PARENTAL EFFECTS AND PROVISIONING UNDER DROUGHT AND ...

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

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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.

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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.

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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.

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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……………………………………….…………….…………………….……….…… 1

1. Introduction……………………………………………………………………………………1

2. Materials and Methods…………………………………...…………………….……………...5

3. Results………………………………………….…………………………………………….11

4. Discussion ………………………….……….………….….………...……….….……….….32

Appendix A Additional Tables: Parental Effects of Seed Position in the Pod and Pod Developmental

Time………………………….……………….…….…………….……………….…….………………...39

Appendix B Additional Tables and Figures: Parental Effects of Drought Stress…………………………41

Appendix C Additional Tables: Parental Effects of Phosphorus Stress………….……………….………51

References………………………………………………………………………………….……………...52

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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 (well-

watered 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.

vii

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 = 2.55 + 26.3x, and for

the stylar position, y = 13.1 - 12.7x.

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 = 1.19 + 36.2x, and

for late developing pods, y = 3.29 + 19.7x.

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 = 4.28 +

18.1x, and for the stylar position, y = 4.91 + 11.2x.

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 =

- 4.61 + 49.4x, and for the stylar position, y = 9.58 - 14.9x.

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 = - 0.57 + 28.1x, and for the stylar position, y = 4.09 + 8.5x.

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.

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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 = 41.4 – 120x.

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


Figure 18. Seedling dry weight in progeny from a well-watered and drought parental (Gen.0) field


Figure 19. Density of root hairs borne on seedling tap roots (# of hairs/ mm2) in progeny from a well-

watered 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


Figure 21. Seedling basal root length in progeny from a well-watered and drought parental (Gen.0) field


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


treatments.

Figure 24. Length of lateral roots borne on seedling tap roots (cm) in progeny from a well-watered and


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.

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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.

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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.

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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

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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.

1

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.

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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

3

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 16-20

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

4

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.

5

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, 0.6-1.5 identified as tap and basal roots, and

0.05-0.6 mm identified as lateral roots borne on tap and basal roots.


2.2.1 Plant Material

The following genotypes were used: DOR364, BAT 477, TLP19, and B98311. 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.

2.2.2 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, 77°56’W), in

2011. Pods on parent plants were tagged and dated at initial pod elongation (growth stage R3), on pods

that were 0.5 – 1 cm long. Pods were tagged with the date every Friday from March 12, 2012 – April 20,

2012. 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

6

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.

2.2.3 Root Measurements

Each replication was randomly assigned to a different position within the greenhouse. Plants were

harvested and roots were evaluated according to section 2.1.

2.2.4 Greenhouse Trails

Pots were filled with media comprising of 50% vermiculite (Whittemore Companies Inc.), 30% medium

(0.3-0.5 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 KNO3, 1 Ca(NO3)2·4H2O, 0.25 MgSO4·7H2O, 0.06 (NH4)2SO4, 0.4 NH4H2PO4

and (in uM) 50 KCL, 25 H3BO3, 2 MnSO2·H2O, 2 ZnSO4·7H2O, 0.5 CuSO4·H2O, 0.5

(NH4)6MO7O24·4H2O, and 50 Fe-NaEDTA. One tablespoon of 1% Marathon pesticide was applied to

each pot on August 24, 2012.

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, 2012. Harvests took place every other day from October 1, 2012, through October 11, 2012.

2.2.5 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 ≤ 0.05. 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.



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 G35346 – 3Q)).

The ALB population is an inter-specific cross between the small seeded SER 16 (P. vulgaris), developed

for drought tolerance, and the large seeded G35346 – 3Q (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:

7

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

2010. Parent plants grown in a terminal drought environment showed a shoot biomass reduction

significant at p < 0.0001, based on a 2 way ANOVA analysis. Parent plants did not display differences in

BRN between treatments.

2.3.2 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.

2.3.4 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, 77°56’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, 2012.

Terminal drought was imposed beginning on June 25, 2012.

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

8

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 2.1.

2.3.5 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 2012. 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, 2012.

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 2.1.

2.3.6 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 ≤ 0.05. 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


The following BILFA (bean improvement for low fertility in Africa) genotypes were used: Bf13572-5,

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.

2.4.2 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).

9

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).

2.4.4 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 2011. Seeds were planted on March 14, 2011 and

plants were harvested on March 3, 2011. 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 (0.3-0.5 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 KNO3, 1 Ca(NO3)2·4H2O, 0.25 MgSO4·7H2O, 0.2 (NH4)2SO4, and (in uM) 50 KCL, 25

H3BO3, 2 MnSO2·H2O, 2ZnSO4·7H2O, 0.5 CuSO4·H2O, 0.5 (NH4)6MO7O24·4H2O, 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 KNO3, 1 Ca(NO3)2·4H2O, 0.25 MgSO4·7H2O, 0.06 (NH4)2SO4, 0.4 NH4H2PO4 and

(in uM) 50 KCL, 25 H3BO3, 2 MnSO2·H2O, 2 ZnSO4·7H2O, 0.5 CuSO4·H2O, 0.5 (NH4)6MO7O24·4H2O,

and 50 Fe-NaEDTA.

2.4.5 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 2011. 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, 2011. Herbicide was applied one week before planting, and drip irrigation was installed on June

17, 2011. 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.

10

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 ≤ 0.05. 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.

11

3. Results


Parent plants were grown in the field at the Rock Springs site (PA) and the Ukulima Root Biology Center

site (South Africa). Pods were tagged and dated at initial pod elongation (growth stage R3), on pods that

were 0.5 – 1 cm long. Pods tagged on March 17, 2012, represented early developing pods, whereas pods

tagged on March 30, 2012, represented late developing pods. Progeny from different positions (stylar and

peduncular) within the pod and different pod developmental times (early and late) on parent plants were

grown in the greenhouse in PA. Progeny plants were evaluated for differences in seed, root, and shoot

traits, and excavated at growth stage R2 for root and shoot trait analyses.

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.

In three of four genotypes, seed weight (of an individual seed) was greater in seeds that developed in

stylar than in peduncular positions within the pod (BAT477 p < 0.001, DOR364 p = 0.002, TLP19 p =

0.01) (Figure 3). Variability in seed weight across genotypes was relatively low. In three of four

genotypes, seed weight was higher in seeds from earlier developing pods (DOR364 p = 0.054, B98311 p

= 0.046, TLP19 p = 0.056) (Figure 4). Variability in seed weight across genotypes was also relatively

low.

TLP19DOR364BAT477B98311

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Seed W

eig

ht

(gra

ms)

P

S

Position

Seed


0.30

0.25

0.20

0.15

0.10

0.05

0.00

Seed W

eig

ht

(gra

ms)

Early

Late

Date

Development

* *

*

*

* *

12

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.


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.

BRN was greater at growth stage R2 in seeds that developed in the stylar end of the pod in BAT477 (p =

0.016) (Figure 5), and in seeds from earlier developing pods in B98311 (p = 0.038) and BAT477 (p <

0.001) (Figure 6). BAT477 had the greatest difference in seed weight between seed positions, and was

also the only genotype with differences in BRN between seed positions. Similarly, B98311 had the

greatest difference in seed weight between pod developmental times, and was also the only genotype with

differences in BRN between pod developmental times. These consistent patterns suggest that differences

seen in BRN may be explained by differences in seed weight. Variability in BRN was consistently low

across genotypes and treatments.

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 =

2.55 + 26.3x, and for the stylar position, y =

13.1 - 12.7x.

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 = 1.19 + 36.2x, and for late developing

pods, y = 3.29 + 19.7x.


