David F. Herridge - Mejora de las leguminosas para incrementar la fijación de nitrógeno

Breeding legumes for
enhanced nitrogen fixation
David Herridge, University of New England
Ross Ballard & Liz Drew, SARDI
Australia

Atletico Madrid 1 – Barcelona 1
Atletico Madrid 2014 Champions
La Liga

This talk…...
• Legume N2 fixation – global picture
• Scope for improving N2 fixation by the agricultural legumes
• Selection and breeding legumes for N2 fixation - an old concept
• Strategies for selection and breeding
• Methods for assessing nodulation and N2 fixation of plant genotypes
• Selection & breeding programs, based on the traits:
– Plant vigour
– Promiscuous nodulation
– Selective nodulation
– Exclusive nodulation
– Nodulation and N2 fixation in the presence of moderate-high soil N
• Hypernodulating plant mutants
• Natural variation
– Plant N metabolites
• Breeding legumes for N2 fixation - what was achieved?
• To conclude…where to now?

Legume N2 fixation – global picture
• Pulse and oilseed legumes on 208 million hectares are estimated to fix
about 26 million tonnes N annually, nominally valued at $40 billion
• A 10% improvement in N2 fixation, in the absence of any impact on grain
yield, would have a value of $4 billion
• If grain yield was also increased by 10% as a result of improved N2
fixation, the economic gain would be >$20 billion annually
Area
(Mha)
Grain
prodn (Tg)
Total crop
N (Tg)
%Ndfa Crop N
fixed (Tg)
Dry beans 30 23 1.9 40 0.7
Total pulses 80 69 5.9 57 3.4
Soybean 104 262 29.5 68 20.1
Groundnut 25 40 3.2 68 2.2
Total crop
legumes
208 371 38.6 68 26.4
Values calculated from FAOSTAT (2014) and Herridge et al., Plant Soil 311: 1-18 (2008).

Scope for improving
N2 fixation by the
agricultural legumes?
• Do legumes fix sufficient N to
meet crop N demand?
• Answer is fundamentally yes,
except for common bean
• However, N2 fixation could be
improved for all species,
particularly in the way the
legume deals with
environmental (soil salinity,
acidity, high N, low P) and
biological constraints (infective
but ineffective soil rhizobia)
0
10
20
30
40
50
60
70
80
90
Group A Group B Group C Group D
%Ndfa
Farmers' fields
Experimental
Group A – common bean
Group B – chickpea, lentil, pea,
cowpea, mungbean
Group C – soybean, groundnut
Group D – fababean, lupin
Data aggregated from 19 publications plus >800
measurements of crops in farmers’ fields (Herridge et
al. (2008); Peoples et al., Symbiosis 48: 1-17 (2009))

Selection and breeding legumes for N2
fixation - an old concept
• Given the potential economic payoff, not
surprising that plant selection and breeding
programs for legume N2 fixation initiated
• Perhaps the earliest was that of Phillip
Nutman, Rothamstead UK, during the
1940s focussed on the clovers (Trifolium).
• Quoting Fraser Bergersen1
“…His research
pre-dated the molecular genetics now available to
modern researchers and used the techniques of
plant physiology and Mendelian genetics to
explore the mechanisms of infection, subsequent
nodule development and the symbiotic fixation of
atmospheric nitrogen…”
• Emphasis shifted to the pulse and oilseed
legumes in the 1960s and 70s, e.g. Lance
Mytton (UK), Don Phillips (US)
1
FJ Bergersen, Biogr Mems Fell R Soc 51
(2005)
Phillip Nutman

