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Graduate Theses and Dissertations Graduate College

2013

Enhancing biological nitrogen fixation in common bean (Phaseolus vulgaris L)
MERCY KASUZI KABAHUMA
Iowa State University, kabahuma@iastate.edu

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KABAHUMA, MERCY KASUZI, "Enhancing biological nitrogen fixation in common bean (Phaseolus vulgaris L)" (2013). Graduate Theses and Dissertations. Paper 13162.

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ENHANCING biological nitrogen fixation in Common bean (Phaseolus vulgaris L.) by Mercy Kasuzi Kabahuma

A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE

Major: Crop Production and Physiology Program of Study Committee: Mark E. Westgate, Major Professor Gwyn Beattie Kathleen Delate

Iowa State University Ames, Iowa 2013 Copyright © Mercy Kasuzi Kabahuma, 2013.

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To my family and friends

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TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ABSTRACT CHAPTER 1. GENERAL INTRODUCTION Hypotheses Expected Outcomes References CHAPTER 2. GENOTYPIC VARIATION IN UREIDE CONTENT IN PHASEOLUS VULGARIS L. Abstract Introduction Materials and Methods Results and Discussion Conclusions References v vi viii 1 7 8 9

19 19 20 21 23 28 29

CHAPTER 3. SHOOT AND ROOT CONTROL OF UREIDE ACCUMULATION AND PARTITIONING IN PHASEOLUS VULGARIS L. GENOTYPES 46 Abstract Introduction Materials and Methods Results and Discussion Conclusions References CHAPTER 4. GENERAL DISCUSSION 46 47 48 50 56 58 72

iv ACKNOWLEDGEMENTS 73 .

v LIST OF FIGURES Chapter 2 Figure 1. Puebla. Total N accumulation in tissues of R32. Figure 2. Biomass of tissues collected from R32. Puebla. Number of nodules on R32. and Eagle bean lines during flowering/early pod set. Total N accumulation in tissues of R32. and Eagle bean lines during flowering/early pod set. R99. Ureide content in tissues of R32. R99. Puebla and Eagle controls and grafts harvested during flowering/early pod set. Figure 5. N concentration in tissues of R32. Puebla and Eagle controls and grafted bean lines during flowering/early pod set. and Eagle bean lines collected during flowering/early pod set. Puebla. Chapter 3 Figure 1. Figure 6. Figure 4. Ureide content of tissues of R32. R99. Figure 3. R99. R99. Figure 2. Figure 3. Puebla and Eagle controls and grafts harvested during flowering /early pod set. Ureide concentration in plant tissues of R32. R99. R99. Puebla. Puebla. Biomass of tissues collected from R32. R99. 61 33 34 35 36 37 38 62 63 . R99. Puebla. and Eagle bean lines during flowering /early pod set. and Eagle bean lines harvested during flowering/early pod set. and Eagle roots of greenhouse-grown plants sampled at flowering.

Ureide content of tissues of R32.and intra-species graft of R32. N concentration in tissues of R32. Puebla. Shoot: Root ratios of controls. Biomass of tissues from R32. Table 4. Table 3. and Eagle bean lines collected during flowering/early pod set.and intra-species graft of R32. and Eagle bean lines collected during flowering/early pod set. Percentage plant ureide and percentage N derived from N2 fixation. self. Puebla. R99. Ureide concentration in controls. Table 6. and Eagle bean lines collected during flowering/early pod set. Table 2. Chapter 3 Table 1. Table 3. and Eagle bean lines collected during flowering/early pod set. Puebla. R99. Table 4.and intra-species graft tissues of R32. R99. and Eagle bean lines collected during flowering/early pod set. R99. self. Nodule effectiveness. 64 39 40 41 42 43 44 45 65 66 67 . Puebla. R99. R99. Total N accumulation in tissues of R32. Ureide concentration in tissues of R32. self. and Eagle bean lines collected during flowering/early pod set. R99. R99. Table 2. Table 5.vi LIST OF TABLES Chapter 2 Table 1. and Eagle bean lines collected during flowering/early pod set. and Eagle bean lines collected during flowering/early pod set.and intra-species graft tissues from R32. Table 7. Puebla. Puebla. Puebla. and Eagle roots sampled at flowering. self. Number of nodules on controls. Puebla. Puebla. Biomass of controls. R99.

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Table 5. Ureide content of controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Table 6. N concentration in controls, self- and intra-specie graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Table 7. Total N accumulation in controls, self- and intra-specie graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Table 8. Percent plant N derived from N2 fixation.

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ABSTRACT Common bean (Phaseolus vulgaris L.) is a herbaceous annual which, in a symbiotic relationship with specific soil bacteria, ‘fixes’ atmospheric nitrogen (N2) into amino form that can be used for plant growth. Efforts to optimize biological nitrogen fixation (BNF) in common beans are critical because of widespread increase in soil degradation in Africa. Among legumes, common beans derive the least percent N2 from N2 fixation. This has been attributed partly to susceptibility of common beans to physical and chemical environmental stresses, inconsistent response to inoculum, and lack of selection for the BNF trait. Improvement in productivity of this leguminous crop could be achieved through identification of genotypes with greatest capacity for BNF and nitrogen assimilation from BNF. Chapter 2 presents phenotypic traits that could possibly be associated with BNF and N assimilation. Bean lines varying in ability to form nodules and fix nitrogen were analyzed for root, stem, leaf, petiole and pod biomass, ureide concentration, nitrogen concentration, and nodule numbers. There was significant variation in ureide accumulation across plant tissues and genotypes. A combination of phenotypic traits, however, could be used to select for improved BNF. Moderate nodule number, leaf ureide content, and total biomass at flowering were consistent with greater BNF. Nodule effectiveness should be considered for increasing % N derived from N2 fixation. In Chapter 3 a grafting technique was used to determine shoot and/ or root control of ureide accumulation and partitioning among four genotypes noted for variation in phenotypic traits related to nitrogen fixation. The extent of nodulation, as modified by super-nodulating scions or non-nodulating rootstocks, only indirectly affected ureide and N accumulation. Plants with a greater number of nodules did not accumulate more nitrogen, indicating most nodules were not effective in fixing N. The results indicate shoot regulation of nodulation, ureide metabolism, and nodule effectiveness would be ideal physiological targets for further investigations aimed at improving BNF and yield.

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CHAPTER 1. GENERAL INTRODUCTION
Legumes, such as common beans (Phaseolus vulgaris L.) have the ability to form a symbiotic relationship with soil bacteria capable of trapping nitrogen gas (N2) from the atmosphere and converting it into ammonia, which can be used by the plant for growth, development and seed production. Atmospheric nitrogen is converted to ammonia by the nitrogenase enzyme in a process known as biological nitrogen fixation (BNF) (Postgate, 1998; Bhatia et al., 2001). The capacity of legumes to fix atmospheric nitrogen gives them an advantage over non-leguminous crops when grown on soils low in nitrogen (N). As such, they are an integral part of most small-landholder cropping systems (Bhatia et al., 2001). The need to improve productivity of legumes as a global source of dietary protein, however, has made it vital to understand the factors that influence nitrogen fixation (Schulze, 2004). BNF is a symbiotic relationship between specific nitrogen-fixing bacteria (rhizobia) and legume plants (Caetano-Anolles and Gresshoff, 1991). Upon perception of flavonoids exuded by legume roots, rhizobia activate nod genes which induce production of Nod factors (Burdman et al., 1997). Nod factors are calcium dependent proteins that promote attachment of the rhizobia to root hair surfaces (Smit et al., 1987). Subsequent curling of the root hairs encloses the attached rhizobia (Heidstra et al., 1994), which induce hydrolysis of the root hair cell wall. Invagination of the plasma membrane by the plant forms an infection thread within which rhizobia multiply and penetrate host cells (Ridge et al., 1985). Once inside the parenchyma cells of the root, rhizobia and infected cells proliferate forming a nodule initial. Whether the nodule initial develops into a functional nodule is determined by the plant and a host of environmental factors. Within the successful nodule, rhizobia are surrounded by a peribacteroid membrane where they differentiate into bacteroids and begin fixing atmospheric nitrogen (Hedtke and Newcomb, 1977). In many regions of the world where common beans are grown, nitrogen fixation is limited by unfavorable soil conditions and temperature and water stress. Temperature has been reported to affect nodulation, survival and persistence of rhizobial strains in soil. Depending on their natural habitat, tolerance of rhizobia to temperature varies across various strains

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(Mohammadi et al., 2012). Additionally, temperatures greater than 40 C inhibit infection of root hairs by rhizobia (Hungaria and Franco, 1993) and nitrogen fixation (Long, 2001). Moreover, nitrogenase activity was lower when common beans formed nodules at 35 C (Piha and Munns, 1987). In their study, Michiels et al. (1994) attributed ineffectiveness of rhizobium symbiosis at temperatures greater than 40 C to deletion of the symbiotic plasmids. Low temperatures also limit nitrogen assimilation (Gibson, 1965), photosynthate assimilation, malate production, and nitrogenase activity (Bordeleau and Prevost, 1994). Moisture stress has greater impact on nodulation and nodule activity than on plant root and shoot metabolism (Albrecht et al., 1984). Rhizobial strains vary in their adaptability to water stress. Busse and Bottomley (1989) demonstrated growth rate of Rhizobium meliloti slowed and cell morphology became irregular as water potential of the growth media decreased from −0.15 and −1.5 MPa. Low soil water potentials inhibit nodulation and growth of rhizobia. For example, nodulation in subterrainian clover (Trifolium subterraneum) was inhibited at soil water potential below -0.8MPa; growth of Rhizobium leguminosarum bv. Trifolii was impaired at water potentials below -1.0MPa (Leung and Bottomley, 1994). Ramos et al. (1999) reported lower leghemoglobin (Lb) levels in nodules, and loss of nodule nitrogenase and sucrose synthase activities due to water stress. Indirect effects of reduced root growth in dry soils decreases the number of potential infection sites for rhizobia (Bordeleau and Prevost, 1994). Also, accumulation of ureides in leaf and nodule tissues brought about by water stress has been associated with inhibition of nitrogen fixation (Sinclair and Serraj, 1995). Zahran (1999) reported the toxic effect of soil salinity on plant growth especially in arid and semi-arid regions. Saline soils in arid areas are dominated by Na+ and Cl- . Na+ displaces Ca2+ from root membranes of non-halophytes altering their functionality. Legumes and rhizobia can be extremely sensitive to low soil pH (Corea and Barneix, 1997). The optimum pH range for rhizobial growth is between 6.0 and 7.0 below which very few rhizobia are capable of growing (Brockwell et al., 1991; Graham et al., 1994). Very few species survive in soils at pH below 5.0 (Mohammadi et al., 2012). McKay and Djordjevic (1993) demonstrated a reduction in production and excretion of nodulation factors in R. leguminosarum bv. Trifolii at pH below 5.0. Furthermore, Zahran (1999) attributed failure of nodulation at pH below 5.0 to failure of rhizobia to survive in such acidic soils. More than 23% of the soils for bean production in East Africa

have pH at or below 5.0 (Beebe et al., 2012).

Limited supply of water also increases resistance of nodules to O2 diffusion. Oxygen concentration less than 40 µM in infected nodule cells is required for active nitrogenase. 1990. the terminal electron acceptor in oxidase phosphorylation. In saline soils. 1984). Abaidoo et al.. The nodule is made up of few and small intercellular spaces and is surrounded by a diffusion barrier both of which limit entry of oxygen into the infected cells (Witty et al. Loss of nodule permeability and depletion of internal O2 concentration has been implicated as a common factor underlying the loss of N fixation under unfavorable environmental conditions. Muller and Pereira. inhibits attachment of rhizobia onto root surfaces (Cartano-Anolles et al. rhizobia secrete calcium-dependent proteins to promote attachment onto root hair surfaces (Gage. 2004).. Campo.. soil bacteria. 1995. Streeter. 1995). Aluminum and manganese toxicity and low levels of calcium inhibit growth of rhizobia and nodulation at soil pH less than 5 (Alva et al... Walsh et al. Low phosphorus levels in acidic soils... Oxygen levels around the bacteroids are kept low by oxygen binding leghemoglobin (Lb) within the nodules.. 1994). 1987). 1989). 1991). 1996).. has a high affinity for oxygen. 1990. which also promotes low levels of oxygen in nodules (Preisig et al. During the initial stages of symbiosis. Bordeleau and Prevost. Plant intake of high levels of nitrate reduces permeability of nodules to oxygen diffusion decreasing oxygen available for respiration (ArreseIgor et al. 1995. 1987. 1986) resulting in reduction of ATP and NAD(P)H which are required for nitrogenase activity (Sprent et al. which gives them their distinctive pink color (Dakora et al. 1988). 1994. for example. . González et al. limiting bacteroid respiration and inducing nodule senescence (Walsh. Numerous studies have documented the negative impact of high nitrate concentration in soil on nodulation and nodule weight (Malik et al.... Minchin et al.. The low concentration of calcium ions in acidic soils. delay nodulation and infection of primary roots (Mullen et al. 1995.3 Soil nutrient concentrations also affect legume hosts.. and their symbiotic relationships (Mohammadi et al. 1987. 1998 and Galvez et al. 1990.. 2005). 1989). permeability of nodules to oxygen is reduced resulting in high levels of oxygen accumulating in nodules (Delgado et al. however. 2012). Nodule cytochrome oxidase. High oxygen levels in infected cells of nodules lowers nitrogenase enzyme activity.