9

8

7

6

5

4

3

2

1

0

Basa

l Root

Num

ber

P

S

Position

Seed


9

8

7

6

5

4

3

2

1

0

Basa

l Root

Num

ber

Early

Late

Date

Development

0.300.250.200.150.10

12

11

10

9

8

7

6

5

Weight Per Seed (BAT477) (grams)

BR

N (

BAT477)

P

S

position

Seed

0.300.250.200.150.10

12

11

10

9

8

7

6

5

Weight Per Seed (grams) (BAT477)

BR

N (

BAT477)

Early

Late

Date

Development

* * *

13

Basal root number (BRN) in greenhouse plants at growth stage R2 and seed weight (per seed) were

correlated in BAT477 at p = 0.174 in seeds from the peduncular position within the pod (R2 = 17.7%, p =

0.174), but were not correlated in seeds from the stylar position within the pod (R2 = 4.6%, p = 0.504)

(Figure 7). BRN and seed weight (per seed) in BAT477 were correlated in seeds from both early

developing pods (R2 = 70.2%, p = 0.001), and late developing pods (R2 = 46.2%, p = 0.015) (Figure 8).

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 = 4.28 +

18.1x, and for the stylar position, y =

4.91 + 11.2x.

Basal root number (BRN) in greenhouse plants at growth stage R2 and seed weight (per seed) were

correlated (all genotypes combined) in seeds from the peduncular position within the pod (R2 = 24.2%, p

< 0.001), but not correlated in seeds from the stylar position within the pod (R2 = 3.1%, p = 0.235)

(Figure 9).

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


0.350.300.250.200.150.10

12

11

10

9

8

7

6

5

4

3

Weight per seed (grams)

BR

N

P

S

Position

Seed


6

5

4

3

2

1

0

Tap R

oot

Dia

mete

r (m

m)

P

S

Position

Seed

*

14

Tap root diameter was greater in progeny at growth stage R2 from the stylar position within the pod in

DOR364 (p = 0.038). (Figure 10). Tap root diameter was not different between seeds from different pod


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.



Root dry weight at growth stage R2 was greater in progeny from the stylar position within the pod in

BAT477 (p = 0.049) (Figure 11). Root dry weight was not different between seeds from different pod


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 = - 4.61 + 49.4x, and

for the stylar position, y = 9.58 -

14.9x.

Root dry weight in greenhouse plants at growth stage R2 and seed weight (per seed) were correlated in

BAT477, in seeds from the peduncular position within the pod (R2 = 46.5%, p = 0.015), but not correlated

in seeds from the stylar position within the pod (R2 = 8.9%, p = 0.446) (Figure 12).

0.300.250.200.150.10

8

7

6

5

4

3

2

1

Weight Per Seed (grams) (BAT477)

Root

Dry

Weig

ht

(gra

ms)

(BAT477) P

S

Position

Seed


9

8

7

6

5

4

3

2

1

0

Root

Dry

Weig

ht

(gra

ms)

P

S

Position

Seed

*

15

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 = -

0.57 + 28.1x, and for the stylar

position, y = 4.09 + 8.5x.

Root dry weight in greenhouse plants at growth stage R2 and seed weight (per seed) were correlated (all

genotypes combined) in seeds from the peduncular position within the pod (R2 = 18.2%, p = 0.002), but

not correlated in seeds from the stylar position within the pod (R2 = 1.2%, p = 0.465) (Figure 13).

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.

Number of lateral roots per basal root at growth stage R2 was greater in progeny from the stylar position

within the pod in DOR364 (p = 0.02) (Figure 14). The number of lateral roots per basal root was not

different between seeds from early versus late pod developmental times.

0.350.300.250.200.150.10

14

12

10

8

6

4

2

0

Weight per seed (grams)

Root

Dry

Weig

ht

(gra

ms)

P

S

Position

Seed


30

25

20

15

10

5

0

Num

ber

of Late

ral R

oots

per

Basa

l Root

P

S

Position

Seed

*

16

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 = 41.4 – 120x.

Number of lateral roots per basal root in greenhouse plants at growth stage R2 and seed weight (per seed)

were negatively correlated at p = 0.163 in DOR364, in seeds from the peduncular position within the pod

(R2 = 18.5%, p = 0.163), but not correlated in seeds from the stylar position within the pod (R2 = 1.7%, p

= 0.69) (Figure 15). In all genotypes combined, the number of lateral roots per basal root in greenhouse

plants at growth stage R2 and seed weight (per seed) were not correlated in seeds from the peduncular

position within the pod or in seeds from the stylar position within the pod.

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.

B98311 BAT477 DOR364 TLP19

Seed Weight (Early>Late) p = 0.046 F = 4.52

Seed Weight (S>P) p < 0.001 F = 106.81

Seed Weight (S>P) p = 0.002 F = 13.05

Seed Weight (S>P) p = 0.01 F = 8.03

BRN (Early>Late) p = 0.038 F = 4.95

BRN (Early>Late) p = 0.016 F = 6.98

Tap Root Diameter (S>P) p = 0.038 F = 4.96

Seed Weight (Early>Late) p = 0.056 F = 4.13

BRN (S>P) p < 0.001 F = 20.31

Number of Lateral Roots per Basal Root (S>P) p = 0.02 F = 6.37

Root Weight (S>P) p = 0.049 F = 4.39

Overall, seeds from stylar (S) positions in the pod and from earlier developing pods had seed and root

traits greater in number or weight relative to seeds from peduncular (P) positions in the pod and later

developing pods. Similar patterns across genotypes between seed weight and BRN suggest differences in

BRN may be directly or indirectly explained by differences in seed weight. For instance, BAT477 and

0.280.260.240.220.200.18

30

25

20

15

10

Weight Per Seed (grams) (DOR364)

Num

ber

of Late

ral R

oots

per

Basa

l Root

(DO

R364)

P

S

Position

Seed

17

B98311 showed the greatest differences in seed weight between pod positions and pod developmental

times, respectively. The same genotypes also displayed the greatest differences in BRN between

treatment groups. Further, genotypes with no difference in seed weight between pod positions and/or pod

developmental times did not have differences in root traits between treatments.

Regression analysis was used to determine whether there were relationships between seed weight and root

traits. Correlations were found between seed weight and BRN in the peduncular position in all genotypes

combined (R2 = 24.2%), in the peduncular position in BAT477 (R2 = 17.7%), and in early and late

developing pods in BAT477 (R2 = 70.2%, R2 = 46.2%, respectively). There was no relationship between

BRN and seed weight from the stylar position within the pod.

Relationships were also found between seed weight and root dry weight in seeds from the peduncular

position in all genotypes (R2 = 18.2%) and in BAT477 (R2 = 46.5%), and a negative correlation was

found between seed weight and the number of lateral roots per basal root in the peduncular position in

DOR364 (R2 = 18.5%).

Root traits measured in the study that did not result in significant differences between either pod position

or pod developmental time treatments included shoot-borne root number, dominant shoot-borne root

number, dominant basal root number, basal root length, basal root diameter, and tap lateral root number.

ANOVA tables of results are included in the appendix.

18


Progeny from parent plants grown in drought and well-watered field environments were evaluated for

seed and seedling (5-day old) traits, and for mature plant and root traits at growth stage R2 in the field at

Rock Springs, PA (2012) and the Ukulima Root Biology Center (URBC) in South Africa, 2012. Drought

was imposed two weeks after planting in the Rock Springs and URBC trials. Parent plants were grown

under a well-watered or moderate drought conditions at the Rock Springs site in 2010. Parent plants

grown in a terminal drought environment showed a shoot biomass reduction of 46% (p < 0.0001).

3.2.1 Seed and Seedling Traits

Progeny from drought and well-watered field environments were evaluated for seed and seedling (5-day

old) traits. Seeds were geminated in roll-up germination paper with 0.5mM calcium sulfate solution, and

placed in a dark germination chamber for 72 hours, then 48 hours under light.

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.

All genotypes had significantly higher individual seed weight in seeds from well-watered parental

conditions (p ≤ 0.038) except ALB67 (p = 0.95) (Figure 16). Reduction in individual seed weight ranged

from 0% in ALB67 to 29% in ALB1.

SER

16

SER

118

SEA5

ALB

96

ALB

91

ALB

67

ALB

6

ALB

5

ALB

24

ALB

23

ALB

213

ALB

18

ALB

120

ALB

10.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

See

d W

eight

(gra

ms)

Well watered

Drought

Gen.0 Treatment

*

*

*

*

* * * *

*

* *

*

*

19

Figure 17. Seedling basal root

number in progeny from a well-

watered and drought parental

(Gen.0) field environment.


differences between treatments.

Seedling BRN was greater in progeny from a well-watered parental environment in six of fourteen

genotypes (p ≤ 0.05) (Figure 17). Reduction in seedling BRN ranged from 7% in ALB5 to 36% in

ALB120.

Figure 18. Seedling dry weight in

progeny from a well-watered and

drought parental (Gen.0) field



treatments.

Seedling dry weight was greater in seedlings from a well-watered parental environment, in ALB1 (p =

0.04) and ALB67 (p = 0.004) (Figure 18).

SER

16

SER

118

SEA5

ALB96

ALB91

ALB67

ALB6

ALB5

ALB24

ALB23

ALB213

ALB18

ALB120

ALB1

12

10

8

6

4

2

0

Seedlin

g B

asa

l Root

Num

ber

Well watered

Drought

Gen.0 treatment

*

*

* *

* *

SER16SER118ALB96ALB67ALB6ALB5ALB1

0.018

0.016

0.014

0.012

0.010

0.008

0.006

0.004

0.002

0.000

Seedlin

g D

ry W

eig

ht

(gra

ms)

Well Watered

Drought

Gen.0 Treatment

*

*

20

Figure 19. Density of root hairs

borne on seedling tap roots (# of

hairs/ mm2) in progeny from a well-


(Gen.0) field environment. Asterisks

represent significant differences

between treatments.

In ALB67, the density of root hairs borne on seedling tap roots was greater in seedlings from a drought

stressed parental environment (p = 0.006) (Figure 19). No genotypes had significant effects between

parental treatments in density of root hairs borne on basal roots.

Figure 20. Seedling tap root length (cm) in

progeny from a well-watered and drought

parental (Gen.0) field environment. Asterisks


treatments.

Figure 21. Seedling basal root length (cm) in

progeny from a well-watered and drought

parental (Gen.0) field environment. Asterisks


treatments.

In ALB67, seedling tap root length was greater in progeny from a well-watered parental environment (p <

0.001) (Figure 20). In three of seven genotypes, seedling basal root length was greater in seedlings from

a parental well-watered environment (p ≤ 0.054), however in ALB1 seedlings basal root length was

greater in seedlings from a parental drought environment (p = 0.018) (Figure 21).


20

15

10

5

0

Seedlin

g T

ap R

oot

Length

(cm

)

Well Watered

Drought

Gen.0 Treatment


7

6

5

4

3

2

1

0

Seedlin

g B

asa

l Root

Length

(cm

)

Well Watered

Drought

Gen.0 Treatment


140

120

100

80

60

40

20

0Densi

ty o

f R

oot

Hairs

Born

e o

n S

eedlin

g T

ap R

oots

(#

Hairs/

mm

2)

Well Watered

Drought

Gen.0 Treatment

*

*

*

*

*

*

21

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


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


Genotypes varied in the length of root hairs borne on the seedling tap root (Figure 22). Some genotypes

(ALB6 and SER16) had longer root hairs on the tap root when seedlings were from a parental well-

watered environment (p ≤ 0.045), whereas other genotypes (ALB1 andd ALB96) had longer root hairs on

the tap root when seedlings were from parental drought (p ≤ 0.015). Genotypes also varied the length of

root hairs borne on seedling basal roots (Figure 23). Some genotypes (ALB67 and SER118) had longer

root hairs on seedling basal roots when seedlings were from a parental well-watered environment (p ≤

0.044), whereas other genotypes (ALB5, ALB96, and SER16) had longer root hairs on seedling basal

roots when seedlings were from parental drought (p ≤ 0.028).

Figure 24. Length of lateral roots

borne on seedling tap roots (cm) in

progeny from a well-watered and

drought parental (Gen.0) field



treatments.

In ALB67, the length of lateral roots borne on seedling tap roots was greater in seedlings from a well-

watered parental environment (p = 0.001) (Figure 24).

SER1

6

SER1

18

ALB9

6

ALB6

7AL

B6AL

B5AL

B1

1.0

0.8

0.6

0.4

0.2

0.0

Length

of R

oot

Hairs

Born

e o

n S

eedlin

g T

ap R

oots

(m

m)

Well Watered

Drought

Gen.0 Treatment

SER1

6

SER1

18

ALB9

6

ALB6

7AL

B6AL

B5AL

B1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Length

of Root Hairs

Born

e o

n S

eedlin

g B

asa

l Roots

(m

m)

Well Watered

Drought

Gen.0 Treatment


2.0

1.5

1.0

0.5

0.0

Length

of Late

ral R

oots

Born

e o

n S

eedlin

g T

ap R

oots

(cm

)

Well Watered

Drought

Gen.0 Treatment*

* *

*

*

* *

* * *

22

3.2.2 Mature Plant Traits

Progeny from parental drought or well-watered field environments were evaluated for mature plant and

root traits at growth stage R2 in the field in well-watered or drought treatments, at Rock Springs, PA

(2012) and the Ukulima Root Biology Center (URBC) in South Africa, 2012. Drought was imposed two

weeks after planting in the Rock Springs and URBC trials.

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.

Soil volumetric water content (VWC) in the Rock Springs site showed relatively consistent differences

between drought and well-watered treatments throughout the study. This site imposed drought through a

rain out shelter system, allowing greater control of drought treatments. Drought was imposed at the

URBC site by eliminating irrigation starting two weeks after planting.

Figure 27. Shoot dry weight in

progeny from the field, from a well-

watered and drought (Gen.0) parental



treatments. Progeny were grown at

the URBC site under drought and

well-watered conditions, and


15-M

ar

9-Mar

7-Mar

5-Mar

2-Mar

29-Feb

27-F

eb

24-F

eb

22-F

eb

0.110

0.105

0.100

0.095

0.090

0.085

0.080

Soil Volu

metr

ic W

ate

r C

onte

nt

%

Drought

Well Watered

Treatment

3-Aug25-Jul23-Jul22-Jul18-Jul16-Jul13-Jul11-Jul9-Jul

0.36

0.32

0.28

0.24

0.20

Soil Volu

metr

ic W

ate

r C

onte

nt

%

Drought

Well Watered

treatment

SER16ALB91ALB6ALB5ALB23

12

10

8

6

4

2

0

Shoot

Dry

Weig

ht

(gra

ms)

Well Watered

Drought

Gen.0 Treatment

*

*

23

Shoot dry weight was greater in progeny from a well-watered parental environment in SER16 (p = 0.008)

and was greater in progeny from a drought parental environment ALB23 (p < 0.001) (Figure 27).

Figure 28. Basal root diameter (mm)

in progeny from the field, from a

well-watered and drought (Gen.0)

parental environment. Asterisks


between treatments. Progeny were

grown at the URBC site under

drought and well-watered conditions,

and harvested at growth stage R2.

Basal root diameter was greater in progeny from a well-watered parental environment in three of five

genotypes (p ≤ 0.006) (Figure 28). Data for ALB23 drought parental environment not available.

Figure 29. Basal root angle of a

representative root angle in progeny

from the field, from a well-watered

and drought parental (Gen.0)







Basal root angle (0 = vertical reference) was shallower in progeny from a drought parental environment in

ALB5 (p = 0.003) and ALB91 (p = 0.075) (Figure 29).


0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Basa

l Root

Dia

mete

r (m

m)

Well Watered

Drought

Gen.0 Treatment

* * *


70

60

50

40

30

20

10

0

Basa

l Root

Angle

Well Watered

Drought

Gen.0 Treatment* *

24

Figure 30. Dominant shoot-borne

root number in progeny from the

field, from a well-watered and

drought parental (Gen.0)







Dominant shoot-borne roots were identified as at least four times larger in diameter than a representative

shoot-borne root on a plant, and the number of dominant shoot-borne roots ranged between zero and six

per plant. Dominant shoot-borne root number was greater in progeny from a drought parental

environment in ALB23 (p < 0.001) (Figure 30). Total shoot-borne root number was not affected by the

parental drought environment. Dominant shoot-borne root angle was also tested, but was not different

between parental treatments.

