PARENTAL EFFECTS AND PROVISIONING UNDER DROUGHT AND PHOSPHORUS STRESS IN COMMON BEAN

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1 The Pennsylvania State University The Graduate School Plant Biology Program PARENTAL EFFECTS AND PROVISIONING UNDER DROUGHT AND PHOSPHORUS STRESS IN COMMON BEAN A Thesis in Plant Biology by Claire M. Lorts 2016 Claire M. Lorts Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2016

2 The thesis of Claire M. Lorts was reviewed and approved by the following: Kathleen Brown Professor of Plant Stress Biology Thesis Adviser Jonathan Lynch Professor of Plant Nutrition Dawn Luthe Professor of Plant Stress Biology Teh-hui Kao Distinguished Professor of Biochemistry and Molecular Biology Chair of the Intercollege Graduate Degree Program in Plant Biology Signatures are on file in the Graduate School. ii

3 ABSTRACT Low soil fertility and drought are primary constraints in common bean (Phaseolus vulgaris) production in low input agricultural systems, and a threat to food security in many developing nations. Common bean genotypes tolerant to drought or low phosphorus conditions have been identified, and root traits associated with tolerance to such stress have been examined. The utility of these root traits in tolerant genotypes is usually tested using seed from a well-watered and high-nutrient parental environment. However, many farmers in developing nations collect seed for the next year s crop from parent plants grown in low phosphorus and/or drought conditions. Thus, it is important to understand how progeny from a stressed parental environment perform under similar stressful conditions. This study investigates the impact of a low phosphorus and/or drought parental environment on progeny seed and root traits. To test whether differences in progeny seed and root traits from stressed parental environments could be explained by differences in parental provisioning of seeds during seed development, we also examined seed and root traits in seeds from different pod positions (stylar versus peduncular) and pod developmental times on the parent plant. Greenhouse, field, and seedling experiments were used to evaluate seed, seedling, and mature root traits in progeny from stressed and non-stressed parental conditions. In parental drought studies, progeny from drought stressed parents had lower individual seed weight, lower basal root number (BRN) in both seedlings and plants at growth stage R2, and lighter total seedling dry weight, shorter seedling basal roots, shorter lateral roots borne on seedling tap roots. The length and density of root hairs borne on seedling tap and basal roots also differed between progeny from parental drought and well-watered environments. At growth stage R2 progeny from parental drought had a smaller basal root diameter, lighter shoot dry weight, fewer shoot-borne roots, and fewer dominant shoot-borne roots. In parental phosphorus (P) studies, progeny from a low P parental environment had lower individual seed P content, fewer shoot-borne roots at R2, and greater BRWN at R2. In studies comparing root traits between seeds from the peduncular (closest to the petiole) versus stylar (farthest from the petiole) positions in the pod, and between seeds from early versus late developing pods, seeds from the peduncular position in the pod at growth stage R2 had lower individual seed weight, lower BRN, lighter root dry weight, smaller tap root diameter, and fewer lateral roots borne on basal roots. In all studies, responses to parental effects varied across genotypes. Seed and seedling root traits had greater consistency across genotypes compared to mature root traits, whereas stronger genotypic effects were seen in mature root traits. Seeds and seedlings showed more consistency in parental effects across genotypes likely due to the exposure to fewer environmental factors, resulting in less variability among measured traits. Overall, progeny from drought stressed parents, progeny from a low P parental environment, and seeds from the peduncular position within the pod had root traits that were lighter, shorter, smaller in diameter, or fewer in number. Parent plants grown under stressful conditions such as low P and drought during seed fill may have had less resources available to allocate into seeds during seed fill, relative to parent plants in well-watered and high fertility environments. Seeds from the peduncular position may have had root traits that were lighter, shorter, or smaller in diameter due to later fertilization within the pod compared to seeds from the stylar position. Thus, most differences in root traits from stressed parents or seeds from the peduncular position were likely explained by lower parental provisioning of seeds during seed fill. In addition to parental effects that suggest lower parental provisioning, possible adaptive parental effects were found in both parental drought and parental low P studies. Greater BRWN in progeny from P stressed parents may be adaptive to low P conditions by increasing the area of soil explored, assisting in potentially greater acquisition of P in low P soils. Longer basal roots in seedlings from parental drought may assist in greater exploration of deeper soil where water is more available under drought conditions. iii

4 Results from this study may be used to help improve food security in developing nations by assisting the selection of genotypes that thrive in nutrient and water deprived soils in current and subsequent generations. This thesis demonstrated profound differences in root phenotypes in response to parental stress, seed position in the pod, and pod developmental time, depending on the genotype. Thus, the parental environment in which seeds are collected must be a factor that is considered in breeding programs and phenotyping initiatives. Genotypes displaying potential adaptations to stress in response to the previous generation should be considered in breeding programs, but genotypes displaying relatively greater reduction in provisioning of progeny in response to parental stress should be avoided. iv

5 TABLE OF CONTENTS List of Tables.... vi List of Figures vii List of Abbreviations xi Acknowledgements......xii PARENTAL EFFECTS AND PROVISIONING UNDER DROUGHT AND PHOSPHORUS STRESS IN COMMON BEAN Introduction 1 2. Materials and Methods Results Discussion Appendix A Additional Tables: Parental Effects of Seed Position in the Pod and Pod Developmental Time Appendix B Additional Tables and Figures: Parental Effects of Drought Stress 41 Appendix C Additional Tables: Parental Effects of Phosphorus Stress.. 51 References v