1996). Olsson.. common beans (Phaseolus vulgaris L. particular attention has been paid to understanding the host plant’s role in regulating nodulation through autoregulation.. 2011). legumes generally assimilate 50 to 70% of their nitrogen via symbiotic nitrogen fixation (Philips and Bennett. 1993. 1991. 1997 and Abd-Alla. 1993. 2005 and Sayuri et al. Because the number of nodules formed could set an upper limit to plant supported BNF. a signal is produced that suppresses formation of new nodules (Renee and Bohlool. Efforts using these lines to improve symbiotic nitrogen fixation have been directed towards .. 1978. Kipe-Nolt and Giller. 1989. Hamaguchi et al. 2003)..4 Despite the numerous factors that compromise nodulation and nitrogen fixation. 1993. 1991). Relative to other legumes. Evidently. Yet there are reports of some varieties of Phaseolus vulgaris L.. and overall plant productivity (Noel et al. 2003). 2013). These mutants have revealed both root and shoot tissues have major roles in control of nodulation and nitrogen fixation (Buttery and Park. and Gamma rays in chickpeas (Cicer arietinum) to develop nodulation mutants.. N-nitroso-N-methylurea (NMU) mutagenesis in soybeans. Becker Underwood (beckerunderwood.. including non-nodulating. Devi et al. This includes development of complex inoculant formulations designed to improve early plant growth. scientists have used Ethyl Methane Sulphonate (EMS) mutagenesis in Phaseolus vulgaris L. Harper et al.. Gremaud and Harper. 1993. And there has been considerable investment in improving the capacity of Phaseolus vulgaris L.. and strains of rhizobia exhibiting high rates of nitrogen fixation (Bliss. et al. once a critical number of nodules are formed. biological nitrogen fixation. 1988. ineffective nodulating.com). 1993. Vance. however. manufactures as series of BioStacked inoculants that contain compounds and biological agents to promote nodulation and improve nitrogen fixation. 1993.. Hardarson and Atkins. Sheng and Harper. 1984. Dasharath and VandenBosch. 1989 and Caetano-Anollés and Gresshoff. Autoregulation is a process within legume plants which controls the number of nodules formed on roots that are already supporting nodules (Bhatia et al. and super-nodulating (with autoregulation deficiencies) lines (Park and Buttery. for fixing atmospheric nitrogen (Peña-Cabriales and Castellanos. Numerous studies have been conducted to determine the origin and control of the autoregulatory signal (Lee et al. 2012). In an attempt to discern the source of the autoregulatory signal. 1993.. Hardarson et al. Melki. Buttery and Park. 1990. 1981 and Bliss. 1997. 2001). for example.) are poor nitrogen fixers (Westermann et al. 1997). 2008). MartinezRomero.

2000). 2010. 2004... Todd et al. Díaz-Leal et al. 2010). Schubert (1986) demonstrated that only half as much energy (ATP) was required to produce ureides than to produce amides. is still under scrutiny (Díaz-Leal et al. Díaz-Leal et al. 2006). however... 2006. 2012). 1991... 2012). Alamillo et al. Witte. 2006. allantoate is transported in the xylem to the leaves and ultimately hydrolyzed to NH4+ and CO2. others such as common bean produce and translocate ureides allantoin and allantoate (known as ureidic plants) (Thomas et al. Todd et al. 1993.. Todd et al. 2006. Boldt and Zrenner 2003. In soybean. 2002. Nodule biosynthesis of purines is not the only source of ureides. There is great interest in understanding how nitrogen fixed by BNF is converted to forms that can easily be transported to other plant parts for use in growth and development. 2006). Alamillo et al. 2006.. Using less carbon and ATP to transport N has given ureidic legumes an advantage over amidic legumes under drought conditions (Todd et al. as non-nodulating legumes have been reported to accumulate ureides (Thomas et al. the proposed mechanism for allantoate degradation is via allantoate amidohydrolase to NH4+. Because ureides have a highly condensed N: C ratio of 1:1..5 developing breeding lines capable of nodulation and nitrogen fixation at high soil nitrate or ammonium concentrations (Herridge and Rose. Xanthine dehydrogenase (XAN) in infected cells converts the purines to uric acid. 1980. 1980. and ureidoglycolate (Tajima et al. Tajima et al. Allantoinase reduces allantoin to allantoate (Webb and Lindell. Ureides (allantoin and allantoate) are synthesized primarily in root nodules via purine biosynthesis and oxidation (Smith and Atkins. 2012). it is considered more energetically favorable in terms of g C g -1 fixed N to transport N as ureides from the nodules to the shoot (Atkins. CO2.. 2010 ... Degradation of ureidoglycolate to glyoxylate and urea is catalyzed by ureidoglycolate urea-lyase (Todd and Polacco 2004. Todd et al. Alamillo et al.. Some leguminous plants translocate fixed N2 as amides asparagine and glutamine (known as amidic plants).. 2006). Todd et al. 2002. Todd et al.. Alamillo et al... the resulting ammonia is then incorporated into organic acids needed for growth (Todd et al. 2004. 2006). 2010). 2011. The specific pathway to NH4+ and CO2 from allantoate in Phaseolus.. Whether transporting N as ureides provides a competitive advantage for common beans is not known. Todd et al. 2006. Urea is eventually metabolized into NH4+ and CO2. Zrenner et al.. which is oxidized to allantoin in adjacent uninfected cells (Smith and Atkins. 2006)..

total N. total tissue ureide levels in plant tissues could include contributions from N2 fixation. 1993. (2000) showed that high concentration of Mn in plant tissues especially under drought conditions was correlated with a lower concentration of ureides in leaves. Similar studies have not been conducted in Phaseolus. Numerous studies have documented genotypic variability for symbiotic N2 fixation in Phaseolus vulgaris (St. If so.. Miranda and Bliss. For example. Because ureides are a known product of N2 fixation.. Vadez and Sinclair (2001) also associated an accumulation of ureides in leaf tissues with depressed N2 fixation. Some of the plant characteristics that have been used to determine superiority for N2 fixation are early plant vigor. (2012) showed accumulation of high levels of ureides in non-nodulated Phaseolus vulgaris L. 1993. 1992. They concluded ureides were recycled from tissues undergoing senescence or from the catabolism of purines (Thomas et al. Clair and Bliss 1991. (2010) showed that non-nodulating Phaseolus vulgaris L. Alamillo et al. Todd and Polacco (2004) illustrated in soybean the dependence of enzymes responsible for ureide catabolism on tissue Mn concentration. nodulation. Puebla 152 (Barrona et al. They suggested selection of lines with a high rate of ureide degradation coupled with Mn-independent ureide degradation would improve N2 fixation under dry conditions.. lines accumulated ureides under drought stress conditions.6 and Díaz-Leal et al.. total biomass accumulation. 1999. and remobilization (Aveline et al. Díaz-Leal et al. Kipe-Nolt. Kipe-Nolt and Giller... The accumulation was attributed to remobilization of nitrogen from older vegetative tissues.. 1980). 1999. Peña-Cabriales and Castellanos.. Purcell et al. It is essential then to include comparisons against non-nodulating lines when evaluating common bean germplasm for superior BNF or proportion of tissue N derived from N2 fixation. Lines with lower leaf Mn levels maintained higher rates of N2 fixation. ureide accumulation in plant tissues . Furthermore. 1993 and Vadez et al. 1993). and ureide content in various plant tissues (Pedalino et al. Furthermore. 2006) have been used as parental lines to select progeny for improved BNF. Bliss.. 1993. 1995). Devi et al. 2000).. Hardarson et al. purine catabolism.. 2013). 1991. at early stages of development and at the onset of the reproduction. Purcell et al. grain yield. Peña-Cabriales and Castellanos. 2012). The impact of drought stress on N2 fixation has been associated with concentration of manganese (Mn) in leaves and accumulation of ureides in aerial plant tissues and nodules (Serraj et al. 1993. 1999) and BAT 477 (Miklas et al.

Accumulation of ureides in aerial plant tissues could be used to identify bean genotypes superior for BNF. To address these objectives. Phaseolus vulgaris L. ureide and N concentration. Shoot and root control of nodulation can be transferred to alter ureide and total nitrogen accumulation and partitioning in common bean. and root tissues were analyzed for biomass. 2005). and regulation in lines with varying characteristics related to N2 fixation would be beneficial in improving BNF in common beans. and the extent to which that control could be transferred via intra-species grafting. leaf. efforts to understand ureide accumulation. 2. biomass accumulation. If so. ureide and N content. Plants were cultivated under controlled conditions favoring nodulation and active nitrogen fixation and harvested during flowering/early pod formation. The objectives of the second study was to determine shoot and/or root control of ureide accumulation and partitioning among four bean genotypes. pod. ureide concentration and content. Hypotheses 1. petiole. . nitrogen concentration and content. lines were selected based on differences in their phenotypic characteristics associated with N2 fixation. The primary objectives of the first study were to identify phenotypic traits consistently associated with variation in capacity for BNF and to identify individual or sets of traits useful for selecting genotypes with superior capacity for N assimilation from BNF. In both studies. The objectives of this study were to identify phenotypic traits related to variation in BNF and N accumulation from BNF. and the number of nodules.7 has been suggested as an indirect indicator of the rate of nitrogen fixation in legumes (Purcell et al. partitioning. Two greenhouse studies were conducted to test these hypotheses. and to determine if these traits could be transferred between genotypes. Stem.. 2000 and King and Purcell. and nodulation were evaluated in selected nodulated and non-nodulated Phaseolus vulgaris lines.

these phenotypic traits will be particularly useful for selecting genotype with superior BNF and N assimilation. N accumulation. there should be predictable relationships between nodulation. . and percent N derived from BNF. These relationships should be evident and consistent among genotypes with varying capacity for nodulation. Grafting super-nodulating shoots onto normally nodulating and non-nodulating root stocks is expected to increase nodulation resulting in increased sites for N2 fixation and ureide synthesis. greater nodulation is expected to promote and increase in tissue ureide content and percent N derived from N2 fixation. ureide accumulation.8 Expected Outcomes Because N2 fixation and ureide synthesis take place in nodulated plants. If so. Also.

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Plants were cultivated under controlled conditions favoring nodulation and active nitrogen fixation and harvested during flowering/early pod formation. Moderate nodule number. Puebla. As such. and non-nodulating R99. GENOTYPIC VARIATION IN UREIDE CONTENT IN PHASEOLUS VULGARIS L. and Eagle) accumulated more ureide and N than the non-nodulated line R99. biomass (Puebla ≥ Eagle ≥ R32 ~ R99). the supernodulating R32. was to identify phenotypic traits of common bean consistently associated with variation in BNF. total N (Puebla ~ Eagle ≥ R32 ≥ R99). Leaves accumulated the greatest biomass and total N and could provide information needed to select for high BNF. leaf. and nodule numbers. Iowa. The objective of this study. of bean genotypes for phenotypic traits associated with variation in BNF. Ames. which had moderate nodule numbers and lowest tissue ureide concentration. ureide concentration. Westgate1 Iowa State University. Puebla. 1 Mercy K. Lines selected for study and their phenotypic characteristics included: Puebla which exhibits rapid biomass and N accumulation. Genotypic rankings for ureide content (Eagle ~ R32 > Puebla > R99). however. There was significant variation in ureide accumulation across plant tissues and genotypes.). derived the greatest percentage of plant N from N2 fixation. Kabahuma1 and Mark E. The nodulated lines (R32. such as common beans (Phaseolus vulgaris L. Plants were analyzed for root. nodule number (R32 > Puebla > Eagle > R99) were not consistently related. Ureide concentrations also varied among tissues and were not consistently related with tissue N values. nitrogen concentration. These results indicate a combination of phenotypic traits should be evaluated when selecting common bean germplasm for improved capacity for BNF. stem. therefore. College of Agriculture and Life Sciences Department of Agronomy A paper to be submitted to Journal of Crop Science Abstract Ureidic legumes. There has been limited characterization. transport N derived from N2 fixation in root nodules to the shoot as ureides. ureide accumulation in aerial plant tissues could be used to identify bean lines with superior biological nitrogen fixation (BNF). petiole and pod biomass. Eagle which has small root system and low percent N from BNF.19 CHAPTER 2. .