Figure 31. Dominant shoot-borne root number in

progeny from the field, from a well-watered and

drought parental (Gen.0) environment.


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


Dominant shoot-borne root number was greater in progeny in ALB120 from a drought parental

environment in ALB120 (p = 0.011) (Figure 31). In ALB120, BRN was also greater (Figure 32) in

ALB96ALB5ALB120

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Dom

inant

Shoot-

Born

e R

oot

Num

ber

Well Watered

Drought

Gen.0 Treatment

ALB96ALB5ALB120

2.5

2.0

1.5

1.0

0.5

0.0

Dom

inant

Shoot-

Born

e R

oot

Num

ber

WW WW

WW D

D WW

D D

Treatment

Gen.0

Treatment

Gen.1*

*

*


3.0

2.5

2.0

1.5

1.0

0.5

0.0

Dom

inant

Shoot-

Born

e R

oot

Num

ber

Well Watered

Drought

Gen.0 Treatment*

25

progeny from a drought parental environment (hashed blue bar), when all progeny were grown in a well-

watered environment (both hashed and solid blue bars) (p = 0.001).

Figure 33. Basal root number in progeny from

the field, from a well-watered and drought

parental (Gen.0) environment. Asterisks


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,


BRN was greater in progeny from a well-watered parental environment only in ALB5 (ALB5, p = 0.001)

(Figure 33). In ALB5, BRN was also greater (Figure 34) in progeny from a well-watered parental

environment (solid blue bar), when all progeny were grown in a well-watered environment (both hashed

and solid blue bars) (p = 0.001).

Figure 35. Tap root diameter (mm) in progeny

from the field, from a well-watered or drought

parental (Gen.0) environment. Asterisks


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,


ALB96ALB5ALB120

5

4

3

2

1

0

Basa

l Root

Num

ber

Well Watered

Drought

Gen.0 Treatment

ALB96ALB5ALB120

5

4

3

2

1

0

Basa

l Root

Num

ber

WW WW

WW D

D WW

D D

Treatment

Gen.0

Treatment

Gen.1

ALB96ALB5ALB120

5

4

3

2

1

0

Tap R

oot

Dia

mete

r (m

m)

Well Watered

Drought

Gen.0 Treatment

ALB96ALB5ALB120

6

5

4

3

2

1

0

Tap R

oot

Dia

mete

r (m

m)

WW WW

WW D

D WW

D D

Treatment

Gen.0

Treatment

Gen.1

* *

*

*

*

*

*

26

Tap root diameter was greater in progeny from a well-watered parental environment in ALB5 (p = 0.03)

and ALB120 (p < 0.001) (Figure 35). In ALB120, BRN was also greater (Figure 36) in progeny from a

well-watered parental environment (solid blue bar), when all progeny were grown in a well-watered

environment (both hashed and solid blue bars) (p = 0.001).

Figure 37. Basal root diameter (mm)

in progeny from the field, from a

well-watered and drought parental

(Gen.0) environment. Asterisks



grown at the Rock Springs, PA site

under drought and well-watered

conditions, and harvested at growth

stage R2.

Basal root diameter was greater in progeny from a well-watered parental environment only in ALB5 (p <

0.001) (Figure 37).

Figure 38. Stomatal conductance in

progeny from the field, from a well-


(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


treatments.

Stomatal conductance was greater in progeny from a well-watered parental environment in ALB5 (p =

0.043) (Figure 38).

ALB96ALB5ALB120

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Basa

l Root

Dia

mete

r (m

m)

Well Watered

Drought

Gen.0 Treatment

*


0.6

0.5

0.4

0.3

0.2

0.1

0.0

Sto

mata

l Conducta

nce (

mm

ol m

⁻² s⁻¹

CO

2)

Well Watered

Drought

Gen.0 Treatment

*

27

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 (well-watered 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.

ALB1 ALB120 ALB18 ALB213 ALB23 ALB24 ALB5 ALB6 ALB91 ALB96 SER16 SER118 ALB67

Seed Weight (WW>D) p ≤ 0.001


Seed Weight (WW>D) p = 0.005










(Seed Weight not significant at p = 0.95)

Length of root hairs borne on seedling tap roots (D>WW) p = 0.015 F = 6.91

Seedling BRN (WW>D) p < 0.001 F = 21.25

Shoot Dry Weight (URBC) (WW>D) p = 0.002 F = 15.46

Length of root hairs borne on seedling basal roots (D>WW) p < 0.001 F = 22.73

Length of root hairs borne on seedling tap roots (WW>D) p = 0.045 F = 4.53

Seedling BRN (WW>D) p = 0.012 F = 6.79

Length of root hairs borne on seedling basal roots (D>WW) p = 0.028 F = 5.52

Length of root hairs borne on seedling basal roots (D>WW) p < 0.001 F = 39.07

Length of root hairs borne on seedling basal roots (WW>D) p = 0.033 F = 5.21

Length of root hairs borne on seedling basal roots (WW>D) p = 0.044 F = 4.55

Seedling Basal Root Length (D>WW) p = 0.018 F = 10.50

Tap Root Diameter (PA) (WW>D) p < 0.001 F = 17.85

Dominant Shoot-borne root # (URBC) (WW>D) p < 0.001 F = 20.15


Basal Root Diameter (URBC) (WW>D) p < 0.001 F = 23.37

Length of root hairs borne on seedling tap roots (D>WW) p = 0.014 F = 7.16

Length of root hairs borne on seedling tap roots (WW>D) p = 0.028 F = 5.57


Density of root hairs borne on seedling tap roots (D>WW) p = 0.006 F = 17.81

Seedling Dry Weight (WW>D) p = 0.04 F = 6.82

Dominant Shoot-borne root # (PA) (WW>D) p = 0.011 F = 3.60

BRN (PA) (WW>D) p = 0.001 F = 12.62

Basal Root Angle (URBC) (WW>D) p = 0.075 F = 3.50


Seedling Basal Root Length (WW>D) p = 0.054 F = 5.71

Seedling Basal Root Length (WW>D) p < 0.001 F = 99.76


Basal Root Diameter (PA) (WW>D) p < 0.001 F = 14.99

Shoot-borne root # (URBC) (WW>D) p = 0.048 F = 4.13

Shoot Dry Weight (URBC) (WW>D) p = 0.008 F = 8.02

Seedling Tap Root Length (WW>D) p < 0.001 F = 55.88

Tap Root Diameter (PA) (WW>D) p = 0.03 F = 4.87

Basal Root Diameter (URBC) (WW>D) p = 0.022 F = 5.90

Length of lateral roots borne on seedling tap roots (WW>D) p = 0.001 F = 34.12

Basal Root Diameter (URBC) (WW>D) p = 0.006 F = 5.06

Seedling Dry Weight (WW>D) p = 0.004 F = 21.00

Basal Root Angle (URBC) (WW>D) p = 0.003 F = 10.97

All genotypes except ALB67 had greater seed weight in seeds from well-watered parental conditions. In

six of thirteen genotypes, seedling BRN was greater in progeny from a well-watered parental

28

environment. In two genotypes, seedling dry weight was significantly different between parental

treatments. In ALB1, seedling basal root length was greater in seedlings from a drought stressed parental

environment, however in ALB67, seedling basal root length was greater in seedlings from a well-watered

parental environment. ALB67 was the only genotype without differences in seed weight between parental

treatments, but displayed differences between parental treatments in seedling traits including seedling dry

weight, seedling tap and basal root length, seedling tap lateral root length, seedling basal root hair length,

and tap root hair density.

Four of thirteen genotypes showed differences in seedling tap root hair length, two resulting in longer root

hairs when from a drought stressed parental environment, and two resulting in longer root hairs when

from a well-watered parental environment. Four of thirteen genotypes resulted in greater seedling basal

root hair length when seedlings were from a drought stressed parental environment, whereas two of

thirteen genotypes resulted in greater seedling basal root hair length when seedlings were from a well-

watered parental environment. Overall, there was very little consistency among genotypes in tap and basal

root hair length. Unlike results in field trails where root traits were generally fewer or smaller in progeny

from drought stressed parents, genotypes varied in length and density of root hairs borne on the tap root

and basal roots.

