Plant Breeding - Mendelian to Molecular ApproachesH. K. Jain and M. C. Kharkwal (eds .)Copyright © 2004 Narosa Publ ishin g House, New Delhi , India 30

Breeding Legumes for Improved Nitrogen Fixation

O.P. Rupela! and D.L.N. Rao2

Abstract

Legumes can meet about 80 % of their N need under favourable conditions. In

addition, they have been estimated to contribute 20 % of the nitrogen needed for

global grain and oilseed production. Thus nitrogen (Nz) fixation by legumes

plays an important role in maintaining soil fertility in different cropping systems.

Methods of measur ing Nz-fixation, a pre-requisite to asses s its levels, genetic

vari ability in the di fferent traits relevant to Nz-fixation, strategies to select and

breed legumes for high Nz-fixation and yield are presented. Where avai lable,

examples of succ essful selection and breeding for high Nz-fixation have been

cited . It is argued that any legume breeding program will benefit by growing

crop s under low soil-N cond itions, which will exert a select ion pressure in favour

of high Nz-fixing plants. Use of simple to sc reen parameters (ha ving high

correlations with yield) for rapid identifi cation of high Nz-fixing parent lines and

segre gat ing populations likely to be favoured by breeders are discussed.

IntroductionLegumes are an integral part of different cropping systems. Their role in sustaining cropproduction through nitrogen (N2) fixation (a symbiotic process occurring in their nodules)is widely accepted. The fact that legumes f ix N2 was explicitly proved in 1886 byHellriegel and Wilfarth in Bernburg and publi shed in 1888 (Nutman, 1987). Unt il then,there was a controversy about the role of nodules. Even today there is a debate on theprocess of nitrogen fixation , but it is largely centered on methodology of its appropriatequantification (Herridge et at., 1994, Danso et al. , 1993) and on methods of optimizing itfor maximum harnessing (Herridge and Ro se, 2000 ; Rupela and Beck, 1990 ). The oilcrisis of the early 1970s and the consequent rise in fertilizer N prices resulted in intensiveresearch on biological nitrogen fixation (BNF) . Subsequently, the mom entum has beensustained due to environmental concerns associated with manufacture of nitrogenousfertilizers and their usage. As people are becoming increasingly environment conscious(Brown et al. , 2000), it is predicted that BNF by legumes would gain more importance inthe near future.

l lnte rnational Crop s Research Institut e for the Semi -Arid Tropics, Patancheru 502 324, India

2Indian Institute of Soil Science , Bhopal 462 038, India

720 O'P. Rupela and D.L.N. Rao

Legumes can potentially fix about 80 % of their own N need and in addition cancontribute to the yield of subsequent crops (Saraf et al., 1998 ; Ahlawat et al., 1998 ;Lauren et at., 1998). But all these potential benefits can be harnessed under certainconditions. Mere inclusion of a legume in a cropping system does not ensure high BNF .There can be three approaches to harness BNF: Increasing the area of legumes sown byfarmers ; improved crop, soil and water management to achieve maximum efficiency ofBNF including Rhizobium inoculation, and selection of host genotypes to ensure a higherproportion of nitrogen fixation in the plant (Pfix) ' This paper discussesinformationlknowledge that will help endeavors towards breeding legumes for high BNF.The different sections include nodule formation/functions , methods of measuringNTfixation (with emphasis on those potentially useful in breeding), genetic variation fortraits relevant to NTfixation and strategies for selection and breeding for high NTfixation.Where available , examples have been drawn from legumes grown on large areas in India.

Like the other biological processes , Nz-f'ix at ion in legumes is sensitive toenvironmental factors such as temperature, moisture and nutrients . Readers are suggestedto refer to reviews on factors affecting Nj-fixation (Howieson et al., 1993; Rawsthome etal., 1985; Serraj et al., 1999a; Streeter, 1988). A good understanding of the limitations ofNz-fixation should be a help in devising an effective breeding program on Nz-fixation .Such limitations due to factors like mineral nitrogen (N) and water have been discussed.

Partners in the Symbiotic ProcessUntil 1992 , there were four genera of root nodulating bacteria: Rhizobium,Bradyrhizobium, Sinorhizobium and Azorhizobium (Elkan, 1992). Also, some new specieshave been identified and the nomenclature of some of the old species have recently beenrevised (Young , 1996) . Four more genera added recently are Mesorhizobium,Allorhizobium, Methytobacterium and Burkhotderia. All the eight genera exceptBurkhotderia belong to a-subclass of proteobacteria and the last one belongs to ~-subclass

of proteobacteria (Young and Haukka, 1996; Van Berkum and Eartly , 1998; De Lajudie etal., 1998; Moulin et al., 200 I) . It should, however, be noted that these are not newbacteria. These were known or associated with their relevant legumes all along, but due tothe advances made in molecular biology techniques, their nomenclature has improved . Forscientists interested in developing NTfixation technologies for use on farmers' fields, thisfast advancing nomenclature of the bacteria presents a complex situation . However, for allpractical purposes, the symbiotic relationship between the bacteria forming root nodulesin a given host legume remains the same as ever and may not affect field orientedbreeding programs on high NTfixation. In this paper, we have used "rhizobia" orRhizobium to mean any bacterialbacterium forming nodules on a given legume .

The process of nodule formation in legumes has been extensively investigated .Comprehensive reviews on different aspects of the subject are available (Caetano-Anollesand Gresshoff, 1006; 1996; Hirsch et al ., 2001; Long, 2001; Oldroyd, 2001 ; Schultze andKondorosi , 1998 ; Spaink, 2000). Some reviewers have referred to the infection of

Breeding for Improved Biological Nitrogen Fixation 721

legumes roots by the root-nodulating-bacteria (rhizobia) as "a beneficial plant disease"and others have referred to rhizobia as "refined parasite of the legumes" . As per presentunderstanding, at least ten steps are involved in the process of formation and functions ofa given nodule . Each of these is gene-controlled interaction between the host-plant and theroot-nodule bacterium. In response to a variety of substances excreted (e.g. flavonoids) bythe host plant roots, rhizobia are attracted to a particular legume species, leading to theformation of specialized root organ , the nodule. A set of bacterial (nod) genes involved inproduction of lipo-chito-oligosaccharides (nod-factors) that act as signaling molecules arealso important for nodulating the specific legumes hosts (Long, 2001). The steps involvedin establishment of successful symbiosis are: multiplication and colonization at the rootsurface (Roc root colonization), adhesion of bacteria to the root hair surface (Roa rootadhesion), curling or branching of host root hairs (Hab and Hac) , root hair branching andcurling, formation of infection thread (Inf), induction of meristems in the host roots for thenodule initiation and differentiation (Noi) , bacterioid release from the infection thread(Bar), bacterial differentiation (Bad) , onset of nitrogen fixation (Nij) , nodule functionpersistence/maintenance (Nop), development of complementary functions associated withN2-fixation (Co/) and its transport (Sprent and Sprent, 1990; Caetano-Anolles andGresshoff, 1996).

The advances in the understanding of rhizobial genetics has been rapid and beenreviewed extensively (Fischer, 1994, Long, 2001; Van Berkum and Eartly, 1998) . Thesymbiotic genes have been located on large plasmids and on the chromosome of rhizobia.Several rhizobial plasmids (such as a 536 kb plasmid NGR234, Freiberg et al., 1997) havebeen sequenced. An international effort (involving at least 10 institutions) initiated in1998 resulted in sequencing of the complete genome of Sinorhizobium meliloti,comprising 3.65-Mb chromosome , and 1.35-Mb pSymA and 1.68-Mb pSymBmegaplasmids (Galibert et al., 2001) . Readers are referred to the cited references fordetails on rhizobial genetics. For this paper, we have chosen to restrict to legumes andlegume x Rhizobium interactions, as they would apply to developing high nitrogen fixinglegume varieties .

Progress in molecular research of BNF has been relatively slow in legume plants . Atleast 80 genes affecting symbiosis have been identified in different legumes (Tsyganov etal., 1998). Four major types of nodulation variants that have been reported for mostlegumes are, nod- (no nodules), nod ±(few or no nodules), fix- (ineffective nodulation),nod++ (super nodulation or hypernodulation) and nts (nitrate tolerant nodulation). Somesalient aspects on these investigations have been listed in Table 1.

Screening Techniques for Improved Nz-FtxationFor a good breeding program on BNF, one would ideally require a high and stable geneticvariability for this trait, a high heritability and a high correlation between the given BNFrelated trait and yield . A rapid and simple to screen method will be an added advantage.There is, however, no single method universally appropriate for measuring NTfixation by

722

Table 1.

O'P. Rupela and DLN. Rao

Nodulation Variants and genes reported for the different legumes

Legume No. of Genesvariants

Inheritance/Comments

Faba bean 8

Groundnut 7

Pigeonpea (3)

Pea

Soybean

Chickpea

Commonbean

70

20

12(3)*

9

syml tosym40nod. nod2

nts, ril-rj8rj2-Rj5

rnl to rn8

nts, syml,sym2, Nie,nnd2

syml tosym5 sym2

GP50 toGP54 GS-6

NS (Notstudied)

Most of the variants were nod-due to several reasons and monogenicrecessive to wild type (mr). Some have nitrate tolerant nodulationbut others have semi-dominance (e.g. sym-18). Other interestingtraits involve ineffective white nodules, resistance to mycorrhizalinfection, premature senescence of nodules, defective root hairs,impaired symbiosome development.

Several super nodulating mutants with tolerance to nitrate (nts) (dueto absence of auto-regulation) . Some were monogenic recessive whileothers such as RJ2 to RJ5 were monogenic dominant. MostNod-lines/plants have light green foliage.

Several variants developed due to induced mutation. Nodulationvariants (from non-nodulation to high nodulation) reported from landraces and bred lines and developed by pure line selection . Mostmutants studied were monogenic recessive and non-nodulating. Kabulitype nod-lines may have light green leaves while the leaves of theDesi type lines have symptoms similar to drought or salinity stress.

Several nod- and fix- (ineffective nodulation) mutants developed .Most were monogenic recessive. Nitrate tolerant supernodulation alsoreported.

Most were Nod- and monogenic recessive (Sym-3) but somemonogenic dominant (Sym-2). Some were fix- (Sym-l , ineffectivenodulation) while others had 3-5 times more nodules than controland tolerant to high nitrate.

All the variants were picked (due to light green foliage) fromsegregating generations (F2, F3) of different crosses involving normalparents. Most were from ICRISAT. Some were reported as havingmore than one gene, duplicate gene, double recessive or trigenic.

Variants ranging from non-nodulation to high nodulation (higherthan parents) were selected from segregating population (F2) ofseveral crosses. Genetics not studied.

Developed from Bhatia et al., 2001; Rupela, 1992; Rupela and Johansen, 1995; Note: Most of thevariants were developed by induced mutations. *Number of variants in parenthesis were developed fromsegregating population of different crosses of normal parents or were occurring in normal land races orbred varieties .

legumes. N-difference (N yield) , 15N, acetylene reduction, xylem solute (ureide) are themost widely used methods . But all these have their own limitations and strengths andcannot provide an accurate measure of Nrfixation for every legume species grown underall possible variations of soil type and cropping environment. However, some of themethods are more likely than others to provide reliable and quantitative estimates ofNrfixation. These are summarized in Table 2. However, for preliminary screening of

Breeding for Improved Biological Nitrogen Fixation 723

large number of germplasm lines, counting nodule number per plant and taking nodulemass per plant, and even a visual rating scale for nodulation (Rupela, 1990; Rupela andJohansen, 1995) may be sufficient.

Biological yield largely determines Nz-fixation and grain yield (Mytton, 1983; Due et

al., 1988; Kumar Rao and Dart, 1987) . Therefore, breeders who operate in low N soils ,

and select for high grain yield, are highly likely to also select for high Nz-fixation . Thus in

breeding programmes a more direct and useful selection trait would be Nz-fixation (or

plant N or seed N yield under low soil-N conditions of growth), provided legumes were

adequately nodulated and functioning .

Genetic Variability for Nodulation and Nz-Fixation

Presence of a large genotypic variability for traits such as nodule number, nodule massand acetylene reduction activity (ARA) per plant have been known since early seventiesand eighties for chickpea, groundnut and pigeonpea (Nambiar et al. , 1988), soybean(Wacek and Brill , 1976), cowpea (Zari et al., 1978), common bean (Graham and Rosas,1977). Using 15Nisotope-based methods , differences among cultivars have been detectedin soybean (Hardarson et al ., 1989; Rennie et al ., 1982), common bean (Rennie andKemp, 1982; Westermann et ai.,1981), groundnut (Giller et al., 1987) , greengram andblackgram (Sampet and Peoples, unpubl. data cited by Peoples and Crasswell, 1992) ,pigeonpea (J V D K Kumar Rao, ICRISAT pers. commun.) and chickpea (Rupela et al.,unpubl. data) . However, an effort to use this variability in breeding for improvedN2-fixation has been limited or non-existent in many of these legumes. Arunachalam et al.(1984) found that ARA and nodule mass have good predictive value for plant growth andyield related traits in groundnut. After analysis of a six-parent diallel cross, Nigam et ai.

(1985) observed that non-additive genetic variance for ARA was predominant ingroundnut. The groundnut line NC Ac 2821 had the highest general combining ability forARA, total nitrogen, leaf area and was proposed as a good parent for breeding programs.The crosses made between the high- and low-nodulating chickpea lines to investigate theinheritance of nodulation indicated segregation for nodulation in F2 populations fromnon-nodulating to nodulating. But the differences in the extent of nodulation weregenerally not reflected in plant growth (Fig. 1, O. P. Rupela, unpublished). This may bedue to ability of the less nodulated plants to effectively use soil N, which needs to beascertained by further studies. Fig . 1 shows two plants with large visible difference fornodulation capacity but not for shoot mass. This, however, should not be interpreted tomean that legumes can yield well without nodules .

