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Review

Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions

by
Krishna B. Bhandari
1,
Hannah L. Rusch
2 and
Deborah J. Heuschele
3,*
1
Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN 37209, USA
2
Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA
3
United States Department of Agriculture—Agricultural Research Service, Plant Science Research Unit, St. Paul, MN 55108, USA
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(12), 2875; https://doi.org/10.3390/agronomy13122875
Submission received: 21 October 2023 / Revised: 20 November 2023 / Accepted: 20 November 2023 / Published: 23 November 2023
(This article belongs to the Section Grassland and Pasture Science)
Review Reports Versions Notes

Abstract

:
Alfalfa (Medicago sativa L.) is considered as the most important forage legume with high biomass yield and nutritional quality for ruminants. The alfalfa leaf cell walls are highly digestible, but stem cell walls of alfalfa are not readily digestible. The cell wall component of alfalfa has a large source of dietary energy, but ruminant animals can digest less than half of this component due to the presence of high lignin content. The main goal of this review is to make a summary of existing knowledge of alfalfa cell wall thickening and lignification patterns and suggest future directions for improving alfalfa stem cell wall digestibility. We describe alfalfa cell wall biochemistry, alfalfa stem morphology, stem tissue degradation, and existing methods to improve alfalfa digestibility, and discuss the potential future strategies for improving alfalfa cell wall digestibility. Information on these will help alfalfa breeders and producers identify superior alfalfa cultivars with improved stem cell wall digestibility. Concentrating future efforts on the selection and identification of traits and associated genes that affect cell wall digestibility could improve alfalfa cell wall digestibility.

1. Introduction

Alfalfa (Medicago sativa L.) is the most important perennial forage legume in the temperate regions of the world [1], produced primarily for high-quality hay and silage for cattle [2]. This key forage is grown on more than 30 million hectares in the world [3]. In the United States, alfalfa is the third largest crop in dollar value produced with an estimated value of more than USD 10 billion annually [4]. Besides high-quality diets for cattle, alfalfa provides several environmental benefits such as carbon sequestration and mitigation of nitrogen leaching [5], and improves pasture soil health [6,7]. Alfalfa is the best-adapted perennial cool-season legume for hayfields [8,9] and is one of the most resilient forage legumes that can withstand prolonged drought by undergoing dormancy and resuming growth after receiving adequate amounts of water [10,11,12]. Alfalfa can extract water from deep within the soil profile [10]. Thus, crop water losses from deep percolation and runoff can be abated through planting alfalfa, especially during an extended drought. In higher precipitation regions, replacing perennial warm-season grass pastures by alfalfa can prolong the grazing season by earlier initiation of spring grazing and provide greater rate and total weight of stocker cattle [13].
Alfalfa is generally considered to have the greatest nutritive value among the forage crops, because it contains 15–22% crude protein, essential vitamins and their precursors, and several minerals such as calcium, phosphorus, and magnesium [14]. The alfalfa leaf fraction is relatively rich in protein and the leaf cell walls are highly digestible [15]. These high protein and mineral contents of alfalfa provide nutrients for milk and muscle growth in dairy and beef cattle when complemented with balanced energy diets [16,17]. The productivity of dairy cows can be increased with high-quality alfalfa with high dry matter digestibility [16]. The main limitation to nutritive value in alfalfa is the lignification of cell walls of the stem portion [18,19], which composes 50 to 70% of the total shoot biomass [20].
The fiber (cell wall) fraction of forage provides a potentially large source of dietary energy, but animals often readily digest and utilize less than 50% of this fraction [21]. The fiber concentration in alfalfa increases within stems as plants mature, but the fiber concentration in the leaves remains low and constant [22]. An increase in the dry-weight ratio of high digestibility leaves to low digestibility stems in alfalfa improves nutritive value [23], and an increase in the leaf-to-stem ratio increases forage dry-matter intake [24]. Additionally, alfalfa stem digestibility can be affected by several factors such as harvest time, genotype, year, and location [25]. By increasing the digestibility of alfalfa stems, available energy at later maturity stages may also increase along with the increase in dry matter yield. Alfalfa populations with increased fiber digestibility have been developed [26] indicating the possibility of increased fiber digestibility in alfalfa stems.
Screening germplasm for natural variation in traits that confer improved digestibility is one way of improving cell wall digestibility. Researchers using an NIRS-based screening approach found large genetic diversity among alfalfa cultivars and genotypes for stem cell wall digestibility in which that trait was negatively correlated with total lignin concentration [27]. Conventional breeding efforts have resulted in reduced lignin cultivars [25]. As well as the evaluation of individual gene knockdowns in the lignin biosynthetic pathway, identified alfalfa lines with increased stem fiber digestibility [28] have been commercially available since 2015 as reduced-lignin alfalfa cultivars [29]. Another type of variation that might lead to increased digestibility is through anatomical changes in the stem. Some anatomical changes which would increase stem cell wall digestibility include thickening of secondary cell wall of xylem which would inhibit lignification, reducing lignification of phloem fibers or pith parenchyma, or increasing proportion of nonlignified tissues [30].
A better understanding of the alfalfa stem cell wall development and lignification patterns is necessary to develop alfalfa cultivars with improved stem digestibility, which would maximize the efficiency of ruminant livestock production. The objective of this review is to summarize the current knowledge of alfalfa stem cell wall thickening and lignification to develop breeding strategies to increase alfalfa stem cell wall digestibility and utilization by ruminants. Alfalfa germplasm with greater cell-wall digestibility during stem maturation will provide producers with more management flexibility to produce high-yielding alfalfa with high nutritive value. This review will help alfalfa breeders identify traits associated with cell wall development and lignification in alfalfa stems for superior alfalfa cultivars.

2. Fiber Digestibility

Fiber in forage is a major source of dietary energy and affects intake and digestibility of forages, but less than 50% of the fiber is readily digested and utilized by ruminant animals [31]. Neutral detergent fiber (NDF) describes highly digestible cell fiber material. Forages with high NDF concentrations and lignified cell walls limit feed intake [32] because the animal feels “full”. The NDF concentration increases rapidly during a rapid decrease in dry matter digestibility after alfalfa matures beyond the vegetative stage. Acid detergent fiber (ADF), on the other hand, describes cell wall material that either is hard to digest or never digests. Forage legumes have more rapidly digestible cell wall than grasses of similar maturity, but the potential extent of cell wall degradation in legumes is generally low [33]. Legume cell walls contain more pectin but less cellulose and hemicellulose compared to grasses [34] which accounts for the initial rapid digestion in legumes. Grasses have vascular bundles distributed throughout the parenchyma of stem cross-sections, whereas the vascular tissues in legumes form a discrete and continuous ring around the stem which expands through cambial activity [35] that impedes ruminal microbe access for digestion. Histological staining for pectin and lignin indicated that tissues may differ more dramatically in their cell wall composition in legumes than grasses [22,36]. Within grass, leaf tissue makes up the greatest proportion of the plant. These leaves have thin-walled, non-lignified mesophyll tissue [37], and mesophyll tissue is found to be completely degraded by rumen microbes [38]. In Alfalfa, where the proportion of stem tissues is larger than grass, the fiber has higher lignin content and thus lower digestibility (40–50%) compared to high fiber digestibility (60–70%) in grasses [31]. Alfalfa has been reported to contain greater lignin and lesser cell wall concentration than grass when alfalfa and grass had the same digestibility [39].
A previous study [22] divided alfalfa stem components into four major categories in relation to cell wall development: chlorenchyma, cambium, secondary phloem, and primary xylem parenchyma consisting of thin, non-lignified primary walls. The pith parenchyma with thin-walled tissue undergoes little cell wall thickening and lignification after stoppage of stem elongation while the collenchyma, epidermis and primary phloem tissues form thick primary walls without lignin at the end of stem elongation. The primary phloem and secondary xylem undergo thickening of secondary cell wall and are highly lignified, after stem elongation is ceased. Alfalfa stems contain diverse tissue types that include thin, non-lignified walls; minimal wall thickening with lignification; thick walls that do not lignify; and thick cell wall that lignify [22]. Tissues which are non-lignified and pectin-rich, such as collenchyma, are considered as rapidly and completely degradable compared to tissues which are lignified and xylan-rich, such as secondary xylem fiber [40]. The non-lignified wall are completely degradable regardless of the thickness, but lignified tissues have variable degradable pattern based on the lignin distribution in the cell wall [19]. Less than 10% of the cell wall has been found to be degradable in alfalfa with thick primary and secondary wall of xylem fiber [19]. Non-lignified epidermis, collenchyma, chlorenchyma, cambium, and primary xylem parenchyma were found to rapidly and completely degrade within the first 8 h of fermentation [19]. Non-lignified secondary walls of the primary phloem fiber completely degrades in 24 h, while the lignified pith parenchyma and secondary xylem fiber were 9.1 to 65.5% degradable even after 96 h, and the primary and secondary xylem vessels were completely nondegradable [19]. However, in grasses, the thick and lignified sclerenchyma tissue were found to be extensively degradable when fermented for 48 h with rumen microbes [36].
Grass cell walls are more degradable than legume cell walls during 72 h to 96 h fermentation [33,41]. Cell wall degradability may be negatively affected by the cross-linkage of matrix components far greater than lignin concentration alone [42]. Although non-lignified alfalfa stems degrade two to five times faster than nonlignified mesophyll grass tissues, lignified alfalfa stem tissues degrade less when compared to reported lignified grass stem sclerenchyma [19]. The differences of cell wall degradation between alfalfa and grass tissues could be associated with the cell wall lignification and polysaccharide composition of individual tissues.
Dietary NDF, regardless of plant source, have been identified as predictors of enteric methane (CH4) production. As digestibility of fiber increases, so does intake and ruminal fermentation, resulting in increased methane production [43]. However, the effect of carbohydrate type (structural or non-structural) on methane production is relatively less important at low intake levels [44]. Studies that have tried to explain how the NDF intake affects methane emissions have been inconclusive. Some independent studies have found that changes in NDF impact methane production without changes in intake [45,46], while others have found no difference in methane production for either changes in intake or NDF [47]. A meta-analysis of trials investigating NDF and methane in beef cattle found that NDF content alone does not explain enteric methane emissions. However, the quality and intake of the feed does impact methane emissions [48]. Therefore, by improving digestibility and nutritive value resulting in improved feed efficiencies will result in a reduction of methane emissions.

