JWBK201-Kole k0304 March 28, 2008 19:45

Faba Bean
Wolfgang Link1 , Moemen Hanafy2 , Nenad Malenica3 ,
Hans-Jörg Jacobsen4 and Srećko Jelenić3
1
Department of Crop Sciences, University of Göttingen, Göttingen, Germany 2 Plant
Biotechnology Department, National Research Center (NRC),Cairo, Egypt 3 Department of
Molecular Biology, University of Zagreb, Zagreb, Croatia 4 Department of Molecular Genetics,
Hannover University, Hannover, Germany

1. INTRODUCTION 1.2 Botanical Description

1.1 History, Origin, and Distribution The intraspecific diversity is mainly described by

of
use of seed size. Persoon (1807) described the
Vicia faba (faba bean) was not among the very first
r o
small-seeded group as V. faba minor (roundish
P
e
domesticated crops. It was probably introduced seeds of up to 0.6 or 0.7 g weight per seed),
into agriculture only in the late Neolithic period
P a g the medium-large-seeded group as V. faba equina

rst
(Körber-Grohne, 1987). Cubero (1974) concluded (single seed weight less than about 1g) and V. faba

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that the center of origin was in the Near East, Iraq, major with its impressive, large, and flat seeds
and Iran, and secondary centers evolved later on weighing more than 1 g per seed. The name “field
in Afghanistan and Ethiopia. Before 1000 BC, the beans” indicates to the small and medium sized
culture of faba beans was already very established types, whereas “broad beans” denotes V. faba
in Europe, including Britain. Large-seeded types major. Today, often “faba bean” is used to address
are of recent origin and they were probably the species in its total diversity. A wild ancestor of
developed only 1000–1200 years ago in East Iraq, faba bean is not known, and the related vetch Vicia
and from there spread to Asia, across North Africa narbonensis is considered as the nearest relative
to Europe, and eventually to America. In China, to V. faba (Zohary and Hopf, 1973). Based on
the crop seems to have arrived only after 1200 AD. restriction fragment length polymorphism (RFLP)
The faba bean reached Mexico and South America and polymerase chain reaction (PCR) data, more
by the Spaniards. From then on, it experienced recently Van den Ven et al. (1993) placed Vicia
there an independent evolution (Körber-Grohne, peregrina and Vicia michauxii into the direct
1987; Bond, 1995). Several gene bank accessions taxonomic neighborhood of V. faba. These related
with promising agronomic features originated vetches have seven chromosomes. V. faba belongs
from Ecuador. These are mostly large-seeded with Vicia villosa and Vicia sativa to the genus
types. The more recent history has to mention Vicia. With Lens culinaris and Pisum sativum,
the comprehensive reports of Muratova (1931), it belongs to the tribe Vicieae and only at the
Sirks (1931) and Hanelt et al. (1972) on genetics, family level of Fabaceae it is united with the
systematics, taxonomy, history, and geographical Phaseolus bean, the soybean and with the lupines
topics (Bond, 1995). (Sitte et al., 2002).

A Compendium of Transgenic Crop Plants: Legume Grains and Forages. Edited by Chittaranjan Kole and Timothy C. Hall


C 2008 Blackwell Publishing Ltd. ISBN 9781405167062
JWBK201-Kole k0304 March 28, 2008 19:45

3-2 LEGUME GRAINS AND FORAGES

The faba bean crop is annual, sown either in

autumn or in spring. It bears a strong, hollow,
tetragonal erect stem, with zero or up to five
(or even more) basal branches arising from basal
leaf axis. There are only noneffective rudiments
of tendrils at the leaf tip. Different from pea,
stipules are very small, with a distinct black stipule
spot on the stipule’s down side. The bean has
got a robust tap root with profusely branched
secondary roots. Nevertheless, the rooting system
is not as voluminous as that of cereals like oats or
wheat. The roots bear the typical nodules formed
by Rhizobium leguminosarum, as expected for it
being a leguminous species. The first, juvenile
leaves bear two leaflets. After four or more nodes I II III IV V VI
the maximum number of leaflets (typically six)
is reached. The first, lowermost inflorescence is Figure 2 Karyogram of Vicia faba [Reproduced from Fuchs
located in the axis of a leave with at least four et al. (1998)]
leaflets, inserted on the stem as low as at the fourth
node or several nodes later (higher). Two to more
than eight flowers per inflorescence occur. The wild seeds per pod and more than 1 g weight per seed.
type flower color is white with a soupcon of pink The contrary is realized in some V. faba minor

of
ssp. paucijuga types from the Hindukush region,
o
traces and a very distinct, satin black spot on both
wing petals (Figure 1). Brown, violet, red, and
P r
showing small, 2 seeded pods with less than 0.3 g

e
weight per seed, growing on small, gracile, tillering
g
further grades of flower colors exist. Totally white

P a
flowers are a pleiotropic effect of an allele for zero plants with very slender leaflets. These types are

rst
suspected to represent the most primitive version

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tannin in the seed testa, accompanied by grayish
testa color instead of buff testa and by absence of V. faba (Cubero, 1974).
of the wild type stipule spot. One to two pods V. faba is one of the best cytogenetically charac-
with three or four seeds are to be expected per terized plants. Its six chromosome pairs contain
inflorescence; one plant may yield far more than as much as 1C approximately 13 pg of DNA,
12 pods, distributed across more than six nodes. In which corresponds approximately to 13000 Mbp.
germplasm used for human consumption, often The first, very large chromosome (about 18 µm
very few, very large pods per plant are realized, length) is metacentric, with one satellite. There are
with more than 20 cm pod length, more than six five similar (approx. 7–9 µm length) acrocentric
chromosomes (Figure 2). The metacentric chro-
mosome I of V. faba probably originated from a
remote fusion of two telocentric chromosomes.
Many cytogenetic phenomena were observed for
the first time by studying V. faba, for instance,
nucleolus formation at the secondary constrictions
during telophase, or the existence of an upper
tolerance limit for chromosome arm length
(Schubert and Oud, 1997; Fuchs et al., 1998).

1.3 Economic Importance

At the worldwide scale, faba bean occupies about

Figure 1 Vicia faba flower with white flag petal and black 2.6 millions ha, which in 2005 represented 4%
spot on wing petal of the total area dedicated to pulses. From the
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FABA BEAN 3-3

worldwide area occupied by faba bean, 41% was amounts to 20% of the total production of faba
concentrated in Asia, 33% in Africa, only 12% bean. It is a common food in the Middle East,
in Europe, and 7% in Oceania as well as in the Mediterranean region, Latin America, China, and
United States. China is the largest grower of faba Ethiopia. The production of green faba bean
bean in the world with 39% of the worldwide area. in the Mediterranean region accounts for 40%
In Africa, faba bean is mostly concentrated in of the worldwide green bean production. The
Ethiopia (15% of the world wide faba bean area). Middle East and Latin America contribute equally
The worldwide production in 2005 was about with about 19% to the worldwide green bean
5.8 million tons of which China produced 43% production. Other uses of faba bean have been
and is, therefore, the largest producer in the world identified. Indeed, haulm from faba bean harvest
(http://faostat.fao.org/site/336/DesktopDefault fetches a premium in Egypt and Sudan and is
.aspx?PageID=336). considered as a cash crop. The haulm can also
Faba bean is a very minor crop in Germany be used for brick making and as a fuel in parts of
(16 000 ha in 2005), in Poland (12 000 ha), and Sudan and Ethiopia (http://faostat.fao.org).
Austria (4000 ha). France did grow a total of
105 000 ha, amounting to one third of the French
pea acreage. In the United Kingdom, faba beans 1.4 Traditional Breeding
were grown in 188 000 ha, this is double the
area grown to pea plus lupins. Spain had in The breeding objectives for this crop totally
2005 about 53 000 ha of faba bean. Mediterranean depend on the economic and agro-ecological
types are sown in late autumn. In parts of the conditions and on the geographical region and
United Kingdom and France, where winter is use. For combine harvest of dry, mature seeds,
relatively mild, autumn sowing of faba beans is to
oof
all pods and even stem and leaves of the crop
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some extent practiced as well using “true” winter have to become mature simultaneously, whereas
beans that can survive winters north of Pyrenees
a g e
for manual harvest of vegetable green bean

P
Mountains and Alps. In the United Kingdom, pods, a long-lasting harvesting period is sought.

Fi rst
more or less, half of the faba beans are such winter For combine harvesting, nonshattering and
types. All types of faba bean can survive very nonlodging habits are essential, whereas they are
mild frosts as young plants (until about −6 ◦ C). of lesser importance for the production of green
Beans from the Hindukush do even show some pods. For the production of feed, mostly small
frost tolerance in later stages. Autumn sowing is as grains are bred, whereas for human consumption,
well realized in North Africa, in south-east China equina and major types are preferred in most areas
(along the Yangtze Valley), and parts of Japan. (except Ethiopia, where small types are used for
Cultivated faba bean is used as human food and food). Moreover, color, taste, and cooking features
as animal feed, mainly for pigs, horses, poultry, are important for vegetable type germplasm. If
and pigeons in developing countries and almost grown for animal feed, primarily mature grain
strictly as animal feed in developed countries. yield and yield stability are sought, and to sustain
For the human consumption, it can be used as high performance, resistance against drought (and
a vegetable, either green or dried. Feeding value winter frost in case of winter bean breeding) and
of faba bean is high; with about 30% of protein against fungi, pathogens, and pests is needed.
it is considered in some areas to be superior to An additional objective is mature grain quality,
field peas or other legumes. It is one of the most depending on the actual animal species to be fed.
important winter crops for human consumption in Normal faba beans show indeterminate growth;
the Middle East. Faba bean has been considered flowers and very young pods grow in competition
as a meat extender or substitute and as a skim milk with the vegetative apex of the stems. Several
substitute. Roasted seeds are eaten like peanuts in alternative growth types have been studied as a
India. The proportion of the dried faba bean used strategy to strengthen the pods as sinks for assimi-
as human food in the developing countries is not lates. The so-called ti-type (terminal inflorescence,
defined but data are available for green faba bean or “topless”) and the so-called stable type (st,
and allow assessing the contribution of faba bean somewhat stunted habit, and very stiff stem) were
in the human nutrition. Its use as green vegetable introduced. Dwarfism is known as well. These
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3-4 LEGUME GRAINS AND FORAGES

phenotypes are all caused by a single recessive conditions, it is often a very serious threat, and
allele (ICARDA 1986). Several corresponding still no convincing source of resistance is known.
cultivars have been bred in Germany, like “Tina” Severe outbreaks are most common in the Nile
(ti), “Tinova” (ti) or like “Boss” (st) and “Mythos” delta, near rivers in China, rainy coastal areas of
(st). Still, these are not widely used, and no such the Mediterranean Basin, and the more oceanic
type is present on the 2005 German list of varieties. climate of western France and western United
On the other hand, very recently, Nadal et al. Kingdom (Tivoli et al., 2006). Differences in
(2005) in Spain proposed the use of the ti-type susceptibility follow a quantitative genetic pattern.
as a solution in broomrape-infested (see below) Several less susceptible bean genotypes are known
conditions (production of young pods for fresh (e.g., some ICARDA lines originating from South
consumption). There has been a remarkable input America). For Ascochyta and Uromyces, specific
into a type named “independent vascular supply” resistances are known and even molecular markers
(ivs). Gates et al. (1983) proposed to circumvent the were developed. The line 29H, amongst others, was
physiological interaction and competition between repeatedly used to improve Ascochyta resistance.
pods and flowers within the same inflorescence Bean rust (Uromyces) resistance is available in
by independent vascular traces to each flower, so many cultivars. Qualitative resistance is common
that direct interaction between flowers and young and widely used by breeders (Sillero et al., 2006).
pods cannot occur, and distinguished this type Phoma and mildew cause further less well-studied
from the “usual” branched vascular pattern. The foliar diseases in V. faba. Few, if any, convincing
conclusion was that selection for ivs would be most sources of resistance against root rot are known.
reliable to improve pod set. By microscopic studies, Material with a zero content of tannin (see below)
Ruckenbauer and Mollenkopf (1983) found that in the seed testa (monogenic recessive feature)
a classification into independent and branched
oof
seems to be more susceptible at germinating and
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vascular bundles is not adequate. In material that emergence than tannin-containing germplasm.
was claimed to express the ivs features, these
a g e Viruses are not a frequent problem for the faba

P
authors found no hints on any structural deviation bean producer; still, viral diseases may occur as

Fi rst
of the vascular traces from “normal” beans. serious epidemic. The bean yellow mosaic virus,
Faba bean, in spite of its high importance in bean leaf roll virus, broad bean true mosaic virus,
several semiarid regions like the Mediterranean and the broad bean stain virus must be named.
Basin, is rather susceptible to drought. Its main Since no direct pesticide protection exists, the
mechanism to deal with this stress is a very genetic strategy must hold. The two latter viruses
early and sensitive stomata closure. Adapted are to some extent seed transmitted and not
material escapes terminal drought by earliness, aphid transmitted. Yet, the beetle Sitona lineatus
whereas no powerful strategy is known to deal (see below) spread these two viruses early in the
with unpredictable, intermittent drought. Several season (Rohloff, 1980). There has been promising
physiological traits may be used to assist in breeding research prior to 1989 in GDR, but these
breeding for drought tolerance. Germplasm from programs were then abandoned (Lötsch, 1989). In
gene banks like ICARDA was used to specifically 1992 and again in 1999, a new, aphid-transmitted
enlarge diversity. Frost resistance is mostly virus (faba bean necrotic yellow virus) occurred at
analyzed as a component of winter survival. Very a devastating level in the Nile Valley. Meanwhile
few genotypes with outstanding frost resistance are resistant genotypes have been identified (e.g.,
known (Stoddard et al., 2006). Similar to drought, “ILB132” Khaled et al., 2000).
physiological traits might help in breeding, such The most important pest is Aphis fabae, the
as fatty acid composition of leaves (Arbaoui and black aphid. It occurs very often at significant
Link, 2006). No molecular tools are as yet available levels, therefore, insecticides are used. In addition
in faba bean breeding for these two traits. to the direct damage, it is spreading viruses. No
The most important fungal foliar diseases are useful resistance is known. Even earlier in the
Botrytis fabae, Ascochyta fabae, and Uromyces season than this aphid, the weevil S. lineatus feeds
viciae-fabae; besides, root rot caused by Rhizoc- on the first, very young leaves. More important
tonia solani, Fusarium species, and other fungi can is the damage of its larvae, which feed on the
occur. Botrytis is seen in a wide range of growing root nodules and thus cause direct damage and
JWBK201-Kole k0304 March 28, 2008 19:45