There is sufficient evidence to show that non-nodulating lines in some legumes (such as

chickpea) do not yield at par with nodulated lines, unless provided with high doses of

N-fertilizers (Rupela, 1992) . In some other legumes (such as groundnut) the

non-nodulating lines do not yield at par with nodulating parents even when high doses of

N is provided (Nambiar et al., 1986).

Tab

le2.

Lis

tof

diff

eren

tm

etho

dsof

quan

tifi

cati

onof

nitr

ogen

fixa

tion

,th

eir

salie

ntfe

atur

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wea

knes

san

dst

reng

ths

--J

N ...M

etho

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alie

ntfe

atur

eW

eakn

esse

sS

tren

gths

Ref

eren

ce

N-d

iffe

renc

eN

on-n

odul

atin

gle

gum

eor

asu

itabl

eS

ubst

anti

aldi

ffer

ence

s-S

impl

e,do

esno

tne

edB

odde

yet

al.,

1984

Kum

arce

real

isus

edas

refe

renc

e.N

itrog

enin

betw

een

N2-

fixi

ngan

dex

pens

ive

equi

pmen

tsR

aoan

dD

art,

1987

thes

eis

dedu

cted

from

the

nitr

ogen

inno

n-f

ixin

gpl

ants

for

thei

r-W

hen

grow

non

the

test

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me

phys

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me

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corr

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xatio

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ther

efor

ea

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ple

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bree

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for

high

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aco

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3663

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1988

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ease

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e15

NR

equi

rea

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accu

racy

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tre

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hear

eran

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ohl,

1986

;:>:

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red

with

atm

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iffe

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valu

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(abo

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ple

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-.l

N VI

726 o.r. Rupela and D.L.N. Rao

Fig. 1. In a cross between two chickpea lines, sixty two days old plants grown on a Vertisol field at ICRISATPatancheru were uprooted for nodulation studies. Segregation for nodulation at F2 ranged from fewnodules in the plant at right [lower than the low nodulating parent F3 Partner 4-14-1 (ICRISATgermplasm accession IC.6679) ] to normal nodulation in the plant at left [close to that of the normalnodulating parent NEC 802 (ICRISA T germplasm accession IC-5770)]. Few plants nodulated betterthan the normal nodulating parent (not shown in the figure) . However, the plant growth did not seemto correlate with nodulation, suggest ing availability of sufficient nitrogen in the field and emphasizingthe need of breeding legumes at low soil-N.

Following Mahalanobis D2 statistic, Dangaria et al. (1994) reported large geneticdivergence (among 32 chickpea genotypes) for nodule number per plant, nodule mass perplant and nodule size. Cultivar K 850 formed highest nodule mass and clusteredseparately . These studies thus indicate the complexity of the Nrfixation related traits.Screening for high Nrfixation traits for chickpea and groundnut can be made in field.Legumes like pigeonpea offer difficulty for Nrfixation studies in field , because theirnodules are loosely attached to roots and generally fall off during excavation of fieldgrown plants. It is perhaps due to this reason that there were no reports in pigeonpea so faron studies of the type reported above for groundnut.

Intracultivar Variability for Nz-Fixation : Plant-to-plant variability for nodulationwithin chickpea cultivars, including occurrence of non-nodulat ing plants in land races andbred cultivars has been reported by Rupela (1992). Consistent variability for nodulation

Breeding for Improved Biological Nitrogen Fixation 727

extent was also subsequently detected within pigeonpea cultivars (Rupela, 1994). Unlikein chickpea, however, non-nodulating plants in pigeonpea were found in segregatingpopulations at Fz (Rupela and Johansen, 1995). In addition to the breeding method usedfor developing a material , absence of any natural selection pressure for nodulation orNj-fixation during its development may be responsible for the occurrence of the differentnodulation types within a material up to release stage. This view gained strength from thefact that during a screening for high nodulating plants at high mineral N in soil, both highand low nodulating plants were observed in 85 out of 90 advanced breeding lines ofchickpea (Rupela, 1994).

Using appropriate screening procedures several different nodulation types [highnodulating (HN), low nodulating (LN), non-nodulating (NN)] have been identified withinseveral chickpea and pigeonpea cultivars (Rupela, 1994). Preliminary studies ofVenkateswarlu and Katyal (1994) also indicated plant-to-plant variability within cultivarsof groundnut. It is , therefore, likely that intra-cultivars variability is present in otherlegumes also. Intracultivars variability for a given trait in a crop species is not new . Singhet at. (1988) identified downy mildew resistant lines from a highly susceptible parent ofpearl millet (open pollinated crop) and M. P. Haware (ex-ICRISAT scientist, personalcommunication) developed «fusarium wilt resistant line from a susceptible culti var(Annigeri) of ch ickpea (self-pollinated crop) . Obviously, the Nod-'(NN) and thelow-nodulating (LN) selections are of academic interest and serve as an importantreference base in Nz-fixation quantification studies . High-nodulating (HN) selectiongenerally grew better than the NN and LN selections of a given cultivar. Yield trials wereconducted only with the LN and HN chickpea selections of G 130 and K 850 at fivelocations in India and a location each in Bangladesh, Nepal and Pakistan (Dudeja et al.,1997; Khanam et al., 1997). At ICRISAT, the HN-selection of cultivar G 130 produced 31% more grains than its LN-selection at low soil N (Nj) level. The HN-selection ofG 130yielded better even at high soil N (Nz) level. [Note: The two contrasting soil N levels wereprepared by applying 0 (Nt) and 100 kg N ha- t (N z)as urea to the preceding sorghum onVertisol field after the whole field was depleted for N by growing cover crops for twoyears] . N is a highly dynamic element and varies through the soil profile due to severalfactors. At sowing, the available N in the top 15 em soil was 1.7 times more in the Nzplots than in Nt plots (8 .7 mg N kg- t soil)] . But the LN and HN selections of anothercultivar K 850 yielded the same under N1 and Nzlevels . This may be due to high rootlength density of the LN-K 850. In a previous pot trial the root length density of LN-K850 was 32 m planr ! that was two-times greater than that of the LN-G 130. Perhaps thecultivar K 850 could scavenge the soil N more efficiently than that of G 130 due to itshigh root length density, and as a result both the HN and LN lines of K 850 yieldedsimilarly. The HN selections, however, yielded higher than the relevant LN selections(Table 3). The se studies thus suggest a great scope of enhancing Nz-fixation in legume sthrough host plant selection.