In both field locations (PA and URBC) basal root diameter and dominant shoot-borne root number were

different between parental treatments in at least one genotype. Differences in shoot dry weight, tap root

diameter, and BRN were only seen in the PA study, whereas differences in basal root angle were only

seen in the URBC study.

Root traits measured in drought studies, but not displaying significant differences between parental

treatments, included tap lateral root number, dominant basal root number, dominant basal root angle,

dominant shoot-borne root angle, nodule number, leaf water potential and stomatal conductance, and root

depth (field trials). There were no treatment effects of seed, shoot, or root traits in ALB67, thus this

genotype was not included in Table 2. Regression slopes were also calculated between significant traits

both within genotypes and all genotypes combined. Results did not show differences between regression

slopes of low and high P treatments. In addition, differences were not found in allometric relationships

between traits. ANOVA tables of results are included in the appendix.

29


Progeny from a low and high P parental (Gen.0) field environment were grown under a low and high P

environment at the Rock Springs, PA field site in 2011, and in the greenhouses in 2011. Parent plants

were grown under high and low P, and seed was collected at the Rock Springs site in 2010. Prior to

planting progeny, seeds were evaluated for individual seed weight and P concentration. Progeny plants

were excavated at growth stage R2, and evaluated for root and shoot traits.

Figure 39. Seed P concentration

(micromoles) in seeds from a high

and low P parental environment

(Gen.0). Asterisks represent


treatments.

Seed P concentration was lower in progeny from a low P parental environment by 13-21% in three of nine

genotypes (SER85 p = 0.077, SER16 p = 0.01, SER43 p = 0.05) (Figure 39). There were no differences

in individual seed weight between progeny from different parental P environments.

Figure 40. Shoot-borne root number

in progeny from the field, from a low

and high P parental environment

(Gen.0). Asterisks represent


treatments. Progeny were grown in

the field under low and high P and


Tioc

anela7

5

SER8

5

SER8

3

SER7

9

SER5

5

SER4

3

SER1

6

Bf13

572-

5

25

20

15

10

5

0

Shoot-

Born

e R

oot

Num

ber

High P

Low P

Treatment

Gen.0

*

*

Tioc

anela7

5

SER85

SER83

SER79

SER55

SER43

SER16

SER15

Bf13

572-

5

30

25

20

15

10

5

0

Seed P

concentr

ation (

uM

)

High P

Low P

Treatment

Gen.0

* * *

30

Shoot-borne root number was greater in progeny from a high P parental environment in SER16 (p =

0.014), Tiocanela75 (p = 0.009) (Figure 40). Dominant shoot-borne root number was also measured but

was not different between parental treatments.

Figure 41. Basal root whorl number

in progeny from the greenhouse

2011, from a low and high P parental

environment (Gen.0). Asterisks



grown in the greenhouse under low

and high P and harvested at growth

stage R2.

Basal root whorl number was greater in progeny from a high P parental environment in Tiocanela75 (p =

0.043) (Figure 41). BRN was also measured but was not different between parental treatments.

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.

BF13572-5 SER15 SER16 Tiocanela75

Seed P Concentration (uM/grams) (HP>LP) p = 0.06



Shoot-borne root # (HP>LP) p = 0.014 F = 6.05

Shoot-borne root # (HP>LP) p = 0.009 F = 8.15

Basal Root Whorl # (LP>HP) p = 0.043 F = 5.33

Tiocanela75SER79SER16

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Basa

l root

whorl n

um

ber

High P

Low P

Treatment

Gen.0

*

31

Seed P concentration was greater in seeds from a high P parental environment in three of eight genotypes,

however seed weight was not different between treatments in any genotype. Tiocanela75 showed a trend

toward greater seed P concentration in seeds from high P parental environments, but variability in seed P

concentration was high in seeds from a low P parental environment. Shoot-borne root number was

greater in progeny from a high P parental environment in two of eight genotypes. In Tiocanela75, BRWN

was greater in progeny from a low P parental environment.

Root traits measured in phosphorus studies, but that did not result in significant differences between

parental treatments, included dominant shoot-borne root number, dominant basal root number, basal root

angle, dominant basal root angle, dominant shoot-borne root angle, basal root diameter, and basal root

number. ANOVA tables of results are included in the appendix. Regression slopes were also calculated

between significant traits both within genotypes and all genotypes combined. Results did not show

differences between regression slopes of low and high P treatments. In addition, differences were not

found in allometric relationships between traits. Soil P levels in the four low P blocks where parent plants

were grown may not have been low enough to elicit responses in the next generation. The four low P

blocks in the parental generation had P levels of 11, 9.5, 11, 9.5 (ppm), where the recommended level of

P for small grains and soybean is 30-50 ppm (from The Pennsylvania State University Agricultural

Analytical Lab).

32

4. Discussion


Parental provisioning of seeds involves the investment of resources including nutrients, carbohydrates,

and protein into seeds by parent plants during seed fill. Allocation of resources from the parent plant to

each seed is usually not equal, and depends on a variety of factors such as the environment during seed

fill, the time of seed development on the parent plant, and the location of the seed on the parent plant. For

instance, Cheplick and Sung (1998) found that heaver seeds from the lower part of the panicle in Triplasis

purpurea had greater seedling shoot and root dry weight. Assuming seeds from the lower part of the

panicle were heavier due to greater parental provisioning and allocation of resources into the seed, it

could be hypothesized that these seeds might have better seedling establishment and vigor due to a greater

availability of resources within the seed. Wulff (1986) found that individual seed weight in Desmodium

paniculatum 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. This thesis explored how root traits

in seedlings and at growth stage (R2) in P. vulgaris differ between seeds from stylar versus peduncular

positions in the pod, and between pods from late versus early developmental times on the parent plant.

Seeds from the stylar (S) position in the pod and from earlier developing pods had greater seed weight

relative to seeds from the peduncular (P) position in the pod and later developing pods in the majority of

genotypes tested. Rocha and Stephenson (1990) found similar differences in seed weight between seeds

from stylar and peduncular positions in the pod in P. coccineus. They suggested that seeds from the stylar

position in the pod may have greater mass due to primary fertilization of ovules at the stylar end of the

pod, thus obtaining more resources from the parent plant during seed fill relative to seeds from the

peduncular position in the pod. Assuming that the vasculature is similar in P. vulgaris, the differences in

seed weight would suggest weaker partitioning of resources to peduncular seeds relative to stylar seeds

due to the order of fertilization within the pod, and may explain differences in seed weight between seeds

from stylar and peduncular positions.

There was no relationship between BRN and individual seed weight in seeds from the stylar position.

However, correlations were found between BRN and individual seed weight from the peduncular position

in all genotypes combined. Correlations were also found between BRN and individual seed weight in

BAT477 in seeds from the peduncular position, seeds from early developing pods, and seeds from late

developing pods. In addition, two of four genotypes had lower BRN in seeds from the peduncular

position. Lower BRN in seeds from the peduncular position may be in part explained by limited

resources in lighter seeds due to lower parental provisioning and allocation of resources into peduncular

seeds. Limited resources in the seed such as nutrients, protein, and carbohydrates necessary for seedling

establishment and growth, could limit development of seedling organs that develop early after

germination such as the tap root and basal roots. Lower BRN in lighter seeds from the peduncular

position was thus consistent with weaker provisioning of peduncular seeds by parent plants.

Positive relationships were also found between individual seed weight and root dry weight at growth

stage R2, but only in seeds from the peduncular position. In addition, basal lateral root number, tap root

diameter, and root weight were greater in seeds from the stylar position in two different genotypes.