Strategies for selection & breeding
legumes for enhanced N2 fixation
• Legume N2 fixation determined by two factors - growth
of the legume (N yield), and the percentage of legume N
derived from N2 fixation (%Ndfa)
• Thus N fixed (kg/ha) = N yield (kg/ha) x Ndfa (%)
• Then N2 fixation can be enhanced by:
– increasing total N yield
– and/or increasing %Ndfa (most crop legumes)
• Of course, the following is also true
• N yield (kg/ha) = N fixed (kg/ha)/Ndfa (%)
• Then N yield (grain yield) can be enhanced by:
– increasing plant N2 fixation

legumes for enhanced N2 fixation
• Legume N2 fixation determined by two factors - growth
of the legume (N yield), and the percentage of legume N
derived from N2 fixation (%Ndfa)
• Thus N fixed (kg/ha) = N yield (kg/ha) x Ndfa (%)
• Then N2 fixation can be enhanced by:
– increasing total N yield
– and/or increasing %Ndfa (most crop legumes)
• Of course, the following is also true
• N yield (kg/ha) = N fixed (kg/ha)/Ndfa (%)
• Then N yield (grain yield?) can be enhanced by:
– increasing plant N2 fixation (common bean)

legumes for enhanced N2 fixation…
• Variation in total N yield and %Ndfa can be generated a number
of ways, associated with the following plant traits:
– Increased plant vigour and adaptation to biotic and abiotic plant-
growth constraints, e.g. low P, acid soils
– Selective nodulation1
– Exclusive nodulation1
– N2
fixation in the presence of moderate-high soil N
(hypernodulation)
– Plant N traits, e.g. %grain protein, petiole %ureides.
• Essentially all selection and breeding programs for legume N2
fixation target one or more of those 6 plant traits.
1
Yates et al. (2011) Plant Soil 348: 255-267.

legumes for enhanced N2 fixation…
• Variation in total N yield and %Ndfa can be generated a number
of ways, associated with the following plant traits:
– Increased plant vigour and adaptation to biotic and abiotic plant-
growth constraints, e.g. low P, acid soils
– Selective nodulation1
– Exclusive nodulation1
– N2
fixation in the presence of moderate-high soil N
(hypernodulation)
– Plant N traits, e.g. %grain protein, petiole %ureides.
• Essentially selection and breeding programs for legume N2
fixation target one or more of those 6 plant traits.
1
Yates et al. (2011) Plant Soil 348: 255-267.

Strategies for selection & breeding - high
N2-fixing legume ideotype…
• The perfect breeding program
would produce a legume that:
– had good vigour with a high
demand for N and tolerance to
biotic and abiotic stresses
– freely nodulated and/or
preferentially nodulated with
highly effective soil/inoculant
rhizobia
– continued to fix N2 in the
presence of soil nitrate

Methods for assessing nodulation and N2
fixation of plant genotypes
• Nodulation assessed visually, by number and by weight
• Plant N yield determined simply as plant biomass x %N
• Acetylene (C2H2) reduction for N2 fixation activity
• Various techniques to assess %Ndfa, including:
– N difference
– natural 15
N abundance
– enriched 15
N isotope dilution
– xylem/stem ureides
• For large-scale selection of plant genotypes for %Ndfa, the
most appropriate methods arguably natural 15
N abundance
and xylem/stem ureides
• The ureide method only relevant to ureide producing
legumes in the tribes Phaseoleae and Desmodieae e.g.
soybean, common bean

Use of the xylem solute method for
assessing breeding material for %Ndfa
Ureides
Amino N
Nitrate
N2
Soil nitrate
N2
• Composition of N solutes in the
xylem stream changes from one
dominated by ureides in strongly
N2-fixing plants to one dominated
by nitrate and amino-N in plants
using soil N.
• Used successfully to assess
populations and as non-
destructive assay of single F2
plants (800 sampled & analysed
in a 4-week period) (Herridge and
Rose, Fld Crops Res. 65: 229-248
(2000))

Comparing methodologies...
• Late generation populations - single sampling of xylem sap during
early pod-fill and natural 15
N abundance of whole shoots , values for 2
seasons at 3 sites (Herridge and Rose, Fld Crops Res. 65: 229-248 (2000))

Trait 1 – plant vigour & adaptation to
plant-growth constraints
• Selection for yield conducted
in low N soils (may include
the plant-growth constraint
such as low soil P) with little
consideration given to the
rhizobia
• Not really necessary to
quantify %Ndfa
• The legume most targeted in
this type of program is the
common bean (Phaseolus
vulgaris).
0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Fixed N
Soil N
No effect of rhizobia
Genotype X
Grain yield and
residual N benefit
Genotype Y