3. Cell Wall Biochemistry

Cell walls make up 23 to 90% of the plant mass [49] and are composed mainly of cellulose, hemicellulose, lignin, and other components, such as pectin and protein. Cell wall digestibility is variable and is negatively related to lignin concentration which is the primary limiting component of cell wall digestion [42]. Cell wall fibers that have high lignin and are linked to other structural carbohydrates are negatively associated with dry matter intake, dry matter digestibility, and animal performance [50].

3.1. Cellulose

Cellulose constitutes the largest portion of cell wall accounting for 40 to 50% of plant dry matter [51]. The yield of the stem and concentration of cellulose in the cell wall component of forage (fiber fraction) increase as alfalfa matures [40]. Although increases in stem mass and cellulose concentration in the cell wall theoretically increase the potential yield of digestible energy [26], the increase in lignin deposition during stem maturation and tight linkages of lignin with cellulose microfibrils substantially reduce microbial degradation of lignocellulosic biomass [52,53]. Rumen microflora have less than 50% access to plant fiber fractions [21] because of the heteromatrix complex formation between low-digestible lignin and high-energy cellulose [54]. The digestibility of cellulose is reduced when cellulose and lignin form these complex structures [55]. However, cellulose and hemicellulose are completely degraded when they do not have any bound lignin [56].

3.2. Hemicellulose

Hemicellulose is the second largest component of the alfalfa cell wall, constituting 15 to 20% of the forage dry matter [57]. Hemicellulose digestibility is more affected by cellulose digestion than lignin, despite the fact that hemicellulose and lignin being covalently linked [58]. Grass hemicellulose is more digestible by ruminants compared to cellulose, whereas the reverse is true for legumes [59]. Hemicellulose is characterized by complex structures built using monosaccharides such as xylose, galactose, mannose, and arabinose. Xylans have the slowest rate and extent of digestion [19,60] of all the hemicellulose components. A very strong negative relationship between xylose concentration and alfalfa in vitro degradability was reported indicating the vital role of xylan in inhibiting alfalfa digestibility [61]. Although xylans in legume cell walls are slowly degradable, its concentration in legumes is less than that in grasses [40]. A study found that xylan from alfalfa cell walls was the least digestible cell wall carbohydrate by sheep [62] and the same can be assumed for cattle. Low digestibility in mature grasses could be due to the linkage of xylan and lignin [62] that occur during plant maturity. No information is available about the effect of binding between xylan and cellulose on the degradation of these carbohydrates in the rumen [63].

3.3. Lignin

Lignin composes approximately 15% of the total dry matter of the alfalfa cell wall component, which limits cell wall degradation and forage digestibility by rumen microbes [64,65]. Lignin binds cellulose [66], xylose, arabinose, and mannose of heteroxylans of hemicellulose [67,68], which increase as alfalfa matures [69]. Cell wall digestibility is limited by lignin through the combination with cell wall components [70,71] and through the inhibition of phenolic acids such as p-coumaric acid, ferulic acid, and sinapic acid on rumen microbes [66,72,73]. These acids have inhibitory effects on rumen fungi, which limits the ability of fungi to degrade fibers in alfalfa and bermudagrass (Cynodon dactylon L. Pers.) [74]. Jung and Fahey [73] found that in vitro digestibility of cellulose and starch was depressed when supplied with p-coumaric, ferulic, salicylic, and vanillin acids. Some studies stated that lignin is indigestible [75,76,77,78] owing to the lack of known ruminal anaerobic fermentation [79], whereas others found that lignin is partially digestible in the abomasum with little change in the intestines [80]. During ruminal digestion of lignin, methane (22–29%), and carbon dioxide (65–69%) gases are produced, suggesting that ruminal digestion is relatively efficient compared to anerobic digesters [81]. Some studies have divided lignin into non-core lignin and core lignin monomers or polymers and number of covalent bonds [18,82]. Non-core lignins are monomers [83] and normally have one covalent linkage between a phenolic compound and either core lignin or hemicellulose [84], whereas core lignins are condensed and polymeric [85] and usually have two or more covalent bonds between monomers within its molecule [83]. Depending upon the lignin covalent links with carbohydrates, lignin can protect about 1.4 times its own mass of cell wall carbohydrates from microbial fermentation [86]. Another study found that lignin can protect two times its own mass of cell wall carbohydrates from microbial fermentation [87].
Alfalfa stems contain a variety of tissues with different patterns of cell wall development. The process of lignification varies between plant tissues. Some tissues, such as mesophyll in leaves, never lignify, whereas secondary xylem and other tissues accumulate high concentrations of lignin. About 6–9% of the dry weight of the whole alfalfa plant and 20% of the cell wall is lignin [88]. The lignin pathway was altered in alfalfa through decreasing the expression of two genes involved in the biosynthesis of coniferyl and sinapyl alcohol, which are considered as the main building blocks of lignin [28]. These changes in lignins were able to reduce 20% lignin within the plant which resulted in 2–5% increase in digestibility of the tissue. In contrast, conventional breeding takes more than 15 years of selection, which has resulted in a 2–3% increase in cell wall digestibility [89]. The relationship between in vivo dry matter digestibility and lignin was found to be significantly negative in alfalfa, when measured as acid detergent lignin [90].

3.4. Pectin

Pectin is a non-fibrous carbohydrate that accounts for 10–12% of the cell wall matrix in alfalfa stems. Pectin is completely digestible and is the cell wall polysaccharide most rapidly degradable by rumen microbes [60,91,92,93]. Selectively increasing easily digestible carbohydrates that make up the alfalfa cell wall, such as pectin, is a method to increase carbohydrate availability and hence improve protein utilization and alfalfa digestibility by ruminants [94,95,96]. About 20–35% and 1.0–10% of extractable pectic polysaccharides on a cell wall basis are present in legumes and in grasses, respectively [57]. Alfalfa leaves contain higher pectin concentrations than stems [60,97], and pectin concentration declines as stems mature [32,97]. However, pectin does not lower ruminal pH via lactate production, an intermediate product derived from microbial starch catabolism in the rumen, despite being a readily degradable source of energy in the rumen [98,99].
Neutral detergent soluble fiber (NDSF), in which pectic polysaccharides are the predominant components, can be used to estimate pectin concentration in alfalfa [97]. Variation in NDSF has been found in alfalfa [97,100]. Similarly, significant genetic variability for NDSF was reported in two alfalfa populations [101,102]. In addition, NDSF concentration was found to be negatively correlated with NDF, ADF, and acid detergent lignin (ADL) concentrations, whereas it positively correlated with in vitro dry matter digestibility (IVDMD) in alfalfa [102]. Similar results were found related to genetic improvement for NDSF concentration in five alfalfa populations, in which NDSF was found to be negatively correlated with total cell wall concentration (CW), and proportions of neutral detergent fiber (NDF), cellulose, and lignin in the CW, and positively correlated with crude protein concentration and IVDMD [103].

4. Alfalfa Morphology, Stem Tissue Development, and Lignification

As a perennial plant, alfalfa may proceed from the vegetative stage to seed production multiple times per growing season and over multiple years. The mass and height of alfalfa increases with plant maturity and the latter is associated with an increase in the number of stem internodes [40]. While the diameter of stem internodes continues to increase with plant maturity, the elongation of stem internodes ceases after approximately 21 days according to some reports and remains stable, or decreases in length over time [22,40]. The alfalfa leaf-to-stem ratio declines over time as stems proliferate with branching and defoliation due to leaf shading and foliar diseases. A defoliation event, such as grazing and mechanical harvest, can effectively reset alfalfa growth back to the vegetative stage.
In tandem with plant morphological development, the patterns of cell wall development of the different alfalfa stem tissues have been observed using microscopy. Alfalfa stem tissues follow different developmental pathways as they mature, which have implications for the degradability of stem cell walls. While some tissues, such as thin-walled chlorenchyma and thick-walled collenchyma, remain non-lignified and thus completely degradable in the presence of rumen microbes at all maturity stages; others like primary phloem fibers and pith parenchyma become lignified once stem elongation ceases and cambial activity begins [19,22,40]. Lignification occurs only in tissues with thickened secondary walls, including pith parenchyma, primary phloem fibers, and xylem tissues, with lignin deposition beginning in the primary cell wall and then proceeding into the secondary cell wall [22]. Xylem primary and secondary tissues immediately lignify making them impervious to degradation by rumen microbes [40]. Secondary xylem tissues arising from cambial activity comprise an increasing proportion of the stem as alfalfa matures, which helps to explain why stem lignin content increases with maturity [40]. At the same time, the concentration of pectin decreases while cellulose and hemicellulose concentrations increase in stem tissues contributing to the decline in alfalfa stem digestibility [40].

5. Methods to Increase Alfalfa Digestibility

Two common ways to increase digestibility of fiber in alfalfa are conventional breeding [31] and genetic engineering as reduced-lignin types [29]. Conventional breeding that used selection traits targeting the stems rather than the total biomass was found to have a greater impact on alfalfa digestibility owing to the presence of highly lignified fiber in stems [104]. Several studies have successfully used traditional breeding methods and genetic techniques to improve the digestibility in alfalfa cultivars. For example, the Hi-Gest alfalfa trait was developed by conventional breeding to improve total forage digestibility through increased leaf to stem ratio [105,106]. Conventional breeding improved fiber digestibility in alfalfa stem cell walls without diminishing dry matter yield, where populations were developed by recurrent phenotypic selection and evaluated by enzyme-released glucose as a proxy trait for fiber digestibility, indicating that selection was effective [107]. Alfalfa plants differed in stem NDF and ADL as a proportion of NDF (ADL/NDF) in another breeding program, in which plants were identified with either low or high rapid and low or high potential IVNDFD [25,30]. In contrast, the HarvXtra alfalfa (reduced lignin) was developed by genetic modification to improve nutritive value mainly through altering ADL and in vitro neutral detergent fiber digestibility (IVNDFD) in the stems [108,109]. Similar leaf-to-stem ratio and biomass yield between HarvXtra and conventional alfalfa cultivars was found through the evaluation of morphological characteristics in a single-location study [109]. One study conducted in multiple locations found higher leaf-to-stem ratio and increased stem digestibility but lower biomass yield in HarvXtra compared to conventional alfalfa [110]. Alfalfa breeding programs should emphasize stems when comparing nutritive value among whole plants, leaves, and stems because of the presence of the larger value of narrow-sense heritability for feeding-value traits in the stems [111], and they are the rate-limiting step for overall forage digestibility [23].

