Morphological Adaptations of Soybean in Response to Early Season Flood Stress

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MORPHOLOGICAL ADAPTATIONS OF SOYBEAN IN RESPONSE TO EARLY
SEASON FLOOD STRESS















By

THOMAS L. HENSHAW


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005


































Copyright 2005

by

Thomas L. Henshaw

































This thesis is dedicated to my family for getting me here and to Erin for keeping me here.
We may be young, but we are on our way.















ACKNOWLEDGMENTS

I would like to thank my committee, Drs. Robert Gilbert, Johannes Scholberg, and

Tom Sinclair, for their availability, patience, and help. I thank all the people in Belle

Glade that made this project possible Ron Gosa, Lee Liang, and Pepe Gonzalez. Special

appreciation goes to Dr. Chen and the University of Arkansas soybean breading program

for providing germplasm and background information.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S ............................... .. ....... .. .. ........... .............. .. vii

LIST OF FIGURES ................................. ...... ... ................. .x

ABSTRACT .............. ......................................... xi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

O v e rv iew .................................................................................. 1
Introduction .........................................................................................................
Background ..... ..................................................... ................
Crop rotations with sugarcane on mineral soils ..........................................2
Soybean (Glycine max. L. Merrill) as a rotational crop..............................3
R rationale ............... .. ....................................................... 4
Selection of genetic material ........ ... ....................................................4
F lood toleran ce .................................................................. ............... .. 5
A p p ro a c h ........................................................ ................ 5
N u ll H y p oth eses.............................. .................................................... ............... .. 6

2 THE EFFECT OF FLOODING AND GENOTYPE ON THE SOYBEAN
(GLYCINE MAX L. MERRILL) RHIZOSPHERE................................. ...............7

R ev iew of L iteratu re ................ .. ...... ............................................... ...............7
Identification of Tolerance to Flood Stress ...........................................................7
Selecting Soybean Genotypes for Flood Tolerance ...........................................11
M materials and M methods ....................................................................... .................. 13
Selection of G genetic M material ................................................... ............. ...... 13
E x p erim ental D esign ............................................... ......................................14
C ro p M an ag em en t ......................................................................................... 15
M easurem ents .................. ....................................... ........... 18
Plant biom ass sam pling ......................... .............. ....... ............... 18
R oot grow th .................................... ................................. ............... 19
Statistical A naly sis ...........................................................20



v









R results and D discussion ........................................................... ........ .......... 21
Primary Root Dry Weight and Dry Matter Partitioning...................................21
Primary Root Length, Average Diameter, Surface Area, and Volume ..............28
Adventitious Root Dry Weight and Dry Matter Partitioning ............................28
Adventitious Root Length, Average Diameter, Surface Area, and Volume .......30
Nodule Dry Weights and Dry Matter Partitioning ...........................................33
Correlations of morphological characters to flood tolerance ............................34
Contrast Between Flood Tolerance Groups ................................ ............... 39
C o n c lu sio n s ..................................................................... 3 9

3 THE EFFECT OF EARLY-SEASON FLOODING AND GENOTYPE ON
SOYBEAN (GLYCINE MAX L. MERRILL) ABOVEGROUND GROWTH AND
T O TA L PL A N T B IO M A SS..................................... ............................................42

R eview of L literature ................ .. