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 LEGUME
PE RS P E C T IV E S

Alfalfa: Back to the Future

Lucerne: The first letter in the legume alphabet

The journal of the International Legume Society

Issue 4 • July 2014
IMPRESSUM

ISSN Editorial Board

2340-1559 (electronic issue)
Editor-in-Chief
Quarterly publication Diego Rubiales
January, April, July and October
(additional issues possible) Assistant Editors
Mike Ambrose (genetic resources), Paolo Annicchiarico (lucerne),
Published by Birte Boelt (seed production), Beat Boller (clovers), Ousmane
International Legume Society (ILS) Boukar (cowpea), Judith Burstin (pea), Marina Carbonaro
(pharmaceutical uses), Branko Ćupina (non-food uses), Vuk
Co-published by Đorđević (soybean), Gérard Duc (faba bean), Noel Ellis (genomics),
Institute of Field and Vegetable Crops, Novi Sad, Serbia Sara Fondevilla (bioinformatics), Bernadette Julier (breeding),
CSIC, Institute for Sustainable Agriculture, Córdoba, Spain Branislav Kovačević (black locust), Kevin McPhee (genetics),
Aleksandar Medović (archaeobotany), Aleksandar Mikić (vetches),
Publishing Director Teresa Millán (chickpea), Fred Muehlbauer (lentil), Ramakrishnan
Diego Rubiales Nair (food uses), Pádraig O‟Kiely (feed uses), Dejan Pajić
CSIC, Institute for Sustainable Agriculture, Córdoba, Spain (scientometrics), Diego Rubiales (biotic stress), Christophe Salon
diego.rubiales@ias.csic.es (phenomics), Marta Santalla (common bean), Petr Smýkal (wild
relatives), Frederick L. Stoddard (abiotic stress), Wojciech Święncicki
Office and subscriptions (lupins), Richard Thompson (Medicago truncatula), Rajeev Varshney
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Apdo. 4084, 14080 Córdoba, Spain Daniel Wipf (symbiosis)
Phone: +34957499215 • Fax: +34957499252
diego.rubiales@ias.csic.es Technical Editor
Aleksandar Mikić
Front cover photo Institute of Field and Vegetable Crops, Novi Sad, Serbia
Paolo Annicchiarico aleksandar.mikic@nsseme.com

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EDITORIAL CONTENTS

his monographic CARTE BLANCHE

T issue on alfalfa
(alias lucerne)
reflects the recent
evolution of AEP (Association
4 Paolo Annicchiarico, Bernadette Julier: Alfalfa: Back to the Future

RESEARCH
5 Christian Huyghe, Luc Delaby, Daniele Rosellini, Fabio Veronesi: Perspectives of alfalfa
Européenne de Recherche sur les in Europe
Protéagineux) into ILS
7 Neal P. Martin: Perspectives of alfalfa in North America
(International Legume Society),
i.e., a wider scientific community 9 Daniel Basigalup, John Irwin, Fugui Mi, Meriem Abdelguerfi-Laouar: Perspectives of
alfalfa in Australia, China, Africa and Latin America
with a focus on both grain and
s
forage legumes. This change
recognizes the large affinities
11 Michael P. Russelle: Environmental benefits of growing perennial legumes in cropping
systems

existing between these groups of 13 Jean-Marie Prosperi, Marie-Hélène Muller, Joëlle Ronfort, Stéphane Fourtier, Jean-Paul
Sampoux, Eric Jenczewski: Alfalfa domestication history, genetic diversity and genetic
crops for genetic and genomic resources
characteristics (as confirmed by
15 Bernadette Julier: From the model legume Medicago truncatula to alfalfa
legume synteny studies and the key
role played by Medicago 17 E. Charles Brummer, Maria J. Monteros: Alfalfa marker-assisted selection
truncatula as a model species) and 19 Daniele Rosellini, Fabio Veronesi: The challenges of genetically engineered alfalfa in
in other respects, e.g.,the Europe
interactions of plants with nitrogen 20 Massimo Confalonieri, Paolo Annicchiarico: Alfalfa genetic variation for response to
fixing bacteria, or the dynamics of tissue culture and in vitro regeneration
associations of legume and non- 21 Paolo Annicchiarico, E. Charles Brummer: Future alfalfa cultivars
legume plants. There is also 23 Paolo Annicchiarico, Luciano Pecetti, Yves Castonguay: Improving alfalfa tolerance to
increasing opportunity for research abiotic stresses
projects devoted to improving 25 John Irwin, David Armour: Improving alfalfa resistance to biotic stresses
simultaneously annual and 27 Luciano Pecetti: Alfalfa as a grazed crop
perennial legumes, such as the 29 Paolo Annicchiarico, Bernadette Julier, Gaëtan Louarn, Amel Maamouri: Alfalfa
FP7-ArimNet project REFORMA intercropping and competitive ability
(Resilient, water- and energy- 31 Carla Scotti, Bernadette Julier: Improving alfalfa forage quality
efficient forage and feed crops for 32 Mark McCaslin, Vincent Béguier: Alfalfa private breeding: American and European
Mediterranean agricultural overview
systems), which provided the 34 Jacques Hacquet, Đura Karagić: Alfalfa management for higher and more sustainable
context for the current editorial seed yields
work. ILS initiatives, including this 36 René Baumont, Valérie Heuzé, Gilles Tran, Maryline Boval: Alfalfa in ruminant diets
journal, will respond to the need
38 JoAnn F.S. Lamb, Michael P. Russelle, Deborah A. Samac, Carroll P. Vance, Hans-
for greater cultivation of legumes - Joachim G. Jung, Craig C. Sheaffer: Alfalfa non-feed uses
of all kind.
EVENTS
Paolo Annicchiarico and 39 25th General Meeting of EGF, Aberystwyth, Wales, UK, 7-11 September 2014
Bernadette Julier 39 18th Symposium of EGF, Wageningen, the Netherlands, 15-18 June 2015
Managing Editors of 39 Symposium of the EUCARPIA Protein Crops Working Group of the Oil and Protein
Legume Perspectives issue 4 Crops Section and Spanish Association for Legumes, Pontevedra, Spain, 4-7 May 2015
39 Symposium of the EUCARPIA Fodder Crops and Amenity Grasses Section, Ghent,
Belgium, 14-17 September 2015

BOOKS
39 Grasslands and Herbivore Production in Europe and Effects of Common Policies, edited
by Christian Huyghe, Alex De Vliegher, Bert van Gils and Alain Peeters (Quæ, Versailles,
France, March 2014, 320 pp)
39 Quantitative Traits Breeding for Multifunctional Grasslands and Turf, edited by Dejan
Sokolović, Christian Huyghe and Jasmina Radović (Springer Science+Business Media, August
2014, 411 pp)

Legume Perspectives Issue

Issue44••July
July2014
2014
3
 Carte blanche
to…
 Alfalfa: Back to the Future
lfalfa (alias lucerne) has played a major agricultural role in

A ages of the human civilization that featured technological

progress and economic stability, such as the most prosperous
periods of the Persian Empire in western Asia, the Roman
Empire in the Mediterranean region, the Han Dynasty in China, the
Arab Empires in the Middle Ages, and the Renaissance in Europe.
These conditions favoured the exploitation of this crop for specialized
forage productions, as well as for enhancing the soil fertility. The
appreciation of this crop by farmers of temperate and subtropical crop-
livestock systems has risen until the second half of the 20th century,
when the low cost of energy and the ignorance of the impact of a high-
carbon economy on climate change favoured the adoption of crop-
livestock systems that are manifestly unsustainable.
...Paolo In West Europe, the maintenance of alfalfa cultivation in vast
areas during this period is remarkable, when considering its much lower
Annicchiarico1 level of EU subsidization compared with other major crops (with
subsidies that were granted only to the industry of dehydration).
and We believe that alfalfa is going to play a crucial role in a new,
environment-friendly chapter of the human civilization in which
agriculture is asked to produce more and better, using far less energy (for
nitrogen fertilizers, provision of extra-farm feed proteins, soil tillage,
weed and pest control, etc.). The return of this crop to its destiny of a
bright future is favoured by its wide range of possible target
environments and utilizations (hay, silage, grazing; high-protein
feedstuff; dual-purpose for forage and energy). ■

Bernadette
Julier2

_________________________________________________________________________________________________________

1Consiglio per la Ricerca e la Sperimentazione in

Agricoltura, Centro di Ricerca per le Produzioni
Foraggere e Lattiero-Casearie, Lodi, Italy
(paolo.annicchiarico@entecra.it)
2INRA, UR4, URP3F, Lusignan, France

(bernadette.julier@lusignan.inra.fr)

Legume
Legume Perspectives
Perspectives Issue
Issue 41 •• July
January
20142013
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 RESEARCH

Perspectives of alfalfa in Europe

by Christian HUYGHE1*, Luc DELABY2, Daniele ROSELLINI3 and Fabio VERONESI3

Abstract: Sustainable agriculture shall The environmental issues are most In Europe, plant and animal productions
reconcile high productivity with preservation complex to identify, because they have both tend to be separated, with specialized farms
of the environment, and at the same time merchant and non-merchant aspects, such as and territories. Alfalfa has the peculiarity to
compete in a global market. Alfalfa can help biodiversity preservation, and because they be inserted in grain crops rotations, and also
to meet the challenges of climate change and have different time horizons. In a short term used in animal farms. While this was
pollution reduction, by favoring crop perspective, the reduction of pollution by considered as a downside, it appears to offer
rotations capable of better preserving the soil nitrates and pesticides is essential and a high potential for future farming needs. In
and controlling weeds. The utilization of prescribed by the European directive on grain farms with specialized short rotations,
alfalfa through grazing may also reduce risks pesticides and quality of the water resources. farmers have to cope with difficulties in
of pollution and production costs. Alfalfa In a mid-term perspective, water availability controlling weeds, which become highly
will keep an important place in European and energy resources for agriculture have to specialized and sometimes resistant to
agriculture, because it sustainably supports be considered. In a long-term perspective, herbicides. The insertion of perennial alfalfa
high quality dairy productions. These unique climate change will dramatically modify the in rotations with a high proportion of winter
features can be further improved through conditions for agricultural activity. As a wheat has proved to be able to control well
technical and varietal innovation. consequence, on the one hand we have to the weed flora. The results reported in Fig. 1
Key words: crop rotation, forage, grazing, anticipate such changes, on the other hand show that the species composition of the
Medicago sativa, sustainable agriculture, weed we should reduce as much as possible the weed community depends on the crops, and
control present emission of greenhouse gas (GHG), the presence of alfalfa in rotations reduces
namely, CO2 from production of agricultural dramatically the risk of specialization of the
inputs, CH4 from ruminants, and N2O from weed flora compared with cereal
application of nitrogen fertilisers. Research monocultures (thereby reducing the risk of
Stakes of the European and innovation are required to meet these generating weed flora resistant to herbicides)
agriculture and worldwide objectives. (2). Moreover, alfalfa in rotation with winter
context cereals has improved the soil structure and
Potential of alfalfa in this has provided a substantial amount of
After a period when an increasing nitrogen for the two crops following alfalfa
production was the only objective of context
(3), thus reducing energy consumption and
agriculture, followed by a decade when the Alfalfa acreage in Europe is devoted either emission of GHG. In the same process, it
environmental issues were the main to production of dehydrated forage, mainly prevents nitrate leaching at the rotation scale.
concerns, it is now well accepted that a in Spain, France and northern Italy, or to on- In organic agriculture, the usefulness of
sustainable agriculture shall reconcile high farm use of forages, mainly as hay. While the alfalfa in weed control and as a source of
productivity with environment preservation. production of dehydrated forage tends to nitrogen fertility at the rotation scale is also
A highly productive agriculture must meet plateau or to decline due to the cost of highly recognized in many European regions.
the food and non-food demand of the energy for dehydration, the on-farm use of Production of feed proteins on the farm or
European societies and also be competitive alfalfa is slightly increasing after a long period in nearby cereal farms makes it possible to
on the world market, considering that most of steady decline. reduce the dependence of animal production
protein sources for supplementation of on imported soybean feedstuff, whose prices
animal feed are imported, and that these show a high volatility. This meets the
products have shown large price variations in demand of farmers to be less exposed to
recent years. risks. In particular for dairy cow nutrition,
alfalfa (which has high protein and calcium
contents) is an excellent companion of maize
silage. However, even though a high protein
_________________________________________________________________________________________________________
production per unit area is a real advantage
1INRA, Scientific Directorate “Agriculture”, Paris, of alfalfa, the high protein solubility and the
France (christian.huyghe@lusignan.inra.fr) difficulty to preserve it when harvested as
2INRA, UMR Pégase, Saint-Gilles, France
3 University of Perugia, Department of Applied
hay (loss of leaves) or silage (protein
degradation) require innovation.
Biology, Perugia, Italy

Legume Perspectives Issue

Issue41••July
January
20142013
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RESEARCH

Which alfalfa crops for the

future European agriculture?
Because of its high agronomic potential
and its ability to produce large amounts of
protein per unit area, alfalfa has a role to play
in European agriculture, even if some
challenges have to be faced. The
achievement of high biomass and protein
production remains a key objective and this
will be obtained mainly by improving the
resistance to biotic (leaf and soil fungal
diseases, nematodes) and abiotic stresses.
Lodging resistance is essential in Europe to
optimize spring harvest, which is the one
providing the highest biomass and protein
production (Fig. 2).
Persistency is not a critical issue for hay or
silage production, as the present duration of
most alfalfa crops (> 3 years) fits correctly in
most cropping systems. It is a key issue,
however, for the grazed crop, which has
gained interest in the last two decades for
extensive livestock systems especially in
southern Europe, in order to reduce the Figure 1. Canonical discriminant analysis of weed communities in 4 groups of plots (a), (b), (c)
environmental impact of intensive animal and (d) representing the four key-steps of rotations including annual cereals and alfalfa. Initial
husbandry and to decrease production cost. data represented the abundance of the various weed species in the crops (1)
Conventional alfalfa varieties generally do
not persist under grazing. Therefore,
Spanish, French and Italian research teams
have started selection programs for grazing
tolerant alfalfa, releasing cultivars with better
survival under grazing. In the near future, References
European varieties specifically adapted to (1) Meiss H, Médiène S, Waldhardt R, Caneill J,
grazing will hopefully enter the seed market Bretagnolle V, Reboud X, Munier-Jolain N (2010)
(4). Perennial lucerne affects weed community
The ability to grow in mixture with a trajectories in grain crop rotations. Weed Res
50:331–340
perennial grass is of interest for animal farms
(2) Munier-Jolain N, Médiene S, Meiss H,
as it makes it possible to achieve a high Boissinot F, Rainer W, Jacques C, Bretagnolle V
biomass production without extra nitrogen (2012) Rôle des prairies temporaires pour la
fertilization, reduced weed damages, and gestion de la flore adventice dans les systèmes
facilitates grazing or the harvest and Figure 2. A stand of the alfalfa crop at the céréaliers. Innov Agron 22:71-84
conservation as silage. Indeed, the water stage of full vegetative development (3) Thiébeau P, Lô-Pelzer E, Klumpp K, Corson
soluble carbohydrates of the associated grass M, Hénault C, Bloor J, de Chezelles E, Soussana
will help to get low pH and improve JF, Lett JM, Jeuffroy MH (2010) Conduite des
légumineuses pour améliorer l‟efficience
conservation. On-farm barn drying is an emerging énergétique et réduire les émissions de gaz à effet
A major effort should be devoted to technique in some European regions, de serre à l‟échelle de la culture et de l‟exploitation
harvesting machinery. For example, mowing especially where high-quality products, such agricole. Innov Agron 11:45-58
machines equipped with roll conditioners as protected-designation-of-origin cheeses, (4) Veronesi F, Brummer EC, Huyghe C (2010)
would make it possible to speed up the are produced. Alfalfa is extremely well Alfalfa. In: Boller B, Posselt U, Veronesi F (eds)
drying process, thus reducing quality losses. adapted for such a production system, as it Fodder Crops and Amenity Grasses. Handbook of
This is of course of major importance for Plant Breeding 5, Springer Science+Business
dries more quickly than other forages in the Media, Dordrecht, the Netherlands, 395-438
hay production, but it is also valuable for drying cells under ventilation.
ensiling with a pre-drying phase, or for In conclusion, we believe that alfalfa will
production of wrapped round bales. keep an important place in European
agriculture. Its favourable features can be
further enhanced through technical and
varietal innovation aimed at improved energy
efficiency and reduced costs. ■

Legume
Legume Perspectives
Perspectives Issue
Issue 41 •• July
January
20142013
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 RESEARCH

Perspectives of alfalfa in North America

by Neal P. MARTIN

Abstract: Harvested alfalfa for hay was 12.3 to develop new markets for alfalfa co- with potential to compete for biofuel
million ha in 2011 in U.S. and Canada. products. In addition there is a need to feedstock production and with efficient
However, acreage is declining due to develop new tools for accelerating and water use.
competition for land area due to high value enhancing breeding programs, and A review of several alfalfa cutting
cash crops and lower yields than corn (silage demonstrating the importance of alfalfa in management studies showed as the interval
and grain), soybean and wheat. Top farmers environmental sustainability. between harvests increased, annual DM yield
within US harvest 2 to 3 times national or increased while nutritive value decreased (1).
regional agricultural statistical averages. Yield Neutral detergent fiber digestibility (NDFD)
Reduced-lignin transgenic alfalfa can increase Yield of alfalfa over the last 60 years did of the diet is an indication of potential DM
forage fiber digestibility for lactating dairy not increase substantially in the absence of intake and milk yield in dairy cows (4).
cows, or allow for 8 to 12 days delayed disease pressure, but new cultivars out Summarizing a range of studies, the authors
harvest. The latter has potential for yielded older checks by as much as 40% in found that a one-unit increase in herbage in
eliminating one or more annual harvests environments where diseases are prevalent vitro or in situ NDFD was associated with a
while increasing yield by over 20%. (2). Average reported whole-farm hay yields 0.17-kg increase in DM intake and 0.25-kg
Key words: fiber digestibility, forage quality, are 50 to 70 % higher than in 1962 and increase in 4 % fat-corrected milk per day.
hay, milk production, yield regularly exceeded corn grain until only 15 During spring and early summer, DM is
years ago (6). In depth analysis of 2007 being produced and nutritive value is
Introduction Census of Agriculture data showed that declining more rapidly (NDF increases and
yields of top tier producers across U.S. in NDFD decreases in early spring) than during
Alfalfa is the most widely harvested forage non-irrigated or irrigated production, late summer (1). Under humid growing
legume worldwide and the fourth most although small in number, are 2 to 3 times conditions early harvest and shorter cutting
valued crop in the U.S., contributing more higher than averages, and matching in some intervals are needed in spring and early
than 10 billion USD annually to the Nation‟s regions the yield of cultivar small plot trials summer, but slower changing nutritive value
farm economy. Farmers harvested alfalfa for (Fig. 1). More on farm programs that in late summer (more arid) fit delaying
hay from 7.8 and 4.5 million ha in 2011, estimate field yield and quality such as harvest to allow additional yield
from U.S. and Canada, respectively. Alfalfa Wisconsin Alfalfa Yield and Persistence accumulation. Utilizing nutritive value
has primary value as an animal feed, but also Program (5) are needed to set yield goals estimates of fiber and fiber digestibility to
diversifies farming production risks, provides closer to the crop genetic potential and to decide on cutting management have resulted
a source of biologically fixed nitrogen for demonstrate to producers, educators, and in quality forage needed to maintain high
subsequent crops in a rotation, reduces soil scientist that alfalfa is an economically feed intakes and digestibility, but have also
erosion, captures nutrients from annual competitive perennial legume feed source, made successful harvest more challenging.
cropping fields that can contaminate surface
and ground water, disrupts pest cycles of
rotation crops, and promotes soil carbon
sequestration.
Competition for land and global climate
change dictate increasing alfalfa yields on
marginal lands. Drought tolerance, salt
tolerance and biotic stress resistance will be
needed on many of western acres. Livestock
producers will need increased yield from
fewer cuts to remain economically
competitive. New research efforts are
needed to develop new harvest, storage and
processing methodologies to maximize
protein utilization produced by the plant and

Figure 1. Number of farms (% of total) that reported annual alfalfa hay yield (13 % moisture)
_________________________________________________________________________________________________________
greater than 18 Mg ha-1 (irrigated) or 13.4 Mg ha-1 (nonirrigated), based on a subset of the
USDA-ARS, U.S. Dairy Forage Research Center, 2007 Census of Agriculture. For states with more than 50 farms, estimates for irrigated were
Madison, USA (darforage13@gmail.com) made by interpolation of best-fit sigmoidal curves

Legume
Legume Perspectives
Perspectives Issue
Issue 41 •• July
January
20142013
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RESEARCH

