parental effects and provisioning under drought and
TRANSCRIPT
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
ii
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
environment. Asterisks represent significant differences between treatments.
Figure 18. Seedling dry weight in progeny from a well-watered and drought parental (Gen.0) field
environment. Asterisks represent significant differences between treatments.
Figure 19. Density of root hairs borne on seedling tap roots (# of hairs/ 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
environment. Asterisks represent significant differences between treatments.
Figure 21. Seedling basal root length in progeny from a well-watered and drought parental (Gen.0) field
environment. Asterisks represent significant differences between treatments.
Figure 22. Length of root hairs borne on seedling tap roots (mm) in progeny from a well-watered and
drought parental (Gen.0) field environment. Asterisks represent significant differences between
treatments.
Figure 23. Length of root hairs borne on seedling basal roots (mm) in progeny from a well-watered and
drought parental (Gen.0) field environment. Asterisks represent significant differences between
treatments.
Figure 24. Length of lateral roots borne on seedling tap roots (cm) in progeny from a well-watered and
drought parental (Gen.0) field environment. Asterisks represent significant differences between
treatments.
Figure 25. Soil volumetric water content in well-watered and drought plots at the URBC site. Each data
point represents the average of 4 replicates from continuous measurements in 2 plots per treatment, at 15
cm below the soil surface.
Figure 26. Soil volumetric water content in well-watered and drought plots at the Rock Springs site. Each
data point represents the average of 2 replicates from continuous measurements in 2 plots per treatment,
at 15 cm below the soil surface.
Figure 27. Shoot dry weight in progeny from the field, from a well-watered and drought (Gen.0) parental
environment. Asterisks represent significant differences between treatments. Progeny were grown at the
URBC site under drought and well-watered conditions, and harvested at growth stage R2.
Figure 28. Basal root diameter (mm) in progeny from the field, from a well-watered and drought (Gen.0)
parental environment. Asterisks represent significant differences between treatments. Progeny were
grown at the URBC site under drought and well-watered conditions, and harvested at growth stage R2.
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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.
x
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 Parental Effects of Seed Position in the Pod and Pod Developmental Time
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.
2.3 Parental Effects of Drought Stress
2.3.1 Plant Material
The following genotypes were used: SER118, SER16, SEA5, all from the Mesoamerican gene pool, and
eleven RILs (recombinant inbred lines) from the ALB population (SER 16 x (SER 16 x 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
2.4.1 Plant Material
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
3.1 Parental Effects of Seed Position in the Pod and Pod Developmental Time
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
TLP19DOR364BAT477B98311
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.
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.
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.
TLP19DOR364BAT477B98311
9
8
7
6
5
4
3
2
1
0
Basa
l Root
Num
ber
P
S
Position
Seed
TLP19DOR364BAT477B98311
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
differences between pod positions.
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
TLP19DOR364BAT477B98311
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
developmental times.
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.
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
developmental times.
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
TLP19DOR364BAT477B98311
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
TLP19DOR364BAT477B98311
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
3.2 Parental Effects of Drought Stress
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.
Asterisks represent significant
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
environment. Asterisks represent
significant differences between
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-
watered and drought parental
(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
represent significant differences between
treatments.
Figure 21. Seedling basal root length (cm) in
progeny from a well-watered and drought
parental (Gen.0) field environment. Asterisks
represent significant differences between
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).
SER16SER118ALB96ALB67ALB6ALB5ALB1
20
15
10
5
0
Seedlin
g T
ap R
oot
Length
(cm
)
Well Watered
Drought
Gen.0 Treatment
SER16SER118ALB96ALB67ALB6ALB5ALB1
7
6
5
4
3
2
1
0
Seedlin
g B
asa
l Root
Length
(cm
)
Well Watered
Drought
Gen.0 Treatment
SER16SER118ALB96ALB67ALB6ALB5ALB1
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
differences between treatments.
Figure 23. Length of root hairs borne on
seedling basal roots (mm) in progeny from a
well-watered and drought parental (Gen.0) field
environment. Asterisks represent significant
differences between treatments.
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
environment. Asterisks represent
significant differences between
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
SER16SER118ALB96ALB67ALB6ALB5ALB1
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
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.
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
represent significant differences
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)
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.
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).
SER16ALB91ALB6ALB5ALB23
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
* * *
SER16ALB91ALB6ALB5ALB23
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)
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.
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.
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.
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*
*
*
SER16ALB91ALB6ALB5ALB23
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
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.
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
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.
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
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.
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-
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.