FABA BEAN 3-5

probably increase root rot (Salt, 1983). Bruchus Meanwhile, three quantitative trait loci (QTLs)
rufimanus, a seed-infesting weevil, is present in for resistance were identified (Torres et al., 2006).
most faba bean fields and stocks. The female Orobanche resistance is a trait that shows all
deposits the eggs in the field onto the very young features to make it a candidate for marker-assisted
pods. Infested seeds are not accepted for human selection. It is a very serious problem, resistance
consumption, thus the beetle is a serious threat for shows quantitative genetic variation, difficult to
this aspect of production. Additional Bruchus and phenotype the trait, and there is only one unique,
Callosobruchus species live in bean seeds (mainly common source of resistance (“F402”). Up to
in the Middle East); several of them complete their now, the level of cooperation among breeders and
cycle in the store, the female laying the eggs onto scientists in the Mediterranean Basin and Nile
the testa of mature dry seeds. The dry seed coat Valley is scanty to realize the importance of this
is a barrier, and not all the larvae can enter and pest and to employ modern breeding techniques.
overcome it; there is no connection to the seed Breeding for improved quality aims mainly
coat’s tannin content. The present breeding of at increase of the seed protein content and
new cultivars with reduced vicine and convicine protein quality. Protein content could easily be
content (see below) of the seed can favor the increased to over 30%. Still, as long as there is
colonization of faba bean by additional weevils no economic incentive to do so, breeders will
such as Callosobruchus maculatus that does not not invest significant effort in achieving this goal.
infest normal-vicine faba beans (Desroches et al., Protein quality is mainly limited due to a low
1995). sulfur-containing amino acids such as methionine
In addition to aphids and beetles, nematodes and cysteine. Classical methods are not promising,
have to be mentioned. Faba bean may be infested since the variation is low and there is a negative
by the stem nematode (Ditylenchus dipsaci) and
oof
genetic correlation between seed protein content
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the cyst nematode (Heterodera goettingiana). The and sulfur-containing amino acids’ content of this
stem nematode is widespread, and its “giant”
a g e
protein (Link et al., 2005). Quality, moreover,

P
race, common in North Africa, is a serious depends on the content of antinutritional factors

Fi rst
pest, especially in cases where nematode-infested like condensed tannins and vicine. Zero-tannin
seeds are sown. Small-seeded beans are generally cultivars, for example those for feeding pigs, do
a poorer host, several resistant genotypes were exist (e.g., “Gloria”). The recessive monogenetic
identified, amongst them is the Ascochyta resistant segregation of this gene and the pure white flower,
line 29H. The cyst nematode is important in many as its pleiotropic effect, make this trait easy to
temperate regions. Obviously no resistances have handle. Low vicine cultivars do as well exist
been found (Sharma et al., 1994). Broomrape, (e.g., “Mélodie”). The trait is again monogenetic
Orobanche crenata, is a parasitic plant, devastating recessive, a morphological marker (white hilum)
pulses and other crops in the Mediterranean Basin and even molecular markers are available. Vicine
and Nile Valley. Hand weeding, use of glyphosate, may have negative effects on human health.
late sowing and breeding is used to fight it. A rare human enzyme deficiency, favism, leads
Breeders have up to now not produced a bean to anemia upon faba bean consumption in
with reliable resistance. Screening is mostly done affected individuals. Also, vicine negatively effects
in fields where this parasitic weed occurs naturally, monogastric animal nutrition like pigs and
which is a difficult test situation. Evaluations chicken. Further antinutritional compounds are
in controlled environments are possible but not of importance in faba beans (Duc et al., 1999).
expensive. These shortcomings make broomrape Classical breeding in faba bean looks back
resistance a problematic trait. Partially resistant on very marked improvements, e.g., for non-
genotypes are available; the resistance trait shows shattering, improved yield and yield stability,
a quantitative genetic variation. The rather highly improved lodging resistance. Still, breeding
resistant genotype F402 identified by Egyptians progress is hampered by the partial allogamy of the
was repeatedly used. Several improved genotypes bean. The pollinators are bumble bees and honey
have been bred from this common source in bees (Link et al., 1994a; Suso and Moreno, 1999).
Egypt (“Giza 402”, “Giza 429”, “Giza 674”) The degree of cross-fertilization is about 50%, with
and in Spain (“Vf1071”, “Vf136”, “Baraca”). a large genotypic and environmental component of
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3-6 LEGUME GRAINS AND FORAGES

Population pollen fertility becomes permanent; offspring

that segregates from a heterozygously restored
plant does not segregate for the pollen sterility
100 loci, p=0.40 q=0.60 0.00 as expected from textbook schemes on CMS
(AA), (Aa)=1; (aa)=0 systems. Molecular genetic findings proposed that
0.50
CMS447 may be the result of an infection with a
Figures indicate 0.75 defect virus, and that restoration corresponds to
inbreeding nontransmittance by seeds (Pfeiffer et al., 1993).
coefficients F
0.88
In Germany, Link et al. (1997) detected two
other CMS systems (CMS199, CMS297), yet due
>0.94
to instability of the sterility, especially due to
spontaneous reversions to pollen fertility, hybrid
breeding still could not be realized in faba bean.
0 20 40 60 80 100 As classical breeding category, line breeding is
Performance, arbitrary scale applied. An important bottleneck in line breeding
is the production of purely selfed seed. In the open
Figure 3 Theoretical distribution of individual plant’s field situation of a breeding nursery, with small
performances in case of partially allogamy like in faba plots and large numbers of genotypes, seed multi-
bean, here with 50% outcrossing of inbred plants and 30%
outcrossing of noninbred plants. The population is shown
plication suffers from uncontrolled contamination
as composition of cohorts of plants with different levels of with cross-pollen, unless spatial isolation and pure
inbreeding [Reproduced from Link et al. (1994b)] lines are used. In cages, pollinators can be excluded
and pure self-fertilization can be enforced. Still,

oof
without pollinator visit, most genotypes admit
r
P
variation and with marked heterosis; heterozygous a need of tripping (a mechanical stimulation
plants show on average less outcrossing than
a g eof the stigma, caused by the pollinator, which

P
homozygous plants. Inbreeding depression for induces successful pollination and fertilization).

Fi rst
grain yield is marked; F1 -hybrids outyield their As a consequence of absence of pollinators, yield
inbred parents mostly by more than 40%. The of purely selfed seed in cages is variable and mostly
partial allogamy and the marked heterosis for very low. Germplasm from Southern Europe and
vigor and productivity cause the genetic variation Northern Africa often shows a lower or no need
of a faba bean population to be very much inflated of tripping. Tripping can be done manually, to
(Figure 3), compared to a situation of pure selfing substitute for the missing pollinators in the cages,
or pure outcrossing. This reduces markedly the thereby allowing true selfing and high seed set,
gain from mass selection for these traits. The but this is a very labor-intensive procedure. A very
reason is that the superiority of the selected plants successful alternative to pure inbred line cultivars
is mainly caused by their high heterozygosity, not is the breeding of synthetic cultivars. Still, due
by a high breeding value; heterozygosity, however, to the limited degree of natural outcrossing, only
is not inherited. A solution is to strictly select about half of the potential hybrid vigor is used in
among entries of identical level of inbreeding, a synthetic cultivar.
preferentially among pure lines.
The first approaches to realize the production
of hybrid cultivars (based on cytoplasmic-genetic 1.5 Rationale for Transgenic Breeding
male sterility) in faba bean trace back to Bond
in Cambridge in 1957 and to Berthelem in The development of gene transfer techniques
Rennes in 1967. Bond worked with the system for faba bean is of commercial interest as they
CMS447, discovered by him in winter beans in might facilitate the production of cultivars with
Newcastle upon Tyne. Berthelem discovered the improved characteristics, such as resistance to
system CMS350 in an English bean population. biotic and abiotic stresses and enhancing the
The system CMS350 was found to be sensitive nutritional value. Chocolate spot, caused by B.
to environmental conditions. The system CMS447 fabae and Ascochyta blight, caused by A. fabae
is very peculiar, since a genetic restoration of are the most widespread and devastating fungal
JWBK201-Kole k0304 March 28, 2008 19:45