Plant vigour & adaptation
to plant-growth constraints
• Arguably, the most inefficient N2-fixing
legume is common bean
• Early published data suggested the
problem was not nodulation, rather N2
fixation efficiency related to H2
evolution (e.g. Hungria & Neves,
1987)1
• Yield reduced in low N soils; strong yield
responses to increasing levels of fertiliser
N
• Graph highlights N responses of selected
genotypes (ICA21573 and ICA20667 - dark
symbols) and commercial checks
(Rainbird, Gallaroy and Spearfelt – open
symbols)2
1
Hungria and Neves, Plant Soil 103: 111-121 (1987)

Plant vigour & adaptation
to plant-growth constraints
• Arguably, the most inefficient N2-
fixing legume is common bean
• Early published data suggested the
problem was not nodulation, rather
N2 fixation efficiency related to H2
evolution (Hungria & Neves, 1987)1
• Yield reduced in low N soils; strong
yield responses to increasing levels of
fertiliser N
• Graph highlights N responses of
selected genotypes (ICA21573 and
ICA20667 - dark symbols) and
commercial checks (Rainbird,
Gallaroy and Spearfelt – open
symbols)2
2
Redden and Herridge, Aust J Expt Ag 39: 975-980 (1999)

Plant vigour & adaptation to plant-growth
constraints
• First serious selection and breeding program was Fred Bliss and
colleagues (U Wisconsin)
• Identified high N2-fixing Puebla 152, then used inbred backcross
breeding with recurrent adapted parents. Selection for yield in low N
soils. Material extensively studied during 1980s-1990s
Source: St Clair et al., Crop Sci 28: 773-778 (1988)

Plant vigour & adaptation to
• Other programs focussed on
enhancing N2 fixation of
common bean under stress
conditions, e.g.:
– drought stress using similar
approach to that used for
soybean, discussed later
(Tom Sinclair and colleagues,
U Florida)1
– Low soil P stress, a critically-
important problem in most
bean-growing areas (Table)2
• Jean-Jacques Drevon, INRA,
targeting common bean in
low P soils……..
1
Devi et al., Plant Soil 364: 29-37 (2013); 2
Broughton et al., Plant Soil 252: 55-128 (2003)

Plant vigour & adaptation to
• Jean-Jacques Drevon, INRA in collaboration with
CIAT (amongst others), targeting common bean in
low P soils……..
• Substantial body of work established variation in N2
fixation of genotypes under low P supply:
– from the core CIAT collection, e.g. Vadez et al.
(1999)1
– From the Spanish collection of landrace material
(Rodino et al. (2009)2
– from crosses of genotypes BAT477 and DOR364,
Tajina and Drevon (2014)3
• Also attempting to understand mechanisms and
develop useful measures for discrimination, e.g. P-
use efficiency
1
Vadez et al., Euphytica 106: 231-242 (1999); 2
Rodino et al., Symbiosis 47: 161-174
(2009); 3
Tajina and Drevon, J Plant Nutrit 37: 532-545 (2014)

Trait 1 – plant vigour & adaptation to
• Five high N2-fixing cultivars released in the early
1990s from the Bliss program, which was
terminated about the same time
• Other programs don’t appear to have released
cultivars
• The 1986-91 IAEA CRP ‘Enhancement of Biological
Nitrogen Fixation of Common Bean in Latin
America’ identified large variation in %Ndfa (0-
73%) and shoot N fixed (0-165 kg/ha). Not sure
whether cultivars were released.

Traits 2, 3, 4 – promiscuous, selective and
exclusive nodulation
0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Fixed N
Soil N
Ineffective
rhizobia
Effective
rhizobia
Genotype X
Grain yield and
residual N benefit
Genotype Y
• Selection for yield conducted in
low N soils
• Type and strain of rhizobia
forming the nodules critical, with
the plant primarily/only
nodulating with highly effective
soil or inoculant rhizobia
• Not really necessary to quantify
%Ndfa
• Example programs:
– Promiscuous nodulation of soybean
in Africa
– Selective nodulation of pea in
Australia
– Exclusive nodulation of soybean in
the US.

0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Fixed N
Soil N
Ineffective
rhizobia
Effective
rhizobia
Genotype X
Grain yield and
residual N benefit
Genotype Y
low N soils
%Ndfa
– Promiscuous nodulation of
soybean in Africa
Australia
the US.
Mpepereki et al., Fld Crops Res. 65: 137-149 (2000).

Promiscuous, selective and exclusive
nodulation
• Exploited ability of certain soybean
genotypes to nodulate effectively
with indigenous ‘cowpea’ rhizobia in
the soil
• Programs in west Africa (IITA) and
southern Africa (Zambia, Zimbabwe,
Tanzania, Malawi)
• In the 1970s, breeders at IITA started
to exploit local cvs that had been
grown for 30 years without
inoculation
• Grafting experiments established
that the trait was associated with the
roots (Pulver et al., Crop Sci 25: 660-663
(1985)).

nodulation
• Graph showing nodulation of promiscuous nodulators (Malayan,
Orba) and US-bred cultivars (Nangju, Agron J:72: 403-406 (1980))
• Hybridisation of promiscuous and high yielding U.S. cultivars
successfully combined grain yield with enhanced nodulation/N2
fixation (Kueneman et al., Plant Soil 82: 387-396 (1984))
(a)
0
50
100
150
200
250
300
350
400
Malayan Orba Bossier Jupiter
Nodulemass(mg/plant)
Not inoculated
Inoculated
Genotype Grain yield (t/ha)
- Fertiliser N + Fertiliser N
Promiscuous
genotypes
TGX326-034D 2.55 2.55
TGX330-054D 1.98 2.37
TGX457-060C 2.30 2.47
Improved check
cultivar
Bossier 0.90 1.60

nodulation
• Cultivars released by IITA
• Although west and southern African programs had
different approaches, end result similar, i.e. genotypes
that nodulate with indigenous soil rhizobia (mainly
Bradyrhizobium spp. but some B. japonicum) and fixing
large amounts N
• Good discussion about the rhizobia by Sanginga (Plant
Soil 252: 25-39 (2003)). Suggested that, because of
occurrence and genetic diversity of soybean-nodulating
rhiziobia, it may be impossible to select/breed plant
genotypes that will always be nodulated effectively
across Africa

0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Fixed N
Soil N
Ineffective
rhizobia
Effective
rhizobia
Genotype X
Grain yield and
residual N benefit
Genotype Y
low N soils
%Ndfa
in Africa
Australia
the US.
Drew et al., Crop & Pasture Science 63: 467-477 (2012)

Promiscuous, selective
and exclusive
nodulation
• After nearly two centuries of vetch and pea cultivation in southern
Australia, soils now contain relatively high populations (102
– 104
/g
soil) of naturalised pea rhizobia
• Program of Ballard, Drew and colleagues1
, funded from the grains
R&D body (GRDC) to:
– Determine compatibility of pea, lentil and fababean cultivars with
inoculant strains and with naturalised soil rhizobia
– Provide advice to farmers about the N2-fixing capacities of the
different cultivars
– Link with pulse breeders to improve N2 fixation of the pulses
1
Drew et al., Crop & Pasture Science 63: 467-477 (2012)

Promiscuous,
selective and
• Assessing individual
genotypes and cultivars to
form symbioses with the
commercial inoculant
strain SU303 and with the
soil populations
• Two of the 10 cvs
assessed appear to have a
problem with both
genetically-diverse soil
rhizobia and the inoculant
strain (also in field…)

Promiscuous,
selective and
• Assessing N2 fixing
capacity of different
cultivars..
• Concept of dual purpose
legumes – for grain and
also residual N for
succeeding crops
• Data for 2013, Pearl and
Kaspa produced much
the same grain yield, but
Pearl contained 60 kg
N/ha more in crop
residues
• With both, %Ndfa high
Leaf type: C = conventional, SL = semi leafless
Plant form: T = tall, MT = medium tall