728 o.r.Rupela and D.L.N. Rao

Table 3. Nodule dry mass (mg plant'"), acetylene reduction activity (~5M C2H 4/planCI-h

-I),

N2 fixed (kg ha-I) and grain yield (t ha-I) of chickpea lines selected for different

nodulation and N2-fixation capacity

Chickpea line Nodule dry mass 11M C2H4 N2 fixed Grain yield(mg planC I)* (planC! h- I ) (kg,ha-I ) (t ha- I )

G 130 HN 163 4.6 23.8 1.34

G 130 LN 93 3.9 13.5 1.08

G 130 (Parent) 117 3.4 17.2 1.22

K 850 HN 219 8.1 30.9 1.49

K 850 LN 88 2.1 23.5 1.33

K 850 (Parent) 197 5.0 24.5 1.36

Rabat-NN (Nod-) 0 0.0 NR 0.88

Annigeri-NN (Nod') 0 0.0 NR 0.81

Mean 110 3.4 22.2 1.19

SEt 4.5 2.16 1.33 0.04

CV% 16.0 63.00 34.00 20.00

Data are means of four locations (Akola, Badnapur, New Delhi, Sehore) in India. Data from Hisar notconsidered due to crop heterogeneity. NR=Not relevant; *=Data is from New Delhi and Sehore for theyear 95/96; **=Data from New Delhi only; HN=High nodulating selection ; LN=Low nodulating selection ;Summarized from: Dudeja et al., 1997

Variability for N2-Fixation Under Stress Conditions: N2-fixation is sensitive toseveral environmental factors including soil-No High nitrogen levels have been reported tosuppress N2-fixation in different legumes (Rawsthome et al., 1985; Peoples et al., 1987;Buttery and Dirks, 1987). Based on several studies (Wu and Harper, 1991; Buttery et al.,1988; Buttery and Gibson, 1990), nodulation and/or Nz-fixation was reduced byapproximately 50 % in different legumes, when N concentration in root environment wasbetween 1.43 and 6.0 mM (approximately equivalent to 20 -90 mg N kg-I soil) in thegrowth medium. The suppression in Nz-fixation was particularly due to the nitrate fractionin the root growth environment (Streeter, 1988). Groundnut grown on an Alfisol withoutfertilizer N application in India derived 61 % of its N requirement through Nz-fixation(proportion of Nz-fixed or Pfixy, and application of 100 kg N ha-I reduced Pfix to 47 %(Yoneyama et al., 1990). The suppressive concentration of mineral-N may be reached inthe root zone with band application of N-fertilizer, at least for some days after application.Mineral N concentrations of 10-32 mg kg-I soil have been recorded at sowing chickpea infarmers' fields around Hisar in Haryana and Gwalior in Madhya Pradesh (Wani et al.,1997). All these studies indicate the need for developing symbiotic lines of legumes thatare tolerant to different stress factors.

Genetic variability for N2-fixation traits has also been reported under droughtconditions . Smith et al. (1988) reported that common bean plants reacted to soildehydration by closing their stomata earlier than soybean plants, which resulted inmaintaining high water content. Nodule growth and activity were more sensitive to

Breeding for Improved Biological Nitrogen Fixation 729

drought in common bean, and decreased earlier during soil dehydration, compared tosoybean. However, Pefia-Cabriales and Castellanos (1993) found, in a comparison ofcommon bean cultivars, that the percentage of N derived from N2-fixation was notaffected by drought stress.

Comparisons across species in N2-fixation response to drought have shown variationsthat indicate a genetic control. De Vries et at. (1989) found that nitrogenase activity perplant and N accumulation in peanut (amide exporter) were less sensitive to droughtcompared with pigeonpea (Cajanus cajan L.) and soybean (ureide exporters) . Sinclair et

at. (1995) has also reported that peanut N2-fixation was relatively insensitive to soildrying, and they identified cultivar variation in drought. Comparison of the percentage ofN derived from Nrfixation under drought stress by chickpea and pea by Beck et al., 1991showed differences between the two species. Wakrim and Wery (1995) showed thatNrfixation in chickpea was depressed under drought conditions, although it had nosignificant effect on yield.

Salinity in the arid and semi-arid regions of the world is a serious threat to agriculture.Production of grain legumes is particularly vulnerable because of their low tolerance tosalinity, combined with the high sensitivity of their BNF system (infection of root hairs byrhizobia and subsequent nodule development) . Salinity does not affect colonisation ofroots by rhizobia as much as initiation and growth of new nodules (Singleton and Bohlool,1984). A soil pH of 8.9 was the critical upper limit for high nodulation in most genotypesof chickpea. At pH 9.0-9 .2, a genotype selected for high-nodulation outperformed theother four used in the study (Rao et al., 2002) . Nodulation was reduced in all the fivechickpea genotypes as the electrical conductivity increased from 1.1 to 8.1, but the highnodulating selection CSG 9372, seemed to have more tolerance and formed about three­times more nodules than the salt tolerant line (CSG 8927), even at 6.2 dS m-1 (Table 4).

Heritability of Nitrogen FixationIn a breeding program, establishing the heritability of relevant traits is a pre-requisite.

Ronis et at. (1985) investigated broad-sense heritability of total and per cent fixed N(using 15N technique), in harvested seed of three F2 soybean populations each of 110plants. Broad-sense heritability for fixed N content of seed ranged from 0.53 to 0.60 .Heritability estimates for per cent fixed N in seed were moderate at 0.37 and 0.43,suggesting that breeding for improved N2-fixation of soybean using appropriate parentsshould be possible. Herridge and Rose (1994) found broad-sense heritability (N2-fixationassessed by ureide analysis) to range from 0 to 0.31 in F2 populations of soybean . For F2derived F3 lines it was 0.36 and for F3 derived F6 and F7 lines it ranged from 0.32 to 0.52.Greder et at. (1986) estimated broad-sense heritability for nodulation (nodule mass) insoybean, which exceeded 0.55 for each population averaged across sites. Their data,together with correlation analyses of nodulation, agronomic and yield traits indicated thatselection for increased nodule mass was warranted.

730 D.P. Rupela and DLN. Rao

Table 4. Shoot and root dry mass of five selected genotypes and nodule number and dr y

mass of nodules of four (the non-nodulating IC C 4918 does not form part of the

analysis) selected genotypes of C. arietinum grown (60 days) in a saline soil (Rao

et al., 2002)

Genot ypes Shoot dry mass (g per pot) LSD Root dry mass (g per pot) LSDP=0.05 P=O.05

ECe (dS m-l ) 1.00 3.20 6.20 8.10 1.00 3.20 6.20 8.10

ICC 4918 0.57 0.61 0.50 0.14 0.10 0.26 0.21 0.15 0.03 0.05(non-nodulating) (among (among

ECe) ECe)

CSG 8890 0.76 0.70 0.50 0.32 0.50 0.37 0.19 0.11(salt-sensiti ve)

BG 256 (check) 1.11 0.90 0.65 0.47 0.46 0.41 0.24 0.13

CSG 9372 1.03 1.22 1.14 0.60 0.38 0.46 0.38 0.22(high-nodulation)

CSG 8927 0.93 0.91 0.60 0.54 0.43 0.47 0.31 0.20(salt-tolerant)

LSD P=0 .05 0.12 (among genotypes) 0.06 (among genotypes)

Number of nodules per pot Dry mass of nodules(mg per pot)

CSG 8890 23.0 10.0 5.6 7.6 6.5 8.9 1.7 0.7 0.5 2.7(salt-sensitivel (among (among

ECe) ECe )

BG 256 (check) 22.2 11.8 13.8 7.6 12.8 5.2 3.3 1.2

CSG 9372 63.2 20.8 21.8 13.0 15.8 5. 1 2.8 1.1(high-nodulation)

CSG 8927 25.2 15.6 7.2 2.6 5.9 4.4 1.4 0.3(salt-tolerant)

LSD P=0.05 6.5 (among genotypes) 2.7 (among genotypes)

Similarly, Arren dell et at. (1985) found in F5 and F6 generation progenies from a crossof Virginia and Spanish cu ltivars of gro undnut, that broad sens e heritabil ity of nodulenumber ranged from 0.25 to 0.57; estimates for ARA (acetylene reduction), shoot weight ,fruit weight ranged from 0.53 to 0.85. The moderate to hig h es timates for these traitsindicated, that superior nodulating and N2 fixing genotypes within the populations studiedcould be readily identified, and that selection for enhanced nitrogen fixation should resultin indirect selection for yield.

Singh et al., 1985 reported considerable variation for total nitrogen, total dry matter andnodu le fresh weight per plant in 49 purelines of mungbean inoculated with cowpeaRhizobium MoS . Combining abi lity analysis in an 8 x 8 diallel of diverse purelinesshowed the importance of both additive and non-additive gene effects in control of thenitrogen fixation traits; non-additive gene effects were more preponderant than additivegene effects. There was considerable transgressive segregation for the three traits in thetwo F2 po pul ations used in this stu dy. The three traits showed very high heritability

Breeding for Improved Biological Nitrogen Fixation 731

estimates , and total nitrogen showed very high positive correlation with total nitrogen andnodule fresh weight. There was no evidence for an antagonistic relationship between grainyield and nitrogen fixation. It was concluded that selection for total dry weight or nodulefresh weight may be done to improve nitrogen fixation in mung bean without adverselyaffecting yield and yield traits.

Legume x Rhizobium InteractionsThere is a range of quantitative variation in nitrogen fixation , which is broadly genetic;but methods of exploiting it have not been fully developed. Difficulties are encountereddue to variable amounts of nitrogen fixation when cultivars are nodulated by differentRhizobium genotypes, and as different environments are encountered. Rapid geneticadvance under selection would result from coincidental selection of plant and Rhizobiumgenotypes (Mytton, 1983) . The extent to which these can be induced to reform theirspecific association is unclear but the mechanism of host strain selectivity as exemplifiedby the cross-inoculation groupings should be of value in achieving this. These objectivescan be pursued by empirical methods, but improvement of legume-Rhizobium associationsand their systems of management would be assisted by the co-ordinated work ofagronomists, microbiologists , physiologists and plant breeders (Mytton, 1983).

How should a field testing program of plant lines be approached? Lie (1981) extendedthe gene-centre concept of Vavilov (1951) to the area of NTfixation, pointing out withexamples from the Pisum and its Rhizobium symbiosis, the variation in response likely tooccur among collections from such gene-centres . With soybean, some evidence of thatvariation is already apparent in the response of Asiatic versus American cultivars tocowpea-type rhizobia (Nangju, 1980; Pulver et al ., 1982) and in the ineffective responseof American cultivars to the fast-growing soybean rhizobia obtained from China. Forbeans, response differences have been found amongst ancestral lines of Phaseolu svulgaris obtained from Mexico (Graham and Temple, 1984) and with other species ofPhaseolus (Ferrera-Cerrato, 1980; Hohenberg et al.. 1982).

Debate on whether to select for increased N2-fixation in the presence of a singlerhizobial strain or with a mixture of strains is likely to continue. Those who advocate amixed-strain inoculum suggest that selection in the presence of multiple rhizobialgenotypes will produce the best host-strain combinations (Barnes et al., 1984) . Thoseadvocating the use of a single-strain inoculum believe that because the most competitiverhizobia are not necessarily the most effective at reducing N2, the presence of multiplerhizobial genotypes may confound the selection of plant genes favouring NTfixation(Phillips and Teuber, 1985, Smith et al., 1982) . Certainly the degree to which lesseffective strains from a mixed population nodulate genetically desirable hosts will slowthe rate of genetic gain when selecting for host genes that favour increased NTfixation. Inthe case of alfalfa, germplasm selected for increased NTfixation in the presence of one R.meliloti strain stimulated greater NTfixation by other strains (Phillips et al., 1985) .

Therefore, the imponderable remains - how will the plants screened in the presence of

732 a.p. Rupela and D.L.N. Rao

mixed rhizobial conditions Interact with various populations of indigenous rhizobia thatwill be encountered in soils at different sites (Gibson, 1962)?

The reported large legume x Rhizobium interaction in Vicia faba L. was used to arguethat most of the genetic variation was under non-additive genetic control (Mytton, et al.,1977) supporting simultaneous selection of Rhizobium and host plant. Other studiesshowed that significant additive and non-additive genetic variance is present for severaltraits related to Nrfixation (Hobbs and Mahon, 1983; Tan, 1981) . Thus, someinvestigators conclude that both legumes and rhizobia should be selected independently(Hobbs and Mahon, 1983). Because rhizobia that nodulate one plant effectively are morelikely to perform similarly on related genotypes than on unrelated plants (Hardarson andJones, 1979; Mytton , 1975), legume selection with one rhizobial strain seems a reasonableapproach to increasing Nrfixation. Scientists following this approach believe that relativerankings of strains in closely related plant materials will not change markedly . Thus,alfalfa plants selected for N2-fixation with a single strain of Rhizobium also improvedsymbiotic performance of several other Rhizobium strains (Phillips et al., 1985).

Another approach that has been followed, ignores host x strain specificity and suggestscreening plant genotypes in a field soil containing high numbers of effective rhizobia,because in a field situation and with current inoculant technology, it is not possible tocontrol the mix of rhizobial strain(s) that nodulate a legume crop, making it difficult toestablish the highly effective, specific-host-strain combination . The studies ofArunachalam et al . (1984), Rao et al. (2002), Rupela (1992 and 1994), Dudeja et ai .(1997) fall under this school. There is also evidence that a superior host genotype selectedon the basis of NTfixation with one or more highly effective strains will express thatsuperiority with others (Buttery and Dirks, 1987 Phillips and Teuber, 1985; Wiersma andOrf, 1992).

In soybean, indigenous B. japonicum strains frequently are only moderately effective atfixing N2, but they prevent superior strains from forming root nodules . Thus, theadvantages of using plant genes to block indigenous strains and permit nodulation bydesirable rhizobial strains is obvious. The rh allele is one plant gene that prevents rootnodule formation by many indigenous strains (Devine and Weber, 1977). A more complexhost plant effect is evident in several primitive soybean introductions (Giycine max (L.)

Merr.) that restrict, but do not prevent, effective root nodule formation by B. japonicumstrains in the 123 serocluster (Cregan and Keyser, 1986). A possibly related, but moreextreme, host plant effect has been observed with the primitive G. max cultivar Peking(Devine, 1984) and the wild soybean (Glycine soja Sieb and Zucc) (Cregan and Keyser,1986), which produce Fix- nodules with the bacterium B. japonicum USDA123 and Fix"nodules with the bacterium R. fredii. Other "promiscuous" G. max lines that apparentlyform Fix" nodules with both B. japonicum and Bradyrhizobium sp. organisms indigenousto African soils are also known (Kueneman et al., 1984). Genetic relationships amongthese phenotypes have not yet been reported . It may, however, be possible to use the

Breeding for Improved Biological Nitrogen Fixation 733

alleles involved in restricting nodulation by indigenous rhizobia, so that more effectivestrains could be supplied as inoculants, to ensure greater nodule occupancy and in turngreater legume BNF and yield.