FABA BEAN 3-7

diseases in all production areas. Viral diseases transgenic plants in the past (Schiemann and
and the parasitic weed broomrape (O. crenata) Eisenreich, 1989; Ramsay and Kumar, 1990; Saal-
are the most important factors contributing to bach et al., 1994; Siefkes-Boer et al., 1995; Jelenic
the losses in faba bean production in the West et al., 2000). The first attempts to transfer foreign
Asia North Africa (WANA) region (Hanounik genes into faba bean were published by Schiemann
et al., 1993; Robertson and Saxena, 1993). Losses and Eisenreich (1989). They used Agrobacterium
in faba bean caused by broomrape are estimated rhizogenes containing the binary vector pGSGluc1
at the range from 50% to 80% in fields with carrying nptII and uidA genes under the control of
medium and high levels of infestations, respectively the bidirectional TR1/2 promoter. GUS-positive
(Gressel et al., 2004). Pests like black bean roots and subsequently transgenic calli lines were
aphid (A. fabae), sitona weevil (S. lineatus), stem obtained. A similar study was performed by
nematode (Ditylenchus dipsaci), and bruchid beetle Ramsay and Kumar (1990), using an A. rhizogenes
(B. rufimanus) also limit the yields, if no pest strain containing pBin19 for inoculation of V. faba
control is employed. Abiotic stresses like cold and cotyledons and stem tissue, leading to successful
drought are also major constraints to yield as transfer of the nptII marker gene. However, no
strong tolerance genes are not available in the transgenic faba bean plants were regenerated in
primary gene pool of V. faba. These constraints any of these studies. Since the lack of efficient
are strong arguments for genetic transformation protocol for regeneration of transgenic plants was
for the improvement of this crop. Recombinant the main obstacle in faba bean transformation,
DNA technology if applied to faba bean, Jelenic et al. (2000) tried to solve this problem by
however, requires the development of efficient application of bacteria carrying shooty types Ti-
and reproducible protocols for both regeneration plasmids, pGV2215, and pGV2235. Neither of the
and genetic transformation. The combination of
oof
two mutants appeared to be useful for faba bean
r
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conventional breeding approaches, plant tissue regeneration.
culture techniques, and recombinant DNA tech-
a g e
Finally, Böttinger et al. (2001) recovered the

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nology will open the necessary avenues needed for first transgenic faba bean plants from transformed

Fi rst
improvement of faba bean in overseeable time. tissues. Their system was based on de novo
regeneration using thidiazuron (TDZ). The second
successful protocol developed by Hanafy et al.
2. DEVELOPMENT OF TRANSGENIC (2005) was based on direct shoot organogenesis
FABA BEANS from meristematic cells of mature or immature
embryo axes.
Faba bean exhibits a rather low competence for in
vitro culture. This is mainly due to the difficulties
in the regeneration from callus tissues and the 2.1 Agrobacterium-mediated
high content of phenolic compounds, which causes Transformation and Regeneration
cell death if not overcome (Bieri et al., 1984; of Transgenic Faba Bean Plants
Selva et al., 1989). Unfortunately, efficient in vitro
techniques are limited for faba bean as compared Böttinger et al. (2001) reported, for the first time, a
to other major economically important crops. method for the production of transgenic faba bean
Faba bean genetic transformation continues to be plants. This Agrobacterium-mediated transforma-
problematic even with the intense efforts of the tion protocol, adopted from a plant regeneration
researchers in this field. As a consequence, among protocol for protoplast derived calli (Tegeder et al.,
the major grain legume crops V. faba was the 1995), was based on the de novo regeneration of
last species where the production of transgenic shoot initials from callus. The transgenic plants
plants had been reported (Böttinger et al., 2001; were recovered by inoculation of stem segments
Hanafy et al., 2005). In contrast, the closely related with Agrobacterium strains EHA 101 or EHA
species V. narbonensis could be relatively easy to be 105, harboring different binary vectors (Table 1).
manipulated in vitro (Pickardt et al., 1991). Calli were induced on MS medium (Murashige
Several investigators worked extensively on and Skoog, 1962) containing 0.5 mg l−1 of each
faba bean transformation and regeneration of TDZ, 2,4-dichlorophenoxyacetic (2,4-D), and
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3-8 LEGUME GRAINS AND FORAGES

Table 1 Summary of successful transgenic faba bean production strategies

Cultivar Explant A. tumefaciens Transgene Selection Referencses

Mythos Etiolated epicotyls EHA 101, EHA 105 uidA, nptII, bar, Kanamycin, Böttinger et al. (2001)
sfa8, lysC Phosphinothricin
Mythos Embryo axes EHA 105 bar, sfa8 Phosphinothricin Hanafy et al. (2005)
Albatross Embryo axes EHA 105 bar, sfa8 Phosphinothricin Hanafy et al. (2005)
Giza 2 Etiolated epicotyls EHA 105 bar, sfa8 Phosphinothricin Hanafy et al. (2005)

naphthylacetic acid (NAA) and 100 mg l−1 containing high concentrations of cytokinins in
kanamycin or 2 mg l−1 DL-Phosphinothricin combination with low auxin, enhanced direct
(PPT) as a selective agent. Afterwards, transgenic shooting without an intermediate callus phase.
shoots were regenerated via organogenesis using In this manner the possibility of somaclonal
a high concentration of TDZ (7.5 mg l−1 ) and variation has been reduced to the minimum. This
0.75 mg l−1 NAA. Finally, plants were recovered by transformation system was an adaptation of the
micrografting. The process required 16–24 months protocol previously reported by Schroeder et al.
to get seed-producing primary transformants. It (1993) in P. sativum. It allowed obtaining seed-
was laborious and time consuming because the bearing primary transformants within approxi-
quantum of regenerable tissue was very limited mately 9–10 months (Hanafy et al., 2005)
and it had relatively low regeneration efficiency,
prone to somaclonal variation.

of
Consequently, in an effort to improve the 2.1.1 Faba bean cultivars and

r o
transformation efficiency of V. faba and to A. tumefaciens strains
overcome the difficulties reported by Böttinger

g e P transformation and regeneration of

a
et al. (2001), an Agrobacterium-mediated trans- Successful
formation system based upon direct shoot

r s t P transgenic faba bean plants has been achieved

Fi
organogenesis has been developed (Hanafy et al., with three cultivars so far: Mythos, Albatross, and
2005). After transformation of meristematic cells Giza 2. Growth media used for tissue culture,
derived from zygotic embryo axes stable and fertile transformation, and regeneration are listed in
transgenic plants were regenerated. The use of Table 2. Mature faba bean seeds were surface ster-
embryonic axes, which were cultivated on media ilized (ethanol and sodium hypochlorite solution),

Table 2 Growth media used for the production of transgenic faba bean plants

Media Composition(b)