6 List of Tables Table 1. Significant seed and root traits from greenhouse trials, organized by genotype. Treatment groups are indicated in parentheses: Pod position (stylar (S)/ peduncular (P)), and pod developmental time (early/ late). Root traits and genotypes that did not result in significant differences between treatments were not included in the table. Table 2. Significant seed, shoot, and root traits from greenhouse and field trials organized by genotype, with p and F values. A two-sample T-test was used in seed weight analyses, thus an F value is not indicated. Location (PA field/greenhouse or URBC) for mature root traits, treatment differences (wellwatered versus drought) indicated in parentheses. Seed weight and seedling BRN were measured in the laboratory in PA. Root traits that did not result in significant differences between treatments were not included in the following table. Table 3. Significant seed and root traits from field trials, organized by genotype. Treatment differences are indicated in parentheses. Only genotypes with differences between treatments were included, thus SER79, SER83, SER85, and SER43 were not included in the following table. Root traits that did not result in significant differences between treatments were also not included in the table. vi

7 List of Figures Figure 1. Diagram of a common bean pod with seeds at the stylar end of the pod, furthest from the petiole, and seeds at the peduncular end of the pod, closest to the petiole. Figure 2. Root classes within the common bean root system, including shoot-borne roots, basal roots, lateral roots, and the tap root. Shoot-borne roots are important in scavenging for topsoil P, and basal roots play roles in both P and water acquisition. Figure 3. Seed weight collected from parent plants, from the stylar (S) and peduncular (P) ends of the pod. Asterisks represent significant differences between pod positions. Figure 4. Seed weight collected from parent plants, from early and late developing pods. Asterisks represent significant differences between developmental times. Figure 5. Basal root number (BRN) in progeny collected from the stylar (S) or peduncular (P) ends of the pod, then grown in the greenhouses. Asterisks represent significant differences between pod positions. Figure 6. Basal root number (BRN) in progeny collected from early or late developing pods, then grown in the greenhouses. Asterisks represent significant differences between developmental times. Figure 7. Relationship between BRN and seed weight (per seed) from stylar (S) and peduncular (P) ends of the pod, in BAT477. The regression equation for the peduncular position was y = x, and for the stylar position, y = x. Figure 8. Relationship between BRN and seed weight (per seed) from early and late developing pods on the parent plant, in BAT477. The regression equation for early developing pods was y = x, and for late developing pods, y = x. Figure 9. Relationship between BRN and seed weight (per seed) from stylar (S) and peduncular (P) ends of the pod, including all genotypes. The regression equation for the peduncular position was y = x, and for the stylar position, y = x. Figure 10. Tap root diameter (mm) in progeny collected from the stylar (S) and peduncular (P) ends of the pod, then grown in the greenhouses. Asterisks represent significant differences between pod positions. Figure 11. Root dry weight (grams) in progeny collected from the stylar (S) and peduncular (P) positions in the pod, then grown in the greenhouses. Asterisks represent significant differences between pod positions. Figure 12. Relationship between root dry weight and seed weight (per seed) from stylar (S) and peduncular (P) ends of the pod, in BAT477. The regression equation for the peduncular position was y = x, and for the stylar position, y = x. Figure 13. Relationship between root dry weight and seed weight (per seed) from stylar (S) and peduncular (P) ends of the pod, including all genotypes. The regression equation for the peduncular position was y = x, and for the stylar position, y = x. Figure 14. Number of lateral roots per basal root in progeny collected from the stylar (S) and peduncular (P) ends of the pod, then grown in the greenhouses. Asterisks represent significant differences between pod positions. vii

8 Figure 15. Relationship between number of lateral roots per basal root and seed weight (per seed) from stylar (S) and peduncular (P) ends of the pod, in DOR364. The regression equation for the peduncular position was y = x. Figure 16. Seed weight (per seed) in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 17. Seedling basal root number in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 18. Seedling dry weight in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 19. Density of root hairs borne on seedling tap roots (# of hairs/ mm 2 ) in progeny from a wellwatered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 20. Seedling tap root length in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 21. Seedling basal root length in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 22. Length of root hairs borne on seedling tap roots (mm) in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 23. Length of root hairs borne on seedling basal roots (mm) in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 24. Length of lateral roots borne on seedling tap roots (cm) in progeny from a well-watered and drought parental (Gen.0) field environment. Asterisks represent significant differences between treatments. Figure 25. Soil volumetric water content in well-watered and drought plots at the URBC site. Each data point represents the average of 4 replicates from continuous measurements in 2 plots per treatment, at 15 cm below the soil surface. Figure 26. Soil volumetric water content in well-watered and drought plots at the Rock Springs site. Each data point represents the average of 2 replicates from continuous measurements in 2 plots per treatment, at 15 cm below the soil surface. Figure 27. Shoot dry weight in progeny from the field, from a well-watered and drought (Gen.0) parental environment. Asterisks represent significant differences between treatments. Progeny were grown at the URBC site under drought and well-watered conditions, and harvested at growth stage R2. Figure 28. Basal root diameter (mm) in progeny from the field, from a well-watered and drought (Gen.0) parental environment. Asterisks represent significant differences between treatments. Progeny were grown at the URBC site under drought and well-watered conditions, and harvested at growth stage R2. viii