Great value has been placed on identifying measurable and inheritable traits associated with greater capacity for N2 fixation (Bliss.. However. soybeans (Glycine max L. Clair and Bliss (1991) used 15 N dilution to identify progeny lines with superior N2 fixation from a cross of high and low N-fixing parents. St.. In this respect. Several studies have revealed genetic variability in N2 fixation activity and total fixed nitrogen in common bean..). 2006 and Zrenner et al. and total biomass at flowering were consistent with greater BNF. Todd et al. Several cultivars were reported to accumulate high percentages of N from N2 fixation under water deficit conditions (Hardarson et al. ureidic legumes transport nitrogen derived from nitrogen fixation to shoots in form of ureides (allantoin and allantoic acid) (Boldt and Zrenner. 2004. 1998). Additionally. 2003. 1993).20 leaf ureide content. Nodule effectiveness also should be considered for increasing %N derived from N2 fixation. Miranda and Bliss (1991) used total seed N to select lines with greater N fixation when grown on N-deficient soils. explored ureide accumulation in common bean tissues under drought stress conditions and reported a lack of relationship between ureide accumulation in drought-stressed tissues and ureide biosynthesis in nodules and nitrogen fixation. Todd and Polacco. Numerous studies have focused on establishing the relationship between tissue ureide concentration and nodule capacity for nitrogen fixation.) (Serraj and Sinclair..). Furthermore.) (Sinclair and Serraj. ureides are also derived from remobilized N in older vegetative tissues (Díaz-Leal et al.. 2012) or recycled from tissues undergoing senescence (Alamillo et al. 1999). Therefore. cowpeas (Vigna unguiculata L. 1993).). For example differences in response of N2 fixation to drought associated with ureide accumulation have been demonstrated in common beans (Phaseolus vulgaris L. peanuts (Arachis hypogaea L.. Furthermore. superiority in symbiotic nitrogen fixation under phosphorus deficient conditions has been observed in some cultivars (Vadez et al. however. 2010). cultivars of common bean with superior capacity for nitrogen fixation are available for selection and breeding purposes. (2010). 1993 and Peña-Cabriales and Castellanos. Introduction Legumes are noted for their ability to fix atmospheric dinitrogen through symbiotic relationships with nitrogen-fixing bacteria (Winkler et al.. 1995 and 1998). . 1988). 2006). 1995). Low ureide accumulation in aerial plant tissues has been associated with less sensitivity of N2 fixation to drought (Sinclair and Serraj. and chickpeas (Cicer arietinum L. Alamillo et al.

Buttery and Park. After two days. 1989.21 Since ureides are the primary transport product of nitrogen fixation from nodules (King and Purcell. R32 and R99 seeds were germinated on paper towels moistened with sterile distilled water in the dark at 20 C. Materials and Methods Plant Materials and Growth Conditions Four common bean genotypes (R99. East Lansing Michigan). germinated seeds were transferred to hydrated peat pellets (Jiffy Products (NB) Ltd. 1990.. Buttery and Park. Because R99 is non-nodulating. . whether this phenotypic characteristic might be correlated with greater N2 fixation and N accumulation in common bean. while R32 is a nitrate-tolerant super-nodulating mutant (Park and Buttery. light photon flux of 150-200 µmol-2s-1 PAR and 16h photoperiod. Vigorous seedlings with healthy unfolded primary leaves were transplanted to 1-L free draining pots filled with commercial potting soil mixture (Sun Gro Horticulture Distribution Inc. Bellevue. Puebla 152. perlite. 1993). and (2) to identify individual or sets of traits useful for selecting genotypes that are superior in assimilating N from BNF. Canada) for five days in a controlled environment chamber at 24 C. The main criterion for selection was variability in biological nitrogen fixation. composted bark.. Eagle. selecting for genotypes with a greater capacity for ureide accumulation in shoot tissues could be useful as a selection strategy to improve N assimilation. 1988 and 2006). Puebla 152 is a black bean landrace from Mexico known for its superiority in symbiotic nitrogen fixation (Barrona et al.. 1990. 2006. however. Puebla. R99 is a non-nodulation mutant (Park and Buttery. and Eagle) were selected for this study (provided by Dr.. it was used as a control to estimate BNF against R32. Eagle is an Andean snap bean commercial cultivar developed in 1971 by Seminis Vegetable Seeds (Navarro et al. 2005). R32. 1989. 1988. 2007). WA containing 15-25% Canadian spagum peat moss. Therefore. Puebla and Eagle. Karen Cichy. It is not clear. 1999). USDA-ARS. R99 and R32 genotypes were both developed in Canada through chemical (Ethyl Methane Sulphonate) mutagenesis of OAC Rico (Park and Buttery. 1993). the objectives of this study were: (1) to identify phenotypic traits of common bean consistently associated with variation in capacity for BNF.

Leaves. stems and pods (except Puebla which lacked pods at the time of harvesting) and roots including nodules were dried at 60°C. One week after transplanting. Tissue extract was boiled in 0. and ground to pass through a 1-mm sieve. Everris Inc. Cleaned roots with nodules were stored in plastic bags at 4 C until nodules were counted manually. leaves. 11.5N NaOH for 8 min.22 vermiculite. This amount of N was enough to enhance growth in R99 (non-nodulating) without inhibiting nodulation in R32. cooled and centrifuged at 10. petioles. Peter’s Excel Water Soluble Fertilizer. stem. pods and roots). petioles..8% nitrate N and 2. Samples were homogenized in 1 mL 0. weighed. cooled. At flowering/early pod set. Valencia. A week after inoculation. Nodule Counts Shoots were detached at the soil surface. 25 mL of fertilizer solution containing 100ppm N (1. ground tissue (leaves. Ames.000g for 5 min.1% urea N..74N . Roots and soil were removed intact from pots and soaked in water to facilitate removal of soil and minimize loss of root nodules.. The supernatant was pipetted into 1. Dublin. Three replicate pots of each line each with a single plant were arranged in a completely randomized design. boiled in 0. nodule number. Puebla and Eagle. CA). tissue ureide concentration. petioles. IA) in a water suspension according to manufacturer’s instructions. dolomite lime and blue chip) and transferred to a greenhouse set at 25 C and 16h photoperiod. Inc. and N concentration. pots were inoculated with a peat-based BioStacked® inoculant (Becker Underwood.1% ammoniacal N. Nitrogen and Ureide Analysis Tissue N concentration was measured in ~125 mg of stems.2N sodium hydroxide (NaOH). Ureide concentration was determined according to Young and Conway (1942) with modifications (de Silva et al. Ureides were extracted from 25 mg of dried. 1996). plants were harvested and dried to measure plant biomass. OH) was added to each pot. pods and roots as percent (%) N using Dumas complete combustion technique (Costech Analytical Technologies.5 mL microfuge tubes and stored at 10 C until ureide determination. boiled at 100°C for 30 min. Nodules were not examined for color to determine whether or not they were active. Samples were stored in tightly sealed glass vials at 20 C until they were analyzed for ureides and N.

tissue ureide concentration. the non-nodulating line. Roots of the . 1). tissue nitrogen concentration. no (or few) nodules formed on R99. As expected. leaves. Plants were cultivated under conditions favoring nodule formation and active nitrogen fixation. Genotypes were ranked for nodule number. Ureide concentration was determined spectrophotometrically (Phoenix Equipment. Inc. Statistical Analysis Single plants from each genotype were considered replicates with five tissues (stems. Genetic Differences in Nodule Number The number of nodules per plant ranged from 0 to more than 2400 (Fig. petioles. biomass in g. and cooled. ureide content in µmoles. pods and roots of four genetically different common bean genotypes.1 (Williams and Abdi. NY) against an allantoin standard at 520 nm within 15 min of addition of KFeCN. while Puebla plants carried more than 300 nodules. N concentration in mg per g dry weight tissue N and total N in mg.23 hydrochloric acid (HCl) and 0.. Rochester. biomass. total ureide content. pods and roots). leaves. and total N with particular emphasis on identifying relationships between these phenotypic characteristics associated with BNF. Results and Discussion Comparison between Genotypes To study the differences in nitrogen assimilation. ureide concentration and content was determined in stems. 2010). Eagle supported a relatively small number of nodules per plant. The results represent the means ± SE of three plants. petioles.33% phenylhydrazine-HCl for 2 min. Data were analyzed by ANOVA and means were compared using Fisher’s LSD test at a probability level of 0. Ureide concentrations are presented in µmol per g dry weight. Color was developed with the addition of concentrated HCl and 1. Plants were harvested at flowering/early pod set to ensure differences in BNF and total N assimilation were well established.67% potassium ferricyanide (KFeCN).

Leaves accounted for approximately 46% of the total biomass in R32. R99 and Eagle. R99. genotypic ranking was Puebla > R32 ~ R99 ~ Eagle and Puebla ≥ R99 ~ Eagle ≥ R32 in petioles. In R32. which had not as yet set pods at the time of harvesting (40 days after planting). Puebla and Eagle tissues was measured in leaves and was closely followed by stems. Puebla was more vegetative with greater growth compared to R32. However. 2003). 2). Ureide Concentration Ureide concentration varied by tissue (Fig. The greatest accumulation of biomass across R32. approximately 56% of Puebla’s total biomass and approximately 36% of Eagle’s total biomass. This might reflect the late reproductive and longer flowering phase of this line. There was no significant difference in pod biomass among R32. These results show that R32. Puebla registered the greatest concentration of ureides in roots (16. ureide concentration in roots of R99 was about 4 times more than the concentration in its shoot tissues.. R99 and Eagle (Table 1). Biomass accumulation varied across genotypes (Table 1). R99’s capacity to absorb and assimilate mineral N was sufficient to support biomass accumulation. Biomass Accumulation well over Genotype ranking for total biomass accumulation was Puebla ≥ Eagle ≥ R32 ~ R99 (Fig. (1986) who reported accumulation of dry matter in the super-nodulating mutant (nts382) was less than that in its normally-nodulating parent (cv. Puebla accumulated significantly greater leaf and stem biomass than R32. Eagle and R99 were further along in development than Puebla. Furthermore.. It was interesting to note the supernodulating line R32 accumulated about the same biomass as the non-nodulating mutant R99 (Fig. Additionally. Among stems and leaves. approximately 41% of total biomass in R99. Evidently. This conclusion is supported by Day et al.24 super-nodulating line R32 had a 200 times more nodules than Eagle.8 µmol/gDW) . 2). the ureide concentration in shoot tissues was six or more times greater than the concentration in roots. Bragg). R99 and Eagle and root biomass accumulation across the four genotypes. 3). It is possible that the large number of nodules initiated by R32 diverted assimilates that would otherwise be used for shoot biomass accumulation (Voisin et al.