Role of alfalfa in animal diets

Alfalfa is the most important forage
legume for dairy cows. It is relatively low in
fiber and high in protein compared to other
forages, and typically results in high intakes
and levels of milk production. The major
disadvantage of alfalfa is low yields when
compared to corn silage and the need for
multiple harvests. Multiple harvests not only
increase the labor and equipment costs for
alfalfa, but also expose the forage to multiple
maturities and harvesting environments,
such as rain damage, that increase the
variability in nutritional quality. Intensive
cutting schedules may also be the root cause
of poor stand survival and reduced yields.
The fiber (cell wall) component of forage
represents a major source of energy; Figure 2. Novel harvest system to alter alfalfa harvest and storage to obtain leaves and stems
however, less than 50% of this fraction is for new animal or products uses. US Dairy Forage Research Center, USDA-ARS
readily digested and utilized by the animal
(3). If alfalfa could be engineered to increase
the extent of fiber digestion, the amount of (PPO1-alfalfa). Proteolysis was inhibited in window for the farmer (uncoupling harvest
energy derived from cattle diets would leaf extracts of the PPO1-alfalfa when a from stage of plant development). Finally,
increase or the potential amount of substrate with similar activity of a o-diphenol such an approach creates a value-added
carbohydrate available for cellulosic ethanol in red clover (caffeic acid) was added (3). stream for products. Protein from harvested
fermentation could increase; each of which Tannins are phenolic compounds that leaves can also be used to produce high
has potential to increase the demand for generally bind with proteins, decreasing the protein supplements in diets for other
alfalfa. Proof of concept reduced-lignin rate and extent of protein digestion. Forage animals, or serve as a raw material base for
transgenic alfalfa hay fed in total mixed diets legumes (e.g. birdsfoot trefoil) that produce proteins destined for more industrial
with corn silage measured increased fiber tannins in leaves or stems have increased applications, while stems could be used in a
digestibility in both lactating dairy cows and stability of the protein in the rumen, thus biomass to bioenergy scheme. Newly
rapidly growing lambs (7). Digestible dry more protein escaping degradation in the constructed facilities will soon begin
matter of one transgenic increased 3.5 % fat rumen. An optimum level of tannins production of fuel from crop residues,
corrected milk over the same plant supported an increase of 5 kg per day of milk primarily corn stover. Widespread stover
population without the reduced-lignin gene from cows fed normal tannin containing harvest could have decidedly negative
by 1.3 kg hd-1 day-1. Late harvested birsdfoot trefoil over alfalfa silage (3). environmental impacts, particularly loss of
transgenic lines had the same NDFD as their Unfortunately, alfalfa does not produce soil organic matter and increased erosion.
control populations harvested 8 to 12 days tannins except in the seed coats. With new Systems that separate leaves from stems
earlier (7). Producers using this trait may be knowledge about tannin biosynthesis (Dixon open the door for wider use in crop
able to delay harvest while maintaining group, Noble Foundation), it may be rotations to provide greater environmental
forage quality, potentially eliminating one or possible to engineer alfalfa to produce and economic stability. ■
more annual harvests while increasing yield tannins that provide protein protection in
by over 20%. the rumen and may also lead to less bloat. References
Immature alfalfa silage is high in protein in (1) Brink G, Hall M, Shewmaker G, Martin N, Undersander D,
which plant proteases can degrade 44% to New harvesting system and Walgenbach R (2010) Changes in alfalfa yield and nutritive value
within individual harvest periods. Agron J 102:1274-1282
87% of forage protein into ammonia, amino value-added products (2) Lamb JS, Sheaffer CS, Rhodes LH, Sulc RM, Undersander DJ,
Brummer EC (2006) Five decades of alfalfa cultivar improvement:
acids, and small peptides during silage Difficulties in managing alfalfa for high impact on forage yield, persistence, and nutritive value. Crop Sci
fermentation (3). Decreasing protein protein and low fiber has inadvertently 46:902-909
(3) Martin NP, Hatfield RD, Mertens DR, Casler MD (2007)
degradation during ensiling and in the rumen resulted in the expansion of corn-based feed Redesigning forages for sustainable dairy production. Proceedings,
would decrease the need for supplemental and silage into feeding rations. Development Intermountain Nutrition Conference, Salt Lake City, US, 23-24
January 2007, 1-16
protein and reduce the loss of nitrogen to of novel alfalfa management and field (4) Oba M, Allen MS (1999) Evaluation of the importance of the
the environment on the dairy farm (8). harvesting equipment that separate high digestibility of neutral detergent fiber from forage: effects on dry
matter intake and milk yield of dairy cows. J Dairy Sci 82:589-596
Recently, the U.S. Dairy Forage Research protein leaves from high fiber stems allows (5) Rankin M (2013) Wisconsin Alfalfa Yield and Persistence
Center has successfully tested the hypothesis recombining these fractions in proportions (WAYP) Program. 2012 Summary Report.
http://www.uwex.edu/ces/crops/teamforage/2012%20WAYP%2
that PPO and o-diphenols inhibit proteolysis that optimize the diet requirements for the 0Summary.pdf
in alfalfa leaf plant extracts (3). Although target animal (e.g., high producing dairy cow, (6) Russelle M (2013) The alfalfa yield gap: a review of the
evidence. Forage Grazingland 11
alfalfa has at least one gene encoding PPO, dry cow, and heifer) (Fig. 2). It also reduces (7) Undersander D, McCaslin M, Sheaffer S, Whalen D, Miller D,
expression has not been detected in any the impact of adverse weather on crop Putnam D, Orloff S (2009) Low lignin alfalfa: redefining the
yield/quality tradeoff. Proceedings, 39th Western Alfalfa & Forage
tissues except developing seed pods. A harvest, increases yields by capturing the Conference, Reno, USA, 2-4 December 2009, 157-161
cloned red clover PPO gene (PPO1) was approximately 30% loss typically occurring (8) Vagnoni DB, Broderick GA (1997) Effects of supplementation
of energy or ruminally undegraded protein to lactating cows fed
constitutively expressed in transgenic alfalfa during harvest, and opens the harvest alfalfa hay or silage. J Dairy Sci 80:1703-1712

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 RESEARCH

Perspectives of alfalfa in Australia, China, Africa and

Latin America
by Daniel BASIGALUP1*, John IRWIN2, Fugui MI3 and Meriem ABDELGUERFI-LAOUAR4

Abstract: The importance of alfalfa in Seed production is mostly concentrated in world. As a centre of diversification, China
Australia, China and some African and Latin South Australia (85%), having reached a total holds a large set of genetic resources. The
American countries is summarized. Present of 7913 t in 2007, 85% of which was country has 13 species of the genera
planting areas, average forage yields, main exported. It is estimated that over 3 M ha of Medicago, mainly distributed in the Western
use (hay or grazing) and crop conditions dryland alfalfa is grazed in Australia by either plateau and in parts of the Changjiang river
(rainfed or irrigated) are briefly reported. sheep or cattle, mostly in NSW. Using basin, roughly between 35-43° N.
Finally, the crop perspectives for the near modeling studies it has been calculated that Nowadays, China grows nearly 3.6 M ha of
future in each region are discussed. an additional 95 M ha could be sown to alfalfa, 77% of which are harvested as hay.
Tolerance to abiotic stresses (drought, dryland alfalfa, especially in NSW and Most fields (80%) produce at least three cuts
salinity, acidity, cold and heat) will play a Queensland. per year, providing forage with crude protein
crucial role in expanding alfalfa over the four Up to 1977, the Flemish type cultivar contents between 14 and 18%. The overall
continents. The importance of landrace Hunter River occupied > 90% of the production (about 60 M t of hay DM in
genetic resources in traditional breeding and Australian alfalfa planting area, but now over 2010) is insufficient for the feeding needs.
biotechnological approaches (transgenesis 50 cultivars are marketed. Pest and disease A total of 62 alfalfa varieties are presently
and marker-assisted selection) for developing resistant cultivars have made a major impact registered in the Chinese forage catalogue
adapted cultivars for those limiting on Australia´s alfalfa production, particularly (24 bred cultivars, 20 landraces, 15
environments are also highlighted. in irrigated hay crops where diseases such as introduced cultivars and 3 domesticated
Key words: Africa, alfalfa, Australia, China, Phytophthora root rot and anthracnose can species). While having different fall
Latin America cause serious losses (6). Most of the modern dormancy classes, all of them are adapted to
Australian cultivars have multiple pest and cold environments. Most cultivars were
disease resistance incorporated. Grazing developed by traditional breeding (including
tolerant cultivars have also been recently crosses between ssp. sativa and ssp. falcata), but
Australia deployed (5). a few recent ones (named Zhongmu No.1, 2
Perspectives. To increase the alfalfa area in and 3) were bred by tissue culture selection.
Alfalfa is Australia‟s most important Australia, there are several traits which will Perspectives. Chinese policies are
temperate and sub-tropical perennial forage have to be bred into future cultivars. emphatically promoting the crop by
legume, being grown extensively from north Tolerance to acid soils is under development articulating activities from government
Tasmania to the Tropic of Capricorn (6). in the US and Australia (5). In sub-tropical business, production farms and scientific
Total Australian alfalfa area (grazing and hay) areas, increased winter activity and the research units. Three research topics have
has been estimated at 3.5 M ha, more than capacity to compete with C4 grasses will be been prioritized, namely: harvesting and
half grown in New South Wales (NSW). Hay important (6). Other valuable traits include processing methodologies, genetic
production has increased by 40% over the drought and salinity tolerance, increased seed improvement of cold and drought tolerance,
period 1990-2005, principally through an size, and non-bloating to bovines. If these and the development of biotechnology
increase in cropping area. In 2005, about traits are addressed, there are good prospects approaches contemplating transgenesis or
250,000 ha were grown solely for hay, with for overcoming the present forage yield marker-assisted selection.
an average yield of 4.96 t DM (dry matter) stagnation.
ha-1, and NSW (40%), Victoria (25%) and Africa
Queensland (18%) as the leading states (8). China Alfalfa is cultivated in just some African
Alfalfa is an important forage crop in countries located in northern and southern
China, where it was introduced from Persia regions. Morocco grows nearly 106,000 ha
in 138-119 BC by the emperor Han Wu as a (22.8% of country‟s total fodder crop land),
_________________________________________________________________________________________________________
source of forage for his horses. Therefore, with an average yield of 5.93 t DM ha-1. In
1INTA Manfredi, Argentina China has more than 2,000 years of history Algeria, alfalfa occupies about 10,000 ha,
(dbasigalup@manfredi.inta.gov.ar) on the crop. At present, it is widely with a mean production of 3-4 t DM ha-1. In
2The University of Queensland, Brisbane,
distributed throughout the country. Just Tunisia, alfalfa is the main forage and
Australia
3Inner Mongolia Agricultural University, Hohhot,
between 1930 and 2012, about 400 alfalfa extends over about 13,000 ha (77% in oases).
China
accessions were introduced from all over the The small area planted in Mauritania is
4ENSA Belfort, El Harrach, Alger, Algeria concentrated in oases. Alfalfa is the fourth

Legume Perspectives Issue 4 • July 2014

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RESEARCH

Figure 1. Irrigated alfalfa in one oasis of Adrar in Algeria, in Figure 2. Alfalfa under rotational grazing for dairy cows in
rotation with wheat (photo kindly provided by Ahmed Chaabena) Argentina

forage crop in Egypt, where it reached 70,000 ha mainly devoted to dairy in the SW, Perspectives. No significant changes in alfalfa
36,000 ha in 2008 all under irrigation. Locally yielding 9-13 t DM ha-1 year-1. Soil acidity planting are expected in Mexico, Peru,
adapted populations (landraces) are and low P content limit the crop expansion Uruguay and Chile. Conversely, the expected
extensively used in Northern Africa oases, in these countries. Chile´s planting area is release of new cultivars adapted to tropical
where alfalfa production is almost 50,000 ha, concentrated in Central and environments could quickly duplicate the
continuous (8-10 cuts year-1) and intervals Southern regions, mostly grown for dairy cropping area in Brazil. In Argentina, the on-
between cuts are very short (1). South Africa systems in association with ryegrass. Peru going development of cultivars tolerant to
is the main alfalfa growing country in the grows nearly 120,000 ha mainly in the coastal abiotic (drought, salinity, acidity) and new
southern hemisphere, with about 300,000 ha areas, with yields of 13-18 t DM ha-1 year-1. biotic stresses (particularly alfalfa enation
(9) mainly under irrigation, and where dairy While intermediate (6-8 cuts year--1) to non- virus), will extend the cropping area.
cow diets contain up to 40% of alfalfa hay. dormant (11 cuts year-1) varieties are used for Transgenic cultivars are being developed
Perspectives. Alfalfa cropping has potential coastal environments, dormant/very with tolerance to herbicides, drought and
for expansion in Africa, particularly under dormant (2-3 cuts year-1) are adopted for the salinity and with delayed leaf senescence. ■
efficient irrigation systems (e.g. pivot high Andean plateau. Mexico grows about
irrigation) and with the development of 400,000 ha of alfalfa, almost all under Acknowledgments
dehydration units at the farm level. Also irrigation. About 65% is used as green We thank Rodrigo Zarza, Fernando Ortega
rainfed cropping of drought-tolerant forage, while the remaining is devoted to hay Klose and Baldomero Alarcón Zúñiga for their
germplasm in semi-arid regions has greater production (with an average yield of 17.5 t valuable information.
potential than hitherto believed (3). The DM ha-1 year-1). With a few exceptions, most
References
evaluation and exploitation of local (2) and of the varieties are imported from USA.
(1) Abdelguerfi A, Laouar M (2002) Les Espèces Pastorales et
exotic (3) genetic resources has crucial Argentina grows 4 M ha of alfalfa, mostly Fourragères, leurs utilisations au Maghreb (Algérie, Maroc,
importance for producing varieties with under rainfed conditions for dairy and beef Tunisie). FAO-RNE, Dokki
(2) Abdelguerfi A, Abdelguerfi-Laouar M (2004) Les ressources
better tolerance to major stresses (e.g., production in the Pampas Region. About génétiques d‟intérêt fourrager et/ou pastoral : diversité, collecte et
drought, salinity, heat) or to frequent cutting. 60% is planted as pure stands, and 40% in valorisation au niveau du Bassin Méditerranéen. Cah Options
Méditerr 62:29-42
The implementation of variety registration, mixtures with temperate grasses. Direct (3) Annicchiarico P, Pecetti L, Abdelguerfi A, Bouizgaren A,
reliable seed multiplication and grazing is still very popular, although hay Carroni A M, Hayek T, M‟Hammadi Bouzina M, Mezni M (2011)
Adaptation of landrace and variety germplasm and selection
commercialization chains are required in (and more recently silage) production is strategies for lucerne in the Mediterranean basin. Field Crop Res
most countries for promoting adapted, gaining importance (4). Other areas (NW, W 120:283–291
(4) Basigalup D (2007) El Cultivo de la Alfalfa en la Argentina.
improved varieties. Similarly in India, warm and Patagonia) are devoted to hay and seed Ediciones INTA, Buenos Aires
southern locations favour early flowering, production under irrigation. The mean (5) Bouton JH (2012) Breeding lucerne for persistence. Crop Past
Sci 63:95-106
while the opposite is the case in the north forage yields are 11 and 20 t DM ha-1 year-1 (6) Irwin JAG, Lloyd DL, Lowe KF (2001) Lucerne biology and
(6). for rainfed and irrigated conditions, genetic improvement - an analysis of past activities and future goals
in Australia. Crop Past Sci 52:699-712
respectively. About 75% of the marketed (7) Köpp MM, Pereira AV, Ferreira RP (2011) Cultivares de alfafa
Latin America seed (9,000 t) is imported, mainly from no Brasil. In: Ferreira RP, Basigalup DH, Gieco JO (ed)
Melhoramento Genético da Alfafa. São Carlos, Embrapa Pecuária
The importance of alfalfa in Latin America Australia and USA. Over 80 cultivars (with Sudeste, Brazil, 301-323

varies greatly among countries. Brazil grows fall dormancy 6 to 10) are marketed, all (8) Martin P (2009) The Australian fodder industry: an overview of
production, use and trade. RIRDC Publ 9/001, RIRDC Barton
only 40,000 ha, mostly for hay production in having multiple aphid and disease resistance. ACT Australia
(9) Mauriès M (2003) Luzerne : culture, récolte, conservation.
the Southern states. The locally adapted cv. Editions France Agricole, Paris
Crioula, with an average yield of 14 t DM ha-1
year-1, is widely used (7). Uruguay has about

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

Environmental benefits of growing perennial legumes

in cropping systems
by Michael P. RUSSELLE

Abstract: Alfalfa offers several fixation range from 40 kg N ha-1 to Nitrate removal capacity is affected by
environmental benefits to farmers and 600 kg N ha-1 and from 10 kg N ha-1 to stand density (plants m-2). As alfalfa stands
society. Reduced nitrate leaching due to 320 kg N ha-1 in mixed alfalfa-grass stands decline with age, disease, winterkill, or
slower water flow and excellent nitrate (6). However, N2 fixation varies within and damage by wheel traffic, nitrate can
removal are valuable in farm fields and at among fields and regions, due to the myriad accumulate in soil and be leached in
remediation sites. Improved N supply to factors that modify it. Because we still can percolating water (3). Where leaching should
following crops reduces energy use, only guess at N2 fixation rates in farm fields, be minimized, the stand should be rotated to
economic costs, and greenhouse gas a key research objective should be to develop an annual or perennial crop that can absorb
emissions, but reliable indicators are needed a reliable technique to use on farms. mineralized N. Alfalfa increases the amount
to identify which fields need supplemental N of mineralizable N in the soil, which is
after alfalfa. Livestock manure is applied to Nitrate removal released earlier, faster, and longer than N
alfalfa on farms, but too little is known about Alfalfa‟s large N requirement is due to high mineralized following maize or wheat (1). If
the benefits and risks. Critical needs are on- herbage yield and protein concentration. the following crop cannot capture this
farm estimates of yield and N2 fixation and Alfalfa does not fill this need solely by N2 mineralized N, nitrate leaching can be
broadened plant improvement objectives to fixation, but rather it absorbs nitrate from significantly higher after alfalfa than after
explicitly include environmental benefits. the soil solution effectively enough to reduce other crops, even in semiarid environments.
Key words: fertilizer replacement, nitrate nitrate leaching losses. Nitrate assimilated
leaching, nitrogen, soil quality, water use from the soil reduces the rate of N2 fixation, Nitrogen supply
thereby reducing N input when plant- This higher N supply reduces the need for
available N supply is high. fertilizer N in crops following alfalfa. On
High yield potential of this C3 forage crop medium-textured soils, alfalfa usually
Introduction results in high water use, although water use provides the entire amount of N required by
In the First Century, Columella wrote that efficiency (WUE, g dry matter g-1 water maize, resulting in substantial economic
“all cattle whatsoever grow fat with [alfalfa]” transpired) is excellent in comparison to savings and greatly reducing the energy cost
and that it “dungs the land.” Today, alfalfa is other C3 crops. Furthermore, water use of maize production (8). On fine- and
known as a premier forage crop, primarily as begins earlier and continues longer than coarse-textured soils it is more common to
a protein source, but its positive most annual crops, such as maize and find a fertilizer N response of first-year
environmental effects are less well soybean. This timing of water use makes maize after alfalfa than on medium-textured
recognized. alfalfa highly effective in reducing nitrate soils (Fig. 1), probably because of slower
Widely acknowledged is the contribution leaching losses. Managed as a harvested nitrification and enhanced denitrification on
of alfalfa to nitrogen (N) availability. forage crop, alfalfa maintained annual nitrate fine-textured soils and greater nitrate
Symbiotic N2 fixation occurs by rhizobia losses through subsurface tile drains to leaching on sands. Still undiscovered are
housed in root nodules and fed by C < 5 kg N ha-1, even when drainage volumes means of identifying fields where the first
compounds from the plant in exchange for were twice the normal amounts (5). In crop after alfalfa will require additional N.
the N-containing amino acids the bacteria contrast, loss under continuous maize or a With alfalfa, decreased fertilizer inputs and
produce. Any factor that affects plant growth maize-soybean rotation averaged overall lower denitrification contribute to
or yield likely affects the amount of N fixed 40 kg N ha-1 with normal and 75 kg N ha-1 greatly reduced net energy requirements (8)
– supply of nutrients (P, K, S, and with high drainage volumes (5). and greenhouse gas emissions (4).
micronutrients), soil pH, inorganic N supply
in the soil, the degree of defoliation at
harvest or by pests, and inadequate or
surplus water availability, to name a few. In Figure 1. Yield response to fertilizer N of
pure alfalfa stands, estimates of annual N2 first-year maize grain yield after alfalfa is
more likely on fine- and coarse-textured soils
than on medium-textured soils, with the
_________________________________________________________________________________________________________
number of N-responsive sites and total sites
in each soil texture class (8)
USDA-ARS, Plant Science Research Unit, St.
Paul, USA (michael.russelle@ars.usda.gov)

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RESEARCH

Only part of this increase could be attributed

to nutrients other than N, and yield increases
of 20 to 40% with manure may be due to
lower photosynthate requirement for
inorganic N assimilation than for symbiotic
N2 fixation. There is a clear need for more
research to document benefits and risks.

Enhancing the benefits

The value of alfalfa in terms of N2 fixation,
nitrate removal, N supply to subsequent
crops, water use, and other benefits is
directly related to forage yield. In most
Figure 2. Alfalfa yield (t dry matter ha-1) on the top 10% of US regions of the USA, alfalfa yield potential is
farms and the yield gap (italics) compared to regional cultivar higher than yields reported by even the best
performance trials, with irrigated (blue shading) and without farmers (Fig. 2). Our ability to generate
irrigation (green shading) (7) reliable assessments of net impacts of alfalfa
on ecosystem services requires better
knowledge of yields, stand lifetime, and soil
Deep rooting Reduced soil erosion N supply. Elimination of yield-limiting
Roots of alfalfa extend vertically about 1 to Perennial forages often are recommended factors will enhance both the economic and
2 cm day-1 at typical soil temperatures, to reduce nutrient and sediment runoff. environmental benefits of this crop.
totaling 1.5 to 2 m annually in temperate Alternating strips of alfalfa and maize that Nearly all of these benefits have been
environments and in a favorable soil matrix follow the contour of a hillslope help reduce demonstrated with commercial alfalfa
(low penetration resistance, adequate soil erosion by surface runoff. Although cultivars. Root system architecture, root
aeration, and appropriate chemical sediment movement is reduced, perennial extension rate, nitrate uptake, stress
conditions). There are anecdotal reports of forages damaged by freezing or drying (e.g., tolerance, and perhaps other characteristics
extremely deep rooting, but measurable during desiccation for hay production) can related to environmental benefits are
alfalfa root activity has been evidenced by release soluble, reactive P into runoff water. moderately heritable. If these traits are
water and nitrate depletion to 5 m and by Where plant growth is limited by cold introduced, plant development companies
uptake of 32P at 5.5 m. Consequently, alfalfa temperatures, soil often is left for several could produce alfalfa with greater public and
is used to prevent or reduce nitrate leaching months without cover or living vegetation. private benefits. ■
from abandoned feedlots and other sites. Cover crops can reduce soil erosion and
High water use by alfalfa has been used to nitrate losses, but they are not widely References
prevent expansion of salt-degraded soils. adopted without supplemental payments or (1) Carpenter-Boggs L, Pikul JL Jr, Vigil MF, Riedell
Replacement of native perennial vegetation regulation. A “living mulch” of Kura clover WE (2000) Soil nitrogen mineralization influenced by
with annual grain crops in arid environments (Trifolium ambiguum) in row crops shows crop rotation and nitrogen fertilization. Soil Sci Soc
Am J 64:2038-2045
has resulted in intrusion of saline promise when competition can be managed. (2) Ceotto E, Spallacci P (2006) Pig slurry applications
groundwater into the root zone. The following year, clover can be grown as a to alfalfa: Productivity, solar radiation utilization, N
Remediation of soils degraded by salinity is forage, or an annual crop can be interseeded and P removal. Field Crops Res 95:135-155
difficult and expensive. An economic means again. This system, first examined by KA (3) Entz MH, Bullied WJ, Forster DA, Gulden R,
of prevention is to plant deeply rooted Albrecht, reduces nitrate leaching and Vessey JK (2001) Extraction of subsoil nitrogen by
alfalfa, alfalfa-wheat, and perennial grass systems.
perennials, such as alfalfa, which probably runoff, but requires further Agron J 93:495-503
simultaneously provide income. Although development to maintain maize yield. It (4) Meyer-Aurich A, Weersink A, Janovicek K, Deen
there have been breeding efforts to increase remains to be determined whether alfalfa can B (2006) Cost efficient rotation and tillage options to
tolerance to salinity, most alfalfa cultivars do be used in this way. sequester carbon and mitigate GHG emissions from
not tolerate moderate salinity conditions. agriculture in Eastern Canada. Agric Ecosyst Environ
117:119-127
As secondarily thickened roots decay, Manure utilization (5) Randall GW, Huggins DR, Russelle MP, Fuchs
improved macroporosity in subsoil facilitates Livestock manure is a valuable resource, DJ, Nelson WW, Anderson JL (1997) Nitrate losses
deeper root penetration of subsequent crops but is applied mostly for annual crops. The through subsurface tile drainage in Conservation
and enhanced water infiltration when Reserve Program, alfalfa, and row crop systems. J
facultative nature of symbiotic N2 fixation Environ Qual 26:1240-1247
macropore flow occurs. A potential hazard is provides the opportunity for alfalfa to utilize (6) Russelle MP (2008) Biological dinitrogen fixation
that macropore flow also can transmit manure N effectively, while other manure in agriculture. In: Schepers JS, Raun WR (eds)
suspended and dissolved materials, such as nutrients also can benefit alfalfa. Manure Nitrogen in agricultural systems. American Society of
surface-applied pesticides, fertilizer, or application methods to alfalfa vary from Agronomy, Crop Science Society of America, Soil
manure. However, this occurs only when the Science Society of America, Madison, USA, 281-359
broadcasting solid or liquid forms, through (7) Russelle MP (2013) The alfalfa yield gap: A review
hydraulic conductivity of the soil matrix is partial incorporation, to shallow injection, of the evidence. Forage Grazingland 11
exceeded, e.g. intensive rainfall or irrigation, but relatively few experiments have been (8) Yost MA, Russelle MP, Coulter JA (2014) Field-
accumulation of runoff, snowmelt and published. Swine manure applied through specific fertilizer nitrogen requirements for first-year
urination by livestock. Serious impacts due irrigation greatly increased alfalfa yield (2). corn following alfalfa. Agron J 106:645-658
to root channels have not been detected.