TNZ MS + 3% sucrose, 0.5 mg l−1 2,4-D, 0.5 mg l−1 NAA, 0.5 mg l−1 TDZ
TNZ1 MS + 3% sucrose, 0.5 mg l−1 2, 4-D, 0.5 mg l−1 NAA, 0.5 mg l−1 TDZ, 500 mg l−1 Betabactyl
TNZ2K MS + 3% sucrose, 0.5 mg l−1 2, 4-D, 0.5 mg l−1 NAA, 0.5 mg l−1 TDZ, 300 mg l−1 Betabactyl, 100 mg l−1
kanamycin.
TNZ2P MS + 3% sucrose, 0.5 mg l−1 2,4-D, 0.5 mg l−1 NAA, 0.5 mg l−1 TDZ, 300 mg l−1 Betabactyl, 2 mg l−1 PPT.
MTN MS + 3% sucrose, 7.5 mg l−1 TDZ, 0.75 mg l−1 NAA.
MB1 MS +3% sucrose, 1 mg l−1 BAP, 1 mg l−1 GA3, 100 mg l−1 coconut milk, 100 mg l−1 Ticarcillin, and 50 mg l−1
Combactam.
CCM Gamborg B5 medium + 30 g l−1 sucrose, 0.5 mg l−1 kinetin, 1 mg l−1 2, 4-D.
SRM MS basal salt medium supplemented with B5 vitamins(a) , 2 mg l−1 NAA, 2 mg l−1 BAP, 150 mg l−1 Ticarcillin, and
100 mg l−1 Combactam.
SIM1 MS basal salt medium supplemented with B5 vitamins, 4.5 mg l−1 BAP, and 2 mg l−1 PPT.
SIM MS basal salt medium supplemented with B5 vitamins, 4.5 mg l−1 BAP, 0.1 mg l−1 NAA, 100 mg l−1 Ticarcilline,
50 mg l−1 Combactam, and 2 mg l−1 PPT.
(a)
Reproduced from Gamborg et al. (1968).
Antibiotics and phosphinothricin are added to the medium after autoclaving. Cultures are kept at 21 ◦ C under cool white fluorescent light
(b)

(80 µmol m−2 s−1 ).

JWBK201-Kole k0304 March 28, 2008 19:45

FABA BEAN 3-9

soaked overnight in sterile tap water with shaking CCM medium at 25 ◦ C in dark for 3–4 days.
and germinated in the dark at 20 ◦ C on 12 MS- After the co-cultivation period, explants were
basal medium. After about 10 days, internodal cultured in a glass container on TNZ1 medium,
segments of the arising main shoots were used in the dark at 20 ◦ C for 3–4 days, for recovering
for co-cultivation with Agrobacterium tumefaciens. without selection pressure. The explants were
The remaining seedlings were kept in dark, and sec- subsequently transferred to TNZ2K- or TNZ2P-
ondary shoots, arising from the cotyledonary buds medium (depending on the selectable marker). The
during the following weeks, were used as explants explants were subcultured every 2 weeks on fresh
source for further transformation experiment. medium for a period of 3–4 months. Resistant
The following A. tumefaciens strains have been calli reaching a diameter of approximately 5mm
successfully used for production of transgenic faba were transferred to MTN-medium. Calli were
bean plants (Böttinger et al., 2001; Hanafy et al., subcultured every 3–4 weeks on this medium for a
2005): (1) EHA101 carrying pGSGluc1, harboring period of more than 12 months. The appearance
uidA and nptII genes, both under the control of of shoot primordia varied between 4 and 12
the bidirectional TR1/2 promoter; (2) EHA101 months. Shoot primordia were transferred to
carrying pAN109, harboring the nptII marker gene elongation medium (MB1) and subcultured every
under the nopaline synthase promoter (Pnos) and 3–4 weeks.
the mutated Escherichia coli lysC gene, coding Healthy resistant shoots, more than 1 cm in
for a feed-back desensitized aspartate kinase III length were excised and grafted onto etiolated
(Shaul and Galili, 1992), driven by the seed specific seedlings of untransformed faba bean of about
bean phaseolin promoter; (3) EHA105 carrying 7–10-day-old (see Pickardt et al., 1991 for details).
pBIOU, harboring the Pnos-nptII marker cassette After 2–3 weeks, when the shoot developed new
and an intron-containing genomic sequence for
oof
leaves, the transgenic plants were transferred to
r
P
the sunflower 2S-albumin 8 (sfa8; Kortt et al., the greenhouse and grown to maturity.
1991), driven by the seed specific usp promoter
a e
g2.1.2
P
from V. faba (Bieri et al., 1984); (4) EHA105

Fi rst
carrying pGlsfa (pGPTV-bar derivative, Becker Transgenic faba bean plants
et al., 1992), harboring the sfa8 gene under the
seed specific legumin B promoter (LeB4) and the Two successful protocols for regeneration of
bar gene under the nos promoter. transgenic faba bean plants have been developed
Bacteria carrying the desirable vector were so far. The first protocol was based on de novo
harvested by centrifugation and resuspended in regeneration using TDZ (Böttinger et al., 2001),
an equal volume of liquid TNZ medium (de novo and the second protocol was based on direct shoot
regeneration protocol) or the bacterial culture was organogenesis from meristematic cells of mature
diluted in a rate of 1:5 with liquid CCM (embryo or immature embryo axes (Hanafy et al., 2005).
axes transformation system). Etiolated epicotyls In both these transformation systems, the control
were cut into segments of 0.2–0.4 cm in length in experiments showed that PPT at 2 mg l−1 totally
the bacterial suspension and incubated for 30 min, suppressed callus development from wild type
then transferred into 250 ml glass containers with faba bean epicotyl segments, cultured on TNZ, or
solidified TNZ medium and co-cultivated for embryo axes cultured on shoot inducing medium
48 h at 20 ◦ C in the dark (de novo regeneration 1 (SIM1).
protocol). With regard to the de novo regeneration pro-
Regarding the embryo axes transformation tocol, in a series of transformation experiments,
system, embryo axes of both mature and immature resistant callus started proliferating after about
seeds were wounded by the removal of the root 1–2 months on the surface of a number of
tips and slicing of the embryo axes to three or explants on TNZ2P medium. Within 3–4 months
four segments longitudinally and inoculated in after culturing the explants on TNZ2P medium,
the bacterial suspension for 15–20 min (immature 4.3–31.6% of the explants produced resistant
embryos) and 30 min (mature embryos) with calli (Figure 4). PPT resistant calli with a
occasional agitation. After infection, the explants diameter of about 5–10 mm were transferred to
(30–40 per plate) were co-cultivated on solid the MTN medium to increase the callus viability
JWBK201-Kole k0304 March 28, 2008 19:45

3-10 LEGUME GRAINS AND FORAGES

Figure 4 Successful transformation and regeneration of transgenic faba bean. (a) Initiation of resistant callus from stem explants
under selection pressure of 2 mg l−1 PPT on TNZ2P- medium. (b) Callus proliferation on MTN medium. (c) Shoot regeneration on
MTN medium. (d) Explant segments derived from embryonic axes. (e) Multiple shoot regeneration under selection pressure (left),
all the control explants (WT) were dead (right). (f) Further selection between PPT resistant and susceptible regenerated shoots on
medium containing 2 mg l−1 PPT. (g) Recovering the transgenic plant by micrografting

to regenerate (Figure 4). Shoot regeneration EHA105/pGlsfa (harboring SFA8 and bar genes)
occurred after 6–12 months on MTN medium alone, or co-transformed with EHA101/pAN109,
(Figure 4c). Shoot primordia were transferred to
oof
which carried a mutated lysC gene from E. coli and
r
P
shoot elongation medium, MB1. Because of the nptII (Böttinger et al., 2001) (Figure 4d). After
very low rooting percentage of the regenerants,
a g e3–4 weeks of culturing on selective medium, all

P
shoots reaching a suitable size (within 2–4 control explants had died. On the other hand,