9 Figure 29. Basal root angle of a representative root angle in progeny from the field, from a well-watered and drought parental (Gen.0) environment. Asterisks represent significant differences between treatments. Progeny were grown at the URBC site under drought and well-watered conditions, and harvested at growth stage R2. Figure 30. Dominant shoot-borne root number in progeny from the field, from a well-watered and drought parental (Gen.0) environment. Asterisks represent significant differences between treatments. Progeny were grown at the URBC site under drought and well-watered conditions, and harvested at growth stage R2. Figure 31. Dominant shoot-borne root number in progeny from the field, from a well-watered and drought parental (Gen.0) environment. Asterisks represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 32. Dominant shoot-borne root number in progeny grown in a well-watered or drought environment (Gen.1), and progeny from a well-watered or drought parental environment (Gen.0). Letters represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 33. Basal root number in progeny from the field, from a well-watered and drought parental (Gen.0) environment. Asterisks represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 34. Basal root number in progeny grown in a well-watered or drought environment (Gen.1), and progeny from a well-watered or drought parental environment (Gen.0), from the Rock Springs site. Asterisks represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 35. Tap root diameter (mm) in progeny from the field, from a well-watered or drought parental (Gen.0) environment. Asterisks represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 36. Tap root diameter (mm) in progeny from the field, grown in a well-watered or drought environment (Gen.1), and progeny from a well-watered or drought stressed parental environment (Gen.0). Asterisks represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 37. Basal root diameter (mm) in progeny from the field, from a well-watered and drought parental (Gen.0) environment. Asterisks represent significant differences between treatments. Progeny were grown at the Rock Springs, PA site under drought and well-watered conditions, and harvested at growth stage R2. Figure 38. Stomatal conductance in progeny from the field, from a well-watered and drought parental (Gen.0) environment. Stomatal conductance was measured the day prior to harvest. Progeny were grown at the URBC site under drought and well-watered conditions. Asterisks represent significant differences between treatments. ix

10 Figure 39. Seed P concentration (micromoles) in seeds from a high and low P parental environment (Gen.0). Asterisks represent significant differences between treatments. Figure 40. Shoot-borne root number in progeny from the field, from a low and high P parental environment (Gen.0). Asterisks represent significant differences between treatments. Progeny were grown in the field under low and high P and harvested at growth stage R2. Figure 41. Basal root whorl number in progeny from the greenhouse 2011, from a low and high P parental environment (Gen.0). Asterisks represent significant differences between treatments. Progeny were grown in the greenhouse under low and high P and harvested at growth stage R2. x

11 List of Abbreviations BRN Basal Root Number BRWN Basal Root Whorl Number DAP Days after planting RIL Recombinant inbred line URBC Ukulima Root Biology Center, Limpopo Province, Republic of South Africa VWC Volumetric water content (of soil) P Phosphorus S Stylar position within the pod P Peduncular position within the pod Gen.0 Parental generation Gen.1 Progeny generation xi

12 Acknowledgements I would like to very much thank my adviser, Kathleen Brown, for her phenomenal support, guidance, constructive advice, and patience throughout my time in the lab and in completing this thesis. I d like to thank Jonathan Lynch and Kathleen Brown for their extensive support and instruction, and for the opportunity to be a part of their outstanding lab. I would also like to thank my committee member, Dawn Luthe, for her time in providing support and advice in my thesis. Thank you to Dr. Teh-Hui Kao and the Plant Biology program for the support and opportunity to be in the program. Thank you to Bob Snyder for his patience and advice in all things laboratory, field, and greenhouse, and thank you to Scott Diloreto for helping me with all my greenhouse experiments. Thank you to all lab members, staff, volunteers, and especially Jimmy Burridge and Katy Barlow for providing advice and help in working with common bean, and for assisting in field and greenhouse harvests. I d like to thank Katy Barlow and Virginia Vere Kapachika Chisale for assisting with my initial yield harvest of the parental generations in the field. Thank you to CIAT and Dr. James Kelly for providing the seed. Thank you to all family and friends who supported me through my time at Penn State. xii