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 RESEARCH

Alfalfa domestication history, genetic diversity and

genetic resources
by Jean-Marie PROSPERI1*, Eric JENCZEWSKI2, Marie-Hélène MULLER1, Stéphane FOURTIER3,
Jean-Paul SAMPOUX3 and Joëlle RONFORT1

Abstract: The domestication history of

alfalfa is poorly known. Here, we summarize
recent results obtained from the
investigation of the genetic diversity available
in the Medicago sativa species complex, using
different molecular markers and
morphological characterization. We conclude
that a large genetic diversity is still available
in the wild form of the species, but original
populations are restricted to a relatively small
geographic area and in some instances
submitted to gene flow from the cultivated
form. A short description of the European
genetic resources is presented.
Key words: diffusion routes, gene flow,
genetic resources, molecular markers,
morphological characterization, wild
Figure 1. The different routes and approximate dates of diffusion of cultivated alfalfa from its
populations
Centre of origin (the symbol (-) correspond to BC dates)

Domestication of alfalfa
Alfalfa was reintroduced into Spain through well as to the hot and dry summers typical of
It is difficult to identify the first traces of North Africa with the Moors. It then crossed continental climates (6, 7).
alfalfa (Medicago sativa L.) domestication. the Pyrenees around the fifteenth century.
Different authors suggested that alfalfa was Thereafter, it was introduced to South Genetic diversity of the
cultivated 9,000 years ago in some rare America (Mexico, Peru, Chile) by the
locations in its centre of origin (Near East to cultivated and wild pools of
Spanish in the sixteenth century, and then to
Central Asia). Sinskaya in 1950 suggested the United States through California at the Medicago sativa
that its distribution spread throughout the beginning of the nineteenth century as The evolutionary history of a wild–
Middle East by 1,000 BC, and from there, to „Chilean clover‟. These major paths of cultivated complex is the outcome of
China and India. But actually, we have no diffusion are summarised, along with a few different factors, among which are the
element to validate these assumptions. The minor ones, in Fig. 1 (6). domestication of the cultivated form, the
first written references mentioned the Feral populations of alfalfa are frequent in geographical and demographic expansion of
introduction of alfalfa to Greece by the a variety of open habitats (roadsides, field the species, and the gene flow between
Medes armies, its spread into Italy and then borders), but wild forms of M. sativa subsp. natural and cultivated populations. To clarify
all over the Roman Empire in Europe. sativa are rare, with the exception of the alfalfa‟s history, we sampled several cultivars
During the Middle Ages, the interest in Iberian Peninsula and its centre of diversity. and landraces, natural populations of M.
alfalfa regressed considerably in Europe. In other west Mediterranean countries sativa subsp. sativa (diploid and tetraploid), as
(southern France, Italy, Greece, and North well as populations of the subsp. falcata and
Africa), plants of M. x varia, are very other perennial species. A special focus was
_________________________________________________________________________________________________________
common as a result of crosses between made on the Spanish wild pool. Molecular
1INRA, UMR 1334, Amélioration Génétique et subsp. sativa and subsp. falcata populations. markers (RAPD, nuclear DNA sequence
Adaptation des Plantes, Montpellier, France The M. sativa subsp. falcata appears more polymorphism and mitochondrial DNA
(jean-marie.prosperi@supagro.inra.fr) frequently as a wild species, from the north variation) and phenotypic characterization
2INRA, UMR1318, Institut Jean-Pierre Bourgin,
of the Mediterranean Sea (Bulgaria, Greece) were used to measure the differentiation
Versailles, France
3INRA, UR 0004, Unité de Recherche to the northern limits of Russia. It is between cultivated and wild types and to
Pluridisciplinaire Prairies et Plantes Fourragères, particularly well adapted to hard winters, as quantify the importance of gene flow.
Lusignan, France

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RESEARCH

From all these data, we noted: the genus and from Asia. The diploid forms (1260 accessions). All together, these
(i) the marked originality of the wild pools, of the M. sativa complex species (i.e. subsp. accessions represent the diversity originating
with specific characteristics such as prostrate glomerata and subsp. coerulea) are also under- from all parts of Europe. Information about
habit, rhizomes, tolerance to drought and represented in gene banks. the ECPGR Central Crop Database for
grazing (3, 7, 8); In order to promote the use and rationalise perennial Medicago species can be obtained
(ii) a significant domestication bottleneck the maintenance of plant genetic resources in from the manager of this database
measured on nuclear DNA polymorphism, Europe, the European Cooperative (http://www.ecpgr.cgiar.org/germplasm_data
with a loss of diversity from wild to Programme for Plant Genetic Resources bases/central_crop_databases/ECCDB_List
domesticated material of 30% on average (ECPGR: http://www.ecpgr.cgiar.org) was .html). ■
(although more data are needed to set up in 1980 and is now coordinated by
strengthen this result). We detected no Bioversity International. References
differentiation between diploid and Within ECPGR, a working group on (1) Jenczewski E, Prosperi JM, Ronfort J (1999)
tetraploid forms (3); forage species brings together 37 countries. Differentiation between natural and cultivated populations
of Medicago sativa (Leguminosae) from Spain: analysis with
(iii) a high level of mtDNA variation, This working group has set up Central Crop random amplified polymorphic DNA (RAPD) markers and
especially in the wild pool. In the wild pool Databases (CCDB) for several groups of comparison to allozymes. Mol Ecol 8:1317-1330
from the presumed area of origin, the forage grass and legume species. A part of (2) Jenczewski E, Prosperi JM, Ronfort J (1999) Evidence
for gene flow between wild and cultivated Medicago sativa
diversity was high but the absence of these CCDB is centralized in the EURISCO (Leguminosae) based on allozyme markers and quantitative
geographical differentiation in our sample database, a web-based catalogue which traits. Amer J Bot 86:677-687
hindered a more precise detection of the automatically receives the National (3) Muller MH, Poncet C, Prosperi J M, Santoni S, Ronfort
J (2006) Domestication history in the Medicago sativa species
centre of domestication (4, 5 and Fig. 2); Inventories of the gene banks from 42 complex: inferences from nuclear sequence polymorphism.
(iv) in cultivated alfalfa, the geographical European countries (http://eurisco.ecpgr.org). Mol Ecol 15:1589-1602
structure of mtDNA and nuclear DNA The ECPGR Central Crop Database for (4) Muller MH, Prosperi JM, Santoni S, Ronfort J (2001)
How mitochondrial DNA diversity can help to understand
variation strongly suggests at least two perennial Medicago species references 7874 the dynamics of wild-cultivated complexes. The case of
independent routes (north and south) of samples (a third from the Russian Medicago sativa in Spain. Mol Ecol 10:2753-2763
dissemination from its centre of origin (3, 5); Federation) maintained over 25 European (5) Muller MH, Prosperi JM, Santoni S, Ronfort J (2003)
Inferences from mitochondrial DNA patterns on the
(v) a specific mitochondrial type in the national gene banks. Some accessions are domestication history of alfalfa (Medicago sativa). Mol Ecol
Spanish wild pool. That suggests an old present as duplicated samples in several gene 12:2187-2199
divergence from the centre of origin and the banks. A primary holder has been identified (6) Prosperi JM, Auricht GC, Génier G, Johnson RC (2001)
Genetic diversity of legumes in the Mediterranean: Medics
fact that these populations were established for each accession (i.e. a gene bank primarily (Medicago L.). In: Maxted N, Bennett S (eds) Plant Genetic
in Spain before the introduction of cultivated responsible for the maintenance of the Resources of Legumes in the Mediterranean. Kluwer
alfalfa (5 and Fig. 2); accession). Nineteen species are represented; Academic Publishers, Dordrecht, the Netherlands, 99-114
(7) Prosperi JM, Jenczewski E, Angevain M, Ronfort J
(vi) an occurrence of crop/wild gene flow some of them including several subspecies; (2006) Morphologic and agronomic diversity of wild genetic
in many locations in Spain, together with the but the tetraploid M. sativa represents more resources of Medicago sativa L. collected in Spain. Genet
persistence of wild Spanish populations than 95% of the total. Referenced accessions Resour Crop Evol 53:843-856
(8) Prosperi JM, Jenczewski E, Ronfort J (1999) The
showing a great agronomic and morphologic include cultivars (1920 accessions), landraces Mielgas: wild Spanish populations of alfalfa. Results of ten
originality (1, 2, 7, 8). (1430 accessions), wild or feral populations years of researches. Proceedings, XIII EUCARPIA Medicago
Taken together, these results show that M. (769 accessions) and breeding materials spp., Perugia, Italy, 13-16 September 1999, 1-10
sativa contains a large genetic diversity and
that there is probably much to gain from the
study, the conservation and the use of the
genetic variability available in the wild
fraction of this species.

Genetic resources and main

gene banks
Numerous gene banks in the world exist
that concentrate on Medicago (National Plant
Germplasm System USA, Australian
Medicago Genetic Resource Centre
Australia, ICARDA Syria, Vavilov Institute
Russia, etc.). For Europe, genetic resources
of perennial Medicago species are maintained
by many countries. Due to past exchanges of
genetic materials among research institutes,
accessions such as cultivars and landraces are
frequently maintained in several gene banks.
Wild perennial species are generally only
maintained by the institute that collected
them. They are poorly represented or not
represented in germplasm collections, Figure 2. Mitochondrial DNA variation between wild and cultivated pools of Medicago sativa
especially those from the centre of origin of in West and East Mediterranean regions. Each mitochondrial haplotype is in a different colour

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

From the model legume Medicago truncatula to

alfalfa
by Bernadette JULIER

Abstract: Large efforts have been devoted Natural populations have been used to available to be incorporated into breeding
to Medicago truncatula genomics. They offer establish mapping populations of programs (Figure 1).
data and tools to identify M. truncatula genes recombinant inbred lines, which have been
involved in genetic variation for traits related mapped with microsatellite markers Identification of genes in M.
to agronomic value in crop species. This developed from EST sequences and used to truncatula
strategy is based on the hypothesis that the detect quantitative trait loci (QTL) for traits
same genes are involved in trait variation in having agronomic interest in crop species. In the first strategy, the QTL detection in
both species. These tools are now used to Fine mapping was used to detect candidate mapping populations of M. truncatula,
develop alfalfa genomics in order to carry genes explaining the QTL. Collections of followed by a fine mapping step, gives access
out genomic studies in the target species. tilling or transposon-tagging mutants have to a small genomic region. Using an in silico
Identification of genes or markers in the been established to detect new alleles of analysis of the genes annotated in the QTL
crop species will contribute to develop tools known genes or new genes involved in region and a literature review of the genes
and theory to implement marker assisted phenotypic variants. known to be involved in the trait under
selection in alfalfa. study, a list of candidate genes can be
Key words: genomics, marker, model From M. truncatula to alfalfa: established. This method was successfully
species, QTL, sequence used for flowering date. One of the
strategies candidate genes, a Constans-like gene,
Many efforts have been devoted to Exploiting genomic knowledge from M. contained sequence polymorphism and
Medicago truncatula Gaertn. genetics and truncatula for the genetic improvement of showed differential expression between the
genomics since 1990. A draft sequence has alfalfa is not simple. Detecting genes two parents (7). Analysis of mutants with
been obtained after sequencing the gene-rich involved in the variation for breeding traits is altered phenotypes (forward genetics), or
genome portions (euchromatin) (9). Its often needed to improve breeding programs. phenotypic analysis of genotypes showing
analysis showed that M. truncatula genome is Two non-exclusive strategies can be devised. differential gene expression (reverse genetics)
more complex than expected from its short In the first one, candidate genes are detected are other ways to detect candidate genes.
genome size. The genome has been shaped in M. truncatula and are then tested for Association genetics in M. truncatula could
by a whole-genome duplication but also by their effect in crop species (Figure 1). In the also help to identify genes or genomic
numerous rearrangements and local second one, the genomics of alfalfa is regions involved in traits. The efficiency of
duplications. These genome changes allowed developed by taking into account the association genetics will depend on the
the emergence of specific genes involved in knowledge from M. truncatula genomics, and linkage disequilibrium (LD) of the
rhizobial nodulation. The number of genes is used to detect genes or markers involved in population under study, which can be long in
over 62,000, and the genome contains traits. These genes or markers become this selfing species.
numerous transposons, retrotransposons and
transcription factors. Resequencing of a set
of 26 accessions has given access to
sequence polymorphism (1). A large diversity
was found, but linkage disequilibrium was
shorter than expected in a selfing species.
Transcriptomics data were used to construct
a genechip array (https:www.affymetrix.com
/products_services/arrays/specific/medicago
.affx) that can be used to detect the role of
genes in specific functions and organs. In
parallel, accessions collected from sites of the
Mediterranean Basin were made available to
the scientific community in gene banks (http://
www1.montpellier.inra.fr/BRC-MTR/).
_________________________________________________________________________________________________________ Figure 1. Strategies to transfer genomics information from M. truncatula to the breeding
INRA, UR4, URP3F, Lusignan, France program of a legume species; several methodologies (boxes) are proposed to achieve each step

Legume Perspectives Issue 4 • July 2014

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RESEARCH

Once a gene has been identified in the population. Fine QTL mapping has not been (5) Herrmann D, Barre P, Santoni S, Julier B
model species, it remains to verify the reported in alfalfa, probably because it would (2010) Association of a CONSTANS-LIKE gene
presence of a homeologue of it in alfalfa require very large mapping populations and to flowering and height in autotetraploid alfalfa.
Theor Appl Genet 121:865-876
which has an effect on the same trait. This in silico analysis of genomics regions is not
(6) Julier B, Flajoulot S, Barre P, Cardinet G,
demonstration can be achieved and exploited feasible yet. However, this in silico analysis, by Santoni S, Huguet T, Huyghe C (2003)
by different methods, such as transgenesis, using the same anchor markers in alfalfa and Construction of two genetic linkage maps in
introgression through backcross, or M. truncatula, can be carried out in the model cultivated tetraploid alfalfa (Medicago sativa) using
association genetics (Fig. 1). With species. On the whole, gene or marker microsatellite and AFLP markers. BMC Plant Biol
transgenesis, the modification of expression detection directly performed in alfalfa 3:9
of an allele in alfalfa is supposed to confer a (through bulk segregant analysis (BSA) or (7) Pierre JB, Bogard M, Herrmann D, Huyghe C,
new trait variation (8), eventually becoming genomic selection) is scarce up to now, but Julier B (2011) A CONSTANS-like gene candidate
that could explain most of the genetic variation
available in a transgenic variety. Introgression the increasing availability of high throughput
for flowering date in Medicago truncatula. Mol Breed
through backcross has the same objective, markers could change this situation in the 28:25-35
but the allele originates from alfalfa. With future. These markers will now be mainly (8) Shadle G, Chen F, Reddy MSS, Jackson L,
association study, the demonstration results developed in alfalfa from sequencing and Nakashima J, Dixon RA (2007) Down-regulation
in the identification of alfalfa allele(s) that resequencing programs. However, any of hydroxycinnamoyl CoA: Shikimate
confer valuable variation, as found for a analysis of their function, position on maps, hydroxycinnamoyl transferase in transgenic alfalfa
Constans-like gene (5). These alleles could sequence polymorphism of their genomic affects lignification, development and forage
then be used in a breeding program assisted regions will benefit from genomic resources quality. Phytochem 68:1521-1529
(9) Young ND, Debelle F, Oldroyd GED, Geurts
by molecular markers, to increase their in M. truncatula.
R, Cannon SB, Udvardi MK, Benedito VA, Mayer
frequency in the synthetic varieties. KFX, Gouzy J, Schoof H, Van de Peer Y, Proost
Perspectives S, Cook DR, Meyers BC, Spannagl M, Cheung F,
Development of alfalfa Current development of alfalfa genomics, De Mita S, Krishnakumar V, Gundlach H, Zhou
genomics now possible because of extensive SG, Mudge J, Bharti AK, Murray JD, Naoumkina
MA, Rosen B, Silverstein KAT, Tang HB,
The second strategy implies the study of knowledge in M. truncatula, offers new
Rombauts S, Zhao PX, Zhou P, Barbe V, Bardou
alfalfa genomics by DNA or cDNA perspective to directly detect genes or P, Bechner M, Bellec A, Berger A, Berges H,
sequencing using the Next Generation markers in the crop species for agronomics Bidwell S, Bisseling T, Choisne N, Couloux A,
Sequencing (NGS) technologies (Fig. 1). traits. All limitations to genetic analysis in Denny R, Deshpande S, Dai XB, Doyle JJ, Dudez
Read assembly is complex because each alfalfa are not yet overcome and more AM, Farmer AD, Fouteau S, Franken C, Gibelin
individual possibly carries 4 different copies specifically, polyploidy and heterozygosity C, Gish J, Goldstein S, Gonzalez AJ, Green PJ,
will lastingly reduce their efficiency. So M. Hallab A, Hartog M, Hua A, Humphray SJ, Jeong
of each gene, in addition to the presence of DH, Jing Y, Jocker A, Kenton SM, Kim DJ, Klee
gene families or repeated elements. The truncatula is still a reference, mainly for all the
K, Lai HS, Lang CT, Lin SP, Macmil SL,
assembly of alfalfa sequences can be -omics data, but also for the identification of
Magdelenat G, Matthews L, McCorrison J,
considered as a re-sequencing, with M. genes involved in agronomic traits. ■ Monaghan EL, Mun JH, Najar FZ, Nicholson C,
truncatula sequence acting as a reference. Noirot C, O'Bleness M, Paule CR, Poulain J, Prion
However, extensive sequence evolution F, Qin BF, Qu CM, Retzel EF, Riddle C, Sallet E,
occurred in the two species, leading to large Samain S, Samson N, Sanders I, Saurat O,
differences in non-coding sequences References Scarpelli C, Schiex T, Segurens B, Severin AJ,
Sherrier DJ, Shi RH, Sims S, Singer SR, Sinharoy
between M. truncatula and alfalfa. Alfalfa (1) Branca A, Paape TD, Zhou P, Briskine R,
S, Sterck L, Viollet A, Wang BB, Wang KQ, Wang
sequencing is currently a scientific challenge Farmer AD, Mudge J, Bharti AK, Woodward JE,
MY, Wang XH, Warfsmann J, Weissenbach J,
that is addressed by a few groups in Europe May GD, Gentzbittel L, Ben C, Denny R,
White DD, White JD, Wiley GB, Wincker P, Xing
Sadowsky MJ, Ronfort J, Bataillon T, Young ND,
and the USA. In the meanwhile, genetic YB, Yang LM, Yao ZY, Ying F, Zhai JX, Zhou
Tiffin P (2011) Whole-genome nucleotide
mapping and QTL detection are feasible. diversity, recombination, and linkage
LP, Zuber A, Denarie J, Dixon RA, May GD,
Theories and software (TetraploidMap, Schwartz DC, Rogers J, Quetier F, Town CD, Roe
disequilibrium in the model legume Medicago
http://www.bioss.ac.uk/knowledge/tetraplo BA (2011) The Medicago genome provides insight
truncatula. Proc Natl Acad Sci USA 108: E864-
into the evolution of rhizobial symbioses. Nat
idmap/) were developed in autotetraploid E870
480: 520-524
species. Microsatellite markers were (2) Castonguay Y, Cloutier J, Laberge S, Bertrand
developed in alfalfa which mostly originated A, Michaud R (2005) A bulk segregant approach
from EST of M. truncatula (6). These to identify genetic polymorphisms associated with
cold tolerance in lucerne. Sapporo, Japan, 10-15
codominant markers with additional
July 2004
dominant markers such as AFLP or DArT (3) Ghesquière M, Barre P, Durand JL, Julier B,
(3) or HRM (4) markers are numerous Litrico I, Maamouri A, Mournet P, Risterucci AM,
enough to build maps of any mapping Sampoux JP, Vignes H (2012) Construction of a
population. The comparison of M. truncatula DArT marker resource for better adapted forage
and alfalfa maps proved that both macro- crops to climate change. Proceedings, 7th
synteny and micro-synteny between the two International Symposium on the Molecular
species are high. Due to autotetraploidy, the Breeding of Forage and Turf, Salt Lake City, USA,
4-7 June 2012, 85
precision of QTL position is lower in alfalfa
(4) Han Y, Khu DM, Monteros MJ (2011) High-
than in a diploid species, so the subsequent resolution melting analysis for SNP genotyping
use of QTL into breeding is still limited to and mapping in tetraploid alfalfa (Medicago sativa
the genetic background of the mapping L.). Mol Breed 29:489-501

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

Alfalfa marker-assisted selection

by E. Charles BRUMMER* and Maria J. MONTEROS

Abstract: Alfalfa improvement has relied on The availability of marker systems that can genes (or quantitative trait loci, QTL) for key
phenotypic selection for genetic improvement. robustly evaluate anything from single markers traits and the subsequent introgression of those
The availability of single nucleotide to genome-wide markers now opens up a markers (and genes) into elite breeding
polymorphisms (SNP) throughout the genome, wealth of opportunities to apply markers to populations. Markers can help increase the
as well as methods, such as genotyping-by- breeding programs in a systematic way. In this frequencies of these genes (alleles), moving
sequencing (GBS), to assay SNPs cost- paper, we suggest three ways markers could them toward fixation, so that all or at least most
effectively will enable successful integration of constructively be applied to selection: (1) seed of synthetic cultivars will contain these
markers into breeding programs. GBS will marker diversity assessment, (2) marker-assisted alleles in the certified seed bag that a farmer
enable breeders to select based on marker trait integration, and (3) genome-wide selection. would purchase. For some traits, such as
breeding values, accelerating genetic gain aluminum tolerance, QTL have been identified
through repeated cycles of selection. Integrating Marker diversity assessment in wild diploid germplasm and transferred to
markers associated with major genes and Perhaps the most extensive use of markers tetraploid populations by interploidy
implementing genomic selection could increase across crop plants has been to characterize hybridization (5). Markers are valuable tools
the speed and efficiency of alfalfa genetic variation. Most alfalfa genetic variation that could be used to track the desirable trait,
improvement. Alfalfa breeding programs may resides within populations, so unsurprisingly, either from diploid accessions or other
need to modify existing methods to fully realize most diversity experiments in alfalfa that shown tetraploid germplasm, through the breeding
the power markers can bring to cultivar that populations with distinct genetic profiles process. Other traits undoubtedly exist where
development. are also highly phenotypically and/or commercial germplasm lacks specific desirable
Key words: alfalfa, genomic selection, geographically differentiated. To date, this type alleles, particularly for quantitative traits
genotyping-by-sequencing, marker-assisted of experiment has had limited practical value to including abiotic stress responses.
selection, SNP breeding programs. Moving beyond these types Many of the major diseases and insect pests
of experiments to explore specific breeding can be relatively easily assayed using classic
Introduction objectives could help breeders more phenotypic screens in which thousands or even
successfully identify unique sources of millions of seedlings are inoculated and
In the time since the first alfalfa genetic maps survivors selected. However, even in these
were published 20 years ago this year, few germplasm, understand genetic diversity within
populations, minimize inbreeding depression, cases, having markers for the major genes
applications of genetic markers to trait selection would enable the small subset of elite parents
in alfalfa have been realized. Despite this and aid in constructing heterotic groups. The
value of markers at identifying heterotic groups and individual genotypes to be screened for the
relative lack of progress, the future indeed looks presence of the resistant/tolerant alleles, and
bright for the potential of markers to make a has often failed in many crops, but selecting
plants within populations to construct de novo perhaps more importantly, determine the allelic
substantial contribution to genetic gain for dosage. Because alfalfa is an autotetraploid
major traits in alfalfa. Despite significant heterotic groups may be more successful. Part
of the problem with using genetically distinct species, dominant alleles can express a trait
investment in a succession of molecular marker despite the presence of three recessive alleles at
systems – RFLP, AFLP, RAPD, SSR – no populations as heterotic groups can be that they
are otherwise not agronomically desirable or that locus. This reservoir of deleterious or
readily assayed, cost effective markers truly undesirable alleles will appear during seed
existed for routine breeding programs until adapted to the target region. We propose a
potentially better approach that involves increase and the resulting cultivar will include
SNP methods were developed in the past plants that may not have any desirable alleles at
several years. Currently, large numbers of SNP starting with agronomically desirable
populations, allocate plants into two pools that locus. Markers could ensure that any
have been identified in alfalfa (3, 7), and selected parental plant would have multiple
markers polymorphic in different germplasm designated as heterotic groups, and then
commence breeding for hybrid performance. copies of that allele, ideally fixed for the correct
have been developed, which can be assayed allele.
individually using high resolution melting Reciprocal recurrent selection has been
successfully used to increase yield in maize (2), Recently, we have used genotyping-by-
analysis (4) or collectively with an Illumina sequencing (GBS) as a high throughput means
Infinium array (8). Further, we are well on the but has not been applied to any
significant extent in alfalfa. We propose that it to assay SNP variation throughout the genome.
way to adapting genotyping-by- sequencing to With GBS, sequences from the end of
alfalfa selection programs, thus generating should be, given the anticipated possibility to
improve yield. restriction fragments are aligned from
inexpensive, genome-wide markers rapidly. different individuals and SNP between
Marker-assisted selection individuals are identified (1). We evaluated allele
_________________________________________________________________________________________________________________________
frequency shifts in bulked populations selected
A second use of markers in a broad sense is for and against whitefly resistance. The GBS
The Samuel Roberts Noble Foundation, Forage
in marker-assisted selection. The common strategy identified 70,000 SNP loci, and one
Improvement Division, Ardmore, USA
(ecbrummer@noble.org)
scenario includes the identification of major location on chromosome 8 was identified as a

Legume Perspectives Issue 4 • July 2014

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RESEARCH

eliminated; in fact, the genotyping cost,

however trivial it eventually becomes, will
almost certainly be an additional cost for the
breeding program. Will the added gain pay for
the added cost? That‟s a question commercial
programs need to answer, but the answer may
be positive if genomic selection results in better
cultivars faster. From an academic standpoint,
the availability of genome-wide markers seems
likely to improve genetic gain for complex
traits.

Perspectives
An aspect that requires additional discussion
Figure 1. By integrating molecular markers, breeders can identify populations with unique genetic is how breeding programs could be rearranged
variation, can identify marker-trait associations to complement phenotypic selection, and are able to to further capture the value of markers (Fig. 1).
implement genomic selection (GS; based on a selection index for multiple traits); In the example here, Current methods are probably not the
four cycles of genomic selection could be completed for every one cycle of phenotypic evaluation; optimum ones given the availability of today‟s
Phenotyping of progenies from selected plants will continue in a GS program, although mainly for marker resources. One potential change could
improving the GS model; Phenotypic evaluation of new germplasmcould also contribute to an updated be a shift from the large population sizes and
model, and genotypes with desirable alleles could enter the next cycle of GS; Prior to any GS cycle, a broad-based synthetics of today‟s breeding
pre-selection of individuals based on phenotypic or marker screens for disease or insect resistances programs to one that is much more focused on
could ensure that a certain trait threshold is maintained during the marker-only phase of the program; fewer, elite clones. The focus would shift from
The resulting populations would then be evaluated under field conditions in the target environments trying to increase allele frequencies for multiple
pest resistances in broad populations to using
likely candidate region for a major resistance about how a trait is structured or which
markers to ensure fixation (or nearly so) of the
locus, based on large SNP allele frequency markers are involved but rather estimates
key alleles in all parental genotypes. More effort
changes across cycles. Similar populations genomic breeding values (GEBV) which
could then be devoted to extensive progeny
selected for resistance to many other diseases capture the effects of a large number of genetic
testing for yield and other key traits,
and pests are available, and this bulking strategy markers. The idea is simply to saturate the
implementing a genomic selection program to
may facilitate the identification of genomic genome, so that any given gene will be in
accelerate genetic gain. New germplasm would
regions associated with resistance. Mapping linkage disequilbrium with at least one marker.
be extensively tested for combining ability
relevant loci in segregating populations and In this way, the breeding value of every marker
before entering the breeding population.
identifying functional SNPs are needed to can be estimated. In order for GS to result in
Hybrid cultivars could also be evaluated to
validate the location of a resistance locus, as in genetic gains, an initial model needs to be
capture non-additive effects. Given the new
our example. Nevertheless, current DNA constructed based on marker genotypes and
genetic marker realities, current alfalfa breeding
sequencing technologies greatly expand the individual plant (or family) phenotypes, so that
methodologies should be revised to most
potential to identify important loci and alleles marker breeding values can be computed. Once
effectively integrate markers, accelerating
and their application to breeding programs. the model has been developed, selection
genetic gain and cultivar improvement. ■
proceeds based on the estimated breeding value
Genome-wide selection of the alleles that each individual in the breeding References
Marker-assisted selection is best applied to population possesses. Because GS is marker- (1) Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler
simply inherited traits, or at least, to traits for only selection, repeated cycles can be ES, Mitchell SE (2011) A robust, simple genotyping-by-sequencing (GBS)
approach for high diversity species. PLoS ONE 6:e19379.
which one or few QTL of known large effect recombined in the greenhouse as fast as (2) Hallauer AR, Russell WA, Lamkey KR (1988) Corn Breeding. In:
have been identified. For most complex traits possible. Because breeding cycles are typically 2- Sprague GF, Dudley JW (eds) Corn and Corn Improvement. American
Society of Agronomy, Crop Science Society of America, Soil Science
such as yield, the QTL-by-QTL strategy seems 5 years long, the number of recombinations Society of America, Madison, USA, 463-564
less effective. Individual QTL effects are from GS per typical breeding program can be (3) Han Y, Kang Y, Torres-Jerez I, Cheung F, Town CD, Zhao PX,
Udvardi M, Monteros MJ (2011) Genome-wide SNP discovery in
typically inflated in most mapping experiments, substantial, offering the prospect of significant tetraploid alfalfa using 454 sequencing and high-resolution melting
and QTL × environment interactions common. gains over traditional selection (6). analysis. BMC Genomics 12:350
(4) Han Y, Khu DM, Monteros MJ (2012) High resolution melting
Manipulating many QTL simultaneously is The limitations of GS are clear. Even if a analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago
difficult, requiring very large population sizes. robust model can be constructed, the multiple - sativa L.). Mol Breeding 29:489-501.
(5) Khu DM, Reyno R, Brummer EC, Bouton JH, Monteros MJ (2013)
Identifying all the QTL of importance is time- rounds of selection and recombination in the Identification of aluminum tolerance QTLs in tetraploid alfalfa (Medicago
consuming, and probably population specific greenhouse still require genotyping, and this can sativa L.). Crop Sci 53:148-163
(6) Li X, Brummer EC (2012) Applied genetics and genomics in alfalfa
as well. Therefore, for these traits, another be costly. Currently, GBS markers can be breeding. Agron 2:40-61
marker-based approach is needed. applied to genotypes for about 10 USD each; (7) Li X, Acharya A, Farmer AD, Crow JA, Bharti AK, Kramer RS, Wei
Y, Han Y, Gou J, May GD, Monteros MJ, Brummer EC (2012)
Genome-wide selection, or genomic selection improved sequencing will undoubtedly bring Prevalence of single nucleotide polymorphism among 27 diverse alfalfa
(GS), was proposed over a decade ago for use that number down further. However, a GS genotypes as assessed by transcriptome sequencing. BMC Genomics
13:568
in livestock breeding (9). In contrast to a typical model is based on the particular allele (8) Li X, Han Y, Wei Y, Acharya A, Farmer A, Ho J, Monteros MJ,
marker-assisted breeding program, in which frequencies present when selection begins. As Brummer EC (2013) The development of a high density SNP array for
alfalfa and its use to evaluate genetic diversity. Plant and Animal Genome
certain genomic regions (QTL) are known (or cycles are conducted, allele frequencies change, Conference, San Diego, USA, 10-14 January 2013
at least hypothesized) to be involved in and an updated model needs to be constructed. (9) Meuwissen THE, Hayes BJ, Goddard ME (2001) Prediction of total
genetic value using genome-wide dense marker maps. Genet 157:1819-
controlling a trait, GS makes no assumptions Therefore, phenotyping really won‟t be 1829

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The challenges of genetically modified alfalfa in Europe

by Daniele ROSELLINI and Fabio VERONESI

Abstract: The application of genetic The European legislation on GM crops Useful traits obtained through genetic
engineering to alfalfa has large potential, and poses serious limitations to the cultivation of engineering that cannot be obtained by
a genetically modified herbicide tolerant transgenic alfalfa, especially with regard to classical breeding are already available and
variety is on the market in the US. In gene flow. Alfalfa is pollinated by insects others are at hand, but it appears
Europe, apart from general public opinion that can fly a long way; consequently, it problematic to adopt GM alfalfa in
issues, the main constraint to the adoption of would be very difficult to completely prevent European agriculture under the present
genetically modified alfalfa varieties is gene transgene flow from GM to non-GM crops regulatory and public opinion scenario. In
flow, because it would be very difficult to and to feral alfalfa plants, which are the future, emerging genetic engineering
completely prevent it. In the future, widespread in many European regions. A techniques such as cisgenesis and gene
however, emerging genetic engineering review study (1) suggests that a distance of targeting may be applied to alfalfa. Cisgenesis
techniques may improve public perception 1000 feet (304.8 m) produces an adventitious utilizes genes from a species, or from
of transgenic crops and allow Europe to take presence of GM seed below 0.5 % in a non- sexually compatible species, for its
advantage of the potential of genetic GM seed crop, which is below the 0.9 % improvement through genetic engineering.
biotechnology for alfalfa improvement in threshold required for the coexistence of Gene targeting can surgically mutate or
Europe. GM crops with conventional and organic replace an allele with another by precise
Key words: coexistence, gene flow, GM ones. For seed production, however, the genome editing. These new techniques may
crops, Medicago sativa, transgenic varieties spatial isolation prescribed in US by the contribute to exploit the potential of
Association of Official Seed Certifying biotechnology for alfalfa improvement in
The application of genetic engineering to Agencies for non-GM seed crops for export Europe. ■
alfalfa has large potential. Many interesting is at least 5 miles (8 km) from GM crops.
prototypes have been produced in several Organic agriculture makes large use of References
labs, and there is a number of traits which alfalfa in rotations and is particularly sensitive (1) CAST (2008) Gene Flow in Alfalfa: Biology,
could improve the productivity, adaptability to the GM crop contamination issue. The Mitigation, and Potential Impact on Production. Council
for Agricultural Science and Technology, www.cast-
and product value of the crop (Table 1, 3). same legal threshold for GM contamination science.org
The first, and only, genetically modified is applied to forages for animals producing (2) Ferradini N, Nicolia A, Capomaccio S, Veronesi F,
(GM) alfalfa on the market to date is organic or conventional products in Europe, Rosellini D (2011) A point mutation in the Medicago sativa
Roundup ready alfalfa (Monsanto and but the organic market may be affected by GSA gene provides a novel, efficient, selectable marker
Forage Genetics International). This variety any level of contamination above zero due to for plant genetic engineering. J Biotechnol 146:147-152
(3) Kölliker R, Rosellini D, Wang Z-Y (2010)
was released in 2005, but in 2006 a group of consumer attitude. Development and application of biotechnological and
organic farmers and nonprofit organizations The complete containment of transgenes molecular genetic tools. In: Boller B, Posselt U, Veronesi
sued the US Department of Agriculture for would be possible if the transgenic crop were F (eds) Fodder Crops and Amenity Grasses. Springer
approving the crop without a thorough completely male-sterile; female sterility Science+Business Media, Dordrecht, the Netherlands,
assessment its environmental and economic would also be desirable, to avoid the 89-113
(4) Rosellini D (2004) Molecular genetics and
impacts. A US federal court stopped its sale, establishment of volunteer and feral GM modification of flowering and reproductive development.
until an environmental impact assessment (5) plants; complete suppression of flowering In: Hopkins A, Wang ZY, Mian R, Sledge M (eds)
allowed again its cultivation. could be obtained by engineering floral Molecular Breeding of Forage and Turf. Kluwer,
The adoption of GM alfalfa in Europe development genes (4). Clonal propagation Dordrecht, the Netherlands, pp 105-126
appears problematic at present. On the of sterile or non-flowering alfalfa producing (5) USDA (2010) Glyphosate-tolerant alfalfa events J101
and J163: Request for nonregulated status. Final
positive side, the development of new, high-value molecules, such as vaccines for environmental impact statement, December 2010
publicly available tools could alleviate some farm animals, might be economically viable.
intellectual property constraints and some
perceived risks; for example, it is now
possible to efficiently transform alfalfa
without antibiotic resistance marker genes, Table 1. A selection of traits introduced into alfalfa by genetic engineering
by using a freely available alfalfa gene (2). Input traits Output traits
This would avoid the environmental release
Herbicide tolerance Protein quality
of antibiotic resistance genes when
cultivating GM alfalfa varieties. Improved phosphorous assimilation Vaccine expression
Drought tolerance Improved digestibility
_________________________________________________________________________________________________________________________

Salt tolerance Improved fermentable sugar yield

University of Perugia, Department of Applied
Biology, Perugia, Italy (roselli@unipg.it) Acid soil tolerance Reduced protein loss in silage and digestion

Legume Perspectives Issue 4 • July 2014

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RESEARCH

Alfalfa genetic variation for response to tissue culture

and in vitro regeneration
by Massimo CONFALONIERI* and Paolo ANNICCHIARICO

Abstract: High in vitro regeneration ability of embryogenesis protocols for alfalfa in vitro showed at least one callus with one or more
alfalfa is rare and subjected to complex genetic regeneration (2). Genetic analyses have somatic embryos. The whole experiment was
control. Identifying new responsive genotypes indicated that the process of embryo formation repeated twice.
that are agronomically valuable is crucial for from vegetative cells is controlled by at least All 53 genotypes were able to produce callus,
broadening the genetic base and raising the two independent dominant genes and different showing high frequency of callus formation.
genetic value of transgenic varieties or in vitro gene interactions (3), while the occurrence of On average, over 78% of the leaflets produced
selections, and widening the genetic maternal effects is controversial. Alfalfa shoot calli, which is a high value compared to various
background of material in functional genomics regenerationviadirectorganogenesis is very rare. earlier studies. In both experiments, genotype
studies. We investigated the response to tissue differences in callus fresh weight per explant
culture and in vitro regeneration of 53 elite, high- Identification of were highly significant and showed a large
yielding genotypes. Good response to tissue agronomically-valuable broad sense heritability (h2 > 0.84). These
culture was fairly common, highly heritable in differences were largely consistent across
the broad sense, and with good consistency germplasm sources experiments (genetic correlation rg = 0.67),
across experiments. Only one genotype also We have searched for new germplasm while showing no relationship with forage
showed good response to subsequent in vitro sources able to combine high agronomic yielding ability as evaluated on the half-sib
regeneration, confirming the need for extensive value with good response to tissue culture progenies of the 53 genotypes (r = -0.01).
screening. and in vitro regeneration, by assessing the Despite the large number of genotypes that
Key words: genetic transformation, genetic latter characteristics in 53 elite genotypes that could produce callus, only one genotype out of
variation, somatic embryogenesis, tissue culture were selected from 4500 genotypes subjected 53 was able to produce somatic embryos and
to stratified mass selection for two-year dry complete the cycle of in vitro regeneration. Only
State of the art matter yield in dense stand (1). The original 10% of the calli of this genotype actually
genetic base of 4500 genotypes was produced somatic embryos (Fig. 1), suggesting
The application of biotechnological established from a set of landraces and that not all cells in the growing calli are
techniques that require the use of in vitro varieties that were highly adapted to the competent for somatic embryogenesis. The
regeneration systems is limited by the small growing conditions of northern Italy. The efficiency of embryo conversion into rooted
number of alfalfa genotypes suitable for in vitro breeding value of the 53 elite genotypes was shoots was 40% for this genotype, a value in
regeneration and by the modest agronomic further assessed based on two-year forage the range of those in earlier reports for
value of these genotypes in terms of yield yield of their half-sib progenies. genotypes responsive to in vitro regeneration.
potential and tolerance to biotic stresses (5). For each genotype, leaflets derived from Interestingly, this genotype was also that
Identifying new, agronomically valuable different apical leaves were excised, surface- ranking first for forage yielding ability.
germplasm with good response to in vitro sterilized, and cultured in vitro on a callus Identifying agronomically valuable germplasm
regeneration has crucial importance for induction medium in a growth chamber. After for in vitro regeneration may require the
broadening the genetic base and raising the three weeks, calli were transferred to the culture screening of large genotype numbers, but its
genetic value of germplasm used for transgenic medium lacking growth regulators for potential outcome is worth the effort. ■
plant production, as well as for widening the regeneration assessment. A genotype was
genetic background of material subjected to considered as embryogenic competent when it References
functional genomics studies (e.g., on gene over- (1) Annicchiarico P (2004) A low-cost procedure for multi-
expression, gene silencing, promoter analysis, purpose, large-scale field evaluation of forage crop genetic
resources. Euphytica 140:223-229
and T-DNA tagging). Also, the availability of (2) Ding Y-L, Aldao-Humble G, Ludlowa E, Drayton M, Lin
recombinant alfalfa in vitro regeneration usually Y-H, Nagel J, Dupal M, Zhaoa G, Pallaghy C, Kalla R,
proceeds via somatic embryogenesis (4). The Emmerling M, Spangenberg G (2003) Efficient plant
regeneration and Agrobacterium-mediated transformation in
low level of plant regeneration, the genotype Medicago and Trifolium species. Plant Sci 165:1419–1427
dependence, and the lack of reproducibility (3) Moltrasio R, Robredo CG, Gómez MC, Díaz Paleo AH,
still represent major shortcomings of somatic Díaz DG, Rios RD, Franzone PM (2004) Alfalfa (Medicago
sativa) somatic embryogenesis: genetic control and
introduction of favourable alleles into elite Argentinean
_________________________________________________________________________________________________________ germplasm. Plant Cell Tiss Org 77:119–124
(4) Saunders J, Bingham E (1972) Production of alfalfa plants
Consiglio per la Ricerca e la Sperimentazione in
from callus tissue. Crop Sci 12:804–808
Agricoltura, Centro di Ricerca per le Produzioni (5) Weeks JT, Ye J, Rommens CM (2008) Development of an
Foraggere e Lattiero-Casearie, Lodi, Italy Figure 1. Somatic embryogenesis in alfalfa in planta method for transformation of alfalfa (Medicago sativa).
(massimo.confalonieri@entecra.it) callus derived from leaf explants Transgenic Res 17:587–597

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

Future alfalfa cultivars

by Paolo ANNICCHIARICO1 and E. Charles BRUMMER2*

Abstract: Alfalfa breeding will be influenced Transgenic vs. non-transgenic GM cultivars for a long time, owing to policy
by technical advances and various socio- restrictions on cultivation of GM material,
economic factors. Transgenic varieties, cultivars difficulties in arranging freedom to operate
where allowed, are likely to justify their Conceptually, transgenic cultivars with components of the GM technology, or
investment for just a few traits. While the full (commonly referred to as genetically the expense of deregulation.
potential of hybrid and semi-hybrid varieties modified or GM crops) could provide
still has to be explored, synthetic varieties numerous new traits to alfalfa produces. Hybrid vs. semi-hybrid vs.
will become less broadly based. Genomic However, the time and investment required synthetic variety
selection mainly based on a Genotyping-by- by current registration and deregulation
Sequencing approach, along with germplasm procedures for GM varieties (where they are A transgene is typically introduced to affect
selection performed in well-defined managed established) can be paid off only if the new a single trait, and breeding to improve other
environments or controlled conditions, will variety features a remarkable agronomic traits – yield, disease resistances, etc. – is still
jointly contribute to breeding cultivars progress. Thus, practically, only few traits needed to generate a commercial cultivar.
targeted to definite environmental conditions will be commercialized. The transgenic Various options exist to develop cultivars,
and utilization modes. Genetic resources and technology is most valuable for traits that whether transgenic or not. Hybrid varieties
plant physiological characteristics will be can be substantially influenced by a single of alfalfa offer a simple means of exploiting
fine-tuned to match specific growing gene. A polygenic trait such as forage heterosis between genetically distant,
conditions. digestibility can be successfully affected by a relatively narrow-based parent populations.
Key words: GM variety, hybrid variety, key gene at a crucial point of the lignin They are produced through a patented male-
selection strategies, semi-hybrid variety metabolic pathway (5). An upcoming sterility technique which is expected to
breakthrough which is foreseeable from produce about 75% hybrid seed (as male-
recent genomic studies in Medicago truncatula sterile and male-fertile parents are planted at
Introduction a seed ratio of 4:1). These varieties have not
(10) is the selection of a transgenic alfalfa
A popular aphorism states that that is able to express a condensed tannins yet shown a distinct yield advantage over
“predictions are very difficult, especially gene in its foliage (thereby reducing cattle traditional synthetic varieties (8), but their
about the future”. Indeed, the future variety bloat and protein degradation in the rumen). potential is expected to rise.
type and selection procedures for alfalfa may The insertion of transgenes for tolerance to Heterosis can also be increased by
be influenced not only by partly abiotic or biotic stresses may also produce producing semi-hybrids (i.e., free hybrids
unforeseeable technical advances but also by definite genetic progress. implying no male-sterile parent) between
future trends for various socio-economic These achievements could increase the genetically distant, well complementing
factors, such as the degree of availability of public awareness of the potential benefits populations which previously underwent
genetic resources and patented technologies, provided by GM alfalfa varieties. Part of the separate selection (4). Sativa × falcata semi-
the structure of global seed systems (e.g. work generating them may also contribute to hybrids aimed to capitalize on the wide
their concentration into a few transnational the breeding of non-GM varieties, when it genetic variation between these subspecies
seed companies), consumers‟ preferences for could highlight the gene or the genomic are limited by the modest intrinsic yield
crop types (e.g. non-GM) and cropping region in which a favourable mutation could potential of the latter subspecies, suggesting
systems (e.g. organic ones), and regional have a major influence on a relevant that developing (or creating de novo) heterotic
policies (e.g. supporting the dual-purpose metabolic pathway (e.g. one affecting the populations from genetically-distant, yet
crop for forage and energy). This brief article lignin type or the expression of condensed adapted sativa germplasm would be a
aims to devise future scenarios without tannins). This information could be preferable starting point. Thus, the full
neglecting this complex context. exploited to detect useful allelic variation or potential of semi-hybrid varieties still has to
to generate it by applying the point mutation be explored. The effective breeding of this
technique to elite non-GM germplasm. variety type will require knowledge and
The open-pollinated reproduction system convenient structuring of the potentially
of alfalfa makes gene flow to native or feral useful genetic diversity, which is easier to
populations likely, which may hinder the attain if molecular diversity and heterosis of
________________________________________________________________________________________________________
acceptance and use of transgenic alfalfa, candidate heterotic populations are
(1) Consiglio per la Ricerca e la Sperimentazione
especially in regions where organically-grown substantially related (2). Genomic selection,
in Agricoltura, Centro di Ricerca per le Produzioni if concurrently foreseen, should very likely
Foraggere e Lattiero-Casearie, Lodi, Italy
alfalfa is widespread. European seed
companies are likely to keep producing non- be specific for each heterotic population.
(2) University of California, Davis, USA
(ecbrummer@ucdavis.edu)