Fi rst
months) were micrografted onto nontransgenic the transformed explants started regenerating (via
root stocks of faba bean to recover whole organogenesis), and about 3–4 shoots appeared
plants. Afterwards, the plants were transferred from each explant on SIM (Figure 4e). The shoots
to soil for acclimatization. Regenerated plants selected for 4–6 months (Figure 4f) were grafted
were transferred to the greenhouse for further in vitro and finally transferred to the greenhouse
plant development and production of T1 seeds. to set seeds (T1 ) (Figure 4g). A total of seven
Some regenerated clones showed morphological stable independent transformants (containing
abnormalities, such as dwarfness and formation SFA8 linked to bar from cv. Mythos and Albatross)
of abnormal flowers and subsequently abnormal have been recovered. Transformation frequencies
(or no) pods. Some plants showed narrow leaves ranged from 0.15% to 2.0%. Regarding the co-
and very weak stems with weak apical dominance. transformation experiments, transformed plants
Only four clones of the cultivars Mytos and Giza (T1 ) were screened for the presence of both
2 produced normal flowers and pods with seeds. T-DNAs by PCR analysis. It was found that only
The time needed to obtain T1 seeds by this process the T-DNA encoding the PPT selectable marker
was about 16–24 months. has been successfully integrated. The time needed
On the other hand, the feasibility of the to obtain T1 seeds by this protocol was about 9–
transformation strategy based on embryo axis 10 months.
transformation was initially evaluated by mon- The transgenic plants were analyzed by testing
itoring the number of regenerated shoots from the expression of bar gene in the greenhouse by
the embryo axis explants, cultured on medium using the leaf painting assay or spraying the young
with high BAP concentration (4.5 mg l−1 ), where plants with the same dilution of the herbicide.
routinely 4–5 shoots were regenerated from each Within two days necrotic spots appeared on the

R
explant. In a series of transformation experiments, untransformed leaves. Ten days after BASTA
the explants (immature or mature embryonic application, the treated transgenic plants and
axes) were inoculated with Agrobacterium strain leaflets showed complete tolerance, in contrast
JWBK201-Kole k0304 March 28, 2008 19:45

FABA BEAN 3-11

R
Figure 5 Herbicide leaf painting test showing the resistance of transgenic leaf to BASTA (300–400 mg l−1 ammonium glufosinate)
application (left)

to completely necrotic leaflets on nontransformed Transformation of pre-excited meristematic

of
plants (Figure 5). cells on the embryo axes is much simpler.

P r o
The major success in legume transformation

e
was achieved by methods based on transfor-
3. FUTURE ROAD MAP
P a g mation of the pre-existing meristems on the

r
Each of the two successful protocols ifor
t
sregen-
embryo axes, cotyledonary nodes, shoot tips,

F
or nodal explants. An Agrobacterium-mediated
eration of faba bean transgenic plants (Böttinger genetic transformation system using pre-excited
et al., 2001; Hanafy et al., 2005) has its advantages meristematic cells on the embryo axes is an
and drawbacks. effective method for the production of transgenic
The major advantage in using de novo regen- faba bean, which provides a useful strategy for
eration protocol is regeneration of nonchimeric an efficient insertion of the economically useful
plants. This process, however, is more time genes into its cultivars. It was demonstrated that
consuming and the main constraints in this the embryo axes of faba bean are competent
protocol are poor regeneration ability, following a for production and multiplication of shoots and
callus phase, reduced fertility and high percentage inherited transformation.
of phenotypic abnormalities in regenerated plants. The production of commercial transgenic faba
This can be ascribed to the fact that explants bean plants requires an efficient regeneration
and callus cells of V. faba tend to produce high system. Although transgenic faba bean plants
amounts of phenolic compounds, resulting in can now be regenerated successfully, the process
subsequent intoxication of the tissue (Bieri et al., is still labor-intensive and therefore, requires
1984; Selva et al., 1989). A possible cause of these improvement.
abnormalities is also due to the long culture time in
vitro (around 7–16 months). The recovery of seed-
producing putative transformed plants under these 3.1 Expected Products
circumstances takes about 16–24 months, which is
a considerably long time period. Transformation Some of the problems of classical faba bean
strategies, which minimize the in vitro culture breeding might be addressed by applying
period and avoid the callus phase would, therefore, strategies of genetic modification. Orobanche sp.
be advantageous. was shown to be sensitive to glyphosate (Lolas,
JWBK201-Kole k0304 March 28, 2008 19:45

3-12 LEGUME GRAINS AND FORAGES

1994; Goldwasser et al., 2002). In this respect, it of the feasibility of nutritional value improvement
would be feasible to introduce genes conferring by transgenic approaches. Böttinger et al. (2001)
resistance to glyphosate to faba bean, like 5- demonstrated the expression and specific activity
enolpyruvylshikimate-3-phosphate synthase from of the mutated lysC gene in transgenic faba bean
the CP4 strain of A. tumefaciens or glyphosate plants. Still, the desired change in amino acid
oxidase from Ochrobactrum anthropi. These genes composition in the transgenic seeds remains to
are already being efficiently used in commercial be demonstrated. In addition, the same group
transgenic crops like cotton and soybeans demonstrated the expression of the methionine-
(Padgette et al., 1995; Saroha et al., 1998). BASTA rich sunflower albumin gene sfa8 in faba bean
(phosphinotrycin-glufosinate ammonium) or transgenic lines using RT-PCR. However, due
bromoxilin herbicide resistance genes could be to the lack of the specific antibody, it was
introduced as well (Cubero and Nadal, 2005). not possible to demonstrate the accumulation
They are not effective against Orobanche sp., but of the corresponding protein in the generated
could be of help against other unwanted weeds in lines. The work of Hanafy et al. (2005) made
the field. Similarly, insect pests of faba bean from another important step forward. By RT-PCR
the orders Lepidoptera, Coleoptera, and Diptera the authors detected the presence of the sfa8
could be controlled by heterologous expression transcripts in the T2 and T3 generation of their
of the Cry proteins from Bacillus thuringiensis transgenic lines. More importantly, by Western
(Schnepf et al., 1998). Moreover, to reduce losses blot analysis the accumulation of the SFA8 protein
caused by aphids the Galanthus nivalis agglutinin was demonstrated in transgenic faba bean lines.
(GNA), a lectin, might be tested in addition to In the two tested generations (T2 and T3 ), the
Cry proteins (Wang et al., 2005). SFA8 expression level remained uniform. The
Nematode resistance could be conferred to faba
oof
exact levels of the foreign protein could not be
r
P
bean by the introduction of the Mi-1.2 gene from determined, due to the lack of purified SFA8.
the wild relative of tomato Lycopersicon peru-
a g e Apart from demonstrating that the transgenic

P
vianum. It was shown that this gene contributes approach could be used to improve protein quality

Fi rst
resistance against nematodes, but also against in faba bean, these two reports also showed that
aphids, when introduced to cultivated tomato the bar gene (PPT acetyltransferase) could be
(Rossi et al., 1998). Heterologous expression of effectively used as a selective agent of transgenic
the same gene in eggplant (Solanum melongena) individuals, instead the unpopular antibiotic
displayed resistance to nematodes (Goggin et al., resistance. Phosphinotricin resistance conferred
2006). A comprehensive list of other cloned or by the bar gene, can be used in the early phase
mapped nematode resistance genes from different of positive clone selection. More importantly,
species has been published recently (Williamson bar-based selection is easily conducted in the
and Kumar, 2006). glasshouse or in the field on fully grown plants.
The problem of faba bean fungal diseases could
potentially be solved by transformation with genes
encoding for plant defensins, a group of small 3.2 Expected Technologies
peptides broadly present throughout the plant
kingdom. These peptides exhibit a wide-range The introduction of molecular biology techniques
antifungal activity, when expressed heterologously revolutionized plant research. In particular, the
(Thomma et al., 2002). For example, the sequencing of the Arabidopsis thaliana genome
Alium cepa antimicrobial protein 1 (Ace-AMP1; has produced a wealth of information on virtually
Cammue et al., 1995), a defensin, was shown to every aspect of plant biology. Also, it has set new
be very efficient in conferring B. resistance to standards that transformed the way plant research
Pelargonium sp. (Bi et al., 1999), powdery mildew is done: high throughput mutagenesis, genome
resistance to Rosa hybrida (Li et al., 2003), and saturated collections of mutants, construction of
most recently, conferring resistance to several fun- dense genetic maps used in positional (map-
gal pathogens in wheat (Roy-Barman et al., 2006). based) cloning, excellent efficiency of in planta
The only two reports so far, describing success- transformation, and availability of the whole-
ful regeneration of transgenic faba bean, raise hope genome microarrays. However, A. thaliana simply
JWBK201-Kole k0304 March 28, 2008 19:45