Any environmental factor affecting BNF will most likely influence Legume xrhizobium interactions. A large number of biotic and abiotic factors affect BNF. Theseinclude water, temperature, salinity/alkalinity/acidity of soil, soil antagonists and toxicchemicals and have been sufficiently reviewed (Sprent et al., 1983, Streeter, 1988,Vincent, 1988). Allelopathy, a direct or indirect harmful or beneficial effect by one planton another through production of chemical compounds in the root rhizosphere (Rice,1974) may be another factor that may influence nodulation and nitrogen fixation . All thesefactors influence performance of a given plant within a cultivar or a given cultivar.Kharkwal et at. (2000) reported that raising two seeds of a given genotype of a legume orof two different legumes sown in a single hill affect nodulation of each other negatively orpositively . Of the three legumes (chickpea, lentil and pea) studied, variety HFP4 of pea(the only one studied) showed enhanced nodulation by 20 to 74% compared to theaccompanying other legumes and the other varieties/legumes generally reducednodulation. Any breeding study therefore has to consider influence of such factors andhave some in-built safeguards to develop high BNF and high yield materials .

Strategies for Improving Nz-FixationResearchers working in this area generally agree that enhanced N2-fixation by the grainlegumes will result from selection and breeding for high N yield, high nitrate tolerance,and specific rhizobial strain x cultivar requirement (Mytton, 1983; Beringer et al., 1988;

Bliss and Miller, 1988). This section examines programs that aim to develop cultivars oflegumes that incorporate one or more of these characteristics .

Legume N-yield: Agronomic and environmental considerations often limit the biomassyield of a legume crop and therefore the capacity of that crop to fix N2. Yield will also bedetermined genetically . Due et at. (1988) evaluated 21 genotypes of faba bean at two sitesnear Dijon, France, over two years. Their data show the strong correlation betweenbiomass N yield and Nrfixation. Where comparisons between genotypes could be madeover sites and years, they suggested a strong genetic basis for N yield and Nrfixation.This, however, can be true under low soil-N and not under high soil-N conditions.

With some species, low N yield may be a typical characteristic. In studies over a rangeof environments and agronomic practices, N yield and Nrfixation by chickpea wereconsistently less than for the other cool season food legumes (Rennie and Dubetz, 1986;Evans and Herridge, 1987; Smith et al., 1987; Beck et al., 1991) . Average values for N

yield were 100 for chickpea, 185 for field pea, 196 for lentil, and 200 kg N ha- l for faba

bean. These studies did not indicate that the inherent capacity of chickpea for either

nodulation or Nrfixation was less than for the other species . It seems that increasing Nyield of chickpea may result in increased Nrfixation.

734 O'P. Rupela and D.L.N. Rao

In the common bean, low N yield is the result of low Nrfixation capacity, rather thanvice versa (Attewell and Bliss, 1985). A breeding program by Bliss and his co-workers atthe University of Wisconsin has produced new genotypes of common bean with higherlevels of Nrfixation, resulting in increased plant vigour and improved N yields (Table 5).Table 5. Summary of data from two experiments from a breeding program to increase N2

fixation by the common bean (Attewell and Bliss, 1985)

Parent .cultivaror line

Experiment 1 Experiment 2

N2 fixation (maturity) Seed yield Maturity Determinate N yield at(mg plant'" ) % N from (g plant') (days) R7

air (mg plant'")

Sanilac 76 12 18 85 Yes 59124-17 583 48 31 110 No 106824-21 216 25 19 91 Yes 104524-55 192 22 23 94 Yes 668Puebla 852 57 38 120 No 1429

Until 1980s most scientists assessed Nrfixation based on the ARA. More recently, 15Nmethods (enriched and depleted) were used (Pereira et al., 1989; Wolyn et al., 1989) .Breeding material was, out of necessity, evaluated under low soil mineral N, because theplant's capacity for NTfixation and not the capacity for growth (N yield) was of principalimportance. In a soil with moderate to high nitrate N, the capacity of the plant to fix N2will not be expressed to the same extent, because of the suppression of N2-fixation by thesoil N. In a species such as chickpea, where N yield rather than Nrfixation per se may bethe problem, evaluation would still be more useful in low N soils, because of the addedcapacity to screen for Nrfixation as well as for N yield. Because N yield and dry matteryield are generally strongly correlated (e.g., Mytton, 1983), a program to enhanceNrfixation in chickpea might involve the following :• Screening a large and diverse germplasm (500-1000 genotypes) of chickpea, inoculated

with highly effective rhizobia, for production of dry matter under low N conditions(preferably in the field, but could also be done in a glasshouse) ;

• Selecting superior genotypes (e.g., top 10 %) for further evaluation;• A second round-of screening is ideally done in the field on a low N-fertility soil, again

with a mixture of highly-effective rhizobia. Assessments should include seed yield andtotal N yield and

• Comparison of elite genotypes over a range of edaphic (particularly soil N fertility) andenvironmental (including diverse rhizobial population) conditions for seed yield, Nyield and N2-fixation, and the latter using 15Nmethods.Figure 2 outlines a program used in chickpea and pigeonpea (Rupela, 1994; Rupela and

Johansen, 1995) for developing high BNF lines . A similar approach was followed byVenkates warlu and Katyal (1994) in groundnut. Genotypes identified through such ascreening protocol are likely to be superior for BNF and seed yield and adapted to thesoils and environments for which they were likely to be used . This protocol would haveon-farm application (Herridge et al., 1994).

Breeding for Improved Biological Nitrogen Fixation 735

Step

2

3

4

Action

Identification of variants, about 3 WAS followedby salvation of plants in PP. Identification ofvariant at physiological maturity in field sown CP.Seed multiplication (selfing in PP)

SPP in pots/rows for confirmation of nodulation(3 WAS in PP, 6 WAS in CP), seedmultiplication(selfing in PP)

,SPP in pots/rows, replicatedfor testing stabilityof traits, seed multiplication (selfing in PP)

Replicated trial for confirmation on Nrfixation,yield assessment, seed multiplication (selfing inPP) --+

Output

Single plant progenies(SPP) of known nodulation trait

Confirmation of SPPs of desiredN2-fixation trait

Stable SPPs of desiredN2-fixation trait

Potentialmaterialwithhigh BNF

Fig. 2. Protocol for selecting nodulation variants of chickpea (CP) and pigeonpea (PP). The screening nurseryfor chickpea can be developed in a field while for pigeonpea, evaluation has to be done in a greenhouse. Seed multiplication can be done both in the greenhouse and field. WAS = weeks after sowing

High N2-fixing genotypes that produced low seed yields or seed of low quality, couldbe used as donor parents in a breeding program (e.g., as with "Puebla" in the commonbean program, Table 5). There may be little scope to adopt this protocol for species suchas field pea and faba bean that are already capable of producing high N yields under fieldconditions, i.e ., 300 kg N ha-1 and fixing substantial amounts of N2 (see Brunner andZapata, 1984; Jensen, 1986). Harvesting this nitrogen into grains may be more importantin these legumes.

Brunner and Zapata (1984) compared 19 mutant lines of faba bean with the parentcultivar for seed yield, dry matter and N yield, and Nrfixation. One line (II-18) wasclearly superior in all characteristics, but it was not clear whether the superiority of theline was because of higher growth and N-assimilation rates or because of a longer cropduration. Nevertheless, its superior performance over a range of environments suggestedthat growth and assimilation rate had been improved by mutagenesis. The authors alsoconcluded that N2-fixation was determined by N yield, rather than PfIX. In a similar studyinvolving pigeonpea, Kumar Rao and Dart (1987) also showed the strong relationship

736 O.P. Rupela and D.L.N. Rao

between N yield and N2-fixation. In that instance , however, N yield appeared to be linkedto crop duration.

Traits Associated with Nz-fixation at Process Level: Although sometimes used ascriteria for increased Nz-fixation, selection of genotypes on the basis of specific traits thatare either directly or indirectly associated with nodulation or nodule (Nz-fixing) activityappears to be of limited value. Heichel et ai. (1989) concluded that the selection of lines ofalfalfa (Medicago sativa L.) for activity of various nodular enzymes , includingnitrogenase, resulted in experimental populations with enhanced or reduced enzymeactivity, but did not result in populations that were different in N2-fixation. The authorsproposed several explanations for their inconclusive findings, including the following:• Short-term measurements of nitrogenase activity using the ARA may not predict

activity over a longer growth period;• The limitations of the ARA may be too great to even compare treatments on a relative,

let alone a quantitative, basis;• Performance of seedlings in a glasshouse may not predict performance in the field over

one or several seasons, and• Enhanced activities of single or several nodular enzymes may be countered by

inadequate or even normal activity of other equally-important enzymes, resulting in nonet change in the rate of Nrfixation.Thus, any program based on screening at biochemical (enzymetic) levels alone may not

be very successful.Effective Symbiosis with Native Rhizobia : Several researchers have argued that it is a

desirable practice to inoculate legumes. It is indeed required for soils where nativeappropriate rhizobia are lacking. But determination of such needs of inoculation is timeconsuming. Also, in most soils where a given legume is grown regularly, any furtherinoculation with laboratory inocula may not enhance nodulation and nitrogen fixation andyield significantly high over non-inoculated control. Exception will be where rhizobiamay die due to environmental conditions, such as high summer temperatures and alsoduring rice phase in the rice-legume cropping systems . Most developing countries shouldselect legumes that nodulate effectively with the native rhizobia, because quality of theirinoculants may be generally poor (Singleton et al., 1997). Soybean from south-east Asianodulate successfully with the indigenous rhizobia in Africa, but the USA bred cultivarsnodulate poorly (Nangju, 1980). Also, the local soybean (Kali Toor) in Madhya Pradeshhas been noted to nodulate more effectively by native rhizobia than some breeding linesinvolving blood from USA bred cultivars (J .A. Thompson, Tamworth, Australia,ex-ICRISAT scientist, personal communication) . Therefore, developing soybean lines thatnodulate and fix N2 quantitatively with native rhizobia may obviate the need forinoculation of such lines, particularly when grown in soils with high population of nativerhizobia. Kueneman et al., 1984 reported a selection program for high nodulationlnitrogen fixation in soybean in Nigeria using native rhizobia (i.e. no inoculation) anddeveloped lines with high yield and N2-fixation.

Breeding for ImprovedBiological Nitrogen Fixation 737

Large difference for Nz-fixation capacity between cultivars has been reported forseveral legume species (see section on 'Genetic variability for nodulation andNz-fixation') . Rupela (1992) reported large variability for nodulation within bred cultivarsand landraces of chickpea.

The protocol of developing high Nz-fixation lines is shown in Figure 2. The highnodulating selections developed from two widely used cultivars (G130, K850) using thisprotocol were evaluated as part of a multilocation (Akola, Badnapur, Hisar, New Delhi ,and Sehore) experiment during 1994/95 and 1995/96. Low nodulating selections fromboth the varieties and the parent lines were used as reference. Non-nodulating selectionsfrom two cultivars (ICC4993 :;: Rabat, a long duration line, and ICC49 I8 :;: Annigeri, ashort duration line) were used as non-Nj-fixing lines, to allow calculation of amount ofNz-fixed by difference method. Indeed the high nodulating pure line selections from thetwo cultivars nodulated significantly higher (Table 3), fixed high nitrogen (measured byARA method at one of the five locations and by difference method at 3 of the 5 locations)besides producing high grain yield . The differences were more pronounced at almost alllocations at low soil-N (N1) than at high soil-N (Nz)conditions . High Nz-fixing selectionswere also made from advanced breeding lines. The final products developed from sixcultivars were evaluated at two soil-N levels, along with their parents . The resultssuggested 0-293 % improvement in nodule mass, with no or insignificant improvement intotal biomass yield (Table 6). It was apparent that this strategy of selecting highnodulating plants and developing their progenies (single plant progenies) into lines (pureline selection) can also be applied for selecting stress tolerant symbiotic lines.

Exclusion ofNodulation by Native Rhizobia: It is generally difficult to displace nativerhizobia by inoculation with laboratory multiplied strains. About 10 % or less nodules areformed by inoculant rhizobia when the population of native rhizobia is high (Rupela andSudarshana, 1990 ; Devine, 1984; Halliday, 1985). Use of undisturbed cores has beensuggested to select rhizobial strains for competitiveness (Sylvester-Bradley et al ., 1983).There are indications that selected, highly effective, and competitive strains can be used toincrease Nz-fixation and yield, particularly where native rhizobia populations are low orineffective (Arsac and Cleyet-Marel, 1986; Beck, 1992) . Successful nodulation byinoculant strains can often be site-specific depending on soil factors and the compositionof the indigenous rhizobial population.

Some groups (Devine, 1984; Cregan and Keyser, 1986) aimed to produce cultivars ofthe host that bypass the resident rhizobia in the soil to become nodulated by moreeffective inoculant strains. This strategy has also been applied to pea. Lie (1978 ; 1981)found that the primitive pea cultivar "Afghanistan" was not nodulated by strains ofrhizobia isolated from temperate soils in Europe, but did form nodules with bacterialstrain Tom from Turkey. This resistance to nodulation has been ascribed to the recessivegene , sym-2 (Holl, 1975). In competition experiments , the European strains causeddifferential blocking of nodulation by Tom (Lie et al., 1988). Subsequent identification of

738 a.p. Rupela and DLN. Rao

rhizobial isolates that could nodulate Afghanistan as well as cultivars bred for Europeanconditions, indicated the presence in the rhizobia of a specific genetic region, termednodX. Fobert et at. (199 1) suggest that the combination of the plant gene, sym-2, and therhizobial gene, nodX, may allow ultimate control of nodulation and prov ide a mechanismfor enhancing Nrfixation, even in the presence of large populations of indigenousrhizobia. This approach assumes that N2-fixation was limited by the effectiveness of thenative rhizobia, which may not always be the case .