13 PARENTAL EFFECTS AND PROVISIONING UNDER DROUGHT AND PHOSPHORUS STRESS IN COMMON BEAN 1. Introduction Common bean (Phaseolus vulgaris) is the primary source of dietary protein in many developing nations, yet produces only 20 to 30 percent of yield potential, primarily due to drought, low nutrient soils, and poor pest and disease control (Wortmann et al., 1998). Many soils used for common bean growth in developing nations within Latin America and Africa are severely deficient in phosphorus (P), and are prone to severe drought. Many farmers in these areas do not have access to fertilizer or water for irrigation, resulting in severely reduced yield due to nutrient and drought stress. Root architectural and morphological traits beneficial for water and P acquisition have been identified, aiding the production of genotypes that thrive in drought or low P soils. Genotypes with these traits have been tested for performance in stressful conditions, but performance of the progeny of plants grown under stress has not been formally tested. Since many farmers in developing nations collect seed for the next year s crop from parent plants grown in low phosphorus and/or drought, it is important to understand how progeny from a stressed parental environment perform relative to progeny from non-stressed parental environment. Several studies have explored how the parental environment impacts progeny traits, independent of the expected genetic contribution of the parent plant. This phenomenon is defined as environmental parental effects. Parental effects have been studied for various abiotic stresses including salinity stress (Amzallag, 1994), shading (Causin, 2004, Galloway, 2005), overall low fertility, nitrogen stress, P stress, and drought, which will be further discussed. Parental effects may include structural or physiological responses in progeny triggered by the parental environment, where responses may or may not be exaggerated when the progeny are grown in similar environmental conditions as the parent plant. Phenotypic plasticity is defined as the capability of an organism to alter its phenotype in response to the current environment. In some cases, parental effects impact the level of plasticity of certain traits in the progeny. In other cases, parental effects are constitutive, independent of the current progeny environment. Many parental effects may serve as a mechanism to precondition progeny adaptation to a similar adverse environment as the parent plant, although this may not always be the case. Parental provisioning of seeds may be reduced due to the stressful environment, resulting in progeny with reduced performance, fitness, and competitive ability. Parental effects are also largely dependent on species and genotype, demonstrated by the present literature. Little is known about parental effects of nutrient and drought stress on root traits. This thesis investigates the effects of parental phosphorus or drought stress on progeny root, seed, and shoot traits when progeny grown under similar stressful conditions. Progeny traits are also examined in seeds that developed in different positions within the pod and from pods that developed early or later on the parent plant. Understanding how parental provisioning of seeds based on pod position and developmental time under normal growing conditions may help eliminate a potential source of parentally-induced variation in root traits in phosphorus and drought studies. 1

14 1.1 Parental Effects of Seed Position in the Pod and Pod Developmental Time Few studies have examined whether seed position within fruiting bodies, or fruit developmental time relative to other fruits on the same parent plant influence progeny growth and traits. Seeds from different pod positions (Figure 1) and/or different pod developmental times may differ in allocation of resources from the parent plant, potentially impacting seed weight and levels of nutrients and resources important during seedling establishment. Rocha and Stephenson (1990) found that seeds from the stylar end of the pod in Phaseolus coccineus had greater mass, likely due to primary fertilization of ovules at the stylar end of the pod, thus having a competitive advantage for parental resources during seed filling (Rocha & Stephenson, 1991). Seeds from pods that developed earlier when resources on the parent plant are plentiful are also hypothesized to have greater allocation of resources into the seed, relative to seeds from pods that developed later on the parent plant. This thesis explores how seed position in the pod and seeds from different pod developmental times affects seed and root traits in common bean. Studies on other species have explored similar questions regarding seed position within the fruiting body and its effects on seedling traits. For instance, Cheplick and Sung (1998) found that seeds from the lower part of the panicle in Triplasis purpurea had greater mass but were fewer in number relative to seeds on the upper part of the panicle. Seeds from the lower part of the panicle with greater mass also had greater seedling shoot and root dry weight, however whether this was due to differences in seed mass or other factors related to seed position on the panicle was unknown. Similarly, Wulff (1986) found that seed weight was correlated with seedling root dry weight, total seedling dry weight, and root length, but did not consider seed position within the fruiting body in the study. Research focusing on seed position within the fruiting body often found differences in seed weight, thus creating difficulty in distinguishing between seed position or weight in explaining results. Susko et al. (2000) distinguished between seed size and seed position within the fruit, examining their effects on seedling traits in Alliaria petiolata. In this study, smaller seeds germinated early, had later primary leaf emergence, and grew taller, whereas seed position affected the time of emergence of the first true leaf. Figure 1. Diagram of a common bean pod with seeds at the stylar end of the pod, furthest from the petiole, and seeds at the peduncular end of the pod, closest to the petiole. 1.2 Parental Effects of Drought Stress There is a diversity of results from research focused on the effects of parental drought on progeny traits, depending on the plant species. Hill et al. (1986) found that parent soybean plants under drought stress during seed fill produced progeny with lower individual seed weight and volume, potentially from limited resources under stressful conditions and a shortened seed filling duration due to drought conditions (Meckel et al., 1984). Another study found that Impatiens progeny from parental drought had reduced shoot biomass when grown in well-watered conditions independent of seed mass, but did not examine root traits (Rigenos et al., 2007). This study also found adaptive responses such as reduced stomatal conductance in progeny grown under drought, but did not find differences in stomatal conductance in response to the parental environment. In contrast, Sultan (1996) found that Polygonum persicaria parent plants grown under drought produced less offspring, but greater mass per seed. These seedlings also had greater seedling biomass and root 2