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
*
SER16ALB91ALB6ALB5ALB23
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 ≤ 0.001
Seed Weight (WW>D) p = 0.005
Seed Weight (WW>D) p ≤ 0.001
Seed Weight (WW>D) p ≤ 0.001
Seed Weight (WW>D) p ≤ 0.001
Seed Weight (WW>D) p ≤ 0.001
Seed Weight (WW>D) p = 0.022
Seed Weight (WW>D) p ≤ 0.001
Seed Weight (WW>D) p = 0.038
Seed Weight (WW>D) p ≤ 0.001
Seed Weight (WW>D) p ≤ 0.001
(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
Seedling BRN (WW>D) p = 0.052 F = 3.98
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
Seedling BRN (WW>D) p < 0.001 F = 18.20
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 BRN (WW>D) p < 0.001 F = 17.35
Seedling Basal Root Length (WW>D) p = 0.054 F = 5.71
Seedling Basal Root Length (WW>D) p < 0.001 F = 99.76
Seedling BRN (WW>D) p = 0.001 F = 13.01
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
3.3 Parental Effects of Phosphorus Stress
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
significant differences between
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
significant differences between
treatments. Progeny were grown in
the field under low and high P and
harvested at growth stage R2.
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
represent significant differences
between treatments. Progeny were
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
Seed P Concentration (uM/grams) (HP>LP) p = 0.03
Seed P Concentration (uM/grams) (HP>LP) p = 0.015
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
4.1 Parental Effects of Seed Position in the Pod and Pod Developmental Time
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.
4.2 Parental Effects of Drought Stress
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.
4.2 Parental Effects of Phosphorus Stress
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
late versus early pod developmental times.
Two-way ANOVA table for individual seed weight in DOR364: Stylar versus peduncular position, and
late versus early pod developmental times.
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
developmental times.
Two-way ANOVA table for root dry weight in BAT477: Stylar versus peduncular position, and late
versus early pod developmental times.
Two-way ANOVA table for tap root diameter in DOR364: Stylar versus peduncular position, and late
versus early pod developmental times.
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.
One-way ANOVA table for seedling BRN in ALB5: Parental drought environment versus parental well-
watered environment.
One-way ANOVA table for seedling BRN in ALB91: Parental drought environment versus parental well-
watered environment.
One-way ANOVA table for seedling BRN in ALB96: Parental drought environment versus parental well-
watered environment.
42
One-way ANOVA table for seedling BRN in SER118: Parental drought environment versus parental
well-watered environment.
One-way ANOVA table for seedling dry weight in ALB67: Parental drought environment versus parental
well-watered environment.
One-way ANOVA table for seedling dry weight in ALB1: Parental drought environment versus parental
well-watered environment.
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
parental well-watered environment.
Seedling Basal root length ser16
43
One-way ANOVA table for seedling basal root length in ALB67: Parental drought environment versus
parental well-watered environment.
One-way ANOVA table for seedling tap root length in ALB67: Parental drought environment versus
parental well-watered environment.
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
environment versus parental well-watered environment.
44
One-way ANOVA table for length of root hairs borne on seedling basal roots in ALB5: Parental drought
environment versus parental well-watered environment.
One-way ANOVA table for length of root hairs borne on seedling basal roots in ALB96: Parental drought
environment versus parental well-watered environment.
One-way ANOVA table for length of root hairs borne on seedling basal roots in ALB67: Parental drought
environment versus parental well-watered environment.
One-way ANOVA table for length of root hairs borne on seedling tap roots in ALB6: Parental drought
environment versus parental well-watered environment.
One-way ANOVA table for length of root hairs borne on seedling tap roots in ALB96: Parental drought
environment versus parental well-watered environment.
45
One-way ANOVA table for length of root hairs borne on seedling tap roots in SER16: Parental drought
environment versus parental well-watered environment.
One-way ANOVA table for length of root hairs borne on seedling tap roots in ALB1: Parental drought
environment versus parental well-watered environment.
One-way ANOVA table for density of root hairs borne on seedling tap roots in ALB67: Parental drought
environment versus parental well-watered environment.
Two-way ANOVA table for basal root diameter in ALB5 (PA site): Parental drought environment versus
parental well-watered environment.
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
parental well-watered environment.
Kruskal-Wallis test for basal root diameter in ALB91 (URBC site): Parental drought environment versus
parental well-watered environment.
Two-way ANOVA table for basal root diameter in SER16 (URBC site): Parental drought environment
versus parental well-watered environment.
Two-way ANOVA table for tap root diameter in ALB5 (PA site): Parental drought environment versus
parental well-watered environment.
47
Kruskal-Wallis test for dominant shoot-borne root number in ALB120 (PA site): Parental drought
environment versus parental well-watered environment.
Kruskal-Wallis test for dominant shoot-borne root number in ALB23 (URBC site): Parental drought
environment versus parental well-watered environment.
Two-way ANOVA table for shoot-borne root number in ALB91 (URBC site): Parental drought
environment versus parental well-watered environment.
Two-way ANOVA table for BRN in ALB5 (PA site): Parental drought environment versus parental well-
watered environment.
48
Kruskal-Wallis test for basal root angle in ALB5 (URBC site): Parental drought environment versus
parental well-watered environment.
Kruskal-Wallis test for basal root angle in ALB91 (URBC site): Parental drought environment versus
parental well-watered environment.
Kruskal-Wallis test for shoot dry weight in ALB23 (URBC site): Parental drought environment versus
parental well-watered environment.
49
Two-way ANOVA table for shoot dry weight in SER16 (URBC site): Parental drought environment
versus parental well-watered 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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