Compendium of Transgenic Crop Plants || Common Bean

JWBK201-Kole k0301 August 6, 2008 8:5

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Common BeanFrancisco J.L. Aragao1, Josias C. Faria2, Maria J. Del Peloso2, Leonardo C. Melo2

and Rosana P.V. Brondani21Embrapa Recursos Geneticos e Biotecnologia, Brasılia, Distrito Federal, Brazil, 2Embrapa Arroz

e Feijao, Santo Antonio de Goias, Goias, Brazil

1. INTRODUCTION

1.1 History, Origin, Botanical Description,and Taxonomy

The Phaseolus genus consists of more than 30species, but only five (P. acutifolius, P. coccineus,P. lunatus, P. polyanthus, and P. vulgaris) havebeen domesticated (Debouck, 1999). The commonbean (P. vulgaris L.) is the most widely cultivatedspecies, occupying more than 85% of the totalarea cropped with Phaseolus throughout the world(Singh, 2001).

The diversity detected in Phaseolus species in re-lation to common bean is organized into primary,secondary, tertiary, and quaternary gene pools(Singh, 2001). The primary gene pool includesthe modern and old cultivars, improved lines,and wild types of P. vulgaris. Within the primarygene pool, there is no intercrossing problems andnormally includes the most cultivated species ofthe genus (P. vulgaris). The secondary gene poolconsists of the species P. coccineus, P. costaricensis,and P. polyanthus (Broughton et al., 2003). Thesethree species intercross easily inter se and alsocross with P. vulgaris without requiring embryorescue, especially when the common bean isused as the female parent (Singh et al., 1991).However, in some crosses partial sterility mayoccur in certain individuals, especially when theP. vulgaris is used as the male parent. The tertiary

gene pool consists of P. acutifolius and Phaseolusparvifolius, which are species that produce fertileprogeny without requiring embryo rescue whenintercrossed. Embryo rescue is required, however,when they are crossed to P. vulgaris. Normally,one or two backcrosses to P. vulgaris are neededto restore fertility. Again, the use of P. vulgarisas female, both in the cross and in the backcrosswith P. acutifolius increases the chance of obtainingfertile individuals in the segregant population.Fertile progeny is not obtained from crossesbetween the quaternary gene pool and P. vulgaris,even using the embryo rescue technique. Thisgermplasm includes the Phaseolus angustissimusand P. lunatus species, which to date have not beenproved to share genes and alleles with P. vulgaris.

There are approximately 30 300 P. vulgarisaccessions in the germplasm bank at CIAT(International Center for Tropical Agriculture),of which approximately 29 000 are cultivatedcommon bean accessions and 1300 are wild type.This bank also contains further 1000 accessionsof secondary germplasm and more than 350accessions of tertiary germplasm (Broughtonet al., 2003). There are thousands of otheraccessions of the primary, secondary, tertiary, andquaternary gene pools distributed in germplasmbanks throughout the world. However, most ofthis variability has not yet been used directly in thecommon bean breeding programs (Miklas, 2000).The introgression and pyramiding of favorable

Compendium of Transgenic Crop Plants: Transgenic Legume Grains and Forages. Edited by Chittaranjan Kole and Timothy C. HallC© 2008 Blackwell Publishing Ltd. ISBN 978-1-405-16924-0


2 TRANSGENIC LEGUME GRAINS AND FORAGES

alleles between and within this germplasm couldincrease the genetic base of the segregantpopulations, maximize genetic gain from selection,and increase the stability of disease resistanceincorporated in the new cultivars.

Although the common bean (P. vulgaris)dispersion process is not fully understood, thereis a general agreement that its origin is in theAmerican continent. Dispersion seemed to benoncentric, having started from Mesoamericaor South America, or independently from bothregions. The agreement among specialists wasreached after most evidence confirmed the theory(Singh, 1989; Singh et al., 1991). The commonbean dispersion process continued. It was taken tothe old world by the Europeans after the discoveryof the United States in the 15th century. Thestudy by Vavilov (1951) supported the hypothesisof origin in the United States for the Phaseolusgenus. The author showed that the center of geneticdiversity of the common bean species P. vulgaris,P. coccineus, P. lunatus, and P. acutifolius is locatedin Mexico and Central America, because it is inthis area that the largest diversity of forms of thesespecies was found. A secondary center of geneticdiversity was identified in a mountain area in Peru.Although the center of diversity and center oforigin do not have the same meaning, the discoveryby Vavilov was a strong indication in favor of theAmerican origin of the common bean (Vieira et al.,1999).

Morphological evidence showed that the wild-type bean, ancestor of the common bean, isextensively distributed in the United States, fromWest Mexico to Northeast Argentina, coveringan almost continuous range of approximately7000 km of mountain areas. Along this area,morphological differences that might affect theadaptation of the wild-type bean to contrastingenvironmental conditions were observed (Kamiet al., 1995). There are botanical differences amongthe wild beans found in Mexico and Central Amer-ica (P. vulgaris var. mexicanus) and those in SouthAmerica (P. vulgaris var. aborigineus). However,there are several traits (climbing plants, with alarge number of small and dehiscent pods, withsmall, hard seeds that are difficult to germinate)that are common to the wild types but not tothe cultivated forms (P. vulgaris var. vulgaris).This species underwent extensive modificationin its characteristics during the domestication

process, and the current cultivars present a smallnumber of flowers, pods, and seeds, which is aconsequence of the smaller proportion of flowersthat reach maturity compared to the wild types. Inaddition, modern cultivars have more restrictedplant growth and more compact form, makingthem shorter and more erect; larger leaves; morerobust stem; larger flowers, seeds and pods; fewerseeds per pod; seeds more permeable to water,that allows uniform germination and shortercooking time; suppression of the seed dispersionmechanism; decrease in the percentage of fibercontent in the pods and neutrality to photoperiod.

The wild beans cross easily with the modernP. vulgaris cultivars, producing fertile F1 andF2 generations, indicating that the wild andcultivated forms belong to the same speciesand no reproductive mechanism has emergedto isolate them. According to Singh (2001), thegenetic differences between the two forms seem toaffect only a small proportion of loci, specificallythe occurrence of the Dl-1 gene in the MiddleAmerican and Dl-2 in Andean wild and cultivatedpopulations.

Studies on the domestication and organizationprocesses of the common bean genetic variabilitywas first based on morphological traits andbiochemical markers. In the late 1980s, after thediscovery of polymerase chain reaction (PCR)methodology, a number of molecular markertechnologies have been developed. The directanalysis of DNA variation allowed a moreprecise estimation of the genetic variation thatcan be accessed at the nuclear and/or organellegenomes, enabling the investigation of differentaspects of the genetic structure of crops species.The characterization of genetic diversity amongcommon bean accessions from divergent genepools using molecular markers, has been shownto be effective to elucidate mechanisms of originand evolution of common beans, to investigate theeffects of domestication process in the reductionof the genetic diversity that has characterizedcommon bean gene pools, and to allow anefficient management and effective exploitationof the germplasm (Gepts, 2004). These studieshave been performed using molecular markers thataccess specific genetic polymorphisms at a knownor anonymous DNA sequence site, includingrandom amplified polymorphic DNA (RAPD)markers that showed to be effective to identify


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races within the Mesoamerican gene pool (Beebeet al., 2001), the use of amplified fragment lengthpolymorphism (AFLP) markers that showed tobe effective to assess the level and direction ofgene flow between wild and domesticated beanpopulations (Papa and Gepts, 2003) and the anal-ysis of variability at specific translated genomicregions that allowed to conclude about the level ofdiversity between wide and domesticated commonbeans (McClean et al., 2004). More recently, usingmicrosatellite markers (simple sequence repeat,SSR), new findings where the intrapopulationdiversity of the Andean gene pool was higherthan within the Mesoamerican gene poll groupswas reported by Blair et al. (2006a), opening newperspectives for the exploration of the geneticvariability at the Andean gene pool.