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Synthetic variety breeding probably still has different targeted growing conditions (e.g., will mainly be applied to seedlings or young
a major role to play in the next several irrigated and drought-stress environments; plants to maximize the number of genotypes
decades. Rather surprisingly, there is little monoculture and association with grasses; that can be evaluated, although careful
information on the optimal selection scheme etc.) (Fig. 1). Increased knowledge on assessment of the relationship between
among the many possibilities (clonal ecophysiological mechanisms that affect seedling and adult plant responses must be
selection; parent or family selection, based alfalfa‟s adaptation to specific conditions done. Finally, there is still a need for better
on half-sib progenies, full-sib progenies, or could help define target traits and field-based phenotyping methods and
different generations of selfing). Modifying morphophysiological types to select (2). equipment for yield and nutritive value, to
synthetic breeding programs to focus on a Alfalfa landraces and natural populations make alfalfa breeding more successful and
few highly selected genotypes may result in of the M. sativa complex have shown cost-effective. ■
better improvement than current broad- remarkable specific-adaptation effects in
based programs. relation to the level of drought, salinity, References
winter cold and utilization regime (mowing (1) Annicchiarico P (2007) Wide- versus specific-adaptation
Selection strategies or grazing) of the target environment (2). strategy for lucerne breeding in northern Italy. Theor Appl
Genet 114:647-657
Marker-assisted selection (MAS) for alfalfa These responses, which are likely to arise (2) Annicchiarico P, Scotti C, Carelli M, Pecetti L (2010)

production traits such as forage yield and from long-standing site-specific selection Questions and avenues for alfalfa improvement. Czech J
Genet Plant Breed 46:1-13
persistence is complicated by tetraploidy and pressures exerted on these populations in (3) Annicchiarico P, Pecetti L, Tava A (2013) Physiological
is unlikely to exploit candidate genes from their site of origin, are associated with and morphological traits associated with adaptation of lucerne
(Medicago sativa L.) to severely drought-stressed and to irrigated
M. truncatula. However, several quantitative environment-specific adaptive traits (3). environments. Ann Appl Biol 162:27-40
trait loci (QTL) for these traits have already Structuring the genetic diversity and its use in (4) Brummer EC (1999) Capturing heterosis in forage crop

been located, although they have proved relation to the target environments allows for cultivar development. Crop Sci 39: 943-954
(5) Guo D, Chen F, Wheeler JS, Winder JA, Selmon S,
largely site-specific (e.g., 9). Genotyping-by- more efficient selection strategies for wide or Peterson M, Dixon RA (2001) Improvement of in-rumen
Sequencing has the potential for cutting specific adaptation (1), and MAS can greatly digestibility of alfalfa forage by genetic manipulation of lignin
contribute to that by digging out and 0-methyltransferases. Transgenic Res 10:457-464
genotyping costs dramatically, opening the (6) Khu DM, Reyno R, Brummer EC, Monteros MJ (2012)
way to genomic selection also for this crop assembling QTL associated with the desired Screening methods for aluminum tolerance in alfalfa. Crop Sci
(7). This approach, whose success depends adaptation pattern. The thorough 52:161-167
(7) Li X, Brummer EC (2012) Applied genetics and genomics
on careful modeling and validation with exploitation of adaptation to specific regions in alfalfa breeding. Agron 2:40-61
respect to the targeted parent germplasm and through local genetic resources and selection (8) Riday H, Wagner S, Viands DR, Lamb JF, Meyer DW,

cropping environments, may ultimately and environments will help regional breeding Smith MA, Brummer EC, Boe A (2008) Sativa by falcata hybrid
variety trials. Proceedings, 41st North American Alfalfa
fundamentally change selection procedures. programs withstand the competition exerted Improvement Conference & 20th Trifolium Conference,
With the decreasing costs of genotyping by large or international programs. Dallas, USA, 1-4 June 2008, www.naaic.org/Meetings/
National/2008meeting/proceedings//Riday2.pdf
data, MAS is now limited by the availability We expect that the development of (9) Robins JG, Hansen JL, Viands DR, Brummer EC (2008)
of robust, multi-location phenotypic data. specific selection methodologies based in Genetic mapping of persistence in tetraploid alfalfa. Crop Sci
More effort will need to be placed on highly controlled environments will increase 48:1780-1786
(10) Verdier J, Zhao J, Torres-Jerez I, Ge SJ, Liu CG, He XZ,
developoing phenotyping platforms suitable in importance in breeding programs, Mysore KS, Dixon RA, Udvardi MK (2012). MtPAR MYB
for this perennial species and capable of especially for tolerance to stresses such as transcription factor acts as an on switch for proanthocyanidin
soil salinity, aluminium (6; Fig. 2), freezing, biosynthesis in Medicago truncatula. Proc Natl Acad Sci USA
reproducing the genotype responses to 109:1766-1771
drought and some diseases.These methods

Figure 1. One of CRA-FLC’s phenotyping platforms; it includes four Figure 2. In vitro selection procedure under development at Noble
environments formed by the combination of two soil types (sandy- to select forage legumes for tolerance to pH or aluminium. Plants
loam or silty-clay) by two possible water regimes (applied via grown under different pH or Al concentrations are phenotypically
microirrigators under a moving shelter) selected on the basis of their root length (photo by Maria J.
Monteros)

Legume Perspectives Issue 4 • July 2014

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Improving alfalfa tolerance to abiotic stresses

by Paolo ANNICCHIARICO1*, Yves CASTONGUAY2 and Luciano PECETTI1

Abstract: Drought, salinity and winter cold are This premise justifies our focus on drought, hydrates in storage organs under severe stress;
the main abiotic stresses that limit alfalfa yields. salt, and low temperature stresses. Actually, (ii) a water-conservation strategy implying less
Greater drought tolerance can be provided by drought is frequently associated with heat stress, water used in initial drought-stress phases due
different adaptive traits, and may be difficult to but little is known on the extent of alfalfa to restricted root development that resulted in
combine with adaptation to water-favourable genetic variation for heat tolerance. Other more water available under severe stress. It also
environments. Breeding for tolerance to abiotic stresses (e.g., soil aluminum and/or showed the difficulty of introgressing these
drought or salinity could largely rely on landrace acidic soils; low soil phosphorous available) may adaptive traits into widely-adapted germplasm,
genetic resources and managed selection well be important for specific regions. suggesting instead the selection of varieties
environments, whereas marker-assisted specifically adapted either to drought-stress or
selection requires thorough investigation by Drought tolerance moisture-favourable conditions (2). This
different possible approaches. Controlled Alfalfa is considered as fairly drought- breeding strategy, useful even for irrigated and
selection environments and molecular markers tolerant, mainly because of its deep root system. moderately-stressed, rainfed environments of
can contribute to selection for cold tolerance. In Investigations on within-species variation for northern Italy (1), could largely exploit drought-
vitro selection is expected to increase in drought tolerance and its underlying tolerant landrace genetic resources (1, 2).
importance, especially for salt tolerance. physiological mechanisms have been, however, Other recent work suggests that drought-
Key words: cold tolerance, drought tolerance, rather limited. In addition, their outcome may tolerant and drought-susceptible germplasm
frost tolerance, marker-assisted selection, salt be limited by insufficient attention to the may share similar physiological mechanisms of
tolerance specific requirements of the perennial species in stress response, the main differences being
terms of evaluation procedures, the choice of quantitative, e.g., for decrease of stomatal
Introduction physiological traits, and the concept and conductance under stress, or production of
measure of water use efficiency (which should osmolytes and antioxidants (7). Inexpensive
Drought is the main stress constraining alfalfa morphological traits, such as lower leaf wilting
production at the global scale, while low winter also take long-term plant survival into account).
Physiological studies including a stress or delayed leaf senescence under stress (2, 7),
temperatures limit alfalfa production and can contribute to selection.
cultivation in northern latitudes and high treatment should be able to mimic the cultivar
adaptive responses across agriculturally relevant Alfalfa marker-assisted selection for drought
elevations. Drought severity is expected to tolerance still requires proper exploration.
increase in most alfalfa cropping regions, owing stress environments, as in Annicchiarico et al.‟s
(2) six-month experiment in large containers Exploiting specific genes involved in tolerance
to decreasing availability of irrigation water and mechanisms is preferable but can be hindered
the combination of higher evapotranspiration (Fig. 1) subjected to progressive drought and
subsequent recovery. This study highlighted the by the complexity of the relevant gene network.
and lower and/or less regular rainfall due to Differences in major adaptive traits between
climate change (6). The increasing adoption of importance of investigating below-ground
organs, suggesting two main tolerance annuals and perennials may limit the
poor-quality, saline irrigation water will enhance exploitation of candidate genes from M.
the impact of salinity stress. Frost will hardly strategies: (i) an osmotic adjustment strategy
lose its importance as a major stress, because based on increasing water soluble carbo-
alfalfa adaptation to climate change will imply
the progressive introduction in northern
latitudes of material with lower autumn
dormancy, which is intrinsically less cold-
tolerant, to take advantage of a longer cropping
season. In addition, climate change is expected
to increase the frequency of extreme climatic
events (6), including early or late frosts,
particularly damaging to plants incompletely
hardened or partially dehardened as a
consequence of unseasonably mild
temperatures.
________________________________________________________________________________________________________

1Consiglio
Figure 1. Growth under severe drought stress of Mamuntanas (left;
per la Ricerca e la Sperimentazione in
drought tolerant Italian landrace), Erfoud 1 (centre; moderately drought
Agricoltura, Centro di Ricerca per le Produzioni
Foraggere e Lattiero-Casearie, Lodi, Italy tolerant Moroccan landrace) and Demnat 203 (right; drought susceptible
(paolo.annicchiarico@entecra.it) Moroccan landrace) in metal containers (55 cm long × 12 cm wide × 75 cm
2Agriculture and Agri-Food Canada, Québec City, deep; 21 plants per container) used for evaluating physiological and
Canada morphological traits of shoots and roots (2)

Legume Perspectives Issue 4 • July 2014

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RESEARCH

accurate phenotypic selection tools. There is a

screening methodology entirely performed
indoors under environmentally-controlled
conditions for the reliable identification of
genotypes with superior tolerance to freezing
(4). This revealed significant increases in cold
tolerance from -25°C to -29°C lethal
temperature for 50% of the plants after 6
selection cycles, associated with higher
accumulations of cryoprotective sugars (Fig. 2)
and superior expression of cold-regulated genes
Figure 2. Starch (A) and sucrose (C) concentrations in crowns of alfalfa plants from the cultivar Apica (4, 5).
and from two advanced cycles of recurrent selection for improved tolerance to freezing AP-TF5 and
AP-TF6 performed within this cultivar; plants were hardened to natural variations in temperature in Perspectives
an unheated greenhouse; reprinted (5) with permission from the Crop Science Society of America Molecular technologies provide new
opportunities to more efficiently exploit
truncatula. In the short term, another useful The greenhouse protocol proposed by Peel et extensive variability in open-pollinated alfalfa.
approach implies the investigation of marker– al. (8) also allows for the phenotypic selection The identification of molecular markers that co-
forage yield association in mapping populations of large plant numbers at the adult stage. segregate with traits of interest would facilitate
phenotyped in severely drought-prone Various recent reports point to the possible the selection of genotypes with desirable alleles
environments. This approach, if properly interest of north-African landraces as valuable and help reduce the number of plants to be
devised (e.g. using material derived from salt-tolerant parent germplasm. Knowledge on phenotypically screened. Next-generation
genetically distant, drought tolerant populations; the mechanisms featuring diverse salt-tolerant sequencing technologies will accelerate the
avoiding space-plant evaluations; etc.), allows germplasm is essential for pyramiding identification of genomic and transcriptomic
concurrent selection of drought-tolerant functionally different salt tolerance genes. sequences that affect tolerance to abiotic
varieties. Managed environments (e.g. by stresses. Identification of polymorphisms
rainout shelters and microirrigators) can reduce Cold tolerance within candidate genes will pave the way to the
the negative impact on selection and development of functional markers to screen a
The capacity of alfalfa to withstand exposure broad range of germplasms. Sustained feedback
phenotyping of year-to-year climatic variability to subfreezing temperature stands out as a core
(1). In vitro selection of alfalfa seedlings is worth interactions between breeding and gene
trait that affects productivity in cold climates. discovery programs will be required, to reap the
being explored for tolerance to drought, salt The use of cultivars adapted to local conditions
and low temperatures, exploiting recent work benefits of molecular genetics for the
and good management practices remain the improvement of alfalfa tolerance to abiotic
from other species (10). best options to minimize winter damage to stresses in the 21st century. ■
stands of alfalfa. Untimely fall harvest increases
Salt tolerance the risk of winterkill and can reduce the yield of References
Alfalfa is moderately sensitive to salinity, spring regrowth by impairing the plant capacity (1) Annicchiarico P, Piano E (2005) Use of artificial environments
reducing its forage yield by one-third with a soil to replenish organic reserves in taproots (3). In to reproduce and exploit genotype × location interaction for
lucerne in northern Italy. Theor Appl Genet 110:219-227
electrical conductivity of 8 dS m-1 in the addition, stubble removal can prevent snow (2) Annicchiarico P, Pecetti L, Tava A (2013) Physiological and
saturation extract. However, genetic variation entrapment and allow exposure of perenniating morphological traits associated with adaptation of lucerne (Medicago
sativa L.) to severely drought-stressed and to irrigated
for salt tolerance exists which involves various roots and crowns to potentially damaging environments. Ann Appl Biol 162:27-40
physiological mechanisms, such as Na+ subfreezing temperatures. (3) Bélanger G, Castonguay Y, Bertrand A, Dhont C, Rochette P,
Couture L, Drapeau R, Mongrain D, Chalifour F-P, Michaud R
exclusion, Na+ compartmentation into cell Declining photoperiod and lower (2006) Winter damage to perennial forage crops in eastern Canada:
vacuoles, and better retention of K+ in plant temperatures in the fall affects plant causes, mitigation, and prediction. Can J Plant Sci 86:33-47
(4) Castonguay Y, Michaud R, Nadeau P, Bertrand A (2009) An
tissues (9). While indirect selection criteria based development and triggers several molecular indoor screening method for improvement of freezing tolerance in
on some of these mechanisms could be changes, including the accumulation of alfalfa. Crop Sci 49:809-818
(5) Castonguay Y, Bertrand A, Michaud R, Laberge S (2011) Cold-
devised, breeding strategies for salt tolerance cryoprotective sugars and changes in gene induced biochemical and molecular changes in alfalfa populations
have essentially relied on direct selection under expression, leading to an increase in freezing selectively improved for freezing tolerance. Crop Sci 51:2132-2144
(6) IPCC (2007) Climate change 2007: Impacts, adaptation and
stress conditions or, more recently mainly in tolerance. Fall dormancy reflects the sensitivity vulnerability. Contribution of Working Group II to the 4 th
China, on the development of transgenic of cultivars to changes in the photoperiod. Assessment report of the Intergovernmental Panel on Climate
Change, 2007. Cambridge University Press, Cambridge, UK
germplasm which exploits stress tolerance However, this trait opposingly affects cold (7) Kang Y, Han YH, Torres-Jerez I, Wang MY, Tang IH,
genes from other species. adaptation and seasonal growth. The Monteros M, Udvardi M (2011) System responses to long-term
drought and re-watering of two contrasting alfalfa varieties. Plant J
Alfalfa plants may be affected by salinity at development of cultivars combining reduced 68:871-889
germination, seedling growth and mature plant fall dormancy and high level of tolerance to (8) Peel MD, Waldron BL, Jensen KB, Chatterton NJ, Horton H,
Dudley LM (2004) Screening for salinity in alfalfa: a repeatable
stages. Field selection in naturally saline soils is freezing would significantly increase alfalfa method. Crop Sci 44:2049-2053
hindered by the marked spatial heterogeneity of productivity in regions with harsh winter (9) Smethurst CF, Rix K, Garnett T, Auricht G, Bayart A., Lane P,
Wilson SJ, Shabala S (2008) Multiple traits associated with salt
the stress. A seed germination test in saline conditions. In spite of evidence that the link tolerance in lucerne: revealing the underlying cellular mechanisms.
water recommended by NAAIC has long been between the fall dormancy response and Funct Plant Biol 35:640-650
(10) Tapingkae T, Zulkarnain Z, Kawaguchi M, Ikeda T, Taji A
adopted as a fast screening criterion for variety freezing tolerance can be dissociated, the (2012) Somatic (asexual) procedures (haploids, protoplasts, cell
evaluation, and contributed largely to the development of commercial products remains selection) and their applications. In: Altman A, Hasegawa PM (eds)
Plant biotechnology and agriculture, pp. 141-162
selection of the salt-tolerant variety Ameristand impeded by insufficient knowledge of the
801S. Another NAAIC test evaluates the forage physiological basis of these traits and the lack of
production of adult plants under stress in pots.

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Improving alfalfa resistance to biotic stresses

by John IRWIN1* and David ARMOUR1

Abstract: The biology of alfalfa has up to now

necessitated its commercialisation as broadly
based synthetics, and the resulting genetic
heterogeneity has provided population
buffering against genetic shifts in pathogen and
pest populations, with new pathotype/biotype
development occurring for only a few diseases
and pests, e.g. anthracnose. The genetics of
resistance is often of complex inheritance,
which may also contribute to its durability.
Molecular markers, genetic linkage maps, and Figure 1. Phytophthora root rot (P. medicaginis), an acute (lethal) alfalfa disease. Plant dying in the
additional genomic resources from the fully field due to Phytophthora root rot (left), an actively growing lesion girdling the tap root (right)
sequenced model legume Medicago truncatula are
being employed to further understand the breeding synthetic varieties will be reduced
genetics of resistance to alfalfa biotic stresses, Biotic stresses
from around 50-100 or more to 15-20. This will
and improve their management. The biotic stresses which negatively impact lead to a greater chance of new pathotypes
Key words: genetic heterogeneity, linkage upon alfalfa productivity and persistence evolving, unless steps are taken to deploy a
mapping, Medicago truncatula synteny, multiple include diseases, insects, nematodes, diversity of resistance mechanisms against
resistances, stability of resistance invertebrates and weeds. The North American individual high risk pathogens. This can only be
Alfalfa Improvement Conference (NAAIC) achieved when genetic mechanisms conferring
Introduction lists 29 biotic stresses for which standard resistance to a pathogen have been identified
screening tests have been developed, including including their chromosomal locations, allowing
Cultivated alfalfa (lucerne) is autotetraploid resistant and susceptible checks for each stress,
(2n = 4x = 32), and belongs to the Medicago deployment of multiple resistances even in a
of which 17 are diseases, 8 insect and 4 narrowly based synthetic. The development of
sativa complex, which includes subspecies sativa, nematode stresses (3, http://www.naaic.org/
coerulea and falcata. Because alfalfa is allogamous molecular markers closely linked to resistance
resource/stdtests.php). Cultivars exhibiting genes would facilitate the breeding of multiple
and individual plants can exhibit high levels of multiple pest and disease resistances are now
self incompatibility and inbreeding depression, pest resistant synthetics based on a small
widely deployed (3). A review of alfalfa diseases number of highly productive parents with good
it is chiefly commercialised as synthetic cultivars indicates that the development of new races
(5). A consequence of this is that alfalfa combining ability and still deploying a diversity
(pathotypes) of pathogens is not a common of resistances against individual pathogens and
cultivars generally include a large number of occurrence for alfalfa. One of the few examples
non-inbred parents (e.g. about 100) or half-sib pests. In breeding alfalfa for sub-tropical
is anthracnose (Colletotrichum trifolii) where three Queensland, where anthracnose and
families, resulting in the cultivar consisting of a pathotypes have been unequivocally established
heterogeneous population of highly Phytophthora root rot (Fig. 1) are major lethal
using single differential alfalfa genotypes (6). constraints to alfalfa productivity, the above
heterozygous plants at the 4th synthetic The reasons for the stability of resistance are
generation (used for commercialisation). Thus strategy is one being pursued (5).
the population buffering afforded by the overall As well as the examples of lethal (acute)
every synthetic alfalfa cultivar varies in its genetic heterogeneity and heterozygosity
proportion of plants resistant to each individual diseases mentioned above, alfalfa is host to a
present in synthetic cultivars, and the diversity wide range of chronic diseases, mostly incited
pest or disease, and if a cultivar possesses and complexity of specific genetic resistance
> 30% resistant plants to a biotic stress, it is by fungi, in the form of crown rots and leaf
mechanisms operating against individual spots. For example, Stagonospora meliloti causes a
considered resistant to that particular stress. In pathogens. Phytophthora medicaginis is a good
alfalfa, biotic stresses necessarily have to be chronic leaf spot (Fig. 2), and a chronic to
example, where several genetically different sometimes lethal crown rot. Almost invariably,
managed through breeding for resistance or systems conditioning resistance have been
tolerance due to economic and environmental the highly winter active germplasm shows high
identified in diverse alfalfa germplasm sources susceptibility to pathogens such as
considerations. This article concentrates on (4).
diseases; however the same general principles Stagonospora, whereas the more dormant
would apply to nematode and insect pests. material of Flemish origin is relatively less
Breeding for biotic stress susceptible. The same applies to the lethal
_________________________________________________________________________________________________________
resistance diseases, with Turkestan alfalfa generally being
The University of Queensland, School of It is likely that in the pursuit of higher the source of resistant breeding material for
Agriculture and Food Sciences, Brisbane, Australia productivity, the number of parents used in diseases such as Phytophthora root rot. Within
(j.irwin@uq.edu.au)

