameter was measured between the second and third node from the bottom of the stem on each of a subsample of 10 stems using calipers. Leaf and stem samples were air-dried at 55°C for 2 d, and leaf-to-stem ratio was computed as the leaf fraction dry weight divided by the stem fraction dry weight.

Each sample was ground separately to pass through a 1-mm screen in a Wiley mill (Thomas-Wiley Laboratory Mills, Philadelphia, PA). All samples were scanned on an NIR spectrophotometer (Model 6150; Pacific Scientific, Silver Spring, MD). For each fraction (leaf and stem) and each year, 60 forage samples were used for calibration purposes by cluster analysis (Infrasoft International, 1993). These calibration samples were chosen among the samples from the divergent selection for ADL/CEL and eight other selection criteria. The calibration samples were analyzed in duplicate for NDF, ADF, and ADL concentrations using the nonsequential detergent fiber system of Goering and Van Soest (1970) as described by Claessens et al. (2004). Calibration samples were also analyzed for IVTD using the ANKOM protocol (Ankom Technology, Fairport, NY) as described by Claessens et al. (2004). Separate NIR spectroscopy calibration equations were developed for each year and for each plant fraction for NDF, ADF, and ADL concentrations and for IVTD values. Predicted values were used in all statistical analysis (Table 1). Predicted values for NDF, ADF, and ADL concentrations were used to estimate hemicellulose concentration by subtracting ADF from NDF and to estimate cellulose concentration (CEL) by subtracting ADL from ADF. Correction for ash concentration was made.


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  		Table 1. Statistics for the near-infrared reflectance spectroscopy calibration equations used to predict trait values in timothy leaf and stem fractions.{dagger}

 
Analysis of variance (GLM Procedure; SAS Institute, 1996, Cary, NC) was conducted on the data from the divergent parental groups or populations, with parental groups or populations and years as fixed effects and blocks as random effects. Because of a significant population x year interaction, the data from each year were analyzed separately. The data are presented for each year. Contrasts were performed between the divergent ADL/CEL parental groups or populations for each plant fraction and each morphological trait.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
In the parental genotypes and the progeny populations derived from them, divergent selection for whole-plant ADL/CEL had a significant direct effect on the ADL/CEL ratio of the stem fraction but not on that of the leaf fraction (Tables 2 and 3). In 1999, the IVTD of the leaf and stem fractions of the low-ADL/CEL parental group was higher than that of the high-ADL/CEL parental group (Table 2). In 2000, the leaf and stem fractions of the low-ADL/CEL parental group and its progeny population had higher IVTD than those of their high-ADL/CEL counterparts (Tables 2 and 3). Differences in IVTD were much greater for the stem fractions (22.0 g kg–1 DM for the parents and 21.5 g kg–1 DM for the progeny populations, averaged over 2 yr) than for the leaf fractions (6.5 g kg–1 DM for the parents and 7.0 g kg–1 DM for the progeny populations). Thus, changes in whole-plant IVTD between divergent ADL/CEL parental groups (Claessens et al., 2004) and progeny populations (Claessens et al., 2005) seem to be due more to changes in the IVTD of stems than that of leaves. A similar conclusion was reached by Casler and Carpenter (1989) after divergent selection of smooth bromegrass for in vitro DM digestibility. These responses to selection are consistent with important influences of stem digestibility on whole-plant digestibility that have been reported by Albrecht et al. (1987) in alfalfa and Bélanger et al. (2001) in timothy.


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  		Table 2. In vitro true digestibility and concentrations of fiber components or their ratios in the stem and leaf fractions of timothy harvested from the 1999 and 2000 spring growth of a field experiment involving two groups of genotypes divergently selected for high and low ratios of lignin to cellulose.{ddagger}

 

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  		Table 3. In vitro true digestibility and concentrations of fiber components or their ratios in the stem and leaf fractions of timothy harvested from the 1999 and 2000 spring growth of a field experiment involving two populations derived from groups of genotypes divergently selected for high and low ratios of lignin to cellulose.{ddagger}

 
Decreased ADL/CEL in the stem fraction of the low-ADL/CEL parental group and progeny population was consistently associated with decreased lignin in both years (Tables 2 and 3) and with decreased hemicellulose concentrations in the low-ADL/CEL parental group in 2000 and in the low-ADL/CEL progeny population in both years (Tables 2 and 3); this resulted in overall decreases in NDF concentration.

The ADL/CEL selection had no consistent effects on plant morphology. The only significant differences observed were for plant area and leaf-to-stem ratio in 2000 (Table 4). In that year, the low-ADL/CEL parental group had somewhat larger plant area and higher leaf-to-stem ratio than the high-ADL/CEL parental group. These differences were small relative to those reported by Durand and Surprenant (1993) for timothy genotypes divergently selected for quality traits (a 444-cm2 difference in plant area). Furthermore, the morphological differences that were observed between the parental groups in our study were not transmitted to the next generation. The two progeny populations did not differ from each other for any of these morphological characteristics in either year.


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  		Table 4. Values for four morphological traits measured on timothy harvested from the 1999 and 2000 spring growth of a field experiment involving two groups of genotypes divergently selected for high and low ratios of lignin to cellulose and their progeny populations.{dagger}

 
These results suggest that the changes in whole-plant fiber composition and digestibility between divergent ADL/CEL parental groups (Claessens et al., 2004) were due only to the modification of the stem fiber composition and mainly to changes in lignin concentration in the stem fraction. Similarly, differences between divergent ADL/CEL progeny populations (Claessens et al., 2005) were also mainly, but not exclusively, due to the modification of the fiber composition of the stem fraction. Many studies have reported high negative correlations between lignin and forage digestibility (Mowat et al., 1969; Casler, 1987; Fonseca et al., 1999; Claessens et al., 2004). The stem fraction has been targeted as the main one limiting digestibility because it can represent up to 70% of forage yield (Bélanger and McQueen, 1998) and its fiber concentration, and, more importantly, its lignin concentration are usually higher than those of leaves (Jung and Engels, 2002). In the plant material evaluated here, selection based on whole-plant ADL/CEL identified parents that differed mainly for lignin concentration, particularly in the stem fraction. This selection led to the development of progeny populations that differed for lignin and total fiber concentrations, also mainly in the stem fraction. The fact that the ADL/CEL selection affected mainly the fiber concentration of the stem fraction without any constant measurable modification to the plant morphology suggests that the divergent selection resulted in changes to the genetic control of fiber metabolism in stems.

The plant cell wall is a complex structure in which all cell wall matrix constituents interact; therefore, finding new selection criteria for digestibility should not be based solely on the concentration of a specific constituent. Our work has shown that phenotypic selection was successful in identifying divergent ADL/CEL genotypes (Claessens et al., 2004) and that ADL/CEL is heritable and has a significant and constant impact on timothy digestibility (Claessens et al., 2004, 2005). The results presented here demonstrate that this impact is achieved mainly through changes in the digestibility of stem tissues. These changes could be due to (i) an increase in the proportion of the most readily digestible tissue types (stem chlorenchyma and phloem) (Ehlke and Casler, 1985), (ii) a reduction of the number of vascular bundles (Shenk and Elliott, 1971), and/or (iii) a reduction of the secondary cell wall thickening in the nodal regions of the stem (Klock et al., 1975). Histologic investigation of the stems of the ADL/CEL divergent populations might identify which phenomena are responsible for the difference in stem digestibility observed between these populations. Meanwhile, ADL/CEL could be used in forage breeding programs as a selection criterion to increase digestibility with no measurable effect on plant morphology and biomass.

Received for publication November 9, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





This Article
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