Over the last years, several research groupshave made efforts in the development of mi-crosatellite markers for common bean, those areconsidered the most informative class of markers.Microsatellites, that present the advantages to beco-dominant, multiallelic, and PCR-based, wereinitially derived from GenBank sequences by Yuet al. (1999, 2000), followed by Guerra-Sanz (2004)and Blair et al. (2003). Methods of microsatellitediscovery based on enriched genomic librarieswere developed by Gaitan-Solıs et al. (2002), Yaishand Perez de la Vega (2003), and Buso et al.(2006). The development of SSR markers derivedfrom bacterial artificial chromosome (BAC) wasconducted in common beans by Caixeta et al.(2005).

Johns et al. (1997) studied the genetic variabilityin the common bean domestication centers usingRAPD markers and morphological traits. Theauthors reported that their RAPD markers werecapable to screen the genotypes into well-definedgroups, which corresponded to the Mesoamericanand Andean domestication centers, whereas themorphological markers were less efficient inidentifying the origin of the germplasm. Theindication is that these data of diverse natureare representing different portions of the totalvariability because they probably suffered distinctevolutionary pressures. According to Gepts (1991)this apparent paradox was related to the differenttype of gene actions that control these markers.Some common bean morphological traits arecontrolled by genes with large phenotypic effectand are thus subject to human selection. On

the other hand, biochemical and molecular traitsrarely have a marked effect on the phenotype andare less likely to suffer selection. Consequently, thechance of perpetuating mutations in biochemicaland molecular traits is smaller than the chanceof maintaining mutations that cause largemorphological alterations, which affect the totalvariability accessed by each one of these markers.

1.2 Economic Importance

Common bean is an important source of proteinin the diet of more than 300 million peoplethroughout the world. It is especially importantin tropical and subtropical developing countries,with significant regional variations concerningtaste and grain type preference. The grains havehigh protein content, high quantity of complexcarbohydrates, fiber, oligosaccharides, and phyto-chemicals, and are also an important source ofiron, phosphorus, magnesium, manganese, andto a lesser extent zinc, copper, and calcium(Broughton et al., 2003).

The common bean grain quality traits includelow dietary fiber content (6%) compared to otherproducts such as wheat (10.5%), maize (9%)(Juliano, 1993), soybean meal (20.6%), oat flakes(6.5%) (Cozzi and Lajolo, 1991), but a greatercontent than brown rice (4%) (Juliano, 1993). Aquantity equivalent to one cup of common beansupplies at least half of the daily requirementof folic acid, which is especially importantduring pregnancy. It also supplies 25–30% of therecommended daily requirements of iron, 25% ofmagnesium and copper, and 15% of potassium andzinc.

The composition of the common bean (P.vulgaris L.) grains provides several health benefitsfor human nutrition. The grains are indicatedin the dietary treatment of several diseases suchas heart diseases, diabetes mellitus, obesity, andcancer (Geil and Anderson, 1994). Beans area good source of food fiber, especially solublefiber (Kutos et al., 2003). The consumptionof soluble fiber-rich foodstuffs has efficientlydecreased total cholesterol serum levels and,consequently, reduced cardiovascular diseases inthe population (Glore et al., 1994).

There are 119 producing countries in theworld if all genera and species included as



common beans in the FAO statistics (FAO,2004) are considered. The common bean (P.vulgaris) is the most widely cultivated speciesof the Phaseolus genus. FAOSTAT places worldcommon bean production at around 22 milliontons cropped in approximately 35 millions ha.Only 10 countries account for approximately 70%of the world production; India is the largestproducer with 14.31%, followed by Brazil with13.39%, Nigeria (10.4%), Myanmar (7.58%), andMexico (5.25%). Only 15% of the total commonbean production worldwide is exported, with fivecountries accounting for 80% of the market: China21%; Myanmar 27%; the United States 10%;Canada 9%, and Argentina 13% (FAO, 2004).

1.3 Traditional Breeding of Common Bean

1.3.1 Objectives in common bean breeding

A sustainable plant breeding program must envis-age the prebreeding, breeding, and postbreedingstages of cultivar development. Extra effort canbe directed to a specific stage requiring greaterprogress at any point of time. The breedingand postbreeding stages normally receive mostattention in a common bean cultivar developmentprogram. On the other hand, the prebreedingstage has been neglected, in part due to the needfor interdisciplinary work, better qualified humanresources, high costs, and long-term returns.Strong prebreeding programs can contribute tothe synthesis of new promising broad genetic basepopulations, to the identification of potentiallyuseful genes and alleles. It would also enhanceknowledge of the germplasm of the species andhelp establishing nuclear collections to ensurecontinuous gains in the plant breeding programs.

The objective of most breeding programs isto increase yield and guarantee yield stability ata production cost that maximizes the economicreturns to the farmers. Under favorable conditions,yield increases through breeding are achievedby accumulating alleles that maximize biomassproduction and foster efficient assimilate distri-bution. In unfavorable environments, stable yieldand reduced production costs can be obtainedthrough breeding for tolerance to biotic andabiotic stresses that cause yield losses. Regardingthe biotic stresses, diseases cause the most losses

and, therefore, always deserve special attentionin the major breeding programs. In additionto improving the level of resistance to themost common disease pathogens that causeanthracnose, common bacterial blight (CBB), rust,angular spot, and common mosaic, a breedingprogram is also expected to develop stableresistance to pathogens of potentially dangerousnew diseases, for example, Curtobacterium blight,scab (caused by Colletotrichum truncatum), Asianrust and soil diseases, especially white mould,root rot, and wilts. There is the challenge ofobtaining resistance to the Bean golden mosaicvirus (BGMV) and to the Bean golden yellowmosaic virus (BGYMV).

The fact that common bean cropping has be-come an important agricultural business changedthe relative importance of the cultivar traits tothe farmers. Growers currently seek cultivars witherect plant canopy (with pods that do not touchthe soil, with short guides and closed branching)to allow mechanical harvesting, with low lossindices, and high grain quality. Cultivars mustalso allow good plant aeration in the field anddecreased disease incidence. Earliness has becomean important trait for growers, since it allows largerflexibility in the cropping system management,greater water and electricity economy in irrigatedcrops, and better chance of escaping seasonaldroughts, insects, and diseases, and also gives afaster return on invested capital. The ever morefrequent water shortage problems associated withthe greater likelihood of paying for irrigation waterindicate that drought tolerance has become anessential trait for future common bean cultivars.Movement of common bean cropping areas tohigher temperature regions and the emergingproblem of global warming have prompted thebreeding programs to work on the development ofhigh temperature tolerant cultivars. There are largecommon bean areas cropped under drought andhigh temperature conditions, where the subsistencefamily agricultural system predominates and thecommon bean plays a fundamental role in the foodsafety of millions of people. Genotypes adapted tothese conditions must be developed.