Genotypic ranking for ureide content was Eagle ~ R32 > Puebla > R99. Eagle’s high level of ureide content in . but the relative importance varied with tissue and genotype. There was little correspondence between nodulation level and ureide concentrations. Ureide concentration ranking in roots was R99 ≥ Puebla ≥ Eagle > R32.1 µmol/gDW in leaves and 52. however. high levels of ureide content in roots of R99 were associated mainly with the high concentration of ureides. This was attributable to concentration of ureides in tissues of non-nodulating R99. Furthermore. Nodule biosynthesis is not the only source of ureides in ureidic legumes especially in non-nodulating common bean. Since plant biomass of R99. These results therefore. R32 and Eagle was not due to differences in developmental stage. Higher ureide concentration levels in stems and petioles of R32 than Puebla could reflect earlier translocation of ureides from roots to shoots of R32 since Puebla lacked pods at the time of harvesting. and R32 were similar. For stems and pods. 2010) have been reported as potential sources of ureide nitrogen.1 µmol/gDW in leaves and 11.. All R99 tissues had detectable levels of ureides with the concentration in roots among the highest of all (Table 2). Remobilized N in older vegetative tissues (DíazLeal et al. the difference in total ureide content was attributable primarily to N2 fixation in the nodulated lines. Ureide content varied by tissue (Table 3).25 followed by 13. 4). Eagle ranked first followed by R32 and lastly Puebla and R99. was dominated by R32 with 39.4 µmol/gDW in stems.. stems accounted for the greatest concentration of ureides in Eagle. Ureide Content Nodulated lines accumulated more ureide than non-nodulated lines on a whole plant basis (Fig. 2012) and nucleic acids or proteins in tissues experiencing drought-induced senescence (Alamillo et al. suggest the difference in whole plant ureide content in the nodulated plants Puebla. Ranking also varied with genotype (Table 2). Excluding pods from this calculation did not change genotypic ranking. Ureide concentration in leaves and petioles. Both tissue biomass and ureide concentration impacted total ureide content. Eagle. With 87. The high level of ureide content in leaves of R32 and Puebla was attributable primarily to greater biomass accumulation.4 µmol/gDW.5 µmol/gDW in stems and the least ureide concentration was noted in petioles.

In leaves. Among stem and pod tissues. Puebla lagged behind Eagle and R32 in genotypic ranking of ureide content. R32 had higher levels of N concentration in roots than Puebla. ranking for genotypes was Eagle > R32 ≥ Puebla ≥ R99. Furthermore. Nitrogen concentration in roots paralleled nodule number. Puebla and R99. In roots. This could be associated with R32 and Eagle being further ahead in development than Puebla therefore translocating ureides to stems and leaves earlier than Puebla. Variation in development among R32. however. Genotypic ranking for total N accumulation was Puebla ~ Eagle ≥ R32 ≥ R99 on a whole plant basis. The higher ureide content level of R32 among leaf tissues and least among root tissues. These results underscore the importance of genotypic differences in ureide content and partitioning. These results are consistent with data reported by Herridge and Peoples (1990) showing early-maturing genotypes had higher levels of relative ureide-N than those of later maturing genotypes. Total Nitrogen Nodulated lines accumulated more N than the non-nodulated line (Fig. Eagle and R99. Except among Eagle tissues. Genotypic ranking in petioles. 5). R99 and Puebla may have influenced ureide content of individual plant tissues. was Eagle ≥ R32 ~ Puebla ≥ R99. ranking was Puebla ~ R99 ≥ Eagle ≥ R32 (Table 3). For the most part. Ranking also varied with genotype (Table 3). Variation of tissue N concentration also was observed across genotypes (Table 4).26 stems was also attributed mainly to a high ureide concentration. Nodulated lines had greater N concentration levels in their tissues than the non-nodulated line. ranking for genotypes in pods was Eagle ≥ R32 ≥ R99. Eagle had the highest level of N concentration compared to R32. R99 had considerable amounts of N indicating N assimilation from inorganic sources. Leaves had the highest concentration of N across all tissues (Fig. the ranking for genotypes was R32 > Puebla > Eagle > R99. 6). Eagle. and Eagle among stem tissues was associated primarily with higher ureide concentration levels in these genotypes. Nitrogen Concentration N concentration varied dramatically among tissues. Among stem tissues. . leaves were closely followed by roots in ranking.

a greater percentage of N from N2 fixation might have been expected (Diaz-Leal. and R32 at 47. % N derived from BNF Puebla derived the greatest percentage of N from N2 fixation at 62. These results suggest little impact of variation among these lines in development on the %N they derived from N2 fixation. Nodule Effectiveness Nodule effectiveness based on the amount of total plant N accumulated per nodule varied more than 300-fold among nodulated lines (Table 7). followed by Eagle at 56. Puebla and Eagle (Table 5). R99. And supernodulation does not necessarily lead to a high percentage of plant N derived from BNF. leaves accumulated the highest N content among tissues of R32. R32 had a higher number of nodules than Puebla and Eagle yet derived the least %N from BNF. while R32 had the least effective nodules. Tissue N varied within each genotype. Interestingly. However.5% (Table 6).2%. Since Eagle and R32 were slightly ahead of Puebla in development. The largest plant Puebla also had the most N in the leaves. 2012). . This range was much greater than the 200fold variation in nodule number. The difference in N accumulation was attributable primarily to N2 fixation. Impact of tissue biomass and N concentration on N content varied depending on genotype and tissue.27 Nodulated lines accumulated approximately 2. This result evidently reflects ineffectiveness of most nodules formed on R32 roots. This was attributed primarily to highest concentration level of N and greatest biomass accumulation in leaves. Genotypic ranking varied across tissues except in roots where no significant difference was noted in N accumulated (Table 5).9%.3 times as much N as the non-nodulating line. the greatest amount of plant N was associated with low nodule number in Eagle. Eagle had the most effective nodules by far. These results indicated a major contribution of BNF to total plant N can be achieved with a moderate number of effective nodules. This calculation assumes the N accumulated by R99 represents the non-symbiotic accumulation in these lines.

which had only 11 nodules per plant. In fact. and total plant N accumulation. Differences in ureide and N accumulation among common bean genotypes examined were not associated with nodulation. These nodules. on average. Eagle displayed superior nodule effectiveness. however. This result suggests that leaves would be the ideal tissue to use for N uptake and assimilation studies in common bean. total ureide. and (2) to identify individual or sets of traits useful for selecting genotypes with superior capacity for N assimilation from BNF. growth. Leaves consistently accumulated the greatest biomass and N across the four genotypes. Further studies should be conducted with Eagle to determine why it exhibits relatively low BNF under field conditions. As such. were highly effective for BNF as nearly 57% of the plant N was derived from them. Nodule effectiveness (plant N/nodule number) should be considered as a trait for increasing %N derived from N2 fixation. In this study. These studies should focus on identifying the physiological and environmental factors that limit BNF in this commercial variety. total biomass and N accumulation should be measured together as criteria for selecting for superior BNF. . Differences in N accumulation generally were associated closely with biomass accumulation. the greatest accumulation of total N and ureide content was observed in Eagle.28 Conclusions The objectives of our study were: (1) to identify phenotypic traits of common bean consistently associated with variation in BNF.

Purine and pyrimidine biosynthesis in higher plants. J. R. Longer. Hardarson. Cigales-Rivero. J. Soybean petiole ureide response to water deficits and decreased transpiration. P. Breeding common bean for improved biological nitrogen fixation. M. A. A. and Park. Pasini. Day. J. M.).). B. Molecular analysis of ureide accumulation under drought stress in Phaseolus vulgaris L. J. D. G. Plant Science 73: 977-983. Pineda. 1993. Buttery. A. R. D. Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L. M. V. Environ. Sanabria. Plant Soil 152: 71–79. Bliss. J.. inoculation and grafting on expression of supernodulation in a mutant of Phaseolus vulgaris L. B. J. A. S. and R. and M. Manrique. R. Effects of nitrogen.29 REFERENCES Alamillo.. Gresshoff. Henson. Peña Cabriales. D. Plant Soil 152: 59 – 70. R. and S. Boldt. L. Buttery. . Field Crops Research 62: 119-128. M. De Silva. F.. Díaz-Leal. J. Crop Science 36: 611-616. Stuthman. Pineda. Park. 2012. Plant Science 70: 375-381. and S. J.. M. Response to selection for seed yield and nitrogen (N2) fixation in common bean (Phaseolus vulgaris L. J. M. Cell. L. Characterization of some non-fixing mutants of common bean (Phaseolus vulgaris L. H. E. J. and P. 1996. 1990. King. Canadian J. Plant... F. Purcell. 33: 1828-1837. Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent. Exp. J. Physiologia Plantarum 117: 297-304. Davis. A. and P. J. Botany 63: 4095-106. Physiologia Plantarum 68: 375-82. Fernández. J. 1993. M. Alamillo. 1993. Bliss. C. D. H. 1986.. and J. Graham. Lambers. Genotypic variation in biological nitrogen fixation by common bean. Tsai. R. Barrona. Canadian J. Zrenner. A. Bateman. Kipe-Nolt. C. Carroll. Pereira. B. M.. L. Gálvez-Valdivieso. W. Díaz-Leal. A. 1999. R. A. 2003. A. 2010. G. A.. Sánchez-Moran. and C. L. J.

. Plant Physiol. Israel. Buttery. J.. Buttery. S. C. R.. 1979. Park. F. . 2005. Transport of Nitrogen in the xylem of soybean plants. D. Park. and F. and B.. R. R. D. D. J. B.. A. and J. 137: 1389-1396. Inheritance of nitrate tolerant nodulation in EMS induced mutants of common bean (Phaseolus vulgaris L. Canadian J. and W. B.). 1988. Sinclair. J. Effects of water stress on N2-fixation and grain yield of Phaseolus vulgaris L. Shirtliffe. Z. T.. R. Annals Botany 82: 229-234. J. Plant Science 76: 73–83. Plant Soil 152:151–155. 1993. Park. cv.. Canadian J. 1989. R.). R. Registration of ineffective nodulation mutant R69 And nonnodulation Mutant R99 common bean genetic stocks. Skroch. S. 1990. Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods. and T. S. Plant Physiol. W. R. 1991. Crop Science 47: 1344-1353. Jung. Evaluation of the relative ureide content of xylem sap as an indicator of N2 fixation in soybeans: greenhouse studies. P.. K. Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids. R. McClure. Vessy. Crop Science 46: 1415-1417.. Park. McClure. J. Plant Physiol. and L. 2007. 93: 495-503. Buttery. and S.. C. Nienhuis. J. 1996. R. J. and B. Nodulation mutants of white bean (Phaseolus vulgaris L. Comparison of growth and N accumulation of common bean (Phaseolus vulgaris L.30 Herridge. King. Plant Science 68:199–202. Quantitative trait loci associated with bacterial brown spot in Phaseolus vulgaris L. Castellanos. F. Serraj. Israel. OAC Rico) and its nodulation mutants R69 and R99. N2 Fixation response to drought in common bean (Phaseolus vulgaris L. Volk. 66: 720-725.) induced by ethyl methane sulphonate. Peña-Cabriales. Purcell. J. and M. I. J. Peoples. J.. 1998. Navarro. Buttery. Selection for increased seed nitrogen accumulation in common bean: implications for improving dinitrogen fixation and seed yield. and J. and R. A. 2006. Bliss. B. Plant Physiol. G. Heredity 80: 486–488. 64: 411-416. S. P. and B. 1980. Plant Breeding 106: 301–311. P. Miranda..

and F. Beck. Van Berkum. 1984. Soybean cultivars ‘Williams 82’and ‘Maple Arrow’ produce both urea and ammonia during ureide degradation.31 Sinclair. and D. Encyclopedia of Research Design. Erismann. J. Weber. D. Annals Botany 92: 557-563 Winkler. A. and J. The effect of different inorganic nitrogen sources and plant age on the composition of bleeding sap of Phaseolus vulgaris. Laso. P. G. (Merr). G. H. Conway. Todd. D.. and F. Feller. Root and Nodule Growth in Pisum sativum L. C. J. Warembourg. J. Williams. Fisher’s Least Significant Difference (LSD) Test. F. Polacco. Dinitrogen fixation sensitivity to drought among grain legume species. D. Sloger. R. Chem. in Relation to Photosynthesis: Analysis using 13C labeling. and C. C.. Todd. and J. 15 N-determmed . C. Randall. P. and nodule mass in greenhouse and field studies with Glycine max L. Jeudy. C. F. P. A. 2010. Exp. Ureide catabolism in nitrogen-fixing legumes. Thousand Oaks. Abdi. J. Clair. Relationship between ureide N and N2 fixation. Pineda. T. On the estimation of allantoin by the Rimini-Schryver reaction. H. J. Plant Physiol.. R. Botany 57: 5-12. Trends Biochemical Sciences 13: 97-100. Botany 55: 867-877. Euphytica 106: 231– 242. D. Viosin. In: N. Update on ureide degradation in legumes. Sage Publications. R. C. Plant Breeding 106: 215–225. 1979. Polacco. and J. Serraj.. Intrapopulation recombination for dinitrogen fixation ability in common bean. Thomas. B. E. G. U. and H... Variability of N2 fixation in common bean (Phaseolus vulgaris L. C.). CA. J. L. P. Exp. R. V. S. H.. New Phytologist 82: 657-669. 1995. Young. 1999. Polacco. Bliss. 2004. 142: 839-853. and H. D. A. aboveground N Accumulation.. and R. 1988. Keyser. Tipton. Nature 378: 344. D. Blevins.. acetylene reduction. and K. G. Piedras. A. C. 2003. D. Cregan. M. C. J. Biol.) under P deficiency is related to P use efficiency.. Salkind (ed. Salon. 77: 53-58. D. Blevins. St.. Vadez. Drevon. 1991. 2006. P. 1942. J.

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and Eagle roots of greenhouse-grown bean lines sampled at flowering. Nodules were counted manually. The values are the mean ± SE of three plants.1 using Fisher’s LSD test. Puebla. There was no attempt made to distinguish between active/non-active nodules. The soil was carefully washed from the roots to minimize loss of nodules.33 Figure 1. Number of nodules on R32. R99. . Means with the same letter above bars indicate nodule number in genotypes is not significantly different at P=0.

34 Figure 2.1 using Fisher’s LSD test. Puebla. Stems (blue bars). The values are the mean ± SE of three plants. pods (purple bars) and roots (brown bars). Tissues were dried at 60 C until a constant weight. Same letters above bars indicate total mass of the genotypes are not significantly different at P=0. leaves (red bars). Biomass of tissues collected from R32. . R99. petioles (green bars). and Eagle bean lines during flowering/early pod set.

petioles (green bars). Puebla.35 Figure 3. leaves (red bars). R99. Ureide concentration in plant tissues of R32. Ureide concentrations were determined spectrophotometrically against an allantoin standard. pods (purple bars) and roots (brown bars). Same letters above bars indicate tissue ureide concentration values within genotypes are not significantly different at P= 0. Stems (blue bars).1 using Fisher’s LSD test. . and Eagle bean lines during flowering/early pod set. Puebla lacked pods at the time of harvesting. The values are the mean ± SE of three plants.

Ureide content in tissues of R32. petioles (green bars). .36 Figure 4. pods (purple bars) and roots (brown bars). Puebla. and Eagle bean lines during flowering /early pod set.1 using Fisher’s LSD test. Same letters above bars indicate total ureide content of the genotypes are not significantly different at P=0. The values are the mean ± SE of three plants. Stems (blue bars). leaves (red bars). R99.

Same letters above bars indicate tissue N values within genotypes are not significantly different at P=0. . R99.1 using Fisher’s LSD test. Stems (blue bars). petioles (green bars). Puebla. The values are the mean ± SE of three plants. leaves (red bars). and Eagle bean lines collected during flowering/early pod set. pods (purple bars) and roots (brown bars). N concentration in tissues of R32. N concentration was determined using Dumas complete combustion technique.37 Figure 5. Puebla lacked pods at the time of harvesting.

Puebla. Total N accumulation in tissues of R32. Same letters above bars indicate total N of the genotypes is not significantly different at P=0. pods (purple bars) and roots (brown bars). and Eagle bean lines harvested during flowering/early pod set. Stems (blue bars).1 using Fisher’s LSD test. R99. Total N was determined using Dumas complete combustion technique.38 Figure 6. The values are the mean ± SE of three plants. . leaves (red bars). petioles (green bars).

6 a 3.5 b A 1.7 c 0. and Eagle bean lines collected during flowering/early pod set. Phaseolus vulgaris Lines Stem R32 R99 Puebla Eagle 1.4 b B 3.39 Table 1.1 b A 0.1. Using Fisher’s LSD test. means in rows followed by the same lower case letter are not significantly different at P= 0. R99.3 b A 1. They were dried under 60 C until a constant weight was reached to obtain their biomass.8 c B AB A AB 1.1 bc A . Biomass of tissues from R32.4 a 3.7 bc B Leaves 3.0 d B 2.3 a B B A B Tissue Biomass (g) Petioles Pods 0.1.2 a 7.4 b B 1.9 c 0.0 b A 1. Puebla.2 ab A Roots 1. The values are the mean ± SE of three plants.3 b 0.5 b A 1. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.6 c A 1.

Using Fisher’s LSD test means in rows followed by the same lower case letter are not significantly different at P= 0.4 c B .1.1 c D 13.0 c C 18. R99.8 a AB 15. Puebla lacked pods at the time of harvesting.4 b A Roots 6. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.1.5 b C 87.0 a B 4.3 c C 11.1 b A 5. The values are the mean ± SE of three plants. Phaseolus vulgaris Lines Stem R32 R99 Puebla Eagle 53. Puebla.2 b AB 7.4 a A Tissue Ureide Concentration (µmol/gDW) Leaves Petioles Pods 39.40 Table 2. and Eagle bean lines collected during flowering/early pod set.1 c C 6.5 c C 19.2 c B 41.0 d C 57. Ureide concentration in tissues of R32.5 c B 52.1 b C 19. Ureide concentrations were determined spectrophotometrically against an allantoin standard.0 b B 0.6 a A 16.4 a A 4.

1.3 a A 28. Ureide concentration and biomass were used to calculate ureide content.3 c A 39. The values are the mean ± SE of three plants.1 a B 62.0 d C 110.1 d B 5.8 c AB .6 b D 96.8 b B 5. R99. Phaseolus vulgaris Lines Stem R32 R99 Puebla Eagle 70. Using Fisher’s LSD test.5 a A 13.3 ab A Roots 9. means followed by the same lower case letter across the same row are not significantly different at P= 0.9 c AB 16.5 c AB 3.1. Puebla.1 a A Leaves 163.1 c B 27.41 Table 3. Ureide content in tissues of R32.6 c B 0.0 bc A 16.8 c C 39. Means in columns followed by the same Upper Case letter are not significantly different at P= 0. and Eagle bean lines collected during flowering/early pod set.3 b BC 149.5 bc C Tissue Ureide Content (µmoles) Petioles Pods 13.2 bc AB 7.

8 b C 25.8 c B 23.2 c A Leaves 47. Using Fisher’s LSD test.1.42 Table 4.0 c C 16.7 b A Roots 35. and Eagle bean lines collected during flowering/early pod set.2 c B 25.0 b A 18. Puebla. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.4 c AB 9.7 c BC .1 c B 6. N concentration in tissues of R32. R99.4 b B 21.3 b B 0. The values are the mean ± SE of three plants.9 a A 48. N concentration was determined using Dumas complete combustion technique.0 c A 0.1.9 a B 39.0 c C 16.0 d C 17.8 a A Nitrogen concentration (mg/gDW tissue N) Petioles Pods 18. Phaseolus vulgaris Lines Stem R32 R99 Puebla Eagle 16. Puebla lacked pods at the time of harvesting. means in rows followed by the same lower case letter are not significantly different at P= 0.0 d C 36.1 a A 24.

0 c B 9.3 b A Roots 54.1.2 a C 282.3 a AB 87. Means in columns followed by the same Upper Case letter are not significantly different at P= 0. The values are the mean ± SE of three plants.3 a A 163.0 bc AB Leaves 196.2 b B 9.1 ab A 35. Using Fisher’s LSD test. Puebla. Phaseolus vulgaris Lines Stem R32 R99 Puebla Eagle 21.1.7 a BC Total Nitrogen (mg) Petioles Pods 5. Total N accumulation in tissues of R32.9 b AB 21. R99.43 Table 5. means in rows followed by the same lower case letter are not significantly different at P= 0.4 c A 0.2 c C 51.1 c AB 13. Total N was determined using Dumas complete combustion technique.1 c B 0.9 b A 23.0 d B 92. and Eagle bean lines collected during flowering/early pod set.9 b A 37.8 c A .5 b A 36.9 ab BC 8.

Non-nodulating R99 was used as control for calculating % ureide and % N from N2 fixation.0 47.5 62.9 % N from N2 fixation 0. Total ureides in nodulated line – Total ureides in non-nodulated line = % Ureide from N2 fixation Total ureides in nodulated line Total N in nodulated line – Total N in non-nodulated line = % N from N2 fixation Total N in nodulated line Phaseolus vulgaris genotypes R99 R32 Puebla Eagle % ureide from N2 fixation 0 80.6 68.44 Table 6.9 .2 56.2 84. Both shoots and roots were considered in this calculation. Percent plant ureide and N derived from N2 fixation.

Non-nodulating R99 was used as control for calculating total N from BNF.73 0. Nodule effectiveness. Phaseolus vulgaris lines Puebla Eagle R32 R99 Total N from BNF (mg) 231 184 124 0 Nodule number per plant 333 11 2400 0 Nodule effectiveness (mg N per nodule) 0.05 N/A . The values are the averages of three plants.45 Table 7.69 16.

). . and nodule numbers among intra-species grafts of these four genotypes. Ames. The extent of nodulation only indirectly affected ureide and N accumulation. 1 Mercy K. and nodule effectiveness would be ideal physiological targets for further investigations aimed at improving BNF and yield in common bean. nitrogen concentration.46 CHAPTER 3: SHOOT AND ROOT CONTROL OF UREIDE ACCUMULATION AND PARTITIONING IN PHASEOLUS VULGARIS L. These results suggest shoot regulation of nodulation. and Eagle (nodulating) than in R99 (non-nodulating). We used a grafting technique to determine shoot and/or root control of ureide accumulation and partitioning among four genotypes of common beans that have been noted for variation in nitrogen fixation related phenotypic traits (R99. ureide metabolism. and accumulation of ureides are regulated in common bean is not understood. Westgate1 Iowa State University. ureide concentration. grafting normal scions onto R32 roots suppressed nodulation. Grafting normal scions onto R99 rootstocks did not affect nodulation. and pod biomass. Ureide content and total N were greater in R32. and R32). transport. The greatest accumulation of nitrogen was in leaves while the least accumulation was observed in petioles. as increasing nodule number did not result in greater accumulation of nitrogen. Super-nodulation was observed when R32 (a super-nodulator) scion was grafted onto normal nodulating rootstocks. Iowa. Puebla. Eagle. Conversely. or total nitrogen. College of Agriculture and Life Sciences Department of Agronomy A paper to be submitted to Journal of Crop Science Abstract Ureides are manufactured predominantly in legume root nodules and are the primary form of organic N transported to the shoots in common bean (Phaseolus vulgaris L. How the production. leaf. but is a fundamental aspect of improving nitrogen fixation in this crop. Puebla. Kabahuma1 and Mark E. stem. total ureide content. Effect of shoot and/or root on ureide accumulation and partitioning was verified by analyzing differences in root.

Hamaguchi et al. and yield (White and Castillo. it might be possible to enhance BNF and ureide accumulation by manipulating shoot and root complements. ureide accumulation could be a useful parameter for identifying genotypes superior in biological nitrogen fixation (BNF). (1993) used grafting to determine control of super-nodulation in soybean and common bean.. 2011). Since ureides are synthesized primarily in nodules (Boldt and Zrenner 2003.)..47 Introduction Many studies have used ureide concentration in aerial plant tissues as a parameter for assessing genetic and environmental effects on nitrogen fixation in legumes (King and Purcell. In their studies White and Castillo (1989.. If found. ureide accumulation. 1993. 2005 and Zrenner et al. . Whether there are consistent relationships between tissue ureide accumulation and N2 fixation in common bean under favorable conditions has not been established. 1992) found that root traits were more important in drought tolerance of common bean than shoot traits. (1997) highlighted the control of hyper-nodulation by a shoot-transmissible factor common to soybean. Regulation of ureide accumulation and partitioning could be addressed using manipulative techniques such as grafting. BNF. mungbean (Vigna radiata L.. Harper et al. Alamillo et al. we tested the impact of reciprocal grafting of shoot scions and rootstocks of bean genotypes varying in their ability to fix atmospheric nitrogen. Several studies have used grafting to investigate various physiological processes such as increasing both seed and clones for breeding purposes (Gurusamy et al. (2010) reported accumulation of ureides in aerial tissues was independent of ureide synthesis in nodules and N2 fixation in drought stressed common bean (Phaseolus vulgaris L. 2006) and both shoot and root tissues participate in the control of nodulation. Harper et al. To this end.. investigating shoot or root control of nodulation (Buttery and Park. 1997. Abd-Alla.) and hyacinth bean (Lablab purpureus L. Hamaguchi et al. They reported a difference between the mechanism controlling nodulation or substances involved in auto-regulation between soybean and common bean.). 1992). King and Purcell (2005) noted a feedback inhibition of N2 fixation by nodule ureides in soybean (Glycine max L. Our goal was to determine the role played by shoots and roots in regulating nodulation. 2010). 1989. 2009).). 2005 and Alamillo et al.. 1989. Todd et al. and N assimilation in common bean.

1988. dolomite lime and blue chip in a greenhouse set at 25 C with a 16h photoperiod. USDA-ARS. WA) containing 15-25% Canadian spagum peat moss. it was used as a control to estimate BNF against R32. Eagle is an Andean snap bean commercial cultivar developed in 1971 by Seminis Vegetable Seeds (Navarro et al.. R32 and R99 seeds were placed in paper towels moistened with sterile distilled water. Grafting Puebla. 1990. R99 is a non-nodulation mutant (Park and Buttery. 2007). The base of the upper hypocotyls (with cotyledons) was cut on opposite sides to form a V-shape that would fit in a 1-cm deep vertical split on the lower half of the hypocotyl. Puebla and Eagle. Vigorous seedlings with healthy unfolded primary leaves were selected and reciprocal grafts using the wedge graft technique were made. Karen Cichy. while R32 is a nitrate-tolerant supernodulation mutant (Park and Buttery. 1989. vigorous seedlings were then transplanted to 1-L free draining pots filled with commercial soil mixture (Sun Gro Horticulture Distribution Inc. After two days. and Eagle) were selected for this study (provided by Dr. perlite. 1993). 1990. pots .. Eagle. light photon flux of 150-200 µmol-2s-1 and a 16h photoperiod.. Puebla 152. Grafted and non-grafted seedlings were returned to the growth chambers for three days and covered with transparent plastic covers to minimize transpiration from the leaves. R32.48 Materials and Methods Plant Materials and Growth Conditions Four Phaseolus vulgaris genotypes (R99. R99 and R32 genotypes were both developed in Canada through chemical (Ethyl Methane Sulphonate) mutagenesis of OAC Rico (Park and Buttery. Because R99 is nonnodulating. 1993). Bellevue. The plants were uncovered after three days. 2006. 1988 and 2006). 1989. One week after transplanting. composted bark. Puebla 152 is a black bean landrace from Mexico known for its superiority in symbiotic nitrogen fixation (Barrona et al. germinated seeds were transferred to hydrated peat pellets (Jiffy Products (NB) Ltd. Hypocotyls of each seedling were detached at mid-point with a razor. vermiculite. The main criterion for selection was variability in biological nitrogen fixation. A week later. These seeds germinated in the dark at room temperature (20 C). Canada) for five days in a controlled environment chamber at 24 C. 1999). Buttery and Park. and Buttery and Park. The graft unions were secured using grafting tape. East Lansing Michigan).

Leaves. Ureide concentration was determined according to Young and Conway (1942) with modifications from Purcell (de Silva et al. Tissue extract was boiled in 0. boiled in 0. Cleaned roots with nodules were stored in plastic bags at 4 C until nodules were counted manually.74N .5 ml microfuge tubes and stored at 10 C until ureide determination. The supernatant was pipetted into 1. Nodules were not examined to determine whether they were active or not. Harvest At flowering/early pod set. petioles. Puebla and Eagle. petioles. cooled and centrifuged at 10. Ames. pods and roots). Nodule Counts Shoots were detached at the soil surface..5N NaOH for 8 min. each with a single plant. Roots and soil were removed intact from pots and soaked in water to facilitate removal of soil and minimize loss of root nodules.. This amount of N was enough to enhance growth in R99 (non-nodulating) without inhibiting nodulation in R32. 11. Samples were stored in tightly sealed glass vials at 20 C until they were analyzed for ureides and N. ground tissue (leaves. IA) in a water suspension according to manufacturer’s instructions.49 were inoculated with a peat-based BioStacked® inoculant (Becker Underwood. boiled at 100°C for 30 min. stem. and ground to pass through a 1-mm sieve. Peter’s Excel Water Soluble Fertilizer. Everris Inc. A week after inoculation. petioles.1% urea N. OH) was added to each pot. leaves. Inc. Ureides were extracted from 25 mg of dried. 25 mL of fertilizer solution containing 100ppm N (1. weighed. Dublin.8% nitrate N and 2. pods and roots as percent (%) N using Dumas complete combustion technique (Costech Analytical Technologies. Nitrogen and Ureide Analysis Tissue N concentration was measured in ~125 mg of stems. 1996). Three replicate pots of each line. plants were harvested and dried to measure plant biomass on a shoot/root basis and ureide concentration. Samples were homogenized in 1mL 0.000g for 5min. were arranged in a completely randomized design. cooled.1% ammoniacal N. stems and pods (except for Puebla which lacked pods at the time of harvesting) and roots (including nodules) were dried at 60°C. CA).2N sodium hydroxide (NaOH). Valencia.

(1996) or a few small whitish nodules (Buttery and Park. Data were analyzed by ANOVA and means were compared using Fisher’s LSD test at a probability level of 0. R32’s supernodulating trait was expressed when its shoots were grafted to normally nodulating common bean genotypes (Puebla and Eagle).33% phenylhydrazine-HCl for 2 min. 2006) followed by Eagle. ureide content in µmoles. The results represent the means ± SE of three plants. Puebla. 2010). cooled. Rochester. leaves. 1997 and Abd-Alla. However. Statistical Analysis Single plants from each genotype and graft were considered replicates with five tissues (stems.67% potassium ferricyanide (KFeCN).50 hydrochloric acid (HCl) and 0. and color was developed with the addition of concentrated HCl and 1. 1993. 1989 and Buttery and Park. Harper et al. . This confirms control of nodulation by a shoot translocatable signal (Park and Buttery. NY) against an allantoin standard at 520 nm within 15 min of addition of KFeCN... 2011). a non-nodulating line with no nodules. Inc. R32 shoots did not induce nodulation when grafted onto R99 roots.1 (Williams and Abdi. Ureide concentrations are presented in µmol per g dry weight. Grafting Eagle shoots onto R32 roots was unsuccessful as roots penetrated through the graft union. 1993. Results and Discussion Impact of Grafting on Nodulation Number of nodules ranged between 0 and 2. biomass in g. Ureide concentration was determined spectrophotometrically (Phoenix Equipment. A seven-fold increase in nodule number in Puebla and a more than 100-fold increase in Eagle was registered when R32 shoots were grafted onto Puebla and Eagle roots. and Park and Buttery. a super-nodulating line with the greatest number of nodules (Table 1). petioles. N concentration in mg per g dry weight tissue N and total N in mg. and R32.400. pods and roots). The ranking of number of nodules formed from least to greatest was R99. as reported by Shirtliffe et al.

whole plant biomass accumulation was significantly depressed when R99 and R32 shoots were grafted onto Puebla roots in comparison to Puebla controls (Fig. Because R99 shoots did not completely inhibit nodulation on R32 roots and in fact increased nodulation in Puebla and Eagle signifies some root control of nodulation. Changing shoots of the R99 mutant did not have a significant impact on whole plant biomass accumulation and yet self-grafting R99 resulted in a significantly smaller root leading to a higher shoot to root ratio (Table 3). This possibility is supported by results from the study carried out by Hamaguchi et al. Bragg. Day et al. changing shoots in R99 had no effect on nodulation. Bragg. They hypothesized shoots of the super-nodulating mutants (En6500 and RBS15) lacked the autoregulatory factor. whole plant biomass and whole plant N content between un-inoculated nts382 and cv.nodules. Total Plant Biomass Accumulation In some cases. They associated the retardation of plant biomass accumulation . 1) and within genotypes (Table 2). (1992) and Buttery and Park (1993). (1986) found no significant differences in accumulation of shoot biomass. While changing the shoot of Eagle resulted in a more than 10-fold increase in nodule number. which was probably restored by grafting shoots of their respective wild type cultivars. It is possible that the autoregulatory effect on nodulation in R32 was restored by grafting Puebla and R99 shoots on R32 roots. but found significant differences between inoculated nts382 and cv. Park and Buttery (1992) reported formation of small ineffective nodules on R99 roots which are similar to the small pale white nodules we noted on some R99 roots in our study. Grafting R99 and Puebla shoots onto R32 root stocks significantly suppressed number of nodules formed on R32 roots (Table 1). Furthermore. Whether the few nodules on R99 roots were an effect of grafting or rhizobial strains in the inoculum applied is unknown. This signifies shoot control of nodulation in R32. Additionally.51 Park and Buttery (1989) and Buttery and Park (1993) showed that R99 roots did not produce nodules with any scion including R32. 1). R32 scions induced increased numbers of nodules on itself and its parental line (OAC Rico). (1993). It is possible that these were pseudo. grafting resulted in significant differences in biomass accumulation across (Fig. Eagle shoots were associated with a decrease in the number of nodules on Puebla roots. when self-grafted and no nodules on R99 roots. Puebla and Eagle genotypes and root control of non-nodulation in the R99 mutant as reported by Pedalino et al. Furthermore.

changing shoots of Eagle stimulated a significant increase in whole plant biomass accumulation. excising the Eagle shoot initiated a self-incompatible response that was not present or effective with other scions or rootstocks. Puebla and R99 shoots on R32 rootstocks resulted in a decrease in ureide concentration in stems. Changing shoots of Puebla resulted in production of pods (Table 2). The nature of this incompatibility is not known. Evidently. (2012) indicated that at the onset of the reproductive phase. Eagle. which showed that super-nodulation of Puebla roots caused by R32 shoots (Table 1) could have been at the expense of shoot and (Table 3) and whole plant biomass accumulation in the Puebla-R32 graft combination (rootshoot) (Fig. . leaves and petioles compared to the control (Table 4). It is possible that grafting accelerated growth by shortening the vegetative period and advancing Puebla plants into reproductive phase earlier than the control and self-grafted plants. Self-grafting R99 significantly increased ureide concentration in stems. grafting R32 shoots onto Eagle root stocks contradicts with this. 1). Leaves followed by stems accumulated the greatest biomass across all treatments (Table 2). Puebla as a shoot and its controls lacked pods at the time of harvesting (40 days after planting).52 and overall growth in inoculated (nodulated) plants to the prolific number of nodules formed on their roots. This result is in line with results from our study. but might be related to the suppression of nodule formation in this genotype. 1). it was interesting to note that R99 had ureides accumulating in its tissues. Even with the lack of nodules. This result indicates slower development of Puebla relative to R32. petioles and pods and decreased concentration in roots. In fact. Greater induction of nodulation on Eagle roots by R32 shoots (Table 1) increased biomass accumulation (Fig. This could in part explain why suppression of nodulation on R32 roots by R99 shoots (Table 1) resulted in significantly greater accumulation of whole plant biomass than R32 controls (Fig. The consistent negative response of Eagle scions to self-grafting on Eagle rootstocks was unique among reciprocal grafts. A study carried out by Díaz-Leal et al. However. and R99. Self-grafting Eagle depressed shoot biomass accumulation. Puebla. 1). Ureide Concentration Genotypes with R32. and Eagle (nodulating genotypes) had higher levels of ureide concentration than genotypes with R99 (non-nodulating) rootstocks (Table 4). This resulted in a lesser shoot to root ratio than the Puebla control. which was associated with decrease in whole plant biomass accumulation and a lower shoot to root ratio than the control.

Remobilization of nitrogen from the oldest vegetative tissues. self-grafting R99 significantly increased biomass accumulation in pods (Table 2) indicating that R99 self-grafts were further in development compared to R99 controls (ungrafted). pods and roots in this graft combination. could in part be the source of ureides in R99 especially in self-grafts of R99. 2). the lack of significant difference in whole plant ureide content and biomass between R99 controls and grafts in which shoots of R99 shoots were replaced with R32.. Ureides are primarily synthesized in root nodules and transported to the shoot where they accumulate in various shoot tissues (Boldt and Zrenner. Significantly greater ureide content was accumulated when R99 (non-nodulating) shoots were grafted onto R32 (super-nodulating) roots (Fig. however. therefore. Furthermore. petioles. 2). Increase in nodule number (sites for ureide synthesis) when R32 shoots were grafted onto Puebla roots coupled with Puebla’s greater ability to symbiotically fix nitrogen could account for the significantly greater ureide and N concentration observed in this graft combination compared to Puebla controls... pods and roots (Table 5) when R99 shoots were grafted onto R32 rootstocks compared to R32 controls. . Conversely. Puebla and Eagle than R99. This was attributable to greater number of nodules in R32. Puebla.53 common bean plants obtain ureides by remobilizing nitrogen from the oldest vegetative tissues. In relation to this. Matsumoto et al. It is possible that self-grafting R99 stimulated its rate of development which shortened the vegetative stage and thus advanced to the reproductive stage earlier. 1999). (1978) reported allantoin content was less in shoots of an A62-2 (non-nodulating) soybean line grafted onto A621 (nodulating) root than in ungrafted A62-1. Puebla has been identified for its superiority in symbiotic nitrogen fixation (Barrona et al. using R32 as a shoot onto Puebla roots registered the greatest increase in ureide concentration in stems. Ureide content was greater in R32. indicate a decrease in nodule number (Table 1) and an increase in ureide content in stems. This increase in ureide content could be attributed to the greater accumulation of biomass in stems. Puebla and Eagle (nodulating) than in R99 (non-nodulating). 2006 and Zrenner et al. They concluded allantoin production was not dependent on the type of shoot and that an increase in nodule number led to an increase in allantoin level. Among the graft combinations in which Puebla was the rootstock. Todd et al. leaves. 2003. 2006). Ureide Content There was great variation in the accumulation of ureides across the treatments (Fig. Our results. pods and roots.