Legume Perspectives Issue 4 • July 2014

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RESEARCH

the M. sativa complex (subsp. sativa, coerulea and

falcata), enormous genetic diversity exists,
including resistances and tolerances to a wide
range of biotic stresses.
Figure 2. Stagonospora leaf spot (Stagonospora
Linkage mapping and marker meliloti), a chronic (non lethal) alfalfa disease
assisted selection
Molecular evidence for the complexity of
genetic mechanisms conferring resistance has
been uncovered for the alfalfa pathogens C.
trifolii, P. medicaginis and S. meliloti. This has
largely been achieved through the development
of genetic linkage maps using molecular
markers of known chromosomal origin, which Table 1. QTLs identified in alfalfa conditioning resistance to three pathogens
are anchored to existing genetic or physical % of phenotypic Total number Chromosome
maps. For resistance to the above pathogens in Pathogen Race variation explained of independent location of Reference
alfalfa, the inheritance of resistance could not by largest QTL QTLs identified QTL
be explained by the segregation of a single 1 32 2 4, 8 (6)
completely dominant tetrasomic gene. C. trifolii 2 28 1 2 (6)
However, QTL analysis revealed multiple loci 4 40 2 6, 8 (6)
contributing to resistance to each of these Armour and
pathogens, with both small and large effects Irwin,
P. medicaginis - 33 4 2, 5, 6 and 7
(Table 1). unpublished
data
Application of M. truncatula – S. meliloti - 17 2 2, 7 (7)
derived technology to alfalfa a independent QTLs explaining > 6% of the phenotypic variation
The extensive genome synteny shared
between M. sativa and the model legume M.
truncatula provides a wealth of additional chromosome 4 which confers resistance to all pool, and this should always be exhaustively
molecular resources which can contribute to three C. trifolii races (8), whereas in alfalfa, searched for resistance or tolerance before
the development and understanding of biotic molecular and inheritance data indicates a more attempting interspecific crosses as outlined
stress resistance in alfalfa. Molecular markers complex genetic control of resistance operates above. ■
developed in one Medicago species will almost (6). The increased genetic complexity of
invariably map to the corresponding autotetraploid alfalfa versus diploid M. References
chromosomal location in the other. truncatula, with greater opportunities for inter (1) Anderson J R, Bentley S, Irwin JAG, Mackie J M,
Importantly, M. truncatula is host to many acute and intra locus interactions, means that findings Neate S, Pattemore JA. (2004) Characterisation of
Rhizoctonia solani isolates causing root canker of lucerne in
alfalfa pathogens and pests, including but not in M. truncatula may not always be directly Australia. Australas Plant Pathol 33:241-247
limited to C. trifolii, P. medicaginis, Aphanomyces applicable to alfalfa. (2) Armour DJ, Mackie JM, Musial JM, Irwin JAG (2008)
eutiches, spotted alfalfa aphid (SAA) (Therioaphis Transfer of anthracnose resistance and pod coiling traits
trifolii f. maculata) and bluegreen aphid (BGA) Perspectives from Medicago arborea to M. sativa by sexual reproduction.
Theor Appl Genet 117:149-156
(Acyrthosiphon kondoi). Together with the ability There are some alfalfa diseases for which no (3) Bouton (2012) Breeding lucerne for persistence. Crop
to develop inbred populations and the worthwhile resistance has been found, and Past Sci 63:734-738.
resources offered by a sequenced and Rhizoctonia root canker (Rhizoctonia solanii AG
(4) Havey MJ, Maxwell DP, Irwin JAG (1987)
Independent inheritance of genes conditioning resistance
assembled genome, M. truncatula is a useful 6) is one such disease (1). This lethal disease to Phytophthora megasperma from diploid and tetraploid
model system to study alfalfa biotic stresses. In seriously limits alfalfa utilisation in western alfalfa. Crop Sci 27: 873-879
M. truncatula, single genes have been identified Queensland, Australia, where an additional area (5) Irwin JAG, Lloyd DL, Lowe KF (2001). Lucerne
which confer resistance to bluegreen and biology and genetic improvement - an analysis of past
of up to 20 M ha could be grown in dryland activities and future goals in Australia. Crop Past Sci
spotted alfalfa aphids on chromosome 3, C. pastures. A transgenic approach may be 52:699-712
trifolii resistance on chromosome 4, and A. required for such a disease, but only if the (6) Mackie JM, Musial JM, Armour DJ, Phan HTT,
eutiches resistance on chromosome 3. However, exhaustive screening of accessions could
Ellwood SE, Aitken KS, Irwin JAG (2007). Identification
of QTL for reaction to three races of Colletotrichum trifolii
the translation of findings from the model exclude the occurrence of resistance within the and further analysis of inheritance of resistance in
system to alfalfa is likely to uncover additional M. sativa complex. Armour et al. (2) were able autotetraploid lucerne. Theor Appl Genet 114:1417-1426
complexities. A further study into to transfer resistance to C. trifolii from Medicago (7) Musial JM, Mackie JM, Armour DJ, Phan HTT,
Aphanomyces resistance in M. truncatula found Ellwood SE, Aitken KS, Irwin, JAG (2007). Identification
arborea (2n = 4x = 32) to alfalfa through the of QTL for resistance and susceptibility to Stagonospora
that the major gene on chromosome 3 offered production of partial hybrids, using male sterile meliloti in autotetraploid lucerne. Theor Appl Genet 114:
broad spectrum resistance, with numerous alfalfa as the female parent. M. arborea, being the 1427-1435
additional race specific resistance loci on other longest-lived Medicago spp, may be a source of
(8) Yang S, Gao M, Xu C, Gao J, Deshpande S, Lin S,
Roe BA, Zhu H (2008). Alfalfa benefits from Medicago
chromosomes; a situation likely to occur in new resistances to biotic and abiotic stresses truncatula: The rct1 gene from M. truncatula confers broad-
alfalfa. A further example of this is the which can be used to improve alfalfa. However, spectrum resistance to anthracnose in alfalfa. Proc Natl
finding of a single gene on M. truncatula there is enormous diversity in the alfalfa gene Acad Sci USA 105: 12164-12169

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

Alfalfa as a grazed crop

by Luciano PECETTI

Abstract: Alfalfa has great potential for livestock systems in the more favorable Ad-hoc selection of grazing-
grazing, but an acknowledged limit to this environments, and to maintain the economic
exploitation is the poor persistence of profitability in marginal areas where little tolerant alfalfa
common, hay-type germplasm under agronomic alternative to extensive livestock The research carried out in the last few
continuous and intensive grazing. Morpho- exists. decades provided better comprehension of the
physiological traits were identified that may morphological and physiological mechanisms
enhance the grazing tolerance. However, direct Possible limits of alfalfa underlying the tolerance to grazing in alfalfa.
selection for tolerance under continuous grazing Several traits were claimed to contribute to
stocking proved more effective than the grazing tolerance, including decumbent growth
indirect selection for positive traits such as A major drawback to the use of alfalfa as a habit, large and deep-set crown, prolific
growth habit and spreading ability, and grazing crop is the recognized poor persistence underground budding, large stubble leaf area
contributed to the release of tolerant varieties. of traditional cultivars under intensive below the grazing height, and high root
Successful attempts were made to combine the exploitation. Although a rational alfalfa grazing carbohydrate reserves. However, indirect
advantages of favorable morphological traits should „mimic‟ hay cutting, with rest intervals of selection for these traits generally proved less
with those of an effective selection method. 30-35 days following short grazing periods of 5- efficient in selecting tolerant alfalfa than the
Ongoing studies are trying to break a possible 7 days (4), prolonged stocking and overgrazing direct selection for persistence under actual
relationship between grazing tolerance, high may frequently be caused by various events, grazing (10). A useful selection method devised
cold-season dormancy, and low vigor. such as, for instance, unfavorable climatic in the USA is based on continuous grazing and
Key words: crown, dormancy, grazing conditions, reduction of pasture areas due to high stocking rate, which causes overgrazing of
tolerance, growth habit, persistence, plant unexpected changes in crop rotations, or the evaluated germplasm and subjects all plants
morphology excessive animal number following a favorable to any possible stress associated with grazing
market conjuncture. There is large evidence (defoliation, tearing, trampling, excretions),
that continuous stocking and intensive grazing thereby minimizing the chance for non-tolerant
Introduction of hay-type alfalfa cause severe stand thinning. genotypes to be selected (9). This method was
Although mainly exploited worldwide for The plants of this kind of cultivars generally eventually adopted as a standard test of
hay-making, alfalfa (Medicago sativa L.) can also have small and shallow crowns, which may evaluation by the North American Alfalfa
be a valuable crop for grazing and, as such, it is easily be damaged by animal trampling and Improvement Conference (2), and resulted in
largely grown in countries such as Argentina, become susceptible to biotic (e.g., fungal the release of varieties with enhanced
the USA, Canada and Australia. An infections) or abiotic (e.g., frost) stresses. persistency that has increased the use of alfalfa
enhancement of pasture productivity through Moreover, the continuous defoliation as a grazing crop (3). Further work proved that
alfalfa cropping is widely recognized (8). determines a progressive depletion of the varieties selected by this method could also
Grazing of alfalfa has traditionally been underground reserves, with further, negative retain their better stand survival under well-
disregarded as a specific crop exploitation in effects on the plant longevity. managed grazing systems (1).
Europe. It was seen, at most, as a by-product of
the hay-making activity in southern countries,
where cool-season regrowths were frequently Table 1. Growth habit, autumn dormancy, tolerance to continuous sheep grazing, and forage yield
„cleaned up‟ by grazing. In recent decades, under a mowing regime of alfalfa experimental and check cultivars in Lodi, Italy (6)
however, alfalfa grazing is also experiencing an Autumn Survival after Dry-matter
increasing interest in South Europe, in parallel Cultivar Growth habit
dormancy (1) grazing (%) (2) yield (t/ha) (3)
with a re-consideration of the role of grazing.
Owing to its acknowledged positive features, Camporegio Prostrate 2 90.4 a 25.02 e
such as perenniality, high forage quality, La Diana Semi-prostrate 2 69.2 b 29.38 d
remarkable drought tolerance, improvement of
soil fertility and low farming costs, alfalfa can Fontegaia Semi-prostrate 3 70.4 b 30.39 cd
substantially contribute both to increase the Mens Sana Semi-erect 3 60.0 bc 32.29 bcd
environmental sustainability of semi-intensive Sunto Semi-erect 4 44.7 c 34.14 bcd
Verbena Semi-erect 4 68.9 b 34.79 bc
_________________________________________________________________________________________________________ Alfagraze (check) Semi-erect 2 71.1 b 33.78 bcd
Consiglio per la Ricerca e la Sperimentazione in Prosementi (check) Upright 6 1.9 d 41.70 a
Agricoltura, Centro di Ricerca per le Produzioni Means followed by different letters differ at P < 0.05 according to Duncan’s test; (1) Scale from 1 = maximum, to
Foraggere e Lattiero-Casearie (CRA-FLC), Lodi, 11 = minimum dormancy, recorded according to NAAIC standard test; (2) Average of two years (average grazing
Italy (luciano.pecetti@entecra.it) period of ca. 150 days year-1); (3) Sum of three years (total of 12 harvests)

Legume Perspectives Issue 4 • July 2014

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RESEARCH

regions with mild winters, such as those with

Mediterranean climate. We have implemented,
therefore, a new path of research also involving
molecular tools, aiming at investigating the
genetic correlations among dormancy,
morphology and grazing tolerance, and
identifying germplasm tolerant to grazing and
characterized by limited dormancy and good
seed yield. ■

References

(1) Bouton JH, Gates RN (2003) Grazing-tolerant

alfalfa cultivars perform well under rotational
stocking and hay management. Crop Sci 43:1461-
1464
(2) Bouton JH, Smith SR Jr (1998) Standard test to
characterize alfalfa cultivar tolerance to intensive
grazing with continuous stocking. In: Fox CC,
Berberet R, Gray FA, Grau CR, Jessen DL,
Peterson MA (eds) Standard Test to Characterize
Alfalfa Cultivars, 3rd edition, p. A-8
https://www.naaic.org/stdtests/Grazing.html
Figure 1. Representative plots of Verbena, Alfagraze (tolerant check), Prosementi (intolerant check) and (3) Henning JC (2000) On-farm use of alfalfa for
Camporegio at the spring regrowth following two years of continuous sheep grazing with high stocking grazing. In: Proceedings 37th North American
rate in Lodi, Italy Improvement Conference, 359-363
(4) Hoveland CS (1994) Alfalfa grazing
management for yield, quality and persistence. In:
Proceedings ,National Alfalfa Grazing
Conference, 17-22
In our breeding program at CRA-FLC, we a contiguous, mown sub-experiment. The (5) Pecetti L, Romani M, Piano E (2006)
tried to implement the standard test by applying prostrate cultivar „Camporegio‟ had outstanding Persistence of morphologically diverse lucerne
continuous stocking and intensive grazing to grazing tolerance (Fig. 1; Table 1), although under continuous stocking and intensive grazing.
germplasm that had preliminarily been selected associated with low forage yield in the absence Crop Past Sci 57:999-1007
for the presence of putatively positive traits, of grazing (Table 1). This confirmed a certain (6) Pecetti L, Romani M, De Rosa L, Piano E
such as large and deep crowns, proliferation of trade-off between plant vigor and trait (2008) Selection of grazing-tolerant lucerne
crown buds and high plant spreading ability, architecture conferring grazing tolerance, cultivars. Grass Forage Sci 63:360-368
(7) Piano E, Valentini P, Pecetti L, Romani M
with the aim of combining the advantages of a already observed in other studies. As also
(1996) Evaluation of a lucerne germplasm
suitable morphology with those of an effective shown in other work (5), the poor persistence collection in relation to traits conferring grazing
selection method. Our activity basically entailed of „conventional‟ upright types under grazing is tolerance. Euphytica 89:279-288
a three-step work, the first one being the likely to offset any advantage in herbage yield (8) Popp JD, McCaughey WP, Cohen RDH,
screening of large germplasm collections and that they may have under a mowing regime. McAllister TA, Majak W (2000) Enhancing
the identification of plants with the required The semi-erect cultivar „Verbena‟ possessed a pasture productivity with alfalfa: a review. Can J
morphology (7). The identified genotypes were good balance between grazing tolerance and Plant Sci 80:513-519
grouped and intercrossed according to potential yielding ability (Table 1; Fig. 1). (9) Smith SR Jr, Bouton JH, Hoveland CS (1989)
Alfalfa persistence and regrowth potential under
„models‟, that is, ideotypes defined according to Verbena has been registered in the Italian
continuous grazing. Agron J 81:960-965
morphology and vigor of the aerial parts and Register of varieties and is currently under (10) Smith SR Jr, Bouton JH, Singh A,
underground organs. These ideotypes ranged registration elsewhere in Europe, while McCaughey WP (2000) Development and
from the very rhizomatous, prostrate type with Camporegio is completing its registration evaluation of grazing-tolerant alfalfa cultivars: a
great spreading ability, to the semi-erect model process in Italy. review. Can J Plant Sci 80:503-512
with remarkable shoot proliferation from a There appeared to be a close, possibly
deep and robust crown (7). The progenies thus genetic, relationship between grazing tolerance,
obtained were evaluated under sheep grazing in marked cold-season dormancy, low stature, low
the second phase with reference to tolerant and vigor and low seed yield (5; 6; Table 1). This
intolerant check varieties (5). The most may have been determined by the large use of
promising germplasm was synthesized into a set germplasm originating from cold regions
of experimental cultivars, which were evaluated (Central Europe or North America) and often
under grazing in the last step of the selection introgressed with M. sativa subsp. falcata (7),
process (6). Besides persistence under grazing which possessed morphological features
in two years (with an average grazing period of complying with our breeding targets. While
ca. 150 days year-1), forage yield was also being adapted to temperate, cool areas, the
assessed for three years (total of 12 harvests) in selected germplasm may be less suitable for

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Alfalfa intercropping and competitive ability

by Paolo ANNICCHIARICO1, Bernadette JULIER2*, Gaëtan LOUARN2 and Amel MAAMOURI2

Abstract: Alfalfa is subjected to interspecific We briefly review alfalfa competitive partially drives the acquisition of other
competition in two situations: (i) intercropping relationships mainly with respect to their resources. Furthermore, as light availability
with a forage grass, where the required long- relevance to breeders. One major issue in this declines exponentially with the distance from
lasting equilibrium between species is frequently context is whether alfalfa varieties targeted to the top of the canopy, minor differences in
threatened by excessive alfalfa competitive mixed cropping with grasses, or targeted to plant size can have major effects on the relative
ability; (ii) competition against weeds, which can cropping under severe weed competition (as in yield of the intercropped species. The amount
be severe in monoculture and requires high most organic systems), could be successfully of captured light defines the potential growth
competitive ability to avoid any chemical weed selected or recommended from testing trials rate and the nitrogen demand of the crops.
control. In both cases, competition for light carried out in the simpler condition of absence Besides energy capture and photosynthesis,
interception is the main mechanism that drives of competition. The response, which depends competition for water and nutrients is also
the species proportion. Vegetative vigour and on the degree of genetic correlation between critical in explaining the dynamics of plant
plant architectural traits can affect the alfalfa selection and target environments, has far- mixtures.
competition dynamics across successive reaching implications, e.g. for the choice of
harvests. Once completely unveiled, these traits phenotyping conditions in marker-assisted Competitive ability and
could be selected for new varieties with selection studies, or procedures for assessing compatibility with grasses
improved adaptation to competition. the variety value for cultivation and use.
Key words: interspecific interference, legume- The companion grass tends to be at
grass mixtures, organic agriculture, plant Competition competitive disadvantage and less persistent
competition, weed competition than alfalfa when associated with highly
Alfalfa-grass intercropping and weed-invaded productive alfalfa varieties under favourable
alfalfa crops are situations of interspecific plant cropping conditions, while the reverse may
Introduction competition which imply different targets. The occur when alfalfa is grown in shallow soils or
Our focus on alfalfa competitive relationships target is maintaining a desirable legume-grass suffers from poor establishment.
is two-fold here, i.e.: (i) intercropping with a balance in the former case, and excluding the In alfalfa, traits related to growth habit, shoot
grass companion, to obtain a long-lasting, weeds in the latter. Competition for resources is development and branching (leaf area
balanced mixture of the two components of one of the major processes controlling plant expansion), and internode length (vertical leaf
the mixture; (ii) competition against weeds, to growth and explaining the dynamics of plant area distribution) showed a prominent role in
avoid any chemical weed control. Legume-grass mixtures. Competition for light is of primary light interception, and mainly contribute to its
mixtures are gaining new interest in Europe and importance, because it determines the energy advantage compared to grasses (4). For legumes
elsewhere, owing to the energy and available for all physiological processes and
environmental costs associated with the
synthesis and use of nitrogen fertilizer required
for grass forage production and the quest for
greatest self-sufficiency in feed proteins at the
farm and the country levels. Legume-grass
mixtures are currently grown over 70% of the
sown grassland in France, exploited as hay,
silage or grazed forage. Alfalfa monoculture is
still prevalent in other countries, e.g. Italy,
where it is the backbone of organic crop-
livestock systems and contributes significantly
to conventionally managed systems.
Alfalfa competitive ability against weeds is
crucial for organic systems and increasingly
important for conventional ones, especially for
the pure stand.