The consumer demand for common beangrain showing higher nutritional, organoleptic,and functional quality has made this area ofresearch a priority for some genetic breedingprograms in the world. The most common agents


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that affect mineral bioavailability are phytates,tannins, fibers, polysaccharides, and oxalates. Thepolyphenols (tannins) and phytic acid are partof the antinutritional compounds found in thecommon bean, and may be involved in thedevelopment of the hard-to-cook effect (Hohllbergand Stanley, 1987), which increases cookingtime, decreases palatability, and reduces proteindigestibility (Reyes-Moreno and Paredes-Lopez,1993). Polyphenol compounds, which consist ofpolyphenol acids and their derived substances,affect the nutritional quality of foodstuffs andtheir biochemical and physiological properties.These polyphenol substances are also partiallyassociated with the changes that occur in the grainsduring storage, and play an important role inthe development of postharvest grain hardening(Bhatty, 1990; Shomer et al., 1990). Mineralavailability, especially iron, is affected by thepresence of tannin and dietary fibers. Commercialacceptance and consumption of common beangrains are influenced by an irreversible darkeningof the grain tegument that depends on thecultivar and occurs gradually after harvesting.Prolonged storage periods, especially at hightemperatures and relative humidity, considerablyincrease the cooking time and darkening of thegrain. These characteristics are also influenced bythe environment, as shown in the study carriedout by Michaels and Stanley (1991) with 20common bean cultivars in three different locations.Breeding for grain quality has long been reported(Meiners and Litzenberger, 1975; Ghaderi et al.,1984). Current research has shown the possibilityof decreasing the common bean antinutritionalfactors, such as phytates and phytic acid, and hasidentified the presence of wide variability withinthe species (Coelho and Lajolo, 1993; Kigel, 1999).

1.3.2 Common bean breeding:tools and strategies

The increase in yield potential of the commonbean (P. vulgaris) crop has been gradual andlow, in spite of the broad variability presentfor most traits, including grain yield (Nienhuisand Singh, 1988). One of the main reasons forthis low increase in yield potential has beenthe consumer market requirement for differentcommercial grain types according to regions.

This requirement has reduced the germplasmresources used and limited the genetic variabilityavailable in the breeding programs. Also, thetendency of breeding new cultivars with erectstem with fewer ramifications has added to thedifficulty of increasing the yield potential ofmodern cultivars. The use of genetically similargenotypes has become more evident after studiesby Singh (1988, 1989), who grouped the commonbean germplasm into 12 genetic pools and laterfurther grouped them into six races (Singh et al.,1991), indicating that breeders have traditionallycarried out hybridization involving materialspredominantly from a single gene pool. Singh(1988) observed that the variability for grain yieldand its primary components and other agriculturaltraits was greater among than within gene pools.Similar result was also obtained by Abreu et al.(1999) assessing several agriculturally importantmorphological traits in common bean.

The genetic diversity studies of wild commonbean ancestors measured by DNA markers suggesta considerable index of diversity spread withinand among the gene pools (reviewed by Blairet al., 2006a). In contrast, the genetic diversityof cultivated common bean is thought to besmaller than that of wild common bean due toa genetic bottleneck that occurred during cropdomestication, dispersal from center of origin,and selection for specific traits (Gepts, 2004; Papaet al., 2005). The introgression of valuable geneticvariants from wild ancestors of common bean bymeans of applying marker-assisted backcrossingor genetic transformation strategies can help inbroadening the genetic base of the elite breedinggene pool, increasing the chances of genetic gainsin the breeding programs.

The use of genotypes from different genepools as parents in crosses to obtain segregantpopulations increases the probability of obtaininggenetic combinations with greater yield andproduction stability potentials. Singh (1988)reported that crosses between cultivars stemmingfrom gene pools from the same center oforigin and presenting similar growth habit andseed size showed greater frequency of desirablerecombinants. This is probably due to the factthat crosses between very divergent parents canlead to low adaptation of the segregant populationand, consequently, difficulties in the selectionprocess. Theoretically, crosses between cultivars



from different gene pools but from the samecenter of origin should contribute to increase gainfrom selection, especially when adapted parentsare used. More recently, an advanced backcrosspopulation derived from a cultivated Andeangenotype and a Mesoamerican wild accessionof common bean was developed and the resultsshowed that this strategy was advantageous, sinceno fertility barriers were observed between thewild and cultivated gene pools, the derived linesthemselves were close to commercial type, and newdiversity for plant vigor and tolerance mechanismswere incorporated into the cultivated background(Blair et al., 2006b). These findings opened newperspectives for the introduction of allelic variantsfrom nonadapted genotypes into cultivars withmodern traits, such as seed types and plantarchitecture.

Kelly et al. (1997) proposed a hierarchicalpyramidal system with three levels of germplasmimprovement for common bean. Germplasmmovement is from the base to the top as geneaccumulation for yield in specific genotypes occurs.Therefore, at the pyramid base the breeder workswith several limiting factors using the full geneticvariability present in the poorly adapted, wild type,interracial, and interspecific germplasm; at theintermediate level the number of limiting factorsis restricted, but the breeder still uses considerablelevels of genetic diversity; and at the top of thepyramid, elite germplasm breeding is carried out,involving agriculturally and economically accept-able materials within each commercial grain type.

A breeding program for common bean shouldbe structured to meet the demand of growers andconsumers of the new cultivars. The breeder shouldmake use of the genetic diversity of the availablegermplasm, of the knowledge of the problemsof the growing regions and of the knowledge ofproblems that might occur with the establishmentof the crop in the different production systemsand growing seasons. The selection criteria shouldemphasize well-defined regional demands suchas preference for grain type, which includescharacteristics such as size, color, shape, shininess,and cooking quality, and allow association ofdesirable traits during the development of inbredlines superior to the cultivars traditionally in use.The intent of increasing production to supplythe external market has required breeding ofspecial common bean types with large grains,

aiming at increasing product acceptance in theinternational market with higher aggregated valueand differentiated price.

Common bean germplasm shows high levels ofvariability in grain type and size. However, themarket requirements for grain type, as alreadycommented, and the need for resistance to diseasehave limited selection progress through restrictionsto germplasm utilization and to the number ofprogeny advanced, therefore increasing the geneticvulnerability (Smale, 1997; Abreu et al., 1999)of the crop. Recurrent selection is a breedingmethod that has been used in common bean andhas enabled the development of superior lines,without reducing the genetic variability. Becauseit involves a cyclic process of recombination andselection, yield alleles accumulate in segregantpopulations while maintaining high variability forother traits offering the breeder ways of exploitingimmediately this variability by obtaining newinbred lines at each selection cycle. Recurrentselection in inbred species involves the stages ofbase population formation, endogamous familyassessment and selection followed by recombi-nation of the superior families. Ramalho et al.(1988) suggested that breeders using this breedingmethod should be aware of the following points:(1) parents used to form the base populationshould be divergent but should also express goodphenotypes for the greatest number of traits ofinterest; (2) recombination will be efficient if thebest families to be intercrossed are identified. Inthis case, recombination certainly will contributeto increase the frequency of favorable alleles in thepopulation; and (3) selection of individual plants isnot efficient, mainly if the trait has low heritability.Selection should be based on endogamous familiesassessed in replicated experiments. The generalrule is to carry out a recombination cycle atevery two generations of assessment; the firstassessment in a single location, because of thesmall quantity of seeds, and the second in at leasttwo locations. Therefore, superior family selectionin each assessment cycle can be based on the jointanalysis of the generations and locations.

The common bean haploid genome is con-sidered one of the smallest among the legumes(Zheng et al., 1991), estimated at 450–650 Mbp(mega base pair) (Bennett and Leitch, 1995)and has 11 chromosomes. According to Vallejoset al. (1992), in common bean the mean ratio


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of the physical distance compared to the mapdistance is 530 kbp cM–1. As the common beangenome has approximately 637 Mbp, it is estimatedthat the total size of the map of this specieswould be around 1200 cM. Several reports in theliterature have demonstrated the efficient use ofDNA markers to construct informative linkagemaps with high coverage for common beans(for more details, see review by Miklas andSingh, 2007). In the early 1990s, genetic mapsfor common bean based mainly on co-dominantrestriction fragment length polymorphism (RFLP)markers (Chase et al., 1991; Vallejos et al., 1992;Nodari et al., 1993a), dominant RAPD markers(Adam-Blondon et al., 1994) and combination ofdifferent classes of markers (Freyre et al., 1998)were constructed. In recent years, due to theincreasing number of microsatellite that becameavailable, these markers have been extensivelymapped in common beans (Yu et al., 2000; Blairet al., 2003, 2006a). The availability of high-density linkage maps based on a set of co-dominant multiallelic microsatellite markers willlead to the construction of an integrate linkagemap for common bean allowing more precisepositioning of markers, widely distributed acrossthe genome, minimizing the occurrence of largeinterval without markers. These maps will expandthe prospects of making comparative analysis,turning more real the advancement in quantitativetrait loci (QTLs) detection, establishment ofsynteny and validations across different mappingpopulations, increasing the potential use of theselinkage maps among the research groups. Inaddition, these co-dominants mapped makerscould be placed on the corresponding BAC clones,that are already available for beans (Kami et al.,2006), allowing us to probe insights into the aspectsof the genome structures and organization of theP. vulgaris genome, to make an assessment of thecorrespondence between the physical and geneticdistances and also to define physical intervals ofdesirable genome sequences in order to sequenceand clone them.

Following the construction of linkage maps,genomic regions involved in the control of simpleas well as complex heritable traits are beingidentified. QTL for important traits of interesthave been detected in the genus Phaseolus usinga variety of molecular marker classes and severaltypes of segregating populations (Frei et al., 2005;

Beebe et al., 2006; Blair et al., 2006b; Miklaset al., 2006). A wide range of quantitativelyinherited traits, such as domestication syndromesrelated traits, complex disease resistance traits,phenological traits, plant architecture, seed weightand yield components, where generally a fewmajor genes control large proportion of the totalvariation, have been identified (Koinange et al.,1996; Park et al., 2001; Tar’an et al., 2002; Blairet al., 2006b). The experimental approaches usedhave been showed to be effective to successfullyidentify genomic regions that have a significanteffect on the expression of QTLs and provideinformation about the number and the magnitudeof the effects of the traits.

However, it should be pointed out thatthe identification of an important QTL in aparticular genetic background and in a specificenvironmental condition may not be assumed toall genetic backgrounds and locations. When thegenotypes are assessed in more than one location,year and/or cropping season, forming differentenvironments, there is a possibility to identify andestimate the genotype by environment interaction(G × E) effects. There are several studies oncommon bean (Ramalho et al., 1988; Takeda et al.,1991; Melo et al., 1997; Tar’an et al., 2003) thatreport strong G × E effects for the main traitsused in breeding in this species. Melo et al. (2002,2004) and Teixeira et al. (2005) studied the QTL× environment interaction and ascertained thatthis interaction was significant for all the analyzedtraits (flowering time, reaction to powdery mildew,angular spot, seed size, plant architecture, andgrain yield), and the correlation among the meansof the families in the different environments wasmostly low, indicating that in the common beanthe phenotypic expressions, and therefore that ofthe QTLs, depended strictly on the environmentand on its specific interaction with each family ofthe population. Since in the common bean crop,G × E interaction is highly significant due tothe diversity of cropping locations and seasons(environments) used, most QTLs are expectedto suffer the effect of this interaction, and beexpressed only under specific conditions. However,some stable QTLs that maintained their significanteffects across more than one environment and wereconsistent with QTLs from previous studies incommon beans, were reported (Melo et al., 2002,2004; Teixeira et al., 2005) for days to flowering



and seed weight traits (Park et al., 2000; Blair et al.,2006b).

Most of the traits of agricultural importance arecontrolled by multiple genes, with small individualeffects, subjected to a pronounced environmentaleffect, and dependent of the genetic background.Significant progress in map construction and QTLidentification has been reported for common beansin recent years. A large set of microsatellite markershas been made available and, in addition, thegrowing pool of expressed sequence tag (EST)sequencing for P. vulgaris genome (Ramırez et al.,2005) is making available molecular markersbased on expressed sequences that could facilitatethe direct establishment of probable relationshipsbetween candidate genes and specific QTLsthrough the development of a transcriptionalmap, opening the interesting opportunity to testthese markers in association mapping experiments.To date, the molecular data generated couldnot be yet effectively used into the breedingprograms. Progress on mapping experiments basedon genetically more informative markers with abroad and regular genome coverage will supportto look for useful homologies of QTL regionsacross independent crosses also helping to betterelucidate aspects of the architecture of theimportant QTLs and also contributing to increasethe map information mainly for traits that do notsegregate within a single population (Brondaniet al., 2006). Furthermore, informative markersassociated to QTLs controlling traits of interestwill allow to explore, in an efficient and effectiveway, the allelic variation at these QTLs openingnew perspectives for expanding the gene pool ofcultivated common beans and identify potentiallyand useful source of genetic diversity to beintroduced into the P. vulgaris cultigen.

As expected by breeders, the use of these markerswill help particularly in the selection process toidentify lines with desirable phenotypes, whichare expressed in most of the environments, thuscontributing to the increase in the efficiency of thecommon bean breeding programs.