0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Fixed N
Soil N
Ineffective
rhizobia
Effective
rhizobia
Genotype X
Grain yield and
residual N benefit
Genotype Y
low N soils
the plant only nodulating with
highly effective soil or inoculant
rhizobia
%Ndfa
in Africa
Australia
– Exclusive nodulation of soybean
in the US (Sadowsky et al. 1995).
Sadowsky et al., Appl. Environ.
Microbiol. 61, 832-836 (1995)

Promiscuous, selective
and exclusive
nodulation
• Soybean in the U.S. where yields depressed as much as 30% by being
nodulated by less effective soil rhizobia (Vasilas and Fuhrmann (1993) Crop
Sci 33:785-787)
• Two programs…Tom Devine (USDA Beltsville) exploited non-nod gene
of soybean, rj1, by isolating strains of bradyrhizobia from the soil that
overcame the restriction. Then package the non-nod gene and infective
bradyrhizobia together in high-yielding cultivars. Program terminated in
early 1990s
• Also at USDA Beltsville (PB Cregan, HH Keyser, MJ Sadowsky &
colleagues) aimed to restrict nodulation of soybean by certain
serogroups of bradyrhizobia, e.g. USDA123, through plant selection.

nodulation
• Early results promising
• However, restricting all less effective bradyrhizobia in a mixed soil
population difficult. Needed a combination of specific genes related to
restriction of specific groups. Also need updating as the soil population
changes with time……
Genotype % nodules occupied by -
USDA123 USDA122,
USDA138
Other
Restrictive plant genotypes
PI371607 3 89 8
PI377578 5 92 3
Check cultivars
Williams 76 20 4
Cregan and Keyser, Crop Sci. 26: 911-916 (1986); Cregan et al., 1989a, Crop Sci. 29: 307-312 (1989a);
Cregan et al., Appl. Environ. Microbiol. 55: 2532-2536 (1989b).

Trait 5 – nitrate tolerance via
hypernodulating mutants or natural
variation
• Objective to produce legumes
that fix greater-than-normal
levels of N2 in the presence of
moderate-high levels of soil
nitrate
• Plant genotypes selected on the
basis of nodulation or %Ndfa.
• Likely not to have an effect on
yield
• The variation in nodulation and
N2-fixation may be:
– induced through mutagenesis
– natural
0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Fixed N
Soil N
Genotype X
Residual N benefit
Genotype Y

Nitrate tolerance via hypernodulating
mutants
• Mutagenesis first used to generate
pea with enhanced nodulation and
some nitrate tolerance (Jacobsen and
Feenstra, Plant Sci Lett 33: 337-344 (1984))
• Subsequently, major focus on
soybean:
– in Australia (Carroll et al., Proc Nat Acad Sci
USA 82: 4162-4166 (1985))
– in the US (Gremaud and Harper Plant
Physiol 89: 169-173 (1989))
– in Japan (Akao and Kouchi, Soil Sci Plant
Nutr 38: 183-187 (1992))
• Later other legumes incl. lotus:
– in Japan (e.g. Yoshida et al., Plant Cell
Physiol 51:1425-1435 (2010))

mutants
• With soybean, ethyl methanesulfonate (EMS) mutagen-
induced hypernodulating mutants of Bragg (Australia),
Williams (USA) and Enrei (Japan) produced and intensively
studied
• Under high soil N conditions, the mutants had 10-20x
nodulation and C2H2 reduction activity of wild-types, but 30-
40% growth reduction and severely restricted root growth
• In the field in the U.S., grain yields reduced by 20-40%,
compared to wild-type, and inconsistent N2 fixation
advantages (Wu and Harper, 1991; Pracht et al., 1994).
• Field data from Australia mixed but more positive…
Wu and Harper, Crop Sci 31: 1233-1240 (1991); Pracht et al., Crop Sci 34: 738-740
(1994).