6. Future Prospective to Improve Alfalfa Digestibility

Slow progress has been made in the development of improved stem cell wall digestibility of alfalfa cultivars because of the qualitative nature of the trait and low heritability [112]. Some of the desirable traits in alfalfa in relation to digestibility are to increase cellulose and decrease lignin in stem cell walls, maintain 20% crude protein content, and 30% non-fiber carbohydrates (NFC), along with the presence of essential amino acids with slower rate of degradation of proteins in rumen [24,113]. However, a comprehensive understanding of the genetic characteristics associated with each nutritive value trait is needed. The literature lacks basic quantification of CW traits (i.e., components, isomers, bonds) and their interrelationships with digestibility, which leads to animal performance. Without the understanding of these interactions, the genetic improvement of alfalfa digestibility will remain stagnant.
Conventional breeding has improved the yield and some aspects of quality in alfalfa to some extent, but the improvement of cell wall digestibility and the plant traits involved has been limited. Alfalfa germplasm contains variability in soluble CW components [101,102] and IVNDFD [25,26], along with heritability [25]. In a study using recurrent selection for 16 h and 96 h IVNDFD variation, the selection for high IVNDFD was able to reduce NDF and the ADF:NDF ratio in stems at late maturity stages, such as late flowering and green pod in alfalfa with no effect on percentage of stems [25]. Thus this study found recurrent selection for alfalfa stem IVNDFD is a successful strategy for improving fiber digestibility and reducing lignin in stems without affecting the leaf-to-stem ratio in total forage. Therefore, stem 96 h IVNDFD and NDF could be good traits to use in a field selection program to develop alfalfa with potentially improved nutritive value because these traits were stable among alfalfa populations in the field environments [25,26], heritable, and contained sufficient trait variation. However, the rate of gain could be greater if the interaction between CW components and digestibility was better understood instead of using dietary fiber (NDF, ADF) as CW component proxies. Further identification of biochemical traits and associated genes impacting the CW components and their ruminant digestibility need to be accurately identified. Moreover, modern cultivars need to be assessed for CW components and their relation to animal improvement. Currently, this type of information is not publicly available.
The utilization of marker-assisted selection or recombinant DNA within an alfalfa breeding program is being used in privately owned companies. These technologies need to be embraced by the public sector. Genetic engineering has been utilized to reduce lignin concentration, but the reduction in lignin content also reduced dry matter yield and other parameters of the plants [28,29]. Continued investigation into moderate reduction in lignin content without negatively affecting plant health, survival, and function is critically important for future efforts in lignin reduction. A shift in lignin composition toward a lower syringyl:guaiacyl monolignol ratio instead of a reduction in total lignin would be another way of improving alfalfa stem digestibility due to more degradable cell wall material that contain this form of lignin [114]. The lignification amount can also be reduced in the secondary xylem through lowering the syringyl to guaiacyl monolignol ratio in lignin to improve the rate of alfalfa stem cell wall digestion [40]. P-coumaric and ferulic acid, the compounds that form cross-linkages between lignin and polysaccharides, are phenolic compounds known to have anti-microbial effects and decrease methane production [115]. Therefore, feeding alfalfa cultivars containing different phenolic compositions as base forages in dairy rations could have positive impacts on the conversion of digestible energy to metabolizable energy concentration of the diet and climate change, especially if they have anti-methanogenic potential. Additionally, manipulating the allocation of lignin to specific regions of the stem cross-section may be the key to increase rumen microbe access to cell wall carbohydrates that digest efficiently. Lignin deposition could be focused on structurally important cells leaving the rest unbound and therefore easily accessible to microbial degradation. Currently, the literature is lacking in information to determine if manipulating lignin deposition is feasible. With a better understanding of how lignin is formed and allocated throughout the plant, single gene modification techniques, such as crispr CAS9, could be applied. However, selecting for decreased total CW concentration in xylem deposition in relation to maturity has been shown to improve overall digestibility [116]. This method is similar to how low-lignin alfalfa varieties express better digestibility at later maturities.
Lower rapidly fermentable carbohydrates, such as pectin, are present in alfalfa compared to corn silage [117]. Thus, further increasing these rapidly fermentable carbohydrates in alfalfa would be another future strategy to induce alfalfa digestibility closer to corn silage levels. The natural variation for rapidly fermented carbohydrates is present in alfalfa [102] and therefore could modified by classical breeding methods. Increasing the amount of cellulose without increasing the amount of lignin would result in an increase in rapid ruminal degradation, as long as the material is accessible to microbial digestion. Additionally, replacing xylan, which slowly digests compared other hemicellulose carbohydrates, with another rapidly digestible polysaccharide, such as galactose, could potentially increase the fiber digestion in alfalfa. While variation in CW digestibility is found through the chemical analysis of alfalfa genotypes [25,26,101], the complexity of how rapidly fermentable CW traits lead to changes in digestibility and animal performance is not well understood.
Digestibility is not solely a plant trait. Therefore, further research is needed on investigating the interactions between rumen microbes and constituents of plant cell walls to improve forage digestibility and feed efficiency. Some of the factors that determine the degradation of plant cell walls include rumen microbial ecology, chemical characterization of plant cells walls, and the methodology of phenotypic selection of high digestibility population. For example, lignin is degraded by white rot fungi [118] by producing multiple enzymes, including lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases [119]. Similarly, termites are also able to degrade lignin. Although the digestive tract of ruminants does not contain these organisms, it is possible to hold these organisms in the digestive tract for degradation of linin in the future [120]. Additives that contain fibrolytic enzymes, consisting mainly of xylanases and cellulases, are commonly used to improve forage silage digestibility [121]. Several studies have shown that carboxylesterase has positive effects in degrading ferulic acid ester linkages and changing forage lignocellulose structure matrix [122,123]. It was reported that the degradation of forage in the rumen was improved through the breakage of the linkage between lignin and cell wall carbohydrates [124]. Improved forage lignocellulose degradation was reported when ferulic acid esterase-producing lactic acid bacteria was inoculated during ensiling [125]. Improved dry matter and crude protein digestibility were found when alfalfa silage was inoculated with the ferulic acid esterase-producing lactic acid bacteria [126]. Developing lignin-degrading enzymes in feed applications or within the plants themselves could result in future biotechnological advances to improve the rumen microbial ecosystems. Identification and isolation of new microbes and mechanisms that can break down the chemical bonds between lignin and cellulose will help increase substantial forage digestibility by ruminants.