1.3.3 Successes in common bean breeding

Common bean production takes place with severalproblems of biotic and abiotic origins. Commonbiotic problems are diseases caused by fungi,

bacteria, and virus, and damage from insect andnematode attack. The most common abiotic prob-lems are low soil fertility, especially deficiencies innitrogen, phosphorus and zinc, and toxicity fromaluminum and manganese. Drought is also oneof the most generalized abiotic problems affectingcommon bean yield in all producing regions ofthe world, especially in the Brazilian northeast,the central and northern Mexican highlands, EastAfrica, and the intermountain regions of theUnited States, where it causes production losses(Singh, 2001). Breeding programs have worked toincrease tolerance to biotic and abiotic stresses toindirectly increase common bean yield potentialand reduce sensitivity to environmental adversity.However, there is another line of work to directlyincrease the common bean productive capacityby increasing its productive potential in optimummanagement conditions, that is, under conditionsof low environmental stress.

Yield increase in most crops can be attributedto the genetic gains obtained through improvedcultivars, to better cropping technologies andagricultural practices, and also to the improvementof cropping environments. It is a combinationof these factors that facilitate maximum yieldper unit of area. Frequently, improved cultivarshave been the main factor for yield increaseand have provided the stimulus to adopt betteragronomic and agrichemical practices, leading toan additional increase in yield (Singh, 1992).

1.3.4 Limitations of conventional breedingand potential of genetic engineering

Some of the challenges of common bean breedingare more difficult to overcome due to restrictionsimposed by limited genetic variability withinthe species and also due to difficulties in traitassessment. One typical example of limitationis the low genetic variability for resistance tothe common BGMV in Brazil, for sources ofresistance is almost nonexistent. The BGMV andBGYMV, which occur in the main common beanproducing areas in Latin America, are transmittedby the whitefly Bemisia tabaci Gen., and havebeen especially important in the dry growingseason (Morales and Anderson, 2001; Morales,2006). Annual crop reductions in the range of90 000–280 000 tons, amount sufficient to feed


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6000–20 000 adults, are estimated. In addition tothe economic problems caused by yield reduction,this virus also brings other social consequences,since it precludes common bean cropping infamily based agricultural systems. Approximately180 000 ha are currently not apt for common beancropping in the dry season in Brazil, due to theoccurrence of BGMV. These areas can be returnedto the productive process after cultivars withadequate resistance level are available. Althoughthere have been advances in scientific knowledge,technological results with direct application forgrowers have not been achieved, suggesting thatfurther research and adjustments of proceduresare required in several areas. Immunity reactionto both the viruses has not yet been found inPhaseolus spp. accessions evaluated by nationaland international research institutions. Toleranceto the disease is controlled by polygenes and,therefore, difficult to be efficiently transferredto commercial cultivars (Pessoni et al., 1997;Park et al., 2001). However, literature reports onthe genetic control of tolerance to BGMV aresometimes conflicting, with some authors alsosuggesting monogenic (Blair and Beaver, 1993) oroligogenic control (Blair et al., 1993). There arealso reports of cultivars that usually show certaindegree of tolerance to the disease, but becomesusceptible and suffer severe yield losses whensubmitted to high infestations of the insect vector.This fact is aggravated by the failure of naturalcontrol measures and by the high cost of chemicalcontrol.

Regarding the implementation of marker-assisted selection (MAS) to supplement thecommon bean breeding programs for diseaseresistance, in the last decade, with the advantageand accessibility of the molecular marker tech-nology, a number of markers for traits relatedto the major disease resistance with significantimpact in the common bean agriculture have beensuccessfully identified, as extensively reviewed byMiklas and Singh (2007). A list of markerslinked to specific genes conditioning resistanceto several pathogens causing bean diseases hasbeen released for CBB, anthracnose, angular leafspot, BGYMV, and bean rust (Kelly et al., 2003).Markers linked to these major genes can be usedto trace the presence of target genes throughMAS procedures, where the genotype carryingthe target allele is, by indirect way, selected for

the desirable phenotypes. Once important genesare tagged with a marker, selection in successivebackcross generations are performed in order toselect individuals in the progeny that possess amarker allele from the donor parent, improvingthe efficiency and facilitating introgression of theresistance genes into elite common bean genotypes(Oliveira et al., 2002). However, due to the constantappearance of new races of a pathogen, there is anecessity to constantly incorporate new resistancegenes into bean lines and cultivars. This problemhas been partially overcome by the pyramidingof multiple genes for prevalent pathogen racesinto selected cultivars with MAS strategy, inorder to achieve durable resistance. Lines resistantto angular leaf spot carrying resistance genesderived from selected cultivars were developedby Oliveira et al. (2005). In addition, advancedMesoamerican bean lines with “Carioca-type”grains (Ragagnin et al., 2005) and black andred bean cultivars (Costa et al., 2006) have beendeveloped with multiple disease resistance genesalso through marker-assisted strategies. However,although significant progress has been made, somelimitations for the extensive use of MAS asroutine in the common bean breeding programstill remain. As early pointed out by Kelly andMiklas (1998), the main limitations of success ofMAS are: (1) the genomic sequence related to themarker is not sufficiently close to the resistant geneallowing that it segregates independently duringthe recombination; (2) most of the markers tightlylinked to the target genes are not universal inall genetic backgrounds, making their utilizationrestricted to the population where the geneswere mapped; (3) new races of the pathogensevolve constantly in different environments and,in addition, the pathogens rapidly overcomethe resistance of the gene identified; (4) newcultivars with advantageous agronomic attributesare currently being released and the MAS processshould be individually performed for each of theaccession, taking time for the introduction of thedesirable allele.

Drought tolerance in common bean, as in otherscrops, has dramatic consequences on production,mainly in developing countries, reducing the grainyield in more than 1.5 million ha of cultivatedarea in the world (Teran and Singh, 2002).Drought tolerance is considered as an example of atrait difficult, expensive, and inaccurate to assess,



because of the complexity of the environmentalcontrol. Water shortage is the climatic factor thatcontributes the most to harvest frustrations. Lowcrop yield due to water shortage in greater orlesser intensity is frequently reported in most ofthe common bean cropping regions. Flowering isthe most vulnerable stage, followed by the grainfilling period. In Brazil, common bean grown fromJanuary to April is highly constrained by watershortage, which leads to low and unstable yield.Considering that 44% of the Brazilian productionis in this period, it is recommended that newcultivars adapted for sowing from January to Aprilshould be developed. As a part of the GenerationChallenge Program initiative, phenotyping sitesare being structured in Brazil in order tocharacterize genetic materials at the phenotypiclevel, testing for adaptability for drought toleranceand the effects on the productivity. The availabilityof these sites will allow several studies on droughtphenotyping in common bean.

Regarding the potential of molecular markers toimprove drought tolerance in common bean, thereis available today a large set of drought toleranceQTLs and candidate genes previously identifiedfor model crops, such as rice (Vinod et al., 2006).These can be used as a starting point to search forhomologous genes in other species, like P. vulgaris.Once candidate genes are identified, one of theways to evaluate their association with the traitof interest is using the approach of associationmapping studies, where the informative markerspotentially identified as associated to droughttolerance traits could be used in MAS. Projectsare being conducted in maize, rice, and beans, alsoas a part of the Generation Challenge Programinitiative. All strategies discussed until now arerelated to common bean genetic variability todrought tolerance. However, other genes, alleles,promoters, etc., related to drought tolerance,are constantly being identified in other species,however, crossing barriers prevent their directtransfer by hybridization. Therefore, geneticengineering could be a powerful tool to obtaincommon bean cultivars more tolerant to drought.