_________________________________________________________________________________________________________

1Consiglio per la Ricerca e la Sperimentazione in

Figure 1. Biomass yield in the first harvest of 2011 of alfalfa genotypes grown in mixtures (one target
Agricoltura, Centro di Ricerca per le Produzioni
Foraggere e Lattiero-Casearie, Lodi, Italy
alfalfa plant surrounded by 2 alfalfa and 4 tall fescue plants) and in pure stands (one target alfalfa
2INRA, UR4, Unité de Recherche Pluridisciplinaire plant surrounded by 6 alfalfa plants from the tall, erect cultivar Orca); the line is for equal biomass
Prairies et Plantes Fourragères, Lusignan, France production in both stands
(bernadette.julier@lusignan.inra.fr)

Legume Perspectives Issue 4 • July 2014

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RESEARCH

(reproducing organic and conventional systems,

respectively) which were established in one
experiment in Lombardy (northern Italy). On
average, landrace and variety germplasms
showed comparable competitive ability against
weeds. Competitive ability of the cultivars was
strictly related to their forage yielding ability in
the absence of weeds (2), with a semi-prostrate
cultivar showing lowest values of both traits
(Fig. 2). This relationship agrees with the crucial
importance attributed to high relative growth
rate for competitive success of genotypes
belonging to erect, vigorous forage species
Figure 2. Forage yield of eight alfalfa cultivars across conditions reproducing organic or conventional grown in relatively favourable environments (5),
cropping systems in two geographic areas (Lombardy, northern Italy; Umbria, central Italy), and when considering that high relative growth rate
genotype × environment (GE) interaction and phenotypic correlation for cultivar yield across also implies high forage yield in the absence of
environments; the graph pools results from the studies (2) and (3) competition. This relationship may be much
looser in less vigorous, subordinate species such
as white clover, whose genotype competitive
in mixture, intercepted light amount also cropping because of greater compatibility with ability mainly relies on the ability to make fine-
determines the maximal amount of nitrogen (hence, lower competitiveness against) grasses is scale exploitation of light and nutrients from
that can be fixed by nodules (6), which can also worth being investigated. A set of early undepleted zones through morphological
reach around 180 kg N ha-1 year-1 in alfalfa pure studies in Lodi indicated the consistent ranking plasticity (1). The high consistency of cultivar
stands (9). of alfalfa cultivars for biomass yield across pure- yield responses across high vs. negligible weed
The root systems of grasses and alfalfa differ stand and mixed-stand conditions (11). competiton conditions contrasts with the low
strongly in terms of architecture (fasciculate vs. However, preliminary results in Lusignan consistency across two Italian cropping regions
taproot), root length density (high vs. low) and suggest at least some degree of genotype (northern vs. central Italy) (3; Fig. 2), suggesting
rooting depth (short vs. long) (7). Grasses are inconsistency between these conditions (Fig. 1). that distinct material for each of these regions
better competitors for water and most soil This experiment, which includes the be bred with no specific selection for organic
nutrients (mineral N, P and K in particular) observation of a number of morphological systems and/or higher competitive ability.
than alfalfa, especially within the topsoil layers, traits, will be used to test whether other alfalfa Compatibility with grass companions, and
but alfalfa explores deeper soil horizons. traits besides lower vigour may be associated ability to outcompete weeds, likely inquire
However, alfalfa is little affected by soil N with greater compatibility with associated different optima of alfalfa competitive ability.
competition under conditions favourable to N2 grasses. We hypothesize that an alfalfa cultivar They are associated with vegetative vigour and
fixation, owing to its ability to fix atmospheric with lower stem height, more branching and plant architectural traits yet to be discovered,
nitrogen (N2). The proportion of N derived good regrowth dynamics after cutting would which could eventually be selected for to
from fixation is even higher in mixtures than in have a reasonable high yielding ability in pure produce varieties with improved adaptation to
pure stands as a consequence of the stand and would be a less aggressive conditions of competition. ■
specialization of each species on a particular N companion crop for the grass in mixed stand,
resource (atmospheric N for alfalfa; soil N for because of less competition for light References
grasses). N2 fixation is intensively affected by interception. Breeding for all other traits that (1) Annicchiarico P (2003) Breeding white clover for increased ability to
water limitation, because this physiological contributes to alfalfa persistency and quality compete with associated grasses. J Agric Sci 140: 255-266
(2) Annicchiarico P, Pecetti L (2010) Forage and seed yield response of
process is one of the most sensitive to water would still be required for these new varieties. lucerne cultivars to chemically-weeded and non-weeded managements
stress. A drought event can thus quickly reduce Choosing highly competitive grass companions and implications for germplasm choice in organic farming. Eur J Agron
33: 74-80
the nitrogen autotrophy of alfalfa, changing can also be important to ensure more balanced (3) Annicchiarico P, Pecetti L, Torricelli R (2012) Impact of landrace
drastically its ability to compete with grasses. associations. germplasm, non-conventional habit and regional cultivar selection on
forage and seed yield of organically-grown lucerne in Italy. J Agric Sci 150:
The relationships between species in a 345-355
mixture are not only competitive: they may Competitive ability with weeds (4) Baldissera TC, Frak E, de Faccio Carvalho PC, Louarn G (2013) Plant
development controls leaf area expansion in alfalfa competing for light.
produce beneficial effects by modifying the Weed density tends to be higher in alfalfa Ann Bot 113:145-157
environment of the other species. This is monocultures than alfalfa-grass mixtures. The (5) Campbell BD, Grime JP, MacKey JML (1991) A trade-off between
scale and precision in resource foraging. Oecologia 87: 532-538
frequent is grass-legume mixtures, because a false seeding technique, which is frequently (6) Corre-Hellou G, Fustec J, Crozat Y (2006) Interspecific competition
portion of the nitrogen fixed by legumes can be adopted in organic agriculture, can be very for soil N and its interaction with N2 fixation, leaf expansion and crop
growth in pea-barley intercrops. Plant Soil 282: 195-208
transferred to the associated grasses. Such useful to limit weed density and aggressiveness (7) Haynes RJ (1980) Competitive ability of the grass-legume association.
effects tend to be more limited in alfalfa-grass in the early, crucial stage of crop establishment. Adv Agron 33: 227-256
(8) Justes E, Thiébeau P, Cattin G, Larbre D, Nicolardot B (2001)
than in clover-grass mixtures (10). Weed competition after the first mowing is Libération d‟azote après retournement de luzerne. Perspect Agric 264:22-
Maintaining a desirable balance between grass limited to species which are able to regrow. 28
(9) Louarn G, Corre-Hellou G, Fustec J, Lô-Pelzer E, Julier B, Litrico I,
and alfalfa may be achieved by strategic choices The genetic variation for ability to compete Hinsinger P, Lecomte C (2010) Déterminants écologiques et
at sowing (choice of alfalfa cultivars, grass with weeds has been poorly studied in alfalfa. physiologiques de la productivité et de la stabilité des associations
graminées-légumineuses. Innovations Agron 11:79-99
species, date of seeding; density, seeding One study whose results are partly summarized (10) Tomm GO, van Kessel C, Slinkard AE (1994) Bi-directional
pattern) and by tactical management options in Fig. 2 revealed substantially consistent transfer of nitrogen between alfalfa and bromegrass: Short and long
term evidence. Plant Soil 164:77-86
(fertilisation, cutting). The selection of alfalfa responses of eight cultivars across conditions of (11) Rotili P (1985) Strutture prative binarie. Considerazioni sui risultati
varieties which are specifically adapted to mixed high or almost nil weed competition sperimentali ottenuti a Lodi. Riv Agron 19: 170-177

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

Improving alfalfa forage quality

by Carla SCOTTI1* and Bernadette JULIER2

Abstract: Morphological and physiological protein content varies from 9% - 11% to 16% - (4). A second strategy is to act on the stem
traits contributing to the nutritive value of 20% from the basis to the top of the stem. The morphology by increasing the node number,
alfalfa forage are examined at harvesting and dynamics of shoot elongation during a decreasing the average internode length and
during a productive cycle. Leaf-to-stem ratio regrowth affects the forage quality through making it more uniform along the stem. That
and the dynamics of development of stem changes in the proportion of leaves and the resulted in higher leaf-to-stem ratio (0.80 vs.
internodes appear the main factors driving the developmental stage of the internodes (3). At 0.76 in experimental synthetics selected for
nutritive value at the plant level. Within- the beginning of regrowth just after harvest, the short and long internodes, respectively) and a
population variation for these traits, in addition biomass of leaves is prevailing as internodes final increase of CP production per plant (5).
to between-population variation, can be have not yet entered the elongation phase. Since the development stage is negatively
exploited to improve digestibility and protein When the stems reach about 30 cm height, the correlated with plant CP concentration and
content. proportion of leaves has decreased but most positively related with fiber fractions content,
Key words: alfalfa, crude protein, fiber internodes are still in the elongation stage. one further strategy is the uncouplement of
fractions, leaf-to-stem ratio Around the phenological stage of 50% green growing and development processes by
bud stem, when the stems can reach 80 cm to selection for high DM production and delayed
The nutritive value of alfalfa forage is mainly 100 cm height, stem biomass becomes triggering to reproductive phase (high
a result of the forage chemical composition and prevalent as a result of internode elongation, vegetative-to-reproductive node ratio).
in particular of the content of crude protein and and most internodes are in a mature stage with Within-population variation for plant and
fiber fractions (neutral detergent fiber, NDF, high lignification; leaf biomass remains stable, stem morphology, multifoliolate trait and rate
acid detergent fiber, ADF, and acid detergent as newly formed leaves are counterbalanced by of secondary wall deposition (1) can account
lignin, ADL) estimating the proportion and the senescence and loss of older leaves triggered for a major fraction of the genetic variation in
composition of cell walls. ADF and ADL by the decline of incidence radiation (4). As a digestibility among alfalfa plants with
contents show strong negative correlation with consequence, protein and fiber contents of comparable biomass production. High-
ruminal digestibility of forage. alfalfa forage are mainly driven by the leaf-to- throughput screening of forage nutritive value
An alfalfa stem at harvest time includes a stem biomass ratio and secondly by the by means of near infrared reflectance
series of internodes separated by nodes carrying developmental stage of internodes. (Fig. 1). spectroscopy (NIRS) is currently used to
a leaf, and a branch (vegetative nodes) and/or a The main strategies adopted to improve exploit such variation. One last strategy for the
flower or pod (reproductive nodes). Proteins alfalfa protein content and digestibility rely on genetic improvement of alfalfa stem quality
are mainly located in the the green and healthy these morpho-physiological traits. One strategy relies on genetic transformation applied to a
leaves. Leaf protein content shows a limited is improving alfalfa tolerance to an early cutting crucial point of the lignin metabolic pathway
variation (25% - 30% of biomass) that is schedule (e.g. cutting at 5% blooming), which (2). Interestingly, alfalfa higher stem digestibility
determined by leaf position along the stem, implies plant selection for early restoration of could positively impact not only on ruminant
with the highest contents in the upper young nitrogen root reserves in order to fuel the shoot nutrition but also on bio-conversion processing
leaves exposed to light. Similarly, fiber content regrowth and ensure sufficient crop persistence for ethanol production. ■
of the leaves is rather stable (20% - 25% NDF,
15% - 18% ADF). Stems, on the contrary, References
show a high variation for fiber content that is (1) Guines F, Julier B, Ecalle C, Huyghe C (2003) Among- and
within-cultivar variability for histological traits of lucerne (Medicago
related to the developmental stage of the sativa L.) stem. Euphytica 130:293-301
internodes (not yet elongating, elongating or (2) Guo D, Chen F, Wheeler JS, Winder JA, Selmon S, Peterson M,
Dixon RA (2001) Improvement of in-rumen digestibility of alfalfa
mature). In the basal internodes, the mature forage by genetic manipulation of lignin 0-methyltransferases.
stage implies a higher content in secondary Transgenic Res 10:457-464
(3) Lemaire G, Cruz P, Gosse G, Chartier M (1985) Etude des
lignified vascular tissues (mainly xylem) than in relations entre la dynamique de prélèvement d'azote et la
elongating internodes. ADF content of stem dynamique de croissance en matière sèche d'un peuplement de
luzerne (Medicago sativa L). Agron 5:685-692
portions can vary from 55% in the basal (4) Rotili P, Gnocchi G, Scotti C, Zannone L (1991) Some
internodes to 40% in the apical ones. In parallel, problems in the alfalfa breeding for quality: Protein content.
Proceedings, EUCARPIA IX Medicago sativa Group Meeting,
Kompolt, Hungary, 11-13 September 1990, 5-33
(5) Scotti C, Gnocchi G, Carelli M, Pintus B, Ursino A, Odoardi M
(2006) Breeding of the alfalfa stem morphology for quality.
_________________________________________________________________________________________________________ Proceedings, EUCARPIA Fodder Crops and Amenity Grasses
1Consiglio Section and Medicago spp Group Meeting, Perugia, Italy, 3-7
per la Ricerca e la Sperimentazione in September 2006, 80-83
Agricoltura, Centro di Ricerca per le Produzioni
Foraggere e Lattiero-Casearie, Lodi, Italy Figure 1. Relationship between forage
(carla.scotti@entecra.it) digestibility or ADF content and leaf proportion in
2INRA, UR4, Unité de Recherche Pluridisciplinaire the biomass (from Lemaire and Allirand,
Prairies et Plantes Fourragères, Lusignan, France Fourrages, 1993)

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 RESEARCH

Alfalfa private breeding: American and European

overview
by Mark McCASLIN1 and Vincent BÉGUIER2*

Abstract: Since the release of the first required for adaptation to primary markets. be commercialized in alfalfa (1). Genetically-
commercial cultivars like Vernal in USA or The primary markets for the FD 2-5 varieties engineered alfalfa with reduced lignin
Europe in France, much progress has been are the U.S., Canada and China. The primary content is in the early stages of government
achieved in private alfalfa breeding. Private markets for the FD 6-10 varieties are the regulatory review in the U.S. and Canada,
breeding is still very active in North America U.S., Argentina, Mexico and the Middle East and several other potential transgenes are
and in Europe but with some difference in (3). All the U.S. based breeding programs being evaluated by U.S. breeding companies
organization (consolidated in USA, diverse in conduct selection nurseries and/or variety for improved forage quality, increased
Europe), and in breeding achievements and trials in most of the primary markets listed biomass production, abiotic stress tolerance
objectives - particularly with the emergence above. and/or novel herbicide tolerance (7, 9). It is
of molecular breeding and transgenesis. All three of the U.S.-based alfalfa breeding likely that transgenic varieties first
Key words: alfalfa breeding, alfalfa markets, companies conduct both conventional commercialized in the U.S. will also be
genetic engineering, molecular breeding breeding programs and research designed to deregulated and commercialized in Canada,
develop and deploy transgenic traits. The Mexico and Argentina.
Introduction breeding focus has generally been on three Although the scope of industry-funded
traits: forage yield, persistence, and forage alfalfa research in North America has
Commercial companies have conducted quality. All three companies have dedicated broadened greatly to now include molecular
alfalfa breeding programs since the late resources to develop molecular breeding breeding, applied genomics, and genetic
1950‟s. Most of this activity has been in tools for alfalfa, although marker assisted engineering – the total industry alfalfa
North America, Europe and Australia. breeding is in early stages of development in research spend has decreased significantly
Today, 746 varieties are registered on the alfalfa compared to corn and soybeans (6). over the last twenty years because of
OECD list (8). Many of these cultivars were All three companies also have multiple U.S. consolidation. This corresponds to a similar
developed by industry breeders, alfalfa research stations and employ several loss of alfalfa research funding in the public
demonstrating the significant and successful PhD level scientists to drive their research sector. Fortunately there has been an
research investments dedicated to this crop efforts. increase in public/private collaboration,
species. Roundup Ready alfalfa, produced in the specifically on long term strategic projects
In this article, we will focus on private U.S. in 2005, was the first transgenic trait to with potential high impact (7).
breeding in North America and Europe.

Private alfalfa breeding in

North America
The North American private alfalfa
breeding effort has consolidated rapidly in
the last two decades. In 2013, only three
large U.S.-based alfalfa breeding programs
(Dow, DuPont/Pioneer and Forage
Genetics International) represent > 95% of
the industry-funded alfalfa improvement
research in North America (7). This U.S.
breeding effort focuses on multiple global
markets and spans fall dormancy (FD)
groups 2-10, with an emphasis on combining
multiple pest resistance and agronomic traits

_________________________________________________________________________________________________________ Figure 1. Variety trial in Pennsylvania including numerous experimental varieties resistant to
1Forage Genetics International, Shoreview, USA potato leafhopper (green color) and some susceptible commercial varieties (yellow color);
2Jouffray-Drillaud/GIE GRASS, Saint Sauvant, resistance to this pest, which is the most damaging one in Eastern US, requires glandular
France (vbeguier@jouffray-drillaud.fr) trichomes whose genes were introgressed into alfalfa from wild relatives

Legume Perspectives Issue 4 • July 2014

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RESEARCH

Private alfalfa breeding in Table 1. Number of alfalfa varieties registered in Europe by country
Europe Number of
The European private alfalfa breeding is Country alfalfa varieties
not as consolidated as in North America. on the list
Each European country has his own Italy 153
registration list and although a variety
registered on a national list can be France 54
commercialized in all Europe (defined here Hungary 31
as the European Union), we still have strong Greece 26
targeting of local seed markets. Italy (where
many varieties derived simply from mass Spain 24
selection of farm landraces) and France are Poland 21
the countries where alfalfa cultivar Bulgaria 18
registration and seed production are most
active (Table 1). But in Eastern Europe too, Czech Republic 18
alfalfa breeding has been very active, mainly Germany 15
due to the efforts of universities and state Romania 15
breeding stations.
The European cultivars originate mainly Slovakia 14
from local landraces developed in varieties in Austria 11
the 1960‟s and 1970‟s, from dormancy
groups 3-5 in north-western continental Switzerland, Denmark, Lithuania, Luxembourg,
Europe, and dormancy groups 6-7 in Latvia, Netherlands, Norway, Portugal, Sweden, < 10
southern Europe. The Flemish landrace is Slovenia
the main contributor of dormancy group 3-
5. For the dormancy 6-7, local landraces Belgium, Cyprus, Finland, Ireland, Iceland,
0
Malta, United Kingdom
from southern European countries have
been used, such as the Aragon landrace in
Spain and Romagnola in Italy.
The dormancy group 3-5 is characterized
by a very good standing ability, and high
resistance to Verticillium wilt (caused by Seed production also will be an important (6) Li X, Brummer E.C. (2012) Applied Genetics
Verticillium albo-atrum). French breeders have feature, as the competition with cereal crops and Genomics in Alfalfa Breeding. Agron 2:40-61
developed varieties with high resistance to gets globally more and more acute. ■ (7) McCaslin M (2008) State of the Seed Industry.
Proceedings, 2008 California Alfalfa Symposium,
stem nematode (caused by Ditylenchus dipsaci)
http://alfalfa.ucdavis.edu/+symposium/proceedi
(5). For the dormancy group 6-7, resistance References ngs/2008/08-37.pdf
to anthracnose (caused by Colletotrichum (1) Fisher M (2011) GM Alfalfa – Examining the (8) McCaslin M (2010) Re-Designing Alfalfa for
trifolii) has been one of the main objectives. Benefits, costs and potential for cross- Improved Dairy Performance. Proceedings, 2010
The latest breeding targets are increased contamination. Crop Sci News May 2011 California Alfalfa Symposium, http://alfalfa.
levels of protein content and digestibility (9). https://www.agronomy.org/files/publications/cs ucdavis.edu/+symposium/2010/files/talks/CAS2
These characteristics are now taken in a-news/gm-alfalfa.pdf 7_McCaslinRedesigning%20Alfalfa.pdf
(2) Flajoulot S, Ronfort J, Baudouin P, Barre P, (9) OECD (2012) http://www.oecd.org/tad/
account for the registration of new varieties
Huguet T, Huyghe C, Julier B (2005) Genetic standardsforseedstractorsforestfruitandvegetables/
on the French list of varieties. The latest diversity among alfalfa (Medicago sativa) cultivars 2012_7_1%20Grasses%20and%20legumes.pdf
challenge is developing new alfalfa synthetics coming from a breeding program using SSR (10) Undersander D, McCaslin M, Sheaffer C,
better adapted to sustainable agriculture, with markers. Theor Appl Genet 111: 1420-1429 Whalen D, Miller D, Putnam D (2009) Low lignin
a particular emphasis on the adaptation in (3) Frey P, McCaslin M, Miller D (2011) alfalfa: Redifining the yield/quality tradeoff.
mixtures with forage grasses. Coexistence for alfalfa seed export market. Proceedings ,2009 California Alfalfa Symposium.
European breeders are not involved in the National Alfalfa and Forage Alliance http://alfalfa.ucdavis.edu/+symposium/proceedi
development of transgenic varieties but try http://www.alfalfa.org/pdf/CSExportSeed.pdf ngs/2009/09-157.pdf
(4) Julier B, Hughe C (1997) Effect of growth and
to use molecular breeding tools (2).
cultivar on alfalfa digestibility in a multi-site trial.
Agron 17:481-489
Perspective (5) Leclercq D, Caubel G (1991) Résistance
With the projected increase in the world variétale de la luzerne au nématodes des tiges
population and the increasing need for feed Ditylenchus dipscaci (Kühn) Filipjev; test d‟évaluation
et application en sélection. Agron 11:603-612
proteins in developing countries, alfalfa
should have a promising future. For
addressing future needs, commercial
breeders have to integrate new tools to be
able to make continued progress on
improving forage yield and forage quality.

Legume Perspectives Issue 4 • July 2014

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RESEARCH

Alfalfa management for higher and more sustainable

seed yields
by Jacques HACQUET1*and Đura KARAGIĆ2

Abstract: Seed yield is a major component

of alfalfa industry. Alfalfa seed set depends
on the activity of insect pollinators. For this
reason, the essence of alfalfa seed crop
management is providing favourable
conditions for flowering and pollination. Key
issues for maximizing seed yields are the
growing location (soil and climatic
conditions), stand density, cutting timing
(aimed to insure favourable weather
conditions during flowering and presence of
pollinators), variety, and control of weeds
pests and diseases.
Key words: alfalfa, crop management,
ecological conditions, pest control,
pollination, seed yield

Introduction
Both crop management and variety
determine alfalfa seed yields (1), but climatic
Figure 1. Correlation between alfalfa seed yield and air temperatures (A), solar radiation (B)
factors and soil conditions can strongly
and precipitation (C) during the seed crop period (May-August) in the period 1999-2001 (6)
influence the yield in the field (4). Alfalfa
seed yields have significantly increased
during the last 30 years. For instance, the
French average yield increased from Site selection and agro-climatic Crop establishment and weed
200 kg ha-1 to 500 kg ha-1, with big factors control
fluctuations depending on year and climate. Suitable areas for alfalfa seed production The field should be free from aggressive
Both seed yield and quality (purity 98%, must combine good edaphic properties and weeds and other weeds species whose seed is
viability minimum 80%, without weed seeds) weather conditions. Soils that guarantee a prohibited or restricted under rules for
have been improved in a context of a deep root development, rhizobium activity certified seed production. At least 5 years are
sustainable agriculture that requires less and nutrient requirements are suitable for necessary between two alfalfa crops on the
chemicals because they are dangerous for the alfalfa seed production. In general, same field, growing cereals or other winter
environment and the pollinating insects. The favourable sites possess calcareous clayey and spring crops during this interval to
main agronomic improvements aim at and well-drained soils with a pH value reduce diverse aggressive weeds (2).
avoiding lodging and providing the best between 7 and 8, and high water-holding The most common option for establishing
conditions for flowering. There are several capacity (to escape drought periods in the alfalfa, particularly in France, is with
steps that need to be considered, which are absence of irrigation) (4, 6, 8). Rainfall and sunflower as a cover crop. Other cover crops
discussed below. low temperature during the pollination are also possible, such as cereals. Another
period result in an excessive vegetative option is a direct sowing without a cover
growth and low pollinator activity. This is crop in the late summer, or in the spring
why seed production must be restricted to (with irrigation if necessary).
sites in which dry and warm weather prevails The row spacing depends on the site
_________________________________________________________________________________________________________
during the peak of the flowering and climate: 30 cm is usually enough, with 50-60
1National Seed Growers Union (FNAMS), INRA harvesting periods (Fig. 1). cm in drier conditions. The sowing rate is
RD150, Lusignan, France usually about 3 kg ha-1, with additional 1 or 2
(jacques.hacquet@orange.fr) kg ha-1 if sown with a companion crop (8).
2Institute of Field and Vegetable Crops, Novi Sad,

Serbia (djura.karagic@nsseme.com)