mutants
• Song et al. (Soil Biol Biochem 27: 563-569
(1995)) reported no N2 fixation or grain
yield advantage of hypernodulators
compared to cv Manark. The
hypernodulators did increase yield of
subsequent cereal
• Zhao et al. (Proc 9th
Aust Agron Conf, pp 375-
378 (1998)) reported intermediate
hypernodulator (PS47) produced
equivalent grain yields to cv Manark and
fixed more N (graphs).
• Residual effects on soil N greater for the
extreme hypernodulator (PS55) while
residual effects of PS47 were similar to
cv. Manark
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0 Fert N 200 Fert N
Grainyield(t/ha)
Manark
PS47 (Intermediate)
PS55 (Extreme)
0
10
20
30
40
50
60
70
0 Fert N 200 Fert N
Xylemrelativeureides(%)

mutants
• According to Novak (Ann Appl Biol 157: 321-342 (2010)) only two
hypernodulating cultivars released commercially
– Nitrobean 60 (mutant of Bragg) in Australia in 1995 by BJ Carroll, L
Song and PM Gresshoff
– Sakukei 4 (mutant of Enrie) in Japan (Takahashi et al., Bull Natl Inst
Crop Sci 4: 17-28 (2003))
• Sakukei 4 renamed Kanto 100 and, according to Jung et al. (Plant
Prod Sci 11: 291-297 (2008)) has the yield potential to be widely-
grown by farmers
• Novak (2010) suggested hypernodulators better used as forage
legumes
• Genetics and physiology of nodulation mutants including the
hypernodulators reviewed by Bhatia et al. (Euphytica 120: 415-432
(2001)) and Novak (2010)

Natural variation in nodulation/nitrate
tolerance
• Program commenced in Australia in 1980 to
improve soybean nodulation and N2 fixation,
particularly in moderate/high N soils
• Became part of the mainstream breeding
program
• Almost 500 genotypes screened in two cycles of
glasshouse culture under high & low N (Betts and
Herridge Crop Sci 27: 1156-1161 (1987))
• From that, 32 genotypes selected for field
screening, including 9 genotypes from Korea

tolerance
• Superior performance of the Korean genotypes confirmed in mod-
high N soil in the field in 1984-6 (Herridge and Betts, Plant Soil 110:
129-135 (1988); Herridge et al., Plant Physiol 93: 708-716 (1990))
Genotype Nodulation %Ndfa Shoot DM
(g/plant)
Grain yield
(t/ha)
Wt
(mg/plant)
No./plant
Nitrate tolerant
Korean 466 376 35 31 46 1.6
Korean 468 254 17 18 43 1.7
Korean 469 176 20 22 42 1.4
Korean 464 319 17 11 48 1.5
Commercial
Bragg 24 2 0 40 2.2
Davis 40 1 0 49 2.2

tolerance
• The four Korean genotypes used as high N2-fixing parents. More than 800
single F2 plants assessed, then single seed descent..
• Higher %Ndfa maintained through first cycle of breeding and selection.
Data means of 3 sites (grain yield) and 2 high soil N sites (%Ndfa) (Herridge
and Rose, Crop Science 34: 360-367 (1994)) (4 genos used as parents)
Line Flowering (d) Grain yield (t/ha) %Ndfa
F6 F7 F6 F7
Bred N-tolerant lines
D22-8 46 2.08 2.29 47 55
A82-3 52 2.00 1.70 49 49
K78-1 57 1.91 2.09 46 54
A46-4 58 2.24 2.20 51 56
Commercial and Korean checks
Forrest 50 2.67 2.66 27 33
Korean 468 43 0.95 0.58 47 52

tolerance
• 1400 lines field tested at F5 and F6 for yield, seed and growth
traits, then for N2 fixation at F7, F8 and F9
• By the end of the second cycle of breeding, the N-tolerant
lines were no different in %Ndfa to advanced material from
mainstream program and check cultivars (Herridge and Rose, Fld
Crops Res 65: 229-248 (2000))
• None released as cultivars primarily because they lacked
sufficient tolerance/resistance to phytophthora and the 18-
year selection/breeding program terminated in 1998
• Addition of the Korean genes to the mainstream breeding
program seen as a hard-to-quantify benefit
• Note that Serraj et al. (J Plant Physiol 140: 366-371 (1992)) also
reported natural variation in symbiotic nitrate tolerance with
cv Tielingbaime.