2. BEAN GENETIC ENGINEERING

Several methods have been developed for insertinggenetic information into plant cells, such as

Agrobacterium-mediated system, direct DNA up-take into protoplasts and particle bombardment.Due to the advances in the methodologies for genedelivery and plant tissue culture, it was assumedthat plant transformation would become a routinefor most important crops. Unfortunately, legumes,in particular common bean (P. vulgaris), becameone of the greatest challenges to transformationefforts. Indeed, the ability to genetically engineercommon bean is still not trivial (Aragao and Rech,2001; Svetleva et al., 2003; Popelka et al., 2004;Veltcheva et al., 2005).

Early efforts to transform common beansdemonstrated its susceptibility to Agrobacterium,and some transgenic tissues or organs, such ascalli, leaves, meristems, cotyledon, and hypocotylhave been achieved (Lippincott et al., 1968;McClean et al., 1991; Franklin et al., 1993; Beckeret al., 1994; Lewis and Bliss, 1994; Brasileiroet al., 1996; Nagl et al., 1997; Karakaya andOzcan, 2001). Mariotti et al. (1989) reportedproduction of transgenic bean plants throughutilization of the Agrobacterium system. However,there was no molecular evidence for genetictransformation or progeny analysis. Transientgene expression using either electroporation orpolyethylene glycol (PEG)-mediated protoplasttransformation was demonstrated (Crepy et al.,1986; Bustos, 1991; Leon et al., 1991; Giovinazzoet al., 1993). Dillen et al. (1995) demonstratedthe applicability of electroporation of intacttissue to introduce and express the gus gene inbean embryonic axes. A gene transfer systemhas been recently developed by Liu et al.(2005) using sonication and vacuum infiltration-assisted Agrobacterium-mediated transformationfor Kidney beans, without any tissue culture step.

During the last two decades, efforts to achievean efficient methodology for bean transformationwere obstructed due to the lack of an efficienttissue culture system to regenerate bean plantsfrom transformed cells. Numerous attempts havebeen made to regenerate bean plants from severaltypes of isolated cells and tissues. Although nosatisfactory results have been achieved, somemethodologies have described shoot organogene-sis (through multiple shoot induction) of the apicaland axillary meristems from bean embryonic axis(McClean and Grafton, 1989; Malik and Saxena,1992; Mohamed et al., 1992, 1993). Cruz deCarvalho et al. (2000) employed the transverse thin


COMMON BEAN 11

cell layer (tTCL) method to optimize the frequencyof shoot regeneration without an intermediatecallus stage. The same authors also showed thataddition of 10 µM silver nitrate (AgNO3) to themedium with benzylaminopurine (BAP) enhancedthe number of shoots that developed per explantand increased shoot elongation.

2.1 Transformation Methods

The particle bombardment method was initiallyproposed by the group of John Sanford (CornellUniversity) with the objective to introduce geneticmaterial into plant genome (Klein et al., 1987;Sanford et al., 1987). Since 1980s, the universalityof application of particle bombardment hasbeen evaluated, demonstrating to be an effectiveand simple process for the introduction andexpression of genes in to bacteria, protozoa,fungi, algae, insects, mammals, plants, and isolatedorganelles, such as chloroplasts and mitochondria(Daniell et al., 1991; Carrer et al., 1993; Kleinand Fitzpatrick-McElligott, 1993; Sanford et al.,1993; Vainstein et al., 1994; Bogo et al., 1996;Rech et al., 1996). The basis of the particlebombardment method is the acceleration of DNA-coated microprojectiles (particles of tungsten orgold) at high speed (about 1500 km h–1) towardliving cells. After penetration in the cell, the DNAdissociates from the projectiles and integrates intothe chromosomes. Several devices (particle guns)have been constructed in order to accelerate thesemicroprojectiles. All these systems are based on thegeneration of a shock wave with enough energy tomove the microprojectiles. The shock wave can begenerated through: a chemical explosion (Sanfordet al., 1987), discharge of gas helium (Sanfordet al., 1991; Finer et al., 1992; Takeuchi et al.,1992), vaporization of a drop of water throughthe electric discharge with high voltage and lowcapacitance (McCabe et al., 1988) or low voltageand high capacitance (Rech et al., 1991), dischargeof compressed air (Morikawa et al., 1989).

Particle bombardment allowed the bombard-ment of intact plant cells facilitating possibletransformation of plants for which a regenerationsystem was not available. The status of Phaseolustissue culture was well reviewed by Nagl et al.(1997), Svetleva et al. (2003), Popelka et al. (2004)and Veltcheva et al. (2005).

Although several authors have claimed theachievement of P. vulgaris regeneration, sofar, only cytokinin-induced shoot organogenesiswas obtained. Multiple shoots are formed inthe peripheral regions of the apical meristem(Aragao and Rech, 1997). Induction of shootformation in bean meristems can be achievedby culturing the mature embryos in the presenceof cytokinins such as kinetin, zeatin, and BAP(Kartha et al., 1981; Martins and Sondahl,1984; McClean and Grafton, 1989; Franklinet al., 1991; Malik and Saxena, 1992; Mohamedet al., 1992, 1993; Aragao and Rech, 1997;Delgado-Sanchez et al., 2006). Compounds suchas thidiazuron (TDZ) and N-(2-chloro-pyridyl)-N′-phenylurea (CPPU) that possess cytokininlikeeffects have also been tried (Mohamed et al.,1992).

Consequently, the apical region of embryonicaxes became an obvious target for the developmentof a system based on the bombardment ofmeristematic cells. Early experiments showedthe applicability of particle bombardment forintroduction and transient expression of genesin apical meristematic cells (Genga et al., 1991;Aragao et al., 1992, 1993; Russel et al., 1993).However, how deep the particle could penetratein order to reach the cells that could generatetransgenic plants has yet to be determined. Thedifferent parts of meristem have been dividedinto layers (L1, L2, and L3). The layer L1 isthe most external and forms the epidermis ofthe differentiated regions. The layers L2 and L3divide preferentially in the anticlinal and periclinalplanes to form the organs. Several studies havedemonstrated that the differentiated de novo shootsare originated from the subepidermal layers (L2and L3) of the apical meristem. However, theL1 layer could participate in their formation(McClean and Grafton, 1989; Franklin et al., 1991,Malik and Saxena, 1992; Mohamed et al., 1992).The shoots are formed in the peripheral regionsof the apical meristem (Aragao and Rech, 1997).The bombardment of P. vulgaris meristematiccells showed that it was possible to efficientlyreach these layers, demonstrating that it would bepossible to achieve transgenic plants (Aragao et al.,1993; Figure 1).