FABA BEAN 3-13

cannot encompass all the diversity of physio- In order to localize QTLs, such as for yield or
logical processes present throughout the plant seed protein composition, segregating populations
kingdom, e.g., the nodule development in legumes. for mapping agronomically important traits will
Therefore, the development of legume-specific be made available. The Legume Crops Genome
plant models is necessary. Today, two cool-season Initiative (LCGI) predicted that within the next
legume models are established: Lotus japonicus and decade the complete Phaseolus sp. and Arachis sp.
Medicago truncatula. Similarly, Glycine max, as the sequences will be ready, as well as partial sequences
economically the most important legume, is the of Pisum sp. and Cicer sp. (Gepts et al., 2005). At
model for the warm-season legumes. The complete the moment, it is hard to predict weather V. faba
genome sequences of the two cold-season model will reach the same level of sequence information
plants should be complete within the next 1–2 by that time.
years (Young et al., 2005). The soybean genome, For the model species L. japonicus and M.
due to its size of 1200 Mb, is relatively far from truncatula, more effort will be put on elucidating
being completely sequenced. their transcriptome, proteome, and metabolome
Translating the knowledge gained through these (Colebatch et al., 2004). These datasets are
model species, to crop legumes, such as V. faba, inevitable to fully comprehend the regulation of
might improve both classical and transgenic synthesis of desired products: proteins, lipids, and
breeding programs. Its 13 059 Mb genome (Zhu carbohydrates, as well as secondary metabolites
et al., 2005) and its rather secondary economic interacting with pathogens, symbionts, as well as
importance, are serious obstacles for sequencing beneficial or harmful insects.
the faba bean genome. So, it is certain that faba As the amount of genomic sequences from both,
bean breeders will have to rely on information the model and crop legumes, will grow, the correct
extractable from sequences of the model legumes,
oof
annotation of gene function is going to be crucial.
r
P
at least for time being. To perform functional annotation, reverse genetics
Genomics of model legumes represents the
a g e
approaches similar to those in A. thaliana are

P
basis for understanding the molecular, as well as also being employed in model legumes, e.g., a

Fi rst
metabolic basis of synthesis and accumulation of TILLING facility (targeted induced local lesions
desired compounds (e.g., storage proteins), and to in genomes; (Till et al., 2003) has been set up
decipher mechanisms by which legumes resist pests with about 5000 L. japonicus mutagenized lines
and unfavorable environmental conditions. Not (Gilchrist and Haughn, 2005).
less important, genomics is the base for generating The relatively close phylogenetic relationship
large numbers of molecular markers that can be of faba bean and the model legumes is a strong
used to assist classical plant breeding. argument in favor of translational genetic strate-
The legume community has the intention to gies. This was further substantiated by one recent
develop several translational genomic tools that phylogenetic analysis of legumes, based on chloro-
will facilitate the exchange of genetic sequence plast maturase K (matK) sequence (Cronk et al.,
information among different legume species. These 2006). In particular, the Papilionoideae subfamily,
“translational tools” include collections of ESTs, to which crop and model legumes belong, was sub-
cDNA libraries from different plant tissues (seed, divided into seven informally named clades. Faba
leaf, nodule, flower, pod), as well as from various bean was classified to the inverted repeat loss clade
environmental conditions, genomic libraries, and (IRLC), together with legume model M. truncatula
generation of dense genetic maps. Due to limited and crop legumes L. culinaris, P. sativum, Cicer
resources, a priority list of legume species was arietinum. The next most related clade Robinioids
defined. Faba bean is positioned reasonably high includes the other model legume L. japonicus.
on that list, but only after common bean, chickpea, The close phylogenetic relationship between
peanut, and peas (Gepts et al., 2005). Eventually, faba bean and model legumes implicates the
sequencing of gene-rich regions for this group of existence of common genomic microstructure and
crop legumes is planned. In addition, it is aimed macrostructure. Macrostructural similarity, also
to produce a set of at least 500 genetic markers referred as synteny implies the presence of the same
per legume crop species, which will be linked genes on the same chromosome of two species. On
to whole-genome sequences of model legumes. the other hand, microstructural similarity, referred
JWBK201-Kole k0304 March 28, 2008 19:45

3-14 LEGUME GRAINS AND FORAGES

as microsynteny or colinearity, indicates conserved genes. For example, three amino acids permeases,
gene order on a particular chromosome of two VfAAP1, VfAAP3, and VfAAP4, from faba bean,
species. For example, it was demonstrated that have been cloned from a seed-specific cDNA
L. japonicus and M. truncatula are structurally library (Miranda et al., 2001). The same group
more conserved than, e.g., Glycine max and M. reported the cloning of two putative peptide
truncatula (Choi et al., 2004). This is of practical transporter homologues VfPTR1 and VfPTR2. In
importance for designing strategies for small scale the future these candidate genes could be used
sequencing projects and comparative mapping on to manipulate the amounts and composition of
faba bean. Another study compared conserved storage proteins in faba bean seeds.
microsynteny between G. max and M. truncatula, The assignment of various linkage groups and
using a hybridization-based approach (Yan et al., even single genes to particular chromosomes
2003). The high degree of microsynteny detected was facilitated by the work on faba bean
in this study, implied that it should be possible to primary trisomics (Torres et al., 1995; Vaz Patto
use the genome sequence of M. truncatula as a et al., 1999). So far five out of six possible
reference for isolating genes in soybean and other trisomics have been generated (Pozarkova et al.,
closely related legumes, e.g., faba bean. 2002). For the large chromosome 1, for which
A further example for using sequence informa- trisomics could not be obtained so far, a linkage
tion gained on model legumes in crop legumes is map has been generated with an average 8 cM
the cross-species application of EST- and genomic- map interval (Cubero and Nadal, 2005). The
derived SSRs (microsatellites) for M. truncatula integration of genetic and physical map of
in most important European legume pulses: pea, faba bean is further improved by techniques of
faba bean, and chick pea (Gutierrez et al., 2005). flow sorting (Dolezel and Lucretti, 1995; Macas
Of 242 M. truncatula simple sequence repeats
oof
et al., 1996) and in situ hybridization (Fuchs
r
P
(SSRs) analyzed, approximately 40% gave cross- and Schubert, 1995). Even more, flow sorting of
amplification in faba bean. Detailed sequence
a g e
V. faba chromosomes, due to their large size, has

P
analysis of these SSR sequences showed a rather become a model for plant chromosome sorting