Trait 6 – plant N metabolites
• Strategy involved selection of
plant genotypes based on
plant N traits such as:
– %grain protein
– %ureides in petioles, leaves
• Assumption of a link between:
– %grain protein and N2 fixation
– %petiole ureides and N2 fixation
under drought conditions
• The USDA, Beltsville, program
of Bob Leffel and colleagues1
with high protein soybean……
0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Residue N
Grain N
Genotype X
Grain N and
system N
benefit
Genotype Y
1
Leffel et al., Crop Science 32: 747-750 (1992)

Plant N metabolites
• Leffel and colleagues1
showed that high
protein soybean (CX797-21, 46% GP)
continued to fix N for 28 days after R5
stage, compared with cv. Harper’s 7
days N2 fixation (Harper 39% GP)
• Just prior to R7 (physiol
maturity), CX797-21 had
fixed 150 kg N/ha vs 83 kg
N/ha for Harper, an 80%
increase
• Selection/breeding for high
%grain protein will also
result in increased N2
fixation
• But…. (Egli and Bruening
(2007)
1
Leffel et al., Crop Science 32: 1428-1432 (1992)
2
Egli and Bruening, Plant Soil 301: 165-172 (2007)

Trait 6 – plant N metabolites…..
• Strategy involved selection of
plant genotypes based on plant N
traits such as:
– grain protein
– ureide concentration in petioles,
leaves
• Assumption of a link between:
– %grain protein and N2 fixation
– %petiole ureides and N2 fixation
under drought conditions
• The U Florida/U Arkansas
soybean program of Tom
Sinclair/Larry Purcell and
colleagues1
……
1
Sinclair et al., Fld Crops Res 101: 68-71 (2007)
0
40
80
120
160
1 2 3 4
LegumeN(kg/ha)
Residue N
Grain N
Genotype X
Grain N and
system N
benefit
Genotype Y

Plant N metabolites….
• Observation that soybean genotypes with lower
shoot/petiole %ureides were more drought tolerant for
N2 fixation (deSilva et al., Crop Sci 36: 611-616 (1996); Serraj
and Sinclair, Crop Sci 36: 961-968 (1996))
• LH graph shows bred line ROI-416F fixing more N than high-yielding parent under increasing
drought. Two lines, 416F and 518F, released for breeding (Sinclair et al., Field Crops Res 101: 68-71
(2007))

Breeding legumes for N2 fixation…
What was achieved during the past 70 years?
• For all the effort, not a lot
• The few cultivar releases include:
– five high N2-fixing cultivars of common bean1
released in South
America in the late 1980s
– exploitation of promiscuously-nodulating soybean in Africa2
• Other releases were:
– two hypernodulating cultivars
– two soybean lines that were drought tolerant for N2 fixation
(NFDT trait)3
• Reasons….
1
Bliss, Plant Soil 152: 71-79 (1993)
2
Mpepereki et al., Fld Crops Res 65: 137-149 (2000)
3
Chen et al., J Plant Reg 1: 166-167 (2007)

• Legume selection/breeding programs specialising in N2
fixation not universal, but scattered across countries and
associated with key individuals
• In rare instances the legume breeders led the teams, e.g. Fred
Bliss, common bean breeding program at U Wisconsin (Bliss
1993), but most programs initiated by biologists in
collaboration with breeders
• Legume breeders don’t consider N2 fixation one of the high-
priority traits for selection. Instead they logically focus on
grain yield and quality, crop duration and on biotic and
abiotic constraints (e.g. Miklas et al. 2006; Gaur et al. 2014).
Bliss, Plant Soil 152: 71-79 (1993)
Miklas et al., Euphytica 106: 231-242 (2006)
Gaur et al., Legumes in the Omic Era, Chapter 4, Springer Science (2014)