Limited resources in peduncular seeds were expected to affect development in seedlings, but were not

expected to persist into later growth stages since older plants are no longer dependent on seed resources

for growth and development. For instance, lower BRN in seedlings from the peduncular position did not

persist at later growth stages. However, root dry weight and tap root diameter were lower at growth stage

R2 in plants that were from the peduncular position. It is possible that since the majority of basal root

development occurs after the seedling stage, basal roots have the opportunity to recover from limited

development by utilizing resources in the growing environment during later growth stages. The tap root

33

may not be as important as basal roots in later growth stages, thus development of the tap root may be

more affected by limited seed resources at younger growth stages. In addition, it was observed that

development of tap roots did not progress as much as basal roots after the seedling stage. For these

reasons, tap roots in plants from the peduncular position may not recover from poor development as a

seedling, persisting in lower diameter in later growth stages, potentially contributing to the overall lower

root weight displayed in later growth stages.

Overall, limiting factors in smaller seeds due to lower parental provisioning resulted in more drastic

differences in root traits among seeds with different weights, whereas heavier seeds from the stylar

position had larger and more numerous roots, exhibiting higher BRN, greater root dry weight, and a larger

tap root diameter compared to seeds from the peduncular position. Seeds from stylar and peduncular pod

positions showed greater differences in seed weight and root traits compared to early and late pod

developmental times. Differences in individual seed weight were relatively consistent across genotypes,

especially between seeds from stylar versus peduncular positions within the pod.


Relative to parent plants grown under well-watered conditions, drought stressed parent plants are stunted

and have fewer resources available to allocate to yield. Thus, resources available to seeds during seed fill

on drought stressed parent plants were expected to be reduced, and seeds from drought stressed parents

were expected to have lower weight and contain fewer resources such as nutrients, protein, and

carbohydrates per seed, compared to seeds from parents grown under well-watered conditions. This

project confirmed that seeds from parent plants subjected to drought were lower in weight, although

concentrations of specific resources within the seed were not measured. Overall, progeny from drought

stressed parents displayed roots that were smaller or fewer in number in both seedlings and mature roots

at growth stage R2. Differences in root traits between parental treatments is likely due to smaller seeds

from less allocation of resources to seeds from drought stressed parent plants. Other possibilities are

discussed below.

4.2.1 Seed and Seedling Traits

All but one genotype had lower seed weight in seeds from drought stressed parent plants.

Similar to lighter seeds from the peduncular position in the pod, reduced seed weight may be the result of

weaker parental provisioning due to drought conditions during seed fill. Soybean seeds from drought

stressed parent plants also had lower seed weight and volume, likely due to limited resources during seed

fill (Hill et al., 1986, Meckel et al., 1984). Assuming lower seed weight is due to weaker parental

provisioning during seed fill, seedlings would have limited resources such as nutrients, carbohydrates,

and protein, which are essential for seedling establishment and growth. Following these expectations,

seedlings from parental drought had lower overall seedling weight and lower BRN.

In six of thirteen genotypes, seedling BRN was lower in progeny from a drought stressed parental

environment, likely due to lower parental provisioning of seeds during seed fill. Seedling BRN was

correlated with seed weight, thus differences in BRN may be explained by poor parental provisioning

during seed fill under drought stressed conditions. Lower BRN in seedlings would be expected to reduce

uptake of essential water and nutrients after seed reserves are depleted, thus reducing overall success of

the plant during later growth stages.

Seedling dry weight was lower in two genotypes and seedling basal root length was shorter in one

genotype when parents were grown in a drought stressed environment. Lower seedling dry weight and

shorter root length were also observed in a study by Sultan (1996) when parent plants of Polygonum

persicaria were grown in a drought stressed environment. Lower seedling dry weight and shorter

34

seedling basal root length is likely explained by limited resources in seeds from drought stressed parents,

providing less energy and nutrient reserves required for seedling development, relative to seedlings from

well-watered parents. However, in a different genotype seedling basal root length was greater in

seedlings from a drought stressed parental environment, suggesting that other factors besides limited

resources in seeds may play roles in seedling basal root length. Longer basal roots may assist in greater

exploration of deeper soil where water is more available under drought conditions. Since the likelihood is

high that progeny develop under similar drought stressed conditions as parents, this response may be an

adaptation to parental drought since longer basal roots may function in acquiring more water at greater

soil depths. This adaptation could possibly be explained through inheritance of epigenetic modifications

affecting basal root length.

Four of thirteen genotypes showed differences in the length of root hairs borne on seedling tap roots, two

resulting in longer root hairs when from a drought stressed parental environment, and two resulting in

longer root hairs when from a well-watered parental environment. Four of thirteen genotypes resulted in

greater length of root hairs borne on seedling basal roots when seedlings were from a drought stressed

parental environment, whereas two of thirteen genotypes resulted in greater length when seedlings were

from a well-watered parental environment. Only one genotype displayed differences in density of root

hairs borne on seedling tap roots between parental treatments. Overall, there was very little consistency

among genotypes in root hair length on tap roots or basal roots.

ALB67 was the only genotype without differences in seed weight between parental treatments, but had

differences between parental treatments in seedling traits including seedling dry weight, seedling tap and

basal root length, the length of lateral roots borne on the seedling tap root, length of root hairs borne on

seedling basal roots, and density of root hairs borne on seedling tap roots. There were no patterns seen in

seedling root hair traits on basal or tap roots, and root hair traits were not associated with seed weight,

suggesting differences in root hairs are not the result of parental provisioning. Lower seedling dry weight,

shorter basal root length, and shorter lateral roots borne on seedling tap roots in progeny from drought

stressed parents would not be advantageous under drought conditions. Thus differences in these traits are

not adaptive responses, and are likely due to lower provisioning of seeds from parents under drought

stress.

4.2.2 Mature Plant Traits

In field trails, plants at growth stage R2 demonstrated root traits that were fewer in number and lower in

weight or size in progeny from drought stressed parental environments. Lower basal root diameter and

dominant shoot-borne root number in progeny from drought stressed parents were found in both field

locations. Shoot dry weight, tap root diameter, and BRN were all lower in progeny from drought stressed

parents in the PA trial. Seedlings from parental drought also had lower BRN. The persistence of lower

BRN through later growth stages, especially when grown under drought, suggests that plants from

parental drought did not outgrow the parental effects present at earlier stages of growth. These results are

also similar to progeny from the peduncular seed position in the pod, which had lower BRN at R2, and

was correlated with seed weight.

Smaller basal root diameter, smaller tap root diameter, fewer dominant shoot-borne roots, and lighter

shoot dry weight in progeny from drought stressed parents may also be due to the failure to outgrow

delayed growth during earlier developmental stages when seedlings from parental drought had lighter

seedling dry weight and lower BRN likely due to limited seed resources from low parental provision

during drought stress. Such parental effects persisted beyond the seedling stage, displaying lower shoot

dry weight and smaller roots at later growth stages relative to progeny from a well-watered parental

environment. However, yield (seeds per pod and pods per plant) was not different between parental

treatments despite differences in shoot dry weight. Yield on progeny were not measured for seed weight,

35

thus it’s possible that although seed number per plant did not differ, total seed weight per plant may have

differed in progeny from different parental treatments.

At the URBC site, progeny from drought stressed parents had lower stomatal conductance and shallower

basal root angles. Lower stomatal conductance in progeny from drought stressed parents may be an

adaptive response across generations, potentially through mechanisms of epigenetic inheritance. Stomatal

conductance in response to a parental drought environment has been tested in Rigenos et al. (2007) in

Impatiens but did not show differences between progeny from contrasting parental treatments. Lower

stomatal conductance at the URBC site in progeny from drought stressed parents may be an adaptive

response in reducing water loss when in a drought environment.

Shallower basal root angle in progeny from parental drought in the URBC study is likely not explained

through parental provisioning because basal root angle is not associated with different energy costs, and

basal root angles are mostly determined post-seedling stage. It is also likely not adaptive since steeper

angles for acquiring water deeper in the soil would have been expected (Ho et al., 2004). It is possible

that shallower roots in progeny from drought stressed parents could be indirectly explained by differences

in other root traits affected by parental drought such as BRN. However, progeny from drought stressed

parents with lower BRN would be expected to have deeper roots instead of shallower roots since earlier

developing whorls tend to be deeper than later emerging whorls (Basu et al., 2007). Differences in basal

root angle may also be a genotype-specific response to parental drought conditions since only one

genotype demonstrated differences in basal root angle in response to the parental environment.