Whereas Eagle and R32 shoots accumulated greater ureides in leaves and roots. changing the shoots of R99 did not have a significant effect on ureide content compared to R99 controls (Fig. Significantly greater whole plant ureide content was noted in R99 self-grafts. It is possible that grafting interfered with the translocation of ureides from roots to shoots leading to a decrease in concentration of ureides in stems and leaves and an increase in concentration in R32 roots (Table 4). self-grafting R99 depressed ureide content in roots and significantly increased them in stems and pods. Self-grafted Eagle genotypes accumulated the least amount of ureide content among Eagle grafts. pods and roots in these graft combinations (Table 2). R99 and R32 shoots onto Puebla roots accumulated the greatest amount of ureides in leaves (Table 5). This result implies that petiole ureide concentration or content would not be the best indicators of BNF in common beans. For R32 rootstocks. and high biomass accumulation in pods (Table 2). Grafting R32 caused ureide content to decrease in leaves and increase in roots (Table 5). This was associated with the greater accumulation of biomass in leaves than stems. but resulted in biomass accumulation in pods greater than that of R99 control plants (Table 2).54 or Eagle scions demonstrates limited control of shoots over ureide content in R99 mutants (Fig. Moreover R32 shoots did not induce nodulation when grafted onto R99 roots (Table 1). This was attributable to high concentration of ureides in stems (Table 4). petioles. Puebla shoots accumulated the greatest levels in roots when grafted onto R99 stocks. A large increase in the number of nodules formed on Puebla and Eagle roots could have increased sites for ureide synthesis thereby increasing ureide concentration in plant tissues of Puebla-R32 and Eagle-R32 (root-shoot) grafts (Table 4). interfered with ureide accumulation. . Petioles generally accumulated the least amount of ureides across all graft combinations reflecting their small biomass. Importantly. 2). Grafting Eagle shoot onto its root. 2) could be associated with the greater number of nodules formed on Puebla and Eagle roots when a super-nodulating genotype R32 was used as a scion (Table 1). Grafting Puebla. Moreover. the scions of these grafts supported the additional sink demand created by these additional nodules. Nonetheless. 2). Significantly greater ureide content noted when R32 shoots were grafted onto Puebla and Eagle roots (Fig. therefore. leaves and stems accumulated the greatest amount of ureides while petioles and roots accumulated the least amount (Table 5).

The increased number of nodules on the Puebla rootstocks was supported by the R32 scions. R32 and Puebla rootstock grafts maintained the greatest concentration of nitrogen in leaves and the least concentration of nitrogen in petioles and stems (Table 6). leaves. R99 self-grafts formed very few.. petioles. self-grafting R99 resulted in a significant increase in nitrogen concentration in stems. leaves and petioles across all graft combinations in which Eagle was used as a root stock. pods and roots. 1999) apparently could be enhanced further with support from the shoot. changing shoots of Eagle plants did not have a significant effect on accumulation of total N.55 Nitrogen Concentration Irrespective of scion. Grafting depressed N concentration in stems. small and white nodules which were ineffective in N2 fixation. Self-grafting R99 significantly increased total N accumulation over the control. This could have been the source of N and ureides observed in the non-nodulating R99 mutants (Fig. Grafting R99 and Puebla shoots onto R32 rootstocks resulted in a significant decrease in concentration of ureides in leaves and roots.. 2007). This was partly attributable to the N derived from nitrogen fixation (Table 8) and greater nitrogen concentration in R32. petioles and pods. Thus. Self-grafting Eagle did not significantly affect its biomass accumulation and nodule formation compared to the Eagle controls. Many plants utilize assimilated N from mineral N before flowering and remobilize it from older tissues after flowering (Hirel et al. total nitrogen accumulation in R32. Lower N content in self-grafts of Eagle . Puebla’s superiority in BNF (Barrona et al. Similar to Buttery and Park’s (1993) findings. Puebla and Eagle rootstock grafts was greater than that in R99 rootstock grafts (Fig 3). which enabled the observed increase in tissue N concentration and content. Save for self-grafting Eagle which had a significantly decreased total N accumulation (whole plant basis). 2). On the other hand. Grafting R32 shoots onto Puebla root stocks led to a rise in concentration of N in R32 stems. Total Nitrogen Accumulation Save for Eagle self-grafts. It is therefore possible the increase in total N partly resulted from grafting having a stimulatory effect on the capacity of R99 to absorb and assimilate mineral N. Puebla and Eagle tissues than R99.

As such.7% in the control to 54. lowered N concentration in leaves and roots.. the . ureide content. petioles. increased from 44. with no discernible pattern among tissues or graft combinations (Table 5). R99 and R32 shoots were grafted onto Puebla roots. Grafts of R32 scions on Puebla roots were noteworthy in that N concentration in all tissues was greater than in the non-grafted Puebla control (Table 6). Thus. leaves. Eagle is known for its relatively low capacity for nitrogen fixation. Despite the lack of pods in the Puebla control and self-grafts. As in the previous study (Chapter 2). 1999). grafting did not have a significant effect on whole plant N accumulation but changed partitioning and accumulation of N in the stems. These lines are being used as parental lines to produce progeny that vary in biological nitrogen fixation in an attempt to identify phenotypic and genotypic markers associated with variation in BNF. and maintained nitrogen concentration in stems and petioles (Table 6).56 was attributed partly to the least nitrogen concentration reported in the tissues of this graft combination (Table 6). and biomass accumulation. This result shows that the stage of development was not a major inhibitor to nitrogen accumulation in Puebla. Grafting R99 shoots onto R32 roots significantly increased total plant N accumulation (Fig. the calculated increase in total N accumulation was attributable to the greater plant biomass at flowering. Ureide accumulation was highly variable. petioles and pods when Eagle. the results of this study on grafted plants confirm the combination of leaves and biomass as the most appropriate phenotypic traits to identify improvements in common bean BNF. This grafting combination produced significantly fewer nodules (Table 1). pods and roots). We used grafting to identify shoot and root traits associated with BNF that might be transferred among genotypes to improve common bean BNF and yield. 3). Additionally.8% in the R32/R99 rootstock/scion combination (data not shown). Conclusions Puebla has been identified for its superior capacity for nitrogen fixation (Barrona et al. leaves of grafted scions consistently accumulated the greatest amount of N and biomass among the tissues sampled (stems. however. The contribution of total plant N from BNF. Our results show that R32 and Puebla scions on Eagle rootstocks increased nodulation.

ureide. overall development. ureide concentration. Further investigations should focus on the cause of reduced nodulation. Harvesting was targeted after onset of flowering yet some plants had formed pods while others lacked pods. biomass. Although few consistent trends in nodule number. These characteristics could be utilized to improve nitrogen fixation in Puebla and Eagle. This information could help improve BNF and productivity of this genotype. future plant sampling should be carried out at the same developmental stage to avoid the potential for developmental bias. extent of nodulation and %N derived from N2 fixation are transferable traits that can be manipulated to improve N accumulation in common beans. ureide content. and total nitrogen of self-grafted Eagle.57 increase in nodulation and ureide content associated with R32 shoots on Puebla and Eagle rootstocks indicates superiority of R32 shoots in terms of supporting ureide accumulation and nodulation. and N accumulation were associated with specific graft combinations. . Although this difference in development did not appear to give podded plants an advantage in terms of ureide and nitrogen accumulation.

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R99.1 using Fisher’s LSD test. petioles (green bars). Biomass of tissues collected from R32. Stems (blue bars). and Eagle controls and grafted plants during flowering/early pod set. leaves (red bars).61 Figure 1. Puebla. . pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Tissues were dried at 60 C until a constant weight. Same letters above bars indicate total biomass of the genotypes are not significantly different at P=0.

leaves (red bars). Same letters above bars indicate total ureide content of the genotypes are not significantly different at P=0. Puebla. petioles (green bars). Stems (blue bars). pods (purple bars) and roots (brown bars). Ureide content in tissues of R32. and Eagle controls and grafts harvested during flowering/early pod set. The values are the mean ± SE of three plants.62 Figure 2. R99. .1 using Fisher’s LSD test.

. and Eagle controls and grafts harvested during flowering/early pod set. leaves (red bars). petioles (green bars). Same letters above bars indicate total N of the genotypes are not significantly different at P=0.1 using Fisher’s LSD test. Stems (blue bars). The values are the mean ± SE of three plants. pods (purple bars) and roots (brown bars). Puebla. N concentration and biomass were used to calculate total N content in the tissues. Total N was determined using Dumas complete combustion technique. R99.63 Figure 3. Total N accumulation in tissues of R32.

Nodules were not analyzed for activity. R99.and intra-species grafts of R32. Puebla.64 Table 1. The values are the mean ± SE of three plants. The soil was carefully washed from the roots to minimize loss of nodules.1. Nodules were counted manually. Using Fisher’s LSD test. self. means in columns followed by the same letter are not significantly different at P= 0. and Eagle roots sampled at flowering. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Number of nodules 2400 a 2833 a 840 b 910 b 0 9 0 3 0 b a b ab b 333 bc 339 bc 185 c 480 b 2400 a 11 c 9 c 716 b 640 b 1194 a . Number of nodules on controls.

and intra-species graft tissues from R32.7 bc B 1.8 c BC 1.3 b A 3.9 c A 1.2 b A 3.8 b A 0.4 b C 2.0 a A 4.5 c BC 0.2 c AB 0. R99.9 a B 7.3 a B 4.1.8 a A 5.1 bc B 1.2 a B 3.0 c C 1.4 c A 1.8 b A Roots 1.4 d A 0.2 b A 2.6 c B 0.65 Table 2.3 b B 1.1 b A 3. and Eagle bean lines collected during flowering/early pod set.6 bc A Leaves 3.0 b A 0.1 c A 0.4 a A 4.7 d BC 1.9 b AB 1.5 c AB 0.5 c A 1.6 a B 4. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/R99 Puebla/Eagle Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Biomass (g) Petioles 0. The values are the mean ± SE of three plants.4 c C 0.9 a BC 6.5 b B 3.6 c A 1.0 b BC 1.9 cd A .9 b B 1.8 d AB Stems 1.9 c BC 1.3 a C 2.4 c C 0.8 b A 1.6 b AB 1.6 b C 1.9 b A 3.5 b A 0. Puebla lacked pods at the time of harvesting.2 a AB 2.1 b BC 4.4 a A 7.1 a AB 4.7 d A 0.5 a B 7.0 c A 1.0 d B 0.6 c BC 1.5 a C 3. self.8 b AB 3.8 a A 3.2 ab AB 0.6 b A 0.7 b A 1.8 a B Pods 1.4 a C 5.5 a A 3.4 c A 1.5 b A 2.9 b A 1.9 c A 0.0 e B 1. Biomass of controls.8 b A 1. They were dried at 60 C until a constant weight was reached to obtain their biomass.0 c C 1.5 b B 1.8 b B 1.3 b C 0.0 d C 3.5 a A 1. Puebla.5 c A 1.6 b A 1. Using Fisher’s LSD test.8 c AB 0.1 b B 2.1 c B 0.3 b B 0.6 b AB 0. means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.4 b AB 0.1.1 ab A 3.5 b B 2.7 a AB 7.6 b AB 2.1 bc A 1. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.2 a AB 1.6 c A 0.9 b B 2.8 c AB 2.

self.9 A 1.8 AB 2.2 B A B B B 7. and Eagle bean lines collected during flowering/early pod set.3 A 3. Puebla.4 A 1.7 A 1. The values are the mean ± SE of three plants.3 A .7 4.and intra-species graft of R32.6 A 10.9 B 1. R99.0 A 5.8 A 6.1 B 1.2 A 5.9 B 2.6 A 1.8 A Shoot: root ratio 4.3 B 9.5 A 1.1 C 5.9 A Shoot 6.3 A 5.1 A 4.0 C 4.6 A 1.9 B 12.1.2 A 7.0 A 4.5 B 1. They were dried at 60 C until a constant weight was reached to obtain their biomass.8 C 7.2 B 5.1 A 1.1 AB 7.8 AB 13.6 AB 1.9 A 1.4 B 14.9 D 9. Puebla lacked pods at the time of harvesting.7 AB 11.9 A 9.9 A 8.3 AB 5. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/R99 Puebla/Eagle Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Biomass ratios (g) Root 1. means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.7 A 7.5 19.5 ABC 8.2 A 7. Using Fisher’s LSD test.0 5.7 BC 6.5 BC 6.66 Table 3. Shoot: root ratios of controls.3 B 1.4 A 1.8 A 1.6 3.3 A 3.5 A 11.5 A 0.6 B 8.