15 length when grown in well-watered conditions, relative to progeny from a well-watered parental environment. Beaton & Dudley (2010) showed a similar positive correlation in Dipsacus fullonum progeny from parental drought, between seed mass and tolerance to drought. 1.3 Parental Effects of Nutrient Stress Research focused on parental effects of nutrient stress have examined parental environments with overall low soil nutrition, low nitrogen, and low phosphorus. Parrish and Bazzaz (1985) found that seeds from a high nutrient parental environment were larger in volume and outcompeted seeds from a low nutrient parental environment. Arssen and Burton (1990) examined Senecio vulgaris progeny of parents from low fertility soils and found that progeny had lower seed mass, lighter seedling biomass, and delayed germination relative to progeny from parents grown in high fertility soil. However, progeny from parents grown under low fertility survived longer in low soil fertility relative to progeny from parents grown in high fertility soil. These results were counter to expectations based on seed mass, thus other potentially adaptive parental effects may explain longer seedling survivorship under low soil fertility. Plants may also respond to a low nutrient environment by increasing the root:shoot ratio, to enhance soil exploration and surface area for nutrient uptake. Seedlings of Polygonum persicaria from a low nutrient parental environment (low NPK) had a greater root:shoot ratio relative to seedlings from a high nutrient parental environment (Sultan, 1996). Seedlings from a low nutrient parental environment had lower total biomass, likely due to poor parental provisioning. However, greater root:shoot ratio in seedlings from stressed parental conditions suggests an adaptive mechanism that may increase seedlings competitive ability to acquire nutrients in low fertility conditions. Similar studies have explored parental effects specifically from nitrogen stress. A study examining Sinapis arvensi found delayed germination in progeny from a low nitrogen parental environment (Luzuriaga et al, 2005). Since S. arvensi evolved in unpredictable environments, delayed germination is likely an adaptive mechanism to wait and tolerate stressful conditions until the environment is more favorable for growth. Latzel et al. (2010) found that, in two Plantago species, progeny from a low nitrogen parental environment showed greater leaf biomass than progeny from a high nitrogen parental environment when grown in low nitrogen conditions, but not when grown under high nitrogen conditions. This suggests a parental effect that preconditions progeny to a low nitrogen environment, resulting in a better regenerative strategy. Few studies have explored parental effects from P stress. Yan et al. (1995) found that seed size and total seed phosphorus were correlated with root dry weight in P. vulgaris 35 days after planting, especially when parent plants were from a low P environment. Another study on parental effects from different soil P applications in wheat found that heavier seeds were correlated with greater seed P content, and that seedling shoot dry weight and root weight at 3 weeks after germination were correlated with seed P content (Derrick & Ryan, 1998). Similarly, Vandamme et al. (2015) found that seed weight and root length were correlated in soybean up to growth stage V3 (three trifoliates), especially when parents grew under low P conditions. Austin (1966) explored parental effects of P stress in watercress (Rorippa nasturtium aquaticum L. Hayek). In this study, progeny from P stressed parent plants had less biomass at 7-9 weeks, but there was no difference between progeny from contrasting parental environments at weeks. However, progeny from stressed parent plants had reduced yield, likely due to poor parental provisioning during seed filling. 1.4 Root System of Common Bean This thesis will explore how parental effects affect different root classes within the common bean root system (Figure 2), including shoot-borne roots, basal roots, tap root, lateral roots, and root hairs borne on 3

16 basal and tap roots. Different measurements were performed depending on the root class, including length, density, diameter, and angle. Figure 2. Root classes within the common bean root system, including shoot-borne roots, basal roots, lateral roots, and the tap root. Shoot-borne roots are important in scavenging for topsoil P, and basal roots play roles in both P and water acquisition. 4

17 2. Materials and Methods 2.1. Root and Shoot Measurements Harvested plants from the field and greenhouse were evaluated for both shoot and root traits at flowering (growth stage R2). Prior to field harvests, stomatal conductance was measured on a representative plant within each subplot. Representative, young but fully expanded leaves was selected for measurements. During harvest, shoots were separated from the root system and dried for shoot dry weight. Roots from greenhouse studies were separated from shoots, washed, and stored in 70% ethanol for future evaluation. Roots from field studies were separated from shoots, washed, and immediately evaluated. The tap roots were measured for diameter 1 cm from attachment, number of lateral roots borne on the tap root, and were measured for length in greenhouse studies. Basal roots were evaluated for BRN, BRWN, diameter of a representative basal root 1 cm from attachment, angle of a representative basal root (0 = vertical reference), length of a representative basal root (greenhouse studies only), number of lateral roots on a representative basal root, number of nodules on all basal roots, and number of dominant basal roots. Dominant basal roots were identified as at least 4 times larger in diameter than a representative root of the same class within a plant. Shoot-borne roots were measured for total shoot-borne root number, length of a representative shoot-borne root (in greenhouse studies only), and number of dominant shoot-borne roots. Dominant shoot-borne roots were identified as at least 4 times larger in diameter than a representative root of the same class within a plant. In field studies, rooting depth was measured using soil cores. Cores were taken once per subplot in between rows using a 60 cm long, 4 cm diameter coring tube (Giddings Machine Co., Windsor, CO, USA) at mid-flowering to estimate root length density in 10 cm segments. Each segment was washed to extract roots, which were then captured in an image using a flatbed scanner (Epson Expression 1680, 400dpi, Seiko Epson Corporation, Suwa, Japan). Images were analyzed for total root length at each depth using root analysis software WinRHIZO (WinRHIZO Pro version 2002c, Regent Instruments Inc., Quebec, Canada). Roots were also categorized by diameter, identified as tap and basal roots, and mm identified as lateral roots borne on tap and basal roots. 2.2 Parental Effects of Seed Position in the Pod and Pod Developmental Time Plant Material The following genotypes were used: DOR364, BAT 477, TLP19, and B All seeds were provided by CIAT (Centro Internacional de Agricultura Tropical, Cali, Columbia), except B98311 which was developed and provided by Dr. James Kelly at Michigan State University. DOR364 and BAT477 have an intermediate erect bush growth habit, and are from the Mesoamerican gene pool. TLP19 and B98311 have a type II growth habit and are of the Mesoamerican gene pool Pod and Seed Development Seeds for greenhouse trials were collected from field sites at the Ukulima Root Biology Center (URBC) in the Republic of South Africa (RSA) (24 6 E, 28 1 S), in 2012 and at the Russell E. Larson Experimental Farm of the Pennsylvania State University at Rock Springs, PA (40 43 N, W), in Pods on parent plants were tagged and dated at initial pod elongation (growth stage R3), on pods that were cm long. Pods were tagged with the date every Friday from March 12, 2012 April 20, Pods were collected from March 17, 2012 representing early developing pods, and March 30, 2012 representing late developing pods. Pods from earlier and later dates were not collected due to a limited number of seeds from pods for future experiments. Seeds were also collected from the stylar and 5