Among traits measured, seed weight and seedling BRN were the most consistently different between

parental treatment across genotypes. Seedlings showed stronger parental effects due to the exposure to

fewer environmental factors, resulting in less variability among measured traits such as BRN. Mature

plants are exposed to greater environmental variability during growth in the greenhouse or field, resulting

in greater variability in measured root and shoot traits.


Parental provisioning of seeds involves the investment of resources including nutrients, carbohydrates,

and protein into seeds by parent plants during seed fill. Allocation of resources from the parent plant to

each seed is not equal, and depends on a variety of factors including limiting P in the parental

environment during seed fill. This thesis explored how seed and root traits in P. vulgaris differ between

seeds from a low P versus high P parental environment.

There were no differences in seed weight between parental treatments, counter to previous research on

parental effects of P stress in common bean, watercress, and soybean, in which differences were found in

seed weight between parental treatments (Austin, 1996, Derrick & Ryan, 1998, Vandamme et al., 2015,

Yan et al., 1995). These results were also different from parental drought trails reported here, where seed

weight was different between parental treatments across the majority of genotypes. Similar individual

seed weights between parental treatments excludes seed weight as a cause of differences in root traits

between parental treatments, thus seed P may be the best explanation for differences. Other resources in

the seed may also have roles in differences in root traits between parental treatments.

Soil P levels in parental field conditions (low P blocks had P levels of 11, 9.5, 11, and 9.5 ppm) may not

have been low enough to cause differences in seed weight, but low enough to result in differences in seed

P concentration in some genotypes. It is possible that genotypes differed in allocation of P into seeds

under low P conditions, in which case parent plants under P stress may have produced fewer seeds per

plant but with similar mass and equal provisioning of seeds compared to seeds from parental high P. This

was seen in a study on parental drought by Sultan (1996), where Polygonum persicaria parent plants

36

grown under drought produced less offspring, but greater mass per seed. Such genotypic differences in

provisioning of seeds in response to low P may suggest suitable candidates for breeding programs

targeting genotype selection for tolerance to low P across generations.

Lower seed P may affect establishment and vigor of seedlings since P is an important nutrient throughout

growth. Thus, it was expected that genotypes displaying limited P in seeds from parental low P

environments would have root traits that were fewer, shorter, or lower in number as a seedling, and root

traits could also be affected at later growth stages if seedling vigor was reduced in seeds with less P.

Differences were found in shoot-borne root number and BRWN between progeny from low versus high P

parental environments. Shoot-borne roots develop during later growth stages. If seed P concentration reduced seedling vigor in progeny from P stressed parents, this could have caused delayed development

of shoot-borne roots in progeny from parental low P, compared to plants from parental high P conditions.

To confirm this, seedlings from contrasting parental P treatments could be evaluated for shoot-borne root

emergence at consecutive growth stages. It is also possible that differences between parental P treatments

in shoot-borne root number changed over time. For instance, Austin (1966) found that progeny from P

stressed parent species had less biomass at 7-9 weeks, but showed no difference between progeny from

contrasting parental environments at 16-20 weeks. Shoot-borne root number could be measured at

different times throughout growth to clarify how parental P stress affects this trait throughout the plant

life cycle.

BRWN was greater in progeny from a low P parental environment in one genotype. The same genotype

did not display differences in seed P concentration between parental P treatments, possibly due to high

variability in data, but demonstrated a trend toward greater seed P concentration in seeds from a high P

parental environment (p = 0.141). Differences in BRWN between parental P treatments in this genotype

were opposite to BRN results in parental drought trails where progeny from parental drought had lower

BRN. Greater BRWN in progeny from low P parents is not explained by lower parental provisioning, but

may function as an adaptation to P stress from the parent generation. Since the likelihood is high that

progeny will grow under similar low P conditions as parents, this response may be an adaptation to a low

P parental environment since greater BRWN has been found to improve P uptake under low P conditions

in common bean (Miguel, 2012, Miguel et al., 2015). Such a response may be controlled by heritable

epigenetic modifications affecting BRWN.

4.4 Conclusions

Overall, individual seed weight was consistently lower in seeds from the peduncular position within the

pod, seeds from late developing pods, and seeds from parental drought. Lower individual seed weight

was expected in seeds from stressed parental environments, and was displayed in seeds from drought

stressed parents in some genotypes. Parent plants under drought may have had limited resources

available to allocate to seeds during seed fill, such as nutrients, carbohydrates, and protein, thus providing

lower provisioning of seeds. Progeny from the peduncular position in the pod and late developing pods

also had lower individual seed weight in some genotypes because seeds from these two treatments likely

received less resources since seeds from the peduncular position may be fertilized last, and late

developing pods receive the remaining resources available from the parent plant, such as nutrients,

carbohydrates, and protein. These seed components are required for successful seedling establishment,

growth, and vigor.

Not all genotypes had lower seed weight in seeds from the peduncular position, late developing pods, and

seeds from drought stressed parents. Genotypic differences in response to drought such as the production

of fewer seeds with greater mass per seed, may in part explain why some genotypes did not have

differences in seed weight between parental treatments. There may also be genotypic differences in

allocation of resources to seeds from the peduncular versus stylar position, and seeds from early versus

37

late developing pods. For instance, some genotypes may produce seeds that are more similar in mass

from different pod positions and pod developmental times, especially if there were fewer seeds per pod

and fewer seeds per plant.

There was a positive relationship between individual seed weight and BRN in all seed positions and seeds

from pod developmental times, but there was no relationship between individual seed weight and seeding

or mature BRN in parental drought studies. This suggests that variation in BRN in seeds from different

pod positions and developmental times on non-stressed parents may be explained through seed weight.

However, since BRN was not correlated with seed weight in progeny from parental drought (see

appendices), this suggests that factors associated with parental drought, especially in genotypes displaying

differences in BRN between parental treatments, may better explain variation in BRN. This may be

explained by differences in allocation of specific resources into the seed or epigenetic modifications to

progeny in response to parental drought, compared to seeds that received lower provisioning due to seed

position in the pod or pod developmental time. Adaptive responses to parental drought may also explain

lower BRN. For instance, one genotype displaying lower BRN in seedlings from parental drought also

had greater seedling basal root length from parental drought. Development of lower BRN may be a

strategy to allow for greater seedling basal root length to allow for deeper soil exploration for limiting

water.

Seeds from high and low P parental environments did not have differences in individual seed weight, but

seeds from a low P parental environment had lower individual seed P concentration in some genotypes.

Not all genotypes had differences in seed P concentration between parental treatments, possibly due to

genotypic differences in allocation of P into seeds under low P. The absence of differences in individual

seed weight between parental P treatments may have been due to a moderate instead of a low P parental

treatment, especially since similar studies on parental effects of P stress found differences in seed mass

(Austin, 1996, Derrick & Ryan, 1998, Vandamme et al., 2015, Yan et al., 1995). However, it is possible

that in response to low P, parent plants produced fewer seeds per plant but with similar individual seed

mass and equal provisioning of seeds.

Overall, progeny from the peduncular position in the pod, late developing pods, parental drought, and

parental low P had root traits that were lighter, shorter, smaller in diameter, or fewer in number. These

results were likely explained by lower parental provisioning of seeds by drought and P stressed parents,

and fewer resources available to seeds from the peduncular position in the pod and late developing pods.

The majority of root traits displaying differences between treatments followed this pattern, except

seedling root hair traits and basal root length from parental drought in one genotype, and BRWN from

parental low P in another genotype. Length and density of root hairs borne on seedling tap and basal

roots from drought stressed parents did not follow any distinct pattern between parental treatments or

across genotypes, thus parental provisioning nor adaptive plasticity likely explain these results.