Legume Perspectives Issue 4 • July 2014

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 RESEARCH

Weeds are easy to control when they are It is very important to insure the best Acknowledgements
young and small, with mechanical or conditions during the flowering period and Project TR-31024 of the Ministry of Education,
chemical methods. New herbicides are to conserve native pollinators, paying special Science and technological Development.
recommended in established crops, mainly attention to their natural habitat and to pest
during alfalfa resting period in winter and and disease treatments that can kill these
according to the country legislation (8). insects. Seed growers must avoid chemical
treatments during flowering or restrict References
Cutting schedule spraying until late evening. (1) Bolanos-Aguilar ED, Huyghe C, Ecalle C,
Hacquet J, Julier B (2002) Effect of variety and
Cutting is the most effective practice to Seed set requires adequate temperature,
environment on seed yield in alfalfa (Medicago
limit the luxuriant growth and to obtain a water supplies and nutrients. Nevertheless, a sativa L.). Crop Sci 42:45-50
flowering period synchronized with large proportion of pollinated flowers will (2) Casals M-L, Hacquet J, Deneufbourg F,
maximum pollinator activity (7). When not lead to pods with seeds, probably as a Chauvel B, Citron G (2009) Inventaire floristique
cutting is too early, lodged plants are not part of a necessary regulation of the plant. et recherche de facteurs agronomiques pour une
suitable for pollination. However, if cutting gestion raisonnée des adventices en production de
is too late, the available soil water is not Harvest and postharvest semences de luzerne. Proceedings, 13ème
Colloque international sur la biologie des
sufficient in the period of regrowth, leading Losses may occur due to various reasons, mauvaises herbes, Dijon, France, 8-10 September
to short plants and reducing the number of such as diverse timing of seed ripeness, 2009
fertile stems (4). Cutting time is probably one lodging, seed shattering, seed sprouting, or (3) Fairey DT, Griffith SM, Clifford PTP (1998)
of the most determining decisions for seed mechanical harvesting or separation. All Pollination, fertilization and pollinating
yield. these factors may also result in poor seed mechanisms in grasses and legumes. In: Fairey
DT, Hampton JG (eds) Forage Seed production,
quality.
Pests and diseases control The optimum stage of ripeness is about
Volume 1: Temperate Species. CAB International,
Wallingford, UK, 153-179
Different pest species must be controlled 85% of brown pods, but the crop may be (4) Hacquet J (1989) Genetic variability and
in each region, for instance alfalfa weevil very heterogeneous. Lodging amount is also climatic factors affecting lucerne seed production.
larvae, alfalfa pod weevils and seed moth in important. In this respect, cultivars which are J Appl Seed Prod 8:59-67
France, mirid bugs, aphids or Diptera (gall sensitive to lodging also tend to have lower (5) Hacquet J (2002) Survey on strategies for pest
midges) in other countries (5, 8). seed yield (7). control in lucerne seed crops in center-west of
Using sweep netting and a network Pre-cutting or desiccation using an France. Proceedings, Sixth International
Conference in Pests in Agriculture (AFPP),
advising system is a good method to monitor chemical desiccant (diquat) are two methods
Montpellier. France. 4-6 December 2002
the evolution of insect populations. A to reduce vegetative bulk, but either option (6) Karagić Đ, Katić S, Mihailović V, Vasiljević S,
threshold for each pest is used to decide requires good timing in relation to crop Milić D (2003) Correlations between alfalfa seed
whether to treat or not. maturity and expected weather conditions. yield and ecological factors. Grassl Sci Eur 8:372-
The main foliar diseases are alfalfa rust Precise adjustments for the combine 376
(Uromyces striatus), Phoma medicaginis and harvester are necessary. For alfalfa, seed (7) Karagić Đ, Katić S, Milić D, Vasiljević S (2007)
Pseudopeziza medicaginis, especially when drying is not useful, except after especially Effect of cutting schedule on seed yield in alfalfa.
summer is wet. Efficient fungicides are wet conditions for harvesting. Usually, seed Proceedings of the XXVI EUCARPIA Fodder
Crops and Amenity Grasses Section and XVI
available, but their use is less frequent than pre-cleaning (on the farm or in the factory) is
Medicago spp. Group Joint Meeting Breeding and
that of insecticides. sufficient to obtain a seed lot with moisture Seed Production for Conventional and Organic Agriculture,
content below 12%. Perugia, Italy, 3-7 September 2006, 124-127
Pollination and seed set Even under optimal management, alfalfa (8) Marshall AH, Steiner JJ, Niemenlaïnen O,
Insect pollination is obligate for alfalfa. seed yields in the same area can vary largely Hacquet J (1998) Legume seed crop management.
The most efficient species are alkali bees between years, because of unpredictable In: Fairey DT, Hampton JG (eds) Forage Seed
climate variation. For example, seed yields in production, Volume 1: Temperate Species. CAB
(Andrena, Eucera, Halictus, Melitta, Melliturga, International, Wallingford, UK, 127-152
Rophites...) and bumble bees, which feed on Serbia may vary from 50 kg ha-1 to 1200 kg
flowers nectar and pollen (3). In Europe, the ha-1 under the same crop management
main pollinators are wild species. If native depending on weather condition, a
populations of alkali bees are not present, reasonable target being represented by an
alfalfa leaf cutting bees (Megachile rotundata) average yield of 700 kg ha-1 - 800 kg ha-1 of
colonies can be added in the seed production high quality seed. ■
fields (a common practice in North
America).

Legume Perspectives Issue 4 • July 2014

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RESEARCH

Alfalfa in ruminant diets

by René BAUMONT1, 2*, Valérie HEUZÉ3, Gilles TRAN3 and Maryline BOVAL4

Abstract: Alfalfa can be used in ruminant Grazed or fresh alfalfa salivation which also results in rumen
diets as hay, silage, dehydrated, or grazed. buffering (7). Alfalfa hay may be finely
Alfalfa is of high interest for ruminant Rotational grazing is the most suitable chopped, or coarse with long fibre.
nutrition as it is generally well ingested and management for alfalfa-based pastures. The Dehydrated alfalfa. Dehydration is the best
well suited to improve crude protein and stocking rates should be high enough to way to conserve alfalfa, as it dries and
mineral content of diets. Compared to remove the herbage within 7-10 days. The stabilizes the forage while preserving its high
grasses, alfalfa has similar digestibility but rest period between grazing should be long protein content, vitamins and overall
higher protein content, and allows for high enough to let alfalfa reach the bud-to-early- nutritive value. Dehydration requires
levels of voluntary intake. Hay or dehydrated flower stage before the next grazing. In the prewilting and chopping in the field,
alfalfa also brings structural fibre in the diet. USA, several trials reported that cows could transportation of the plant and drum-drying
The best stage to combine alfalfa biomass graze 20 kg DM day-1 of alfalfa pasture and (between 600°C and 800°C) down to 10 %
production and nutritive value for yield about 25 kg day-1 milk without moisture. After drying, dehydrated alfalfa
conservation is bud stage or beginning of supplementation (5). Grazed or fresh alfalfa may be compacted into big square bales,
flowering. at vegetative or mid-bud stage may cause ground to make alfalfa meal, or ground and
Key words: cell-wall, crude protein, bloat in sheep and cattle. Supplementing passed through a screw die to make pellets.
digestibility fresh forage, intake, hay, silage cattle and sheep with grass, cereal grains or Pellets are often standardised to a certain
anti-foaming agents (such as poloxalene) can protein content (such as 17% or 18 %).
Alfalfa (Medicago sativa L.) can be grazed, alleviate this problem, and is essential in Dehydrated alfalfa can partially replace
fed as green forage, offered as hay or silage, intensively grazed situations (4). concentrates in dairy cow diets. Providing 3
or dehydrated. The high dry matter (DM) Fresh alfalfa can also be fed indoors. It is kg of dehydrated alfalfa to cows fed on grass
yield, protein and calcium content of alfalfa, the ideal complement to maize silage to silage, maize silage, barley grain and soybean
as well as its high palatability and voluntary enrich the diet in protein and minerals and to meal, resulted in similar animal performance,
intake, make it a suitable forage for all classes provide fibre for chewing behaviour. An higher feed intake (+2.5 kg DM day-1),
of ruminants. inclusion level of 50 % of the diet results in higher milk protein yield (+1 g kg-1) and
Alfalfa yields more protein per unit area overall higher DM intake. lower soybean meal inclusion (-350 g day-1)
than soybean, and has a well-balanced amino (8).
acids profile for ruminants that compares Conservation of alfalfa Silage. Given its low soluble carbohydrate
favourably with that of soybean. Alfalfa Alfalfa may be cut for conservation several content, alfalfa has to be supplemented with
provides higher amounts of minerals (mainly times a year (up to 12 in irrigated subtropical carbon sources, such as ground cereal grains,
calcium, but also magnesium, potassium, environments). The best stage for cutting is or conservation additives such as organic
sulphur, iron, cobalt, manganese, and zinc) at bud stage or beginning of flowering as the acids (formic acid, formic acid +
and vitamins (beta-carotene) than other nutritive value declines after that with the formaldehyde, propionic acid) to improve
fodders (4). The energy content of alfalfa, decrease of the % of leaves in the aerial the preservation of alfalfa when ensiled at
though slightly lower than that of some biomass (see Scotti and Julier in the same low DM content. Wilted silage at 30 % DM,
forage grasses, should not be overlooked (2). issue). After the first cut, it is advisable to or wrapped bale silages at more than 40%
A recent synthesis on alfalfa utilisation for wait for the young shoots to be 35 to 50 cm DM (3) are also suitable to conserve alfalfa,
animal nutrition was achieved in the long before the next cut (10). as they reduce the risk of bad preservation
Feedipedia project (5). Hay. Best quality hay is obtained by cutting and consequent high protein degradability in
during a dry period so that the swathe dries the rumen. Mixed-crop silages combining
quickly. Raking has to be done at 60 % DM. alfalfa with a forage grass are also a way to
Baling should be done at 82 % DM. The achieve a conserved forage of high nutritive
main difficulty in hay making for alfalfa is to value.
preserve the leaves that are the most
_________________________________________________________________________________________________________
nutritive parts of the plant, especially in Factors of variation of alfalfa’s
1INRA, UMR 1213 Herbivores, Saint-Genès- terms of proteins (10). feed value
Champanelle, France (baumont@clermont.inra.fr) Alfalfa hay is particularly valuable for
2Clermont Université, VetAgro Sup, UMR
ruminants. The high content of structural Updates in nutritive value of alfalfa were
Herbivores, Clermont-Ferrand, France
fibre is rapidly digested by rumen microflora provided for dehydrated alfalfa and different
3Association Française de Zootechnie,
and helps prevent acidosis. This effect is due types of alfalfa forages (2). The main factors
AgroParisTech, Paris, France affecting the feed value of alfalfa are
4INRA, UR143, Unité de Recherches to its intrinsic buffering effect and to the
stimulation of ruminative chewing and discussed below.
Zootechniques, Petit-Bourg, France

Legume Perspectives Issue 4 • July 2014

36
 RESEARCH

Table 1. Effect of growth stage and harvest on the feed value of fresh alfalfa (2) Hay making has a negative effect on alfalfa
Voluntary digestibility, owing to the loss of leaves
Dry OM during raking and baling (Table 2). Alfalfa
Crude protein NDF intake in cow
matter digestibility
(g kg -1 DM) (g kg -1 DM) (g kg-1 hays are very well consumed by animals
(%) (g g-1)
LW0.75) when they are barn-dried or field-dried under
Growth stage in 1st cycle good weather conditions. When made under
Vegetative (30 cm) 14.4 246 423 0.77 156 unfavourable weather conditions, however,
Vegetative (60 cm) 15.6 225 449 0.72 152
they show low digestibility and voluntary
Bud 17.6 193 488 0.66 143
Early flowering 18.9 178 513 0.63 140 intake, making wrapped bale silage preferable
Age at second harvest (Table 2).
5 weeks 19.3 222 487 0.68 161 Feed value of dehydrated alfalfa. The feed value
9 weeks 22.5 179 512 0.59 136 of dehydrated alfalfa increases with its crude
Age at third harvest protein content, which is associated with
5 weeks 21.0 241 468 0.69 147 increased OM digestibility as a consequence
9 weeks 24.9 204 476 0.62 140 of decreased cell-wall content (Table 3).

Table 2. Effect of conservation type on alfalfa feed value assessed for the same fresh forage Conclusion and prospects
harvested at 7 weeks in the second vegetation cycle (2)
Alfalfa has high interest for ruminant
Dry OM Voluntary
Crude protein NDF nutrition because of its feed quality and its
matter digestibility intake in cow
(g kg -1 DM) (g kg -1 DM) flexibility of utilization. However, alfalfa
(%) (g g-1) (g kg-1 LW0.75)
Fresh forage 19.7 198 513 0.65 144 proteins are highly and rapidly degraded in
Dry OM Voluntary the rumen. This can lead to poor nitrogen
Crude protein NDF
Silages matter digestibility intake in cow utilization and excessive nitrogen losses in
(g kg -1 DM) (g kg -1 DM)
(%) (g g-1) (g kg-1 LW0.75) urine. Decreasing the rate of alfalfa protein
- Direct cut with degradation in the silage and the rumen is a
21.0 187 466 0.64 135
preservative challenge to further improve its high-protein
- Wilted 33.5 198 488 0.62 139
crop value. Prospects of using polyphenol-
- Wrapped bale 55.0 183 504 0.61 133
Dry OM Voluntary oxidase present in red clover or tannins
Crude protein NDF present in other legumes like sainfoin (1) are
Hays matter digestibility intake in cow
(g kg -1 DM) (g kg -1 DM) under investigations. Saponins present in
(%) (g g-1) (g kg-1 LW0.75)
- Barn dried hay 85 181 539 0.61 144 alfalfa might contribute to reduce methane
- Field dried, sunny
85 177 555 0.60 136 emissions during rumen fermentation, but
weather results on this issue are still controversial (6).
- Field dried, rainy ■
85 172 585 0.55 128
weather
References
Table 3. Feed value of dehydrated alfalfa as a function of its crude protein content (9) (1) Aufrere J, Dudilieu M, Poncet C, Baumont R (2013) Mixing
sainfoin and lucerne to improve the feed value of legumes fed to
Crude protein Crude protein NDF OM digestibility sheep by the effect of condensed tannins. Anim 7:82-92
Dry matter (%)
content (% DM) (g kg-1 DM) (g kg-1 DM) (g g-1) (2) Baumont R, Dulphy JP, Sauvant D, Meschy F, Aufrere J,
Peyraud JL (2007) Chapitre 8. Valeur alimentaire des fourrages
< 16 91.4 151 503 0.60 et des matières premières: tables et prévision. In: Alimentation
17-18 90.6 175 474 0.62 des bovins, ovins et caprins, Tables INRA 2007, Editions Quae,
18-19 90.6 184 461 0.63 149-179
(3) Demarquilly C, Dulphy JP, Andrieu JP, (1998) Valeurs
22-25 89.8 233 379 0.70 nutritive et alimentaire des fourrages selon les techniques de
conservation: foin, ensilage, enrubannage. Fourrages 155:349-
369
(4) Frame J, Charlton JFL, Laidlaw AS (1998) Temperate forage
Effect of growth stage and harvest. Cell wall age at about the same rate during the second legumes. CAB International, Wallingford
(5) Heuzé V, Tran G, Boval M, Lebas F, Lessire M, Noblet J,
content increases while crude protein, cycle as during the first cycle (Table 1). After Renaudeau D (2012) Alfalfa (Medicago sativa). Feedipedia.org. A
organic matter (OM) digestibility and the second cut, regrowths become leafier, programme by INRA, CIRAD, AFZ and FAO.
http://www.feedipedia.org/node/275
voluntary intake decreases with alfalfa aging resulting in a lower decrease of feed value (6) Niderkorn V, Baumont R (2009) Associative effects between
in each growing cycle (Table 1). Compared with age. forages on feed intake and digestion in ruminants. Anim 3: 951–
to temperate grasses like perennial ryegrass Effect of the conservation as silage or hay. Silage 960
(7) Robinson (1998) Proceedings, 28th California Alfalfa
or cocksfoot, the cell wall content of alfalfa preserves chemical composition of fresh Symposium,, Reno, USA, 3-4 December 1998
is lower, but the OM digestibility is similar as alfalfa and decreases slightly the OM (8) Thénard V, Mauriès M, Trommenschlager JM (2002) Interest
of dehydrated alfalfa in maize and grass silage diets for dairy
a result of lower cell wall digestibility in digestibility and the energetic value (Table 2). cows. INRA Prod Anim 15:119-124
alfalfa. In contrast, the voluntary intake of Cell-wall content can even be slightly (9) Sauvant D, Perez JM, Tran G (2004) Tables de composition
et de valeur nutritive des matières premières destinées aux
alfalfa is always higher than that of grasses, as decreased due to partial hydrolysis of animaux d'élevage: porcs, volailles, bovins, ovins, caprins, lapins,
a result of the lower cell wall content and hemicelluloses during silage fermentation. chevaux, poissons. INRA Editions, Paris
lower rumen fill effect. However, voluntary intake of silage is lower (10) Suttie JM (2000) Hay crops-legumes and pulses. In: Hay and
Straw Conservation-For Small-Scale Farming and Pastoral
The highest digestibility is obtained at the than that of fresh alfalfa, because of the Conditions. FAO Plant Production and Protection Series 29.
beginning of the first vegetation cycle. The difficulty to obtain well-preserved silages, FAO, Rome

feed value of alfalfa regrowths decreases with especially at low DM content.

Legume Perspectives Issue 4 • July 2014

37
RESEARCH

Alfalfa non-feed uses

by JoAnn F. S. LAMB1*, Hans-Joachim G. JUNG2, Michael P. RUSSELLE1, Craig C. SHEAFFER2, Deborah
A. SAMAC1 and Carroll P. VANCE1

Abstract: Non-feed uses for alfalfa such as would include concentrations and seasonal Phytoremediation
biomass energy and phytoremediation could yields of leaf protein, stem biomass, and/or
increase alfalfa acreage and improve farm stem cell wall polysaccharides, depending on Nitrogen laden ground water is a well
profitability. The new bio-energy alfalfa and which the energy conversion platform is being documented human health risk and practical
production system increased forage yield and used. Increasing stem yield in alfalfa can be as remediation methods are needed when over-
ethanol production. New alfalfas with simple as harvesting forage at a later maturity fertilization of annual crops with inorganic N
enhanced nitrogen cycling capacities would than early bud, which is typically used in hay threatens ground water quality. Although alfalfa
protect water quality and enhance alfalfa‟s value production. can obtain most of the N required for growth
in crop rotation systems. An experimental bio-energy alfalfa population through symbiotic N2 fixation, it is also very
Key words: bio-energy, ethanol production, was created through selection for large, erect, effective at removing nitrate-N from the sub-
leaf crude protein, nitrate uptake, nitrogen (N) non-lodging stems when the alfalfa was in soil (Fig. 1) (2, 5).
cycling, phytoremediation, symbiotic N2 bloom. Management protocols have also been At the site of an anhydrous ammonia spill
fixation modified to (i) reduce population stand density where nitrate-N concentration of the ground
to allow the development of larger crowns and water exceeded drinking water standards, a
decrease light competition to keep leaves from conventional cultivar and an ineffectively
Introduction nodulated population (incapable of symbiotic
senescing and (ii) harvest at later maturity stages
Alfalfa plays an essential role in sustainable to maximize both leaf and stem yield. The bio- N2 fixation) were irrigated with the N
agriculture. Its perennial growth habit and energy alfalfa under bio-energy management containing ground water for three years.
extensive root system permits the crop to aerate harvested at the green pod maturity stage Cumulative N removal from the site was over
soil, access deep water resources, capture increased stem yield by 56% and doubled the three times greater by the ineffectively
nutrient run-off and mitigate soil erosion. Using ethanol yield compared to commercial cultivars nodulated alfalfa than would be expected with
alfalfa in rotation interrupts pest and pathogen grown under standard hay management annual cereal grains (5), and soil nitrate-N
cycles and through symbiotic N2 fixation with practices. Leaf crude protein yields were similar concentrations were rapidly reduced to low and
soil bacteria, provides up to 100% of the N between the hay and bio energy production stable levels. With these results in mind, we
nutrient requirement of the subsequent crop. systems for two of three of the site-year successfully created a selection protocol that
Non-feed uses are needed to increase and combinations (4). In another comparison produced new, contrasting cultivars that can
diversify revenue streams for growers and allow between the bio-energy and high quality hay either reduce N losses to ground water by
alfalfa a greater role in sustainable agricultural cultivars under the two management schemes, increased nitrate-N uptake capacity, or decrease
systems. New uses include alfalfa as a renewable stem yields increased 40%, and potential fertilizer N requirements in production systems
biomass feedstock that can help meet the ethanol yield increased between 50% and 97% through enhanced symbiotic N2 fixation (3).
energy demands of the future (2), and as an when harvested at full bloom compared to early These new cultivars could strengthen the crop‟s
inexpensive and efficient remediation tool to bud stage (1). The bio-energy alfalfa under the role in sustainable agriculture by protecting
remove sub-soil nitrate from polluted sites (2). bio-energy production system shows great water quality and enhancing its value in crop
potential for helping meet the energy demands rotation systems. ■
Alfalfa biomass energy system of the future.
A biomass energy production system using
References
alfalfa would separate leaves from stems
(1) Lamb JFS, Sheaffer CC, and Samac, DA (2003) Population
creating two products. The stems would be density and harvest management effects on leaf and stem yield in
processed to produce electricity (combustion) alfalfa. Agron J 95:635-641
(2) Lamb JFS, Jung H-JG, Sheaffer CC, Samac DA (2007) Alfalfa
or biofuel [ethanol (fermentation) or bio-oil leaf protein and stem cell wall polysaccharide yields under hay and
(pyrolysis)], and the leaves would generate a biomass management systems. Crop Sci 47:1407-1415
(3) Lamb JFS, Russelle MP, Fenton DM (2008) Field-based
secondary income as valuable protein feed for selection method creates alfalfa populations that differ in nitrate
livestock (Fig. 1). Therefore, the key traits of nitrogen uptake. Crop Sci 48:450-457
(4) Lamb JFS, Jung H-JG, Riday H (2014) Growth environment,
interest for an alfalfa bio-energy germplasm harvest management and germplasm impacts on potential ethanol
and leaf crude protein yields in alfalfa. Biomass Bioenerg 63:114-
125
_________________________________________________________________________________________________________
(5) Russelle MP, Lamb JFS, Montgomery BR, Elsenheimer DW,
1United States Department of Agriculture, Miller BS, Vance CP (2001) Alfalfa rapidly remediates excess
inorganic nitrogen at a fertilizer spill site. J Environ Qual 30:30-36
Agricultural Research Service, Plant Science
Research Unit, Saint Paul, USA
(joann.lamb@ars.usda.gov)
2University of Minnesota, Department of Figure 1. The alfalfa bio-energy system with two product streams, stems for energy and leaves
Agronomy and Plant Genetics, Saint Paul, USA for animal feed as well as value added products with enhanced nutrient cycling

Legume Perspectives Issue 4 • July 2014

38
 EVENTS

25th General Meeting of the European Grassland Federation

EGF at 50: the Future of European Grasslands
Aberystwyth, Wales, UK, 7-11 September 2014
http://www.egf2014.org/

18th Symposium of the European Grassland Federation

Grassland and Forages in High Output Dairy Farming Systems
Wageningen, the Netherlands, 15-18 June 2015
http://www.europeangrassland.org/events.html

Symposium of the Protein Crops Working Group, Protein Crops Section, and
Spanish Association for Legumes
Plant Proteins for the Future
Pontevedra, Spain, 4-7 May 2015
http://www.symposiumproteincrops.org/

Symposium of the Fodder Crops and Amenity Grasses Section

Ghent, Belgium, 14-17 September 2015
http://www.eucarpia.org/activities/meetings.html

BOOKS

Grasslands and Herbivore Production in Europe and

Effects of Common Policies
Editors: Christian Huyghe, Alex De Vliegher, Bert van Gils and Alain Peeters
Publisher: Quæ, Versailles, France, March 2014; pp. 320; ISBN: 978-27-592-2157-8
http://www.quae.com/fr/r3371-grasslands-and-herbivore-production-in-europe-and-effects-of-common-policies.html

Quantitative Traits Breeding for Multifunctional Grasslands and Turf

Editors: Dejan Sokolović, Christian Huyghe and Jasmina Radović
Publisher: Springer Science+Business Media, Dordrecht, the Netherlands, August 2014; 411 pp; ISBN: 978-94-017-9044-4
http://www.springer.com/life+sciences/agriculture/book/978-94-017-9043-7
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