0 a A 34.0 b C 14.8 b A 19.8 bc BC 11.4 6.0 b C 22.1 b B 13.4 b B 28.8 c B 11.3 c C 25.4 bc B 18.9 b C 18.5 b C 5.0 c E 4.0 a A 33.9 a C 40.0a A 87.4 a B 49.0 a A 47. Puebla.6 c C 26. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.9 a A 20. self. means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.1 b B 28.3 c B 89. The values are the mean ± SE of three plants.0 b B 14.8 a BC 18.2 a BC 21.7 b A 15.0 c C 31. Ureide concentration in controls.and intra-species graft tissues of R32.1 c C 21.7 bc D 32.3 d C 32.67 Table 4.5 c B 9.4 b B 81. R99.1.7 7.1 c B 20.7 b B 3.8 b B Stems 53.4 b A 8.7 c C 13. Ureide concentrations were determined spectrophotometrically against an allantoin standard.5 a A 19.6 c C 22.8 a B 22.1 a A 22./gDW) Petioles Pods 52.0 c B 7.5 b B 80.1 a A 16.7 a B 12.1 b BC 15.3 b A 3.6 a A Leaves 39.7 d B 4.8 c C 32.2 b A 11.5 b C 16. and Eagle lines collected during flowering/early pod set.3 a A 20.8 a B 5.1 c B 48.7 b B 0.8 c D 0.9 b C 4.7 b C 30.9 c BC c ABC cA bC b AB Roots 6.0 b C 28.1 b A 0.1 5.6 a A 2.8 b B 49.6 c B 21.1 c B 4.0 b B .2 a A 45.4 c C 14.9 d B 10.9 b A 57.8 a B 18.5 d B 17. Using Fisher’s LSD test.0 a B 16.0 d D 0.3 4.4 a A 4.0 c A 20.1.0 bc B 19.4 a A 4.0 b B 3.4 c C 14.0 b C 23.0 a A 41.0 d D 12.6 bc B 0.2 c B 3. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/R99 Puebla/Eagle Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Ureide concentration (µmol.1 c B 6.5 a B 46.4 a A 56.6 d B 4.8 c B 6. Puebla lacked pods at the time of harvesting.

means followed by the same lower case letter across the same row within the same rootstock are not significantly different at P= 0. R99.8 a A Leaves 163.8 c A Stems 70.1 b C 76. Using Fisher’s LSD test.5 b B Roots 9.2 b A 98.0 c C 42.7 bc C 80.6 c AB 18.3 a A 7.8 a B 129.9 c D 72.0 d D 113.5 bc B 27.9 a B 191.7 a B 115.8 c B 72. Puebla. Ureide concentration and biomass were used to calculate ureide content.0 c A 16.8 c A 34.2 bc B 78.6 a A 28. Ureide content in controls.5 b C 268.4 a A 27.4 c B 6. The values are the mean ± SE of three plants.1.2 2.0 d C 7. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.7 c B 81.7 a A 13.8 b B 0.0 d B 0.3 c A 38.0 b B 39.1 4.4 c C 16.6 b B 12.68 Table 5.7 dc A 110.4 cd B 0.3 b B 5.1 b BC 50.5 bc C 13.7 c B 26.8 b D 181.9 a A 17.8 b B 99.3 c B 1.6 d A .1 d BC 12.8 c C 24.3 b B 0.6 b A 149.1 a A 97.0 c B 3.7 a B 386.5 a A 105.3 d B 13.3 c B 27.3 3.3 ab A 4.1 c C 45.8 e BC 5.1 a A 70.7 a A 96.7 b B 88.9 c BC 10.0 b B 67.0 2.2 c C 32.2 a A 68.2 c A 17.9 a A 62.7 a BC 87.0 c B 5.and intra-species graft tissues of R32. Phaseolus vulgaris lines Root/shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Ureide content (µmoles) Petioles Pods 13.5 b B 29.1 a B 6.3 a A 0. and Eagle bean lines collected during flowering/early pod set.2 d AB bA c AB dB bB 39.2 a AB 13.6 c B 54.3 b B 56.0 c A 18.6 b AB 27.0 bc BC 35.7 c B 8.5 d C 20. self.9 b A 42.8 a A 37. Puebla lacked pods at the time of harvesting.0 d A 16.1.0 d C 7.2 a A 28.0 e B 21.1 b AB 139.8 d B 19.0 a A 197.8 c B 63.1 bc A 0.5 c B 31.

Using Fisher’s LSD test.0 b A Stems 16.4 b B 24.1 a B 0.2 a A 18. Puebla.1 c B 23. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.9 a C 0.4 ab C 34.9 c AB Roots 35.6 d A 23.2 c C 12.3 a C 36.5 a B 33.9 b A 21.1 c A 15.5 b A 6.0 c A 10.0 d B 0.8 a A 16.4 a B 32.1.8 b B 21.7 b A 17.2 c A 12.6 a A 45.0 a AB 25.3 d B 8.6 b A 0.4 c A 18.5 c A 15.4 a B 47.0 d B 33.8 b A 16.4 c B 27. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Nitrogen concentration (mg/g) Leaves Petioles Pods 47.and intra-species graft tissues of R32.1 b B 33.5 b AB 13.9 a BC 43.7 c A 14.2 c B 17.3b B 0.5 a A 26.0 b BC 16.0 b A 31.9 a B 34.5 c B 16.6 c C 8.8 a A 48.6 a B 39.0 b D 13.0 b B 22. means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.7 c BC 17.2 c B 18.4 c A 25.0 d B 32.8 c AB 16.5 d A 17.3 b A 8.8 c AB 15.69 Table 6.3 c CD 15. R99.1 a A 49.7 c BC 8.6 ab A 0.7 ab BC 11.9 b A 31.5 b B 14.1.0 d B 17.5 b AB 29. self.2 b B 18.7 b B 16.1 b B 16.8 b A .1 a A 15.3 b C 36.4 d A 17.0 e D 16.0 c C 32.7 a C 42.9 b AB 14.3 a A 39.5 a A 22.5 b A 36.2 b C 20. The values are the mean ± SE of three plants.2 b C 23.9 b B 13.9 c A 15. N concentration in controls.6 a A 32. N concentration was determined using Dumas complete combustion technique.0 c A 6.7 b BC 17. Puebla lacked pods at the time of harvesting.2 b AB 25. and Eagle plants collected during flowering/early pod set.8 a C 27.4 a A 17.2 ab B 30.0 c B 6.2 a BC 53.5 b B 22.6 b B 18.2 b BC 32.

1.7 b A 57.8 bc B 0.8 c B 30.7 b A 0. means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.0 d C 119.3 bc AB 0. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1 b A 52.2 c B 20. N concentration and biomass were used to calculate total N content in the tissues.4 a A 25.9 c AB 19.8 a AB 276.9 b B 42.1 a A 274.3 a B 236.8 c B 17.8 b AB 35.7 b A 54.8 b A 35. self.5 c B 9.0 d C 31.8 c A 13.0 b A 61.5 a A 182.7 cd C 37.3 bc AB 51.3 b A 9.6 a A 53.2 b A 8.9 a A 68.4 c A 3. and Eagle plants collected during flowering/early pod set.6 b A Stems 21.7 bc B 61.2 bc A 31.3 b A 90.4 b B 22.2 d A 4.8 d B 21.6 a B 87.0 c B 62.8 a C 239.8 b BC 29.9 b AB 55.7 b A 48. The values are the mean ± SE of three plants.8 c A .9 c B 47.3 a A 282. R99.2 cd BC 47.9 b A 47.0 c B 13.and intra-species graft tissues of R32.7 b A 73.5 bc B 89.4 bc B 92.8 c A 13. Puebla lacked pods at the time of harvesting.9 c C 8.2 b B 0.9 c AB 16.70 Table 7.9 b B 32.3 a A 264.0 b A 36.1.7 c AB 9.6 a AB 259.2 b B 10.0 b AB 30.9 b A 59.2 b A 9.0 d C 25.1 a A Roots 54.6 ab B 59.7 bc C 25.9 c A 4.5 b A 36.1 bc A 7.9 a AB 191.0 d C 0.7 a B 39.5 b A 10.5 a ABC 163.8 c A Leaves 196.5 b A 23.7 b A 5.1 b A 12.6 b A 53.5 b A 6. Using Fisher’s LSD test.4 c A 13.3 a C 188.2 c AB 0.0 b A 34.0 bc AB 18.6 a BC 177.7 b B 38. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla R99 control R99/R99 R99/Eagle R99/Puebla R99/R32 Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32 Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32 Total nitrogen (mg) Petioles Pods 5. Total N accumulation in controls.8 d BC 15.3 a A 60.2 a A 111.4 c A 19.1b A 61. Puebla.

0 60. Puebla and Eagle plants. Percent N derived from N2 fixation. Phaseolus vulgaris Lines used as scions R99 R32 Puebla Eagle % N from N fixation 0.6 .3 69.2 27. Averages of total N accumulated by R99 were subtracted from averages of total N accumulated by R32.71 Table 8. Non-nodulating R99 was used as control for calculating % N from N2 fixation.

and total biomass at flowering were consistent with greater BNF in this study. .72 CHAPTER 4: GENERAL CONCLUSIONS Nitrogen uptake and assimilation are contributors to plant growth and development. Nevertheless. leaf total N content. Biomass and N accumulation. nodule effectiveness might be useful for selecting genotypes with superior capacity for BNF. Also nodule number as low as 11. the variability in phenotypic traits among genotypes and plant tissues made it complex for identification of a trait or traits related to N assimilation from BNF. Identification of a trait or sets of traits associated with superiority for BNF and N assimilation from BNF is important in improving BNF. Results from this study will be used to focus phenotypic analysis of an Andean Diversity Panel (400+ lines) for genomic marker development in a related USAID-funded project. However. biomass accumulation was a vital contributor to N accumulation and this was associated with high capacity for BNF. Studies described in this thesis were aimed at determining genotypic variation in BNF and N assimilation from BNF and determining the transferability traits related to BNF and N assimilation from BNF.

Dr. I will forever be indebted. Karen Cichy (Michigan State University) for providing seed and literature for the research study. strength. endurance. USAID. Steven Beebe (CIAT International Center for Tropical Agriculture) for providing seed for research. For the academic and technical advice. Gwyn Beattie and Dr. Special thanks go to Whitney Bouma (Iowa State University) for the technical assistance offered during the course of my research. I will forever be grateful. encouragement. mercy. To my family and friends both in Uganda and the US. my major advisor without whom my success at Iowa State University would not have been realized. I am in awe. Appreciation goes to Dr. Dr.73 ACKNOWLEDGEMENTS To God Almighty for the wisdom. knowledge. My program of study committee members. love. Kathleen Delate for the academic and technical assistance rendered during the course of my stay at Iowa State University. Mark Westgate. Thanks also go to Nicholas Howell (ISU Horticulture farm) and the Agronomy Farm manager (Iowa State University) for all the help in the field studies. Dr. Larry Purcell (University of Arkansas) for allowing us to learn from his laboratory and providing literature related to our study. You stood with me physically and emotionally enabling me to finish this race even stronger than I started. words of wisdom and always seeing potential in me. . Gratitude goes to the funders. grace and favour He has blessed me with and for bringing me thus far. for the financial assistance.