Table 6. Mean values on nodule mass and total dry matter of single plant progenies selected

for large differences in nodulation capacities wi thin advanced breeding lines of

chickpea, ICRISAT, Patancheru, post-rainy season 1995/96.

Parent No. Type Nodule mass (mg/plant) Total dry matter « ha- t) Nodula-of of N) N2 Mean Nt N2 Mean tion

selecti means improve-2 ment3(%)on

ICCV 2 117±IS.9 74±IS.9 096±1O.1 2.2±O.IS 2.4±O.IS 2.3±O.1O 4a89230 b 114±12.3 SS±12.3 08S±7 .1 2.6±O.12 2.4±O.12 2.S±O.07

ICCV 6 a 129±IS.9 78±IS.9 103±1O.1 2.0±0 .IS 2.4±O.IS 2.2±O.1O89302 b I28±9 .2 62±9.2 09S±4 .1 2.I±O.08 2.4±O.08 2.3±O.04

ICCV S a 076±I S.9 21±IS.9 049±1O.1 2.I±O.15 2.4±O.15 2.3±O.1O 64,N \91016 b 079±9.5 39±9 .5 059±4.5 2.0±0.09 2.4±O.09 2.2±O.05 129.N2ICCV 3 a 036±IS.9 21±15.9 029±1O.1 1.5±O.15 1.9±O.15 1.7±O.1O 293,N191019 b 092±1O.8 S2±1O.8 072±S.8 2.0±O.1O 2.4±O.1O 2.2±O.06 273.N2ICCV 5 a On±15.9 47±15 .9 060±1O.1 1.8±O.15 2.4±O.15 2.I±O.1O 88,Nt91026 b 085±9.S 26±9.5 056±4.5 1.9±O.09 2.4±O.09 2.2±O.OS

ICC 2 a 065±15.9 21±IS.9 043±1O.I 2.3±O.IS 2.9±O.15 2.6±O.1O 41,Nt49S8 b 060±12.3 33±12.3 047±7.1 2.0±0.12 2.7±O.I2 2.4±O.07 99.N2ICC S003 (Control) 091+IS .9 46+IS.9 069+10.1 1.3±O.IS 2.2±O.IS 1.8±O.1O

I . Data for selected parents where selections were significantly different (P < O.OS) from parents oramong themse lves for nodule mass; 2. a =mean values for the parent, b = mean values for selectionsfrom the parent; 3. Percentage of increase in nodule mass of a selection at Ni or N2 over its parent ;4. - = No improvement; S. Low (Ni) and high (N2) soil N level . representing those that can be foundin farmers' fields, were created by applying 0 (Ni ) and 100 kg N ha'J (N2) as urea to the precedingsorghum on Vertisol field after the whole field was depleted for N by growing cover crops for twoyears. N is a highly dynamic element and varies through the soil profile due to several factors. Atsowing, the available N in the top IS em soil was 1.7 times more in the N2 plots than in Ni plots(8.7 mg N kg-I soil); Source: Rupela 1997.

Induced Mutations : Breeding through induced mutations is a well established strategyto improve legumes (Micke, 1984) . High soil-nitrate tolerant (called supemodulating bysome authors) lines of pea (Jacobsen and Feenstra, 1984) and soybean (Carroll et al.,1985a) involving mutations have been reported. Supemodulating lines have been reportedfor soybean cultivars Bragg, Williams, Elgin 87 and Enrei (Herridge and Rose, 2000).

Breeding for Improved Biological Nitrogen Fixation 739

These mutants formed up to 10-20 times more nodulation and AR activity than that of thewild-types in the presence of 5 or 5.5 mM nitrate. However, their yield was 30-40 %lower than the wild types and they had restricted root growth. The super-nodulatingsoybean mutants seemed to be controlled by a single Mendelian recessive gene operatingthrough the shoot (Delves et ai. , 1986; Lee et al., 1991). There was no evidence of host xrhizobial strain specificity affecting expression of the supemodulation trait (Carroll et al.,

1985b; Gremaud and Harper, 1989). A selection and breeding program for high NTfixingand high yield involving supemodulating mutants in Australia suggested that lines withintermediate nodulation (between normal lines and supemodulating mutants) yieldedequal to agronomic cultivar Manark and fixed substantially high N2. At this stage,however, no supemodulating cultivars have been released (Herridge and Rose, 2000) .

The FutureThere has been limited success in selection and breeding for high N2-fixation.Development of soybean lines with effective nodulation due to native rhizobia in Africa(Mpepereki et al., 2000), high N2-fixing cultivars of common bean released in SouthAmerica (Bliss , 1993) , pure lines selections from advanced breeding lines of chickpeawith high nitrogen fixat ion ability (Rupela, 1997) are some examples where materials areavailable for researchers and/or farmers to evaluate and verify their potential. One likelyreason of slow progress may be due to the fact that it would take a multidisciplinary teamto achieve success in this complex trait, in addition to the complex trait 'yield' (it may benoted that breeding legumes for high yield ,per se, has not been as successful as incereals). Also, any breeding program has to combine several traits (such as pest anddrought resistance) along with yield to ensure that the resultant materials are potentiallyacceptable to farmers . Breeding for high NTfixation is feasible and should also be on theresearch agenda of breeders.

Much of the breeding work is conducted at research stations . Soils at research stationsare likely to have higher soil nitrogen than at farmers' fields . High soil-N is known tosuppress nitrogen fixation by legumes (Streeter, 1988; Wani et al., 1997). For promotingN2-fixing traits, breeders should grow their legumes at low soil-N (preferably 10 5gmineral N g-I soil) fields, prepared specially for the purpose. Breeders generally handlelarge numbers of genotypes and materials. In some materials, genes for NTfixation maybe co-segregating with genes for the other traits. It is likely, therefore, that traitcombinations associated with enhanced N2-fixation will be identified, if appropriateassessment methods are applied to the segregating populations. If genetic variation forNTfixation existed in breeding populations, the high NTfixing lines would be producedas a normal outcome. The role of the N2-fixation specialist in the main stream breedingprogram might be to identify sources of genes for particular symbiotic traits and toprovide technical guidance and support (Bliss, 1993) during development of desiredproducts. Recent developments in the field of genomics (particularly on Medicago andLotus) would provide a better understanding of the expression and regulation of symbiotic

740 O'P. Rupela and DLN. Rao

genes. It should also open up opportunities for biotech assisted germplasm enhancementand bio-informatics assisted gene mining and utilization. These developments may lead toa better targeted breeding of legumes for high BNF than hitherto possible.

AcknowledgementsWe thank Drs. Y.S . Chauhan, H.D. Upadhyaya and L.J. Reddy for comments on themanuscript.

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