However, greater BRWN from parental low P and greater seedling basal root length from parental

drought may be an adaptive response to parental stress. In addition to lower parental provisioning,

adaptive parental effects were also expected since the likelihood of progeny establishment in the same or

similar environment as parent plants is high. 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 limited 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. Since the

likelihood is high that progeny develop under similar stressed conditions as parents, responses may be an

adaptation to parental stress. These responses may be caused by epigenetic modifications of progeny

during development on the parent plant, affecting traits that assist in the acquisition of P under low P, and

the acquisition of water under drought conditions.

38

Inconsistencies and variability in traits across experiments and locations may have been due to phenotypic

plasticity in root traits especially in field conditions, and the use of a combination of seeds from different

pod positions and pod developmental times, which have been shown in this thesis to highly affect seed

and root traits. The Rock Springs, PA field site, and the URBC field site differed in environmental

factors such as soil type, rainfall, and average temperature, which may have contributed to the variability

found in phenotypic responses to parental stress. Genotypic differences in responses to parental stress

and provisioning may also explain variation in results. In addition, phenotypic responses to parental

drought in ALB RILs may not be representative of P. vulgaris responses to parental stress since the ALB

population is from an interspecific cross with P. coccineus, and should be considered when interpreting

results from parental drought studies.

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. Breeding programs and experimental sites often evaluate new cultivars for stress tolerance

using seed that developed in a well-watered, high fertility parental environment. Due to the differences in

genotypic and phenotypic responses to parental stress, the parental environment should be considered in

breeding programs and experimental sites that evaluate new cultivars for tolerance to stress. Genotypes

displaying potential adaptations to stress in response to the previous generation should be considered in

breeding programs targeting areas prone to drought or low P, where farmers often use seeds from the

previous year’s crop. In addition, genotypes displaying relatively greater reduction in provisioning of

progeny in response to parental stress should be avoided in breeding programs. Genotypes with

alternative strategies that avoid lower provisioning of seeds under stressful conditions, such as the

production of fewer seeds per plant but greater seed mass per individual seed, should be identified. Such

responses would be beneficial to farmers using seeds from stressed parent plants.

Results also have implications for phenotyping initiatives that focus on identifying common bean

genotypes with root trait variants beneficial under stressful conditions. Phenotyping initiatives

identifying genotypes with phenotypic variability expected to be beneficial under certain stresses often

use seed from non-stressed parental environments. 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 when exploring phenotypic variation in root traits across common bean genotypes.

39

Appendix A

Additional Tables: Parental Effects of Seed Position in the Pod and Pod Developmental Time

Two-way ANOVA table for individual seed weight in B98311: Stylar versus peduncular position, and

late versus early pod developmental times.

Two-way ANOVA table for individual seed weight in BAT477: Stylar versus peduncular position, and


Two-way ANOVA table for individual seed weight in DOR364: Stylar versus peduncular position, and


Two-way ANOVA table for individual seed weight in TLP19: Stylar versus peduncular position, and late

versus early pod developmental times.

40

Two-way ANOVA table for BRN in B98311: Stylar versus peduncular position, and late versus early pod


Two-way ANOVA table for root dry weight in BAT477: Stylar versus peduncular position, and late


Two-way ANOVA table for tap root diameter in DOR364: Stylar versus peduncular position, and late


Two-way ANOVA table for number of lateral roots borne on basal roots in DOR364: Stylar versus

peduncular position, and late versus early pod developmental times.

41

Appendix B

Additional Tables and Figures: Parental Effects of Drought Stress

One-way ANOVA table for seedling BRN in ALB1: Parental drought environment versus parental well-

watered environment.

One-way ANOVA table for seedling BRN in ALB120: Parental drought environment versus parental

well-watered environment.







42

One-way ANOVA table for seedling BRN in SER118: Parental drought environment versus parental


One-way ANOVA table for seedling dry weight in ALB67: Parental drought environment versus parental


One-way ANOVA table for seedling dry weight in ALB1: Parental drought environment versus parental


One-way ANOVA table for seedling basal root length in ALB1: Parental drought environment versus

parental well-watered environment.

One-way ANOVA table for seedling basal root length in SER16: Parental drought environment versus


Seedling Basal root length ser16

43

One-way ANOVA table for seedling basal root length in ALB67: Parental drought environment versus


One-way ANOVA table for seedling tap root length in ALB67: Parental drought environment versus


One-way ANOVA table for length of lateral roots borne on seedling tap roots in ALB67: Parental drought

environment versus parental well-watered environment.

One-way ANOVA table for length of root hairs borne on seedling basal roots in SER118: Parental

drought environment versus parental well-watered environment.

One-way ANOVA table for length of root hairs borne on seedling basal roots in SER16: Parental drought


44

One-way ANOVA table for length of root hairs borne on seedling basal roots in ALB5: Parental drought






One-way ANOVA table for length of root hairs borne on seedling tap roots in ALB6: Parental drought




45

One-way ANOVA table for length of root hairs borne on seedling tap roots in SER16: Parental drought




One-way ANOVA table for density of root hairs borne on seedling tap roots in ALB67: Parental drought


Two-way ANOVA table for basal root diameter in ALB5 (PA site): Parental drought environment versus


Two-way ANOVA table for basal root diameter in ALB120 (PA site): Parental drought environment

versus parental well-watered environment.

46

Kruskal-Wallis test for basal root diameter in ALB5 (URBC site): Parental drought environment versus


Kruskal-Wallis test for basal root diameter in ALB91 (URBC site): Parental drought environment versus


Two-way ANOVA table for basal root diameter in SER16 (URBC site): Parental drought environment


Two-way ANOVA table for tap root diameter in ALB5 (PA site): Parental drought environment versus


47

Kruskal-Wallis test for dominant shoot-borne root number in ALB120 (PA site): Parental drought


Kruskal-Wallis test for dominant shoot-borne root number in ALB23 (URBC site): Parental drought


Two-way ANOVA table for shoot-borne root number in ALB91 (URBC site): Parental drought


Two-way ANOVA table for BRN in ALB5 (PA site): Parental drought environment versus parental well-


48

Kruskal-Wallis test for basal root angle in ALB5 (URBC site): Parental drought environment versus


Kruskal-Wallis test for basal root angle in ALB91 (URBC site): Parental drought environment versus


Kruskal-Wallis test for shoot dry weight in ALB23 (URBC site): Parental drought environment versus


49

Two-way ANOVA table for shoot dry weight in SER16 (URBC site): Parental drought environment


50

Scatterplot of seedling BRN versus individual seed weight in seedlings from a parental drought

environment and seedlings from a parental well-watered environment, including all genotypes. There

was no relationship between seedling BRN and individual seed weight in seedlings from a parental

drought environment or in seedlings from a parental well-watered environment.

Scatterplot of seedling BRN versus individual seed weight in seedlings from a parental drought

environment and seedlings from a parental well-watered environment, including genotypes that displayed

differences in BRN between parental treatments (ALB1, ALB120, ALB5, ALB91, ALB96, and SER118).

There was no relationship between seedling BRN and individual seed weight in seedlings from a parental

drought environment or in seedlings from a parental well-watered environment.

0.450.400.350.300.250.200.150.10

14

12

10

8

6

4

2

Individual Seed Weight (grams)

Seedlin

g B

RN

Well Watered

Drought

Gen.0 Treatment

0.450.400.350.300.250.200.150.10

15.0

12.5

10.0

7.5

5.0

Individual Seed Weight (grams)

Seedin

g B

RN

Well Watered

Drought

Gen.0 Treatment

51

Appendix C

Additional Tables: Parental Effects of Phosphorus Stress

Kruskal-Wallis test for BRWN in Tiocanela75 (Greenhouse): Parental low P environment versus parental

high P environment.

Two-way ANOVA table for shoot-borne root number in Tiocanela75 (Field): Parental low P environment

versus parental high P environment.

Kruskal-Wallis test for shoot-borne root number in Tiocanela75 (Greenhouse): Parental low P

environment versus parental high P environment.

52

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