Fi rst
high variability in the microsatellite motif itself, in general. Also, by using flow fractionation of
and much more conserved flanking regions. chromosomes (Dolezel et al., 2001), it should be
However, amplified microsatellites did not show a possible to prepare chromosome-specific bacterial
size polymorphism in different parental genotypes artificial chromosome (BAC) libraries. These
of faba bean used in the study. This outcome sets BACs could be screened with marker-derived
limits to their use in mapping projects. Still, the probes flanking QTL regions. Sequencing the
effort to produce a large number of cross-legume positive BAC candidates could then lead to the
markers will continue. identification of genetic basis of a particular QTL.
The germplasm of elite faba bean cultivars was In a recent flow sorting approach, microsatellite-
reported to have a rather broad genetic base, enriched chromosome-specific DNA libraries were
as demonstrated by a recent amplified fragment generated, and used for developing of novel
length polymorphism (AFLP) analysis (Zeid et al., DNA markers (Pozarkova et al., 2002). Also, first
2003). So, cultivar improvement by using this attempts were undertaken to fine-map genes or
intraspecific variability presents an important QTLs responsible for resistance to fungal diseases
alternative to the currently unfeasible interspecific (Avila et al., 2003; Roman et al., 2003) or parasitic
hybridization. This approach is currently used plants (Roman et al., 2002). Still, the saturation
to genetically improve faba bean against several of the faba bean linkage map is rather difficult to
important sources of biotic stress, such as fungal achieve due to its huge genome size. A list of link-
diseases caused by rust (U. viciae-fabae), mildew age maps currently available for the V. faba genome
(Peronospora viciae), chocolate spot (B. fabae), has been published recently (Torres et al., 2006).
Ascochyta blight (A. fabae), or the parasitic species As a conclusion, it can be stated that the
broomrape (O. crenata). In addition to these rather major hindrances in generating transgenic faba
classical breeding approaches, the application of bean plants have been overcome. Since the wild
DNA markers and other molecular tools led to progenitor of V. faba has not been determined
precise mapping and isolation of several faba bean yet (Cubero, 1974), faba bean is considered as
JWBK201-Kole k0304 March 28, 2008 19:45

FABA BEAN 3-15

a genetically isolated species, unable to naturally 10th Congress of the European Association for Research on
cross with other Vicia species (Bond et al., 1985). Plant Breeding, EUCARPIA, Wageningen, p. 295.
Bond, D.A. (1995) Faba bean. In: Smartt, J. and Simmonds,
As a consequence, its gene pool seems rather
N.W. (eds.) Evolution of Crop Plants. Longman, Essex,
restricted. Therefore, genetic improvement of faba pp. 312–316.
bean via transgenic approaches is a possible step Bond, D.A., Lawes, D.A., Hawtin, G.C., Saxena, M.C. and
forward. Since, only recently, reproductive and Stephens, J.S. (1985) Faba bean (Vicia faba L.). In:
efficient techniques for regeneration of transgenic Summerfield, R.J. and Roberts, E.H. (eds.) Grain Legume
Crops. William Collins Sons, London, pp. 199–265.
V. faba plants have been established, the number Böttinger, P., Steinmetz, A., Schieder, O. and Pickardt, T.
of transgenes that have been introduced into the (2001) Agrobacterium-mediated transformation of Vicia
faba bean genome is so far limited to five: bar, faba. Molecular Breeding 8, 243–254.
uidA, nptII, sfa8, and lysC (Table 1). Nevertheless, Cammue, B.P., Thevissen, K., Hendriks, M., Eggermont,
these efforts demonstrate a proof of principle K., Goderis, I.J., Proost, P., Damme, V.J., Osborn, R.W.,
Guerbette, F., Kader, J.C. and Broekaert, W.F. (1995) A
that transgenic technology can be used as one of potent antimicrobial protein from onion seeds showing
the approaches to improve faba bean breeding. sequence homology to plant lipid transfer proteins. Plant
There are numerous traits that would be suitable Physiology 109, 445–455. Q1
for improving economic faba bean production. Choi, H.K., Mun, J.H., Kim, D.J., Zhu, H., Baek, J.M., Mudge,
However, to complete the whole procedure of J., Roe, B., Ellis, N., Doyle, J., Kiss, G.B., Young, N.D.
and Cook, D.R. (2004) Estimating genome conservation
generating a commercial faba bean line, starting between crop and model legume species. Proceedings of the
with the newly developed transgenic plant, then National Academy of Sciences of the USA 101, 15289–15294. Q2
outcrossing the new trait into economically Colebatch, G., Desbrosses, G., Ott, T., Krusell, L., Montanari,
valuable genotypes, and eventually getting the O., Kloska, S., Kopka, J., Udvardi, M.K. (2004)
Global changes in transcription orchestrate metabolic
approval for registration of the transgenic line, is

of
differentiation during symbiotic nitrogen fixation in Lotus

o
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P a
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JWBK201-Kole k0304 March 28, 2008 19:45

Abstract:

Vicia faba L. is an old world grain legume, combine

harvested for food and feed in Europe, North and
East Africa, Asia, and in the north west of South
America. It is an annual, diploid plant (x = 6),
with a large genome (1C ∼ 13 pg of DNA), large
seeds (0.3 up to >2 g per seed), and a protein
content of about 30%. Being a small agricultural
species, a relatively large producer in Europe is the
United Kingdom with more than 150 000 ha. On
the global scale, China is the largest producer (39%
of global area of 2.6 million hectares). Important
fungal diseases are Botrytis fabae, Ascochyta fabae,
and Uromyces viciae-fabae. Important pests are
Aphis fabae and Bruchus rufimanus. Broomrape
(Orobanche crenata) is an important parasitic
weed in the Mediterranean basin and the Nile
valley. Traditional breeding is hampered by the
partial allogamous, entomophilous reproduction,
with degrees of outcrossing between 20% and
75%, depending on the actual genotype and the
environment. The faba bean cannot be crossed
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with related species, and it is known as recalcitrant
crop as to in vitro technologies.
a g e
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Keywords:

Vicia faba L., breeding, resistance, breeding

method

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JWBK201-Kole k0304 March 28, 2008 19:45

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they are correct?]
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Q15. [Author: To conform to our house style, in a reference, names of all the authors are to be mentioned.

Fi rst
We have located the names of all the authors on Google and added them to reference “Rossi, et al.
1998” here. Please can you confirm that the names are correct? Similarly, we have added author
names in other references as well, which are highlighted in green. Could you please confirm that
they are correct?]
Q16. [Author: To conform to our house style, in a reference, names of all the authors are to be mentioned.
We have located the names of all the authors on Google and added them to reference “Saalbach,
et al. 1994” here. Please can you confirm that the names are correct? Similarly, we have added
author names in other references as well, which are highlighted in green. Could you please confirm
that they are correct?]
Q17. [Author: To conform to our house style, in a reference, names of all the authors are to be mentioned.
We have located the names of all the authors on Google and added them to reference “Schnepf,
et al. 1998” here. Please can you confirm that the names are correct? Similarly, we have added
author names in other references as well, which are highlighted in green. Could you please confirm
that they are correct?]
Q18. [Author: To conform to our house style, in a reference, names of all the authors are to be mentioned.
We have located the names of all the authors on Google and added them to reference “Till, et al.
2003” here. Please can you confirm that the names are correct? Similarly, we have added author
names in other references as well, which are highlighted in green. Could you please confirm that
they are correct?]

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JWBK201-Kole k0304 March 28, 2008 19:45

Q19. [Author: To conform to our house style, in a reference, names of all the authors are to be mentioned.
We have located the names of all the authors on Google and added them to reference “Yan, et al.
2003” here. Please can you confirm that the names are correct? Similarly, we have added author
names in other references as well, which are highlighted in green. Could you please confirm that
they are correct?]
Q20. [Author: To conform to our house style, in a reference, names of all the authors are to be mentioned.
We have located the names of all the authors on Google and added them to reference “Young, et al.
2005” here. Please can you confirm that the names are correct? Similarly, we have added author
names in other references as well, which are highlighted in green. Could you please confirm that
they are correct?]

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