18 peduncular positions within pods on the same parent plants. Only seeds from pods with complete filling (all seeds are filled within a pod), and at least four seeds per pod were collected. Seeds from stylar and peduncular positions were collected from a variety of pod developmental dates Root Measurements Each replication was randomly assigned to a different position within the greenhouse. Plants were harvested and roots were evaluated according to section Greenhouse Trails Pots were filled with media comprising of 50% vermiculite (Whittemore Companies Inc.), 30% medium ( mm) commercial grade sand (Quikrete Companies Inc., Harrisburg, PA, USA), and 20% perlite (Whittemore Companies Inc.), by volume. All components were mixed evenly throughout each pot. Pots were fertigated daily through drip irrigation with 2 liters of ¼ strength Epstein s nutrient solution, containing (in mm) 1.5 KNO 3, 1 Ca(NO 3) 2 4H 2O, 0.25 MgSO 4 7H 2O, 0.06 (NH 4) 2SO 4, 0.4 NH 4H 2PO 4 and (in um) 50 KCL, 25 H 3BO 3, 2 MnSO 2 H 2O, 2 ZnSO 4 7H 2O, 0.5 CuSO 4 H 2O, 0.5 (NH 4) 6MO 7O 24 4H 2O, and 50 Fe-NaEDTA. One tablespoon of 1% Marathon pesticide was applied to each pot on August 24, Seeds were weighed individually then surface sterilized with 10% bleach solution for 1 minute and rinsed with deionized water. Trials were planted in the greenhouses located at The Pennsylvania State University, University Park PA (4049 N, 7749 W). Two seeds per pot were directly planted into 19-liter pot, and one seedling was selected for uniform growth at 3 days after emergence. Plants were grown under greenhouse lights (Quantum Meter, Apogee instruments inc., Model LQS 50-3M), programmed to turn on at 6:00 AM and off at 6:00 PM. There were five replications per genotype per treatment, each placed in randomly selected locations in the greenhouse. Replications were planted every other day to allow for staggered harvests. Seeds were planted every other day from August 10, 2012, through August 20, Harvests took place every other day from October 1, 2012, through October 11, Statistical Analysis A randomized complete block design was used in greenhouse studies. Replications were also blocked in time (to allow time between harvests) and space. Statistical analyses were performed using Minitab 16 Statistical Software (State College, PA: Minitab, Inc., 2010). Data was analyzed using a two-way ANOVA, with a significance level set at p Log transformed data were used if normality assumptions were not met. If log transformed data were not normally distributed, data was analyzed using a Kruskal-Wallis test. Regression analysis was used to test allometric relationships between traits. 2.3 Parental Effects of Drought Stress Plant Material The following genotypes were used: SER118, SER16, SEA5, all from the Mesoamerican gene pool, and eleven RILs (recombinant inbred lines) from the ALB population (SER 16 x (SER 16 x G Q)). The ALB population is an inter-specific cross between the small seeded SER 16 (P. vulgaris), developed for drought tolerance, and the large seeded G Q (P. coccinius). All seeds were provided by CIAT (Centro Internacional de Agricultura Tropical, Cali, Columbia). The following ALB RILs were used: 1, 120, 18, 213, 23, 24, 5, 6, 67, 91, and 96. All genotypes were measured for individual seed weight and seedling BRN. The following subset of genotypes were used to measure seedling traits: 6