7. Conclusions

This review on alfalfa stem cell wall digestibility was prompted by the availability of the very limited literature on stem cell wall development and lignification patterns in alfalfa. The interaction between stem development and potential stem digestion are intertwined. Therefore, the continued investigation into how modern alfalfa genotypes allocate cell wall components within the stem structure is needed to better understand the degradation cell wall components in the digestion process. This information is needed to develop future strategies for improving alfalfa stem cell wall digestibility and is currently lacking. Although conventional breeding efforts to increase alfalfa stem fiber digestibility have been done through genetic engineering and the trait has been commercially available for many years now in alfalfa cultivars as reduced-lignin types, there have been and still are efforts to capitalize on natural genetic variability for that trait to breed improve alfalfa digestibility. The variability in cell wall components such as cellulose, lignin in alfalfa germplasm, and their allocation patterns allow greater opportunities to improve alfalfa digestibility through the selection and identification of individual genes that may affect cell wall digestibility. Gene modification utilizing gene editing technology to decrease lignin content or increase cellulose available for digestion without increasing lignin could be potential future strategies to improve alfalfa digestibility and ruminant utilization. Additionally, increasing rapidly fermentable carbohydrates, such as pectin and cellulose, available in the cell wall using natural genetic variability or gene modification could improve cell wall digestibility.

Author Contributions

Conceptualization, D.J.H. and K.B.B.; writing—original draft preparation, K.B.B.; literature review—K.B.B. and H.L.R.; writing—review and editing, K.B.B., H.L.R. and D.J.H. All the authors revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by USDA-ARS, grant number USDA-ARS 5062-12210-003-00D.

Data Availability Statement

Data Sharing not applicable.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA does not imply its approval to the exclusion of other products that also can be suitable. The USDA is an equal opportunity provider and employer. All experiments complied with the current laws of the United States, the country in which they were performed.

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Bhandari, K.B.; Rusch, H.L.; Heuschele, D.J. Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions. Agronomy 2023, 13, 2875. https://doi.org/10.3390/agronomy13122875

AMA Style

Bhandari KB, Rusch HL, Heuschele DJ. Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions. Agronomy. 2023; 13(12):2875. https://doi.org/10.3390/agronomy13122875

Chicago/Turabian Style

Bhandari, Krishna B., Hannah L. Rusch, and Deborah J. Heuschele. 2023. "Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions" Agronomy 13, no. 12: 2875. https://doi.org/10.3390/agronomy13122875

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