• Also, difficult to screen the N2 fixation-associated phenotypic
traits, e.g. Miklais et al. (2006)1
“… incorporating selection criteria
for SNF such as nodule mass, nitrogenase activity and xylem
ureide content into breeding schemes while attending to other
breeding objectives remains a challenge…”.
• The answer may lie with development of molecular markers, e.g.
QTLs, ESTs etc, of the N2 fixation traits of interest (see Purcell
20072
; Vance 20073
etc)
• How to do that?
1
Miklas et al. Euphytica 147: 105-131 (2006)
2
Purcell, Nitrogen Fixation in Crop Production, Agron Mono 52 (2009)
3
Vance, Nitrogen Fixation in Crop Production, Agron Mono 52 (2009)

• “..Molecular techniques are radically altering the way that
plant breeding is being performed…” (Broughton et al. (2003))1
• Need a convergence of molecular biologists/plant geneticists
and mainstream plant breeders, e.g.:
– ‘Phaseomics’ program, involving molecular biologists, plant
physiologists/biochemists, plant breeders from 11 countries
(Broughton et al. 2003)1
– CIAT-TSBFI Working Group on BNF (Serraj 2004)2
• Arguably a model program is that of the ‘Genetics and
Ecophysiology of Grain Legumes’ group, INRA Dijon (Bourion et
al. 2010)3
1
Broughton et al., Plant Soil 252: 55-128 (2003); 2
Serraj (Ed) Symbiotic Nitrogen
Fixation pp. 113-143 (2004); 3
Bourion et al., Theor Appl Genet 121: 71-86 (2010)

To conclude…where to now?
• 70 years of productive research to identify N2 fixation problems
and variation and sources of useful N2 fixation associated traits
• Not a lot to show in terms of released cultivars
• Development of molecular markers for N2 fixation, similar to
those for insect and disease resistance/tolerance, may bring N2
fixation into mainstream legume breeding (key issue)
• Need institutional commitment
• Integrated programs already commenced:
– Soybean in Brazil (Nicolas et al., Field Crops Res 95: 355-366 (2006))
– Pea in France (Bourion et al., Theor Appl Genet 121: 71-86 (2010)
– Common bean (International) (Ramaekers et al., Mol. Breeding 31:
163-180 (2013))
– Various legumes at the CGIAR Centres, ICRISAT, CIAT etc

To conclude…where to now?
• 70 years of productive research to identify N2 fixation problems
and variation and sources of useful N2 fixation associated traits
• Not a lot to show in terms of released cultivars
• In the meantime, simplest screening of breeding material is in
low N (+ low P, saline, acid, dry etc) soils with or without
inoculation for:
– grain yield (measured)
– % grain protein (measured)
– above-ground biomass (visually assessed if possible)
– biomass N yield (AG-biomass x % grain protein, used as surrogate
for % N shoot)
• If specific trait involved, e.g. NFDT (Sinclair et al. 2007), will need
genetic marker

Final word
• A risk that in the future, research/plant
improvement institutions will have the
scientists to run the gels and construct the
genetic linkage maps but none with the basic
physiological/agronomic knowledge of
symbiotic N2 fixation to determine and source
the traits of interest and provide the context
(also sentiment of Purcell, Nitrogen Fixation in
Crop Production, Agron Mono 52 (2009))

David F. Herridge - Mejora de las leguminosas para incrementar la fijación de nitrógeno

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (11)

Similar to David F. Herridge - Mejora de las leguminosas para incrementar la fijación de nitrógeno

Similar to David F. Herridge - Mejora de las leguminosas para incrementar la fijación de nitrógeno (20)

More from Fundación Ramón Areces

More from Fundación Ramón Areces (20)

Recently uploaded

Recently uploaded (20)

David F. Herridge - Mejora de las leguminosas para incrementar la fijación de nitrógeno