19 ALB1, ALB5, ALB6, ALB67, ALB96, SER118, and SER16. The following subset of genotypes were used in field trials: ALB23, ALB5, ALB6, ALB91, SER16. Parent plants were grown under a well-watered or moderate drought conditions at the Rock Springs site in Parent plants grown in a terminal drought environment showed a shoot biomass reduction significant at p < , based on a 2 way ANOVA analysis. Parent plants did not display differences in BRN between treatments Seed and Seedling Trials Seeds were weighed then surface sterilized with 10% bleach solution for 1 minute and rinsed with deionized water. Seeds were then placed 2 inches apart in 79 lb roll-up germination paper (Anchor Paper Co., St. Paul, MN) and placed into a 500 ml beaker with 30 ml of 0.5mM calcium sulfate solution. The beaker of seed roll-ups were then placed in a dark germination chamber at 28 C for 72 hours, then 48 hours under light. Seedlings were preserved in 70% ethanol for further analyses. Seedlings were evaluated for total seedling dry weight, basal root number (BRN), basal root whorl number (BRWN), tap root length, basal root length, length of lateral roots borne on the tap root, length and density of root hairs borne on the tap root, and length and density of root hairs borne on basal roots. Four seedling replicates were used per genotype per parental treatment for analysis of all traits. Root lengths of the tap root and a representative basal root were measured, and a representative lateral root borne on the tap root was measured for length. Representative areas were also selected on both the tap and basal roots for root hair length and density. Roots were stained with 0.05% toluidine blue dye to observe root hairs under the dissecting microscope (SMZ-U, Nikon, Tokyo, Japan), and a 1mm segment for both root hair length and density were captured with an attached camera (NIKON DS-Fi1, Tokyo, Japan). Images were used to evaluate root hair length and density using Image J (version 1.32j National Institutes of Health, USA). The number of root hairs per 1mm representative section was used to measure root hair density, and the length of three representative root hairs were measured within images for tap and basal roots Field Trials: Rock Springs, PA Trials were located at the Russell E. Larson Experimental Farm of the Pennsylvania State University at Rock Springs, PA (40 43 N, W) using two rain-out shelters to impose drought treatments. The soil was a Murrill silt loam 12 (fine-loamy, mixed, semi-active, medic Typic Hapludult). Rain-out shelters were covered with clear greenhouse plastic (0.184 mm, Griffin Greenhouse and Nursery Supply, Morgantown, PA), moving over plants when precipitation was sensed, then reversing direction to expose the plots at the end of a rainfall event. Two control plots were located adjacent to rain-out shelters. Both rain-out shelter plots and control plots were 88 ft (26.8 m) x 28 ft (8.5 m). Each plot contained 24 3-row by 2m subplots. There were 4 subplots per genotype per parental treatment in each plot. Rows were planted 60cm apart, and plants were planted 10cm apart. Prior to planting, plots were deep chiseled, harrowed, and scored in early June. Herbicide was applied one week before planting, and standard agronomic pest control was implemented when needed. Trials were planted on June 11, 2012 and a drip irrigation system was installed on June 20, Terminal drought was imposed beginning on June 25, Soil moisture was monitored bi-weekly using a TDR-100 multiplexed time-domain reflectometry system (Campbell Scientific Inc., Logan, UT). Two 20cm probes were buried directly under a row at 15cm and 40cm, in 6 evenly distributed locations within each plot. Stomatal conductance was measured using an 7

20 open system infrared gas-exchange system (LiCor 6400, Li-Cor, Lincoln, NE). Three representative plants per subplot were selected for measurement of stomatal conductance of a representative leaf. Soil cores were taken on August 8, 2012, plants were harvested from August 13-14, 2012, and roots were immediately evaluated according to section Field Trials: Ukulima Root Biological Center (URBC), South Africa Trials were located in a pivot-irrigated field plot at the Ukulima Root Biology Center (URBC) in the Republic of South Africa (RSA) (24 6 E, 28 1 S) in a loamy sandy soil, in There were two field locations within the pivot, one was used as a drought treatment and the other a well-watered treatment. Each location had 4 plots with 3-row by 2m subplots. Within each plot there was one subplot per genotype and parental treatment. Rows were planted 76cm apart, and plants were planted 10cm apart. Prior to planting, plots were deep chiseled, harrowed, and scored in early January. Herbicide was applied one week before planting, and standard agronomic pest control was implemented when needed. Trials were planted on January 19-20, 2012 and drought was imposed starting on February 2, Soil moisture was monitored bi-weekly using a TDR-100 multiplexed time-domain reflectometry system (Campbell Scientific Inc., Logan, UT). Two 20cm probes were buried directly under a row at 15cm and 40cm, in 2 randomly distributed locations within each plot. Stomatal conductance was measured the day prior to harvest using an open system infrared gas-exchange system (LiCor 6400, Li-Cor, Lincoln, NE). Three representative plants per subplot were selected for measurement of stomatal conductance of a representative leaf. Soil cores were taken on March 13, 2012, plants were harvested on March 15, 2012, and roots were immediately evaluated according to section Statistical Analyses Statistical analyses were performed using Minitab 16 Statistical Software (State College, PA: Minitab, Inc., 2010). Data was analyzed using a two-sample T-test or a two-way ANOVA with a significance level set at p Log transformed data were used if normality assumptions were not met, and if log transformed data were still not normally distributed, data was analyzed using a Kruskal-Wallis test. Regression analysis was used to test allometric relationships between traits. 2.4 Parental Effects of Phosphorus Stress Plant Material The following BILFA (bean improvement for low fertility in Africa) genotypes were used: Bf , SER15, SER16, SER43, SER55, SER79, SER83, SER85, and Tiocanela75. BILFA are genotypes screened for tolerance to drought and poor soil nutrition. Parent plants were grown in the field under low and high P at the Russell E. Larson Experimental Farm of the Pennsylvania State University at Rock Springs, PA in 2010, and seeds were collected from high and low P plots Root Analyses Plants were harvested at flowering (growth stage R2) and roots were immediately evaluated according to section 2.1. In addition, leaf P content and yield (pods per plant, seeds per pod, and weight per 100 seeds) were measured. Leaf P content was measured using Murphy-Riley method (Murphy and Riley, 1962) and a Lambda 25 Spectrometer (Perkin-Elmer). 8

21 2.4.3 Seed Weight and P Analysis Seeds were dried at 60 C for two days, weighed, and ground with a Wiley mill, ashed at 500 C for ten hours, then dissolved in 100 mm of hydrochloric acid to prepare samples for testing phosphorus concentration using a Lambda 25 Spectrometer (Perkin-Elmer), based on the Murphy and Riley colorimetric method (Murphy and Riley, 1962) Greenhouse Trials Plant and root trait data were collected from trials in the greenhouses located at The Pennsylvania State University, University Park PA (4049 N, 7749 W) in Seeds were planted on March 14, 2011 and plants were harvested on March 3, During harvest, shoots were separated for leaf area and dry weight analysis, and roots were stored in 70% ethanol and evaluated according to section 2.1. Plants were grown under greenhouse lights (Quantum Meter, Apogee instruments inc., Model LQS 50-3M), programmed to turn on at 6:00 AM and off at 6:00 PM. There were five replications per genotype, per P treatment. Two seeds per pot (one seedling was selected for uniform growth at 3 days after emergence) were directly planted into 19 liter pots containing media with 1% alumina phosphate (Al-P) providing either low P (0.2 um) or sufficient P (150 um) (methodology from Lynch et al., 1990) mixed into the media of 50% vermiculite (Whittemore Companies Inc.), 40% medium ( mm) commercial grade sand (Quikrete Companies Inc., Harrisburg, PA, USA), and 10% perlite (Whittemore Companies Inc.), by volume. All components were mixed evenly throughout each pot. Pots with low P Al-P were fertigated when necessary through drip irrigation with 2 liters of ¼ strength Epstein s nutrient solution, containing (in mm) 1.5 KNO 3, 1 Ca(NO 3) 2 4H 2O, 0.25 MgSO 4 7H 2O, 0.2 (NH 4) 2SO 4, and (in um) 50 KCL, 25 H 3BO 3, 2 MnSO 2 H 2O, 2ZnSO 4 7H 2O, 0.5 CuSO 4 H 2O, 0.5 (NH 4) 6MO 7O 24 4H 2O, and 50 Fe-NaEDTA. Pots with high P Al-P were fertigated daily with 2 liters of ¼ strength Epstein s nutrient solution, containing in mm) 1.5 KNO 3, 1 Ca(NO 3) 2 4H 2O, 0.25 MgSO 4 7H 2O, 0.06 (NH 4) 2SO 4, 0.4 NH 4H 2PO 4 and (in um) 50 KCL, 25 H 3BO 3, 2 MnSO 2 H 2O, 2 ZnSO 4 7H 2O, 0.5 CuSO 4 H 2O, 0.5 (NH 4) 6MO 7O 24 4H 2O, and 50 Fe-NaEDTA Field Trials Trials were located at the Russell E. Larson Experimental Farm of the Pennsylvania State University at Rock Springs, PA (40 44'N, 77 53'W) in The soil was a Murrill silt loam 12 (fine-loamy, mixed, semi-active, medic Typic Hapludult). Four blocks of high P and four blocks of low P were used and soil was tested for P levels by the Agricultural Analytical Services Lab and The Pennsylvania State University prior to planting. In 2010, parent plants were grown in four blocks of low P and four blocks of high P. Low P blocks 1,2,3, and 4 had P levels of 11, 9.5, 11, 9.5 (ppm), respectively, and high P blocks 1,2,3, and 4 had 63, 87, 51.5, 71.5 (ppm), respectively. High P blocks 1,2, 3, and 4 had P levels of 74, 114, 108, and 106 ppm, respectively. In 2011, progeny were grown in four blocks of low P and four blocks of high P. Low P blocks 1,2,3, and 4 had P levels of 14,14,14, and 15 ppm, respectively. Each block had one replication per genotype, per parental P treatment that consisted of 3 2m rows. Rows were planted 76 cm apart, and plants within rows were planted every 10 cm. A 1m buffer was planted around the border of each block. Prior to planting, plots were deep chiseled, harrowed, and scored in early January. Trails were planted on June 8, Herbicide was applied one week before planting, and drip irrigation was installed on June 17, Standard agronomic pest control was implemented when needed. Trials were harvested on August 8, 2011, and plants were immediately analyzed for shoot and root traits according to section 2.1. In addition, yield (pods per plant and seeds per pod, and weight per 100 seeds) were measured. 9

22 2.4.6 Statistical Analyses A randomized complete block design was used in both field and greenhouse studies. Replications in greenhouse studies were blocked in time (to allow time between harvests) and space. Replications in field experiments were blocked in space. Statistical analyses were performed using Minitab 16 Statistical Software (2010), State College, PA: Minitab, Inc. Data were analyzed using a two-way ANOVA with a significance level set at p Log transformed data were used if normality assumptions were not met, and if log transformed data were still not normally distributed, data was analyzed using a Kruskal-Wallis test. Regression analysis was used to test allometric relationships between traits. 10