Russel et al. (1993) were able to achievetransgenic navy bean (cv. Seafarer) plants usingan electrical particle acceleration device. It was



(a)

(e)

(f)(d)

(b)

(c)

Figure 1 Bean transformation via microparticle bombardment. (a) Embryonic axes expressing the gus gene 24 h after bombardment.(b–c) Scanning electron micrograph showing the morphology of the shoot apical meristem regions of two varieties presenting theapical dome exposed (b) and partially covered by primordia leaves (c). (d) Transmission electron micrograph showing tungstenmicroparticles (arrows) penetrating up to the third cell layer from the meristematic apical region. (e) Transgenic shoot developingfrom a multiple shooting apical meristem. (f) Leaves from transgenic plantlets (right) and control (left). Bars represent 100 µm in band c, and 2 µm in d

the first report of P. vulgaris transformation, pre-senting molecular evidences of transgenic progeny.However, the frequency of transgenic plantsobtained was much lower (0.03%) and varietylimited. In addition, the tissue culture protocoldescribed was time consuming, involving several

temperature treatments and medium transfersof the bombarded embryos before recovery oftransgenic shoots. Kim and Minamikawa (1996)achieved transformation bombarding embryonicaxes, obtaining stable transformed bean plants(cv. Goldstar).


COMMON BEAN 13

(a) (b)

(c) (d)

Figure 2 Occurrence of chimerism during the process of obtaining transgenic shoots from particle-bombarded apical meristematicregion (a). Chimerical transgenic branch (b), leaf (c), and root (d) presenting gus gene expression

A reproducible system to achieve transgenicbean plants routinely was developed. The beantransformation system was also based upon thedevelopment of a tissue culture protocol ofmultiple shoot induction, shoot elongation androoting (Figure 1). The average frequency oftransformation (the total number of putativetransgenic plants divided by the total number ofbombarded embryonic axes) was 0.9% (Aragaoet al., 1996; Aragao and Rech, 1997). In addition,they have been able to transform several varietiesof P. vulgaris, including those that were consideredrecalcitrant to transformation in previous studies.Molecular analysis and progeny test of severalgenerations of transgenic lines revealed thepresence of a small number of integrated copies ofthe foreign genes and segregation in a Mendelianfashion in most of them. This is extremelyimportant in order to accelerate the introductionof these plants in a breeding program as well as theproduction of transgenic commercial varieties.

The shoots regenerated from the bombardedmeristematic apical region may include all, some,

or none of the cells that received the transgenes.Consequently, undesirable periclinal, mericlinal,or sectorial chimeras can be produced in theadventitious shoots (Figure 2). Chimeric organshave been observed in transgenic common beanplants, such as leaves and roots (Figure 2). Thislimitation could probably be removed by using amore appropriated selection agent. It is difficultto have an efficient selection for transformedcells because only a few cells from the meristemare transformed and generally cannot be reachedby the selective agents. Although antibiotics andherbicides, such as kanamicin and glufosinateammonium (GA) have been used (Aragao et al.,1996, 2002), this selection is not efficient.Recently, this group has developed a novelsystem for selecting transformed meristematiccells based on the use of imazapyr, a herbicidalmolecule capable of systemically translocating andconcentrating in the apical meristematic regionof the plants. This selectable marker coupledwith an improved multiple particle bombardmentprotocol, resulted in a significant increase in the



recovery of fertile, transgenic material comparedwith standard soybean transformation protocols(Aragao et al., 2000). This selection system isbeing successfully used to introduce genes intodry bean and chimerical plants have not beenobserved.

Genetically modified common bean havealso been obtained by using linearized vectors(Vianna et al., 2004; Bonfim et al., 2007).Although the frequency of transformation is lower(0.2–0.6%), the use of linear vectors is impor-tant to eliminate undesirable antibiotic selectivegenes.

Particle bombardment has been considered auniversal method to introduce macromoleculesinto any living cell, not limited by genotype orvariety (Sanford, 1990). However, the morphologyof the explants utilized during bombardmentmay greatly influence the successful recuperationachievement of transgenic bean plants (Aragaoand Rech, 1997). In some cultivars, the embryonicaxes revealed the apical meristematic regionpartially exposed, whereas only the central regioncould be visualized (Figure 1). The number ofmeristematic cells, which could be reached bythe microparticle coated-DNA, will be drasticallyreduced. Consequently, the efficiency of trans-formation could also be reduced. Several studieshave shown that de novo shoot differentiation inembryos of bean grown on cytokinins appearedin the peripheral layers of the meristematic ring(McClean and Grafton, 1989; Franklin et al.,1991; Malik and Saxena, 1992; Aragao and Rech,1997). Thus, based on these concepts, cultivarswith a nonexposed apical meristematic regionare not suitable for transformation using particlebombardment, considering that removal of the leafprimordia is not practical.

Although the common bean is susceptible toAgrobacterium, only recently stable transforma-tion mediated by Agrobacterium was demon-strated. Liu et al. (2005) reported the developmentof an Agrobacterium-mediated transformation sys-tem based on sonication and vacuum infiltrationof Agrobacterium tumefaciens (LBA4404) intogerminated kidney bean embryos (var. GreenLight). Inoculated germinating seeds of kidneybean were sown in soil pots and from a total of525 surviving plants, 16 were transformed (Liuet al., 2005). The transgenes were detected up tothe second generation.

2.2 Introduction of Useful Traits

The methionine-rich 2S albumin gene from Brazilnut (be2s1 gene) was the first agronomicallyimportant gene expressed in common bean tissues(Aragao et al., 1992). Mature embryos weretransformed in order to have transient expressionof a methionine-rich albumin gene from Brazilnut (be2s1 gene), which could be detected byWestern blot and enzyme-linked immunosorbentassay (ELISA), 24 h later (Aragao et al., 1992).Further, stable genetically engineered commonbean lines containing the be2s1 gene were obtainedaiming to improve the methionine content in theseeds. The transgene was stable and correctlyexpressed in homozygous R2–R6 generations. Intwo of the five transgenic lines, the methioninecontent was significantly increased by 14% and23% over the nontransgenic plants (Aragao et al.,1999). However, 2S albumin from Brazil nutwas identified as an allergen (Nordlee et al.,1996; Koppelman et al., 2005). Indeed, transgenicsoybean containing the Brazil nut 2S albuminwas allergenic to patients. Consequently, thedevelopment of a transgenic common bean varietywith improved methionine content was aborted.

Russel et al. (1993) introduced the bargene that confers resistance to the herbicidephosphinothricin and the coat protein gene frombean BGMV in an attempt to achieve virus-resistant plants. The introduced bar gene showedto confer strong resistance in transgenic beanplants to the herbicide in the greenhouse. However,bean plants containing the BGMV coat proteingene did not show resistance to the virus (D.Maxwell, personal communication).

The 5′ regulatory sequences from severalgenes have been transient and stably studiedin bean tissues transformed using the particlebombardment. Regulatory regions from the seedsspecific promoter from Brazil nut 2S gene werestudied in bean embryos (Grossi de Sa et al.,1994; Vincentz et al., 1997). Transgenic bean plantswere produced containing the β-glucuronidasegene (gus) under the control of the canavalingene promoter (Con A) from jack bean. Eitherthe organ and maturation stage-specific promoterregulation was studied in seeds of transgenic plants(Kim and Minamikawa, 1996, 1997).

Transgenic bean lines containing the bargene, which encodes phosphinothricin acetyl


COMMON BEAN 15

(a)

(b)

(c) (d)

Figure 3 Field trials with genetically modified common bean plants. (a) Transgenic lines (t) tolerant to 400 g ha l−1 of glufosinateammonium compared to a nontransgenic line (nt) treated with the herbicide. (b) Transgenic plants resistant to the BGMV (left). (c)Lines resistant to BGMV being tested in the field. (d) Effect of virus infection on pods of nontransgenic plants (right) and transgenic(left) plants, both inoculated

transferase, and resistant plants to the herbicideGA were generated (Russel et al., 1993; Aragaoet al., 2002) and tested in field (Aragao et al., 2002;Figure 3). Two transgenic events were tolerant to500 g ha l−1 of GA, under green house conditions,with no visible symptoms and developmentalgrowth. Field evaluation has shown that the plantstolerated up to 400 g ha l−1 of GA (Aragao et al.,2002; Figure 3).

In order to obtain common bean plants resistantto BGMV, the genes Rep-TrAP-REn and BC1from the virus were cloned in antisense orientationunder the control of the CaMV 35S promoterand used to transform bean. Two transgenic lineswere obtained both of which had delayed andattenuated viral symptoms (Aragao et al., 1998).Using the strategy of transdominace, transgenicbean lines were obtained with a vector contained

the mutated rep (AC1) gene from the BGMV.The mutated rep gene codes for a mutated AC1(REP) protein with amino acid codon change inthe putative NTP-binding motif (D262R). Oneline exhibited resistance to the virus. However, theresistance was studied during several generationsand depended on the inoculation level (Faria et al.,2006). More recently, the concept of using RNAinterference (RNAi) construct to silence the AC1viral gene has been explored to generate highlyresistant transgenic common bean plants (Bonfimet al., 2007). Eighteen transgenic common beanlines were obtained with an intron-hairpin con-struction to induce post-transcriptional gene si-lencing against the AC1 gene and one line exhibitedhigh resistance (approximately 93% of the plantswere free of symptoms) upon inoculation at highpressure (more than 300 viruliferous whiteflies per



plant during the whole plant life cycle) and at avery early stage of plant development.

Transgenic kidney bean plants were obtainedexpressing a group 3 LEA (late embryogenesisabundant) protein gene (ME-leaN4) from Brassicanapus (Liu et al., 2005). Plants showed enhancedgrowth ability under salt and water stress. Theincreased tolerance was also reflected by delayeddevelopment of damage symptoms caused bydrought stress. In addition, transgenic lines thatpresented high level of ME-leaN4 gene expressionshowed higher stress tolerance than lines withlower expression level (Liu et al., 2005).

2.3 Field Evaluation

Studies on the behavior of transgenic commonbean plants under field conditions have beenconducted. The first field trail was carried outfrom November 2000 to February 2001 in DistritoFederal, Brazil to evaluate the resistance of theT2 generation of an event tolerant to glufosinateammonium (Aragao et al., 2002; Figure 3).Since then, several transgenic lines resistant tothe BGMV have been evaluated in the field atEmbrapa Rice and Bean (Goias, Brazil) (Figure 3).In these studies, it is appraised the interactions ofthe transgenic plants with microorganisms, insects,and others plants from of the agricultural andnatural environment. Moreover, the stability offoreign genes expression, gene flow, and factorsrelated to their interaction with the complexphysiology of these plants exposed to naturalstress in tropical are being evaluated. Furthermore,food biosafety analysis is being carried out by theBiosafety Network from Embrapa to determinedifferences in nutritional and antinutritionalcompounds as well as verify presence of toxicmolecules.

3. FUTURE PROSPECTS

Several species of the genera Phaseolus, suchas P. coccineus, P. accutifolius, and P. angularis(Nagl et al., 1997), have been regenerated in thestrictu sensu, e.g., plants regenerated de novo fromundifferentiated cells originated from differenti-ated cells. The extrapolation of these technologiesto P. vulgaris would be a breakthrough toward

the development of a transformation systemto accelerate the generation of new transgeniccommercial varieties. The ideal system seemsto be the regeneration of fertile mature plantsthrough somatic embryogenesis or organogenesis,preferentially from mature embryonic axes, whichhas been considered the most adequate tissuefor transformation either by Agrobacterium,electroporation and particle bombardment. Theimprovement in the bean transformation technol-ogy is essential for obtaining a larger number oftransgenic lines, increasing the probability to selecttransformation events presenting the desirablephenotype. Depending on the introduced trait,only one of 30–40 independent transgenic plantsobtained has the desirable transgene expressionwithout undesirable characteristics, such as mul-tiple transgene loci and nontransference of theforeign genes to the first generation.

Since the first report of transformation of P.vulgaris in 1993, a few groups have transformedbean to introduce useful traits. Difficulties that stillexist in obtaining transgenic plants might accountfor this fact. Nevertheless, during the last twodecades, efforts to achieve efficient methodologiesfor regeneration and transformation of commonbean plants advanced. In parallel, the availabilityof characterized genes (coding and regulatorysequences) has increased as a direct result ofseveral structural and functional genomics projectsin plants.

An increased understanding of common beangenome associated with contributions frombiotechnology will provide an opportunity forbreeders to accelerate the development of newvarieties with valuable agricultural traits. Commonbean has a relatively small genome of about630 Mb with 11 haploid chromosomes andgenomics studies are in progress because of itssocial and economic value (Vallejos et al., 1992;Blair et al., 2003; Broughton et al., 2003; Nodariet al., 1993b; Pedrosa et al., 2003). The Phaseomicsproject (http://www.phaseolus.net) was started in2001 by an international consortium of laborato-ries with an aim of developing high yielding beanvarieties that have high protein quality and arestress and disease resistant. They plan to initiallysequence large scale ESTs from different tissues ofP. vulgaris, which will be followed by sequencing,and analysis of the bean genome. At thismoment, about 47 300 nucleotide sequences from


COMMON BEAN 17

genus Phaseolus are released (about 26 200 fromP. vulgaris) and these numbers are increasingrapidly. These facts generated an excellent scenariofor introduction of useful traits as well as studygene function in P. vulgaris plants. There is aconsiderable interest in the introduction of genesfor several useful traits in common bean, suchas virus, insect, bacteria, and fungi resistance,environmental stress tolerance, and improve nu-tritional properties. In addition, manipulation ofplant architecture and phenological characteristicsmight facilitate management, increasing yield,quality, and diversity. Several traits might bemanipulated by genetic engineering, such as plantlife cycle and the transition from vegetative toreproductive growth, controlling flowering time.Indeed, several genes controlling phenotypicaltraits have already been cloned and characterizedin distinct organisms. This information can beused to either express heterologous genes orsuppress the expression of homologous genesin common bean plants. Biotechnological toolscomplement those from classical breeding andhave the potential to accelerate the generation ofnew varieties containing genes from agronomictraits, which are difficult to be found in the primaryor secondary gene pool.

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Compendium of Transgenic Crop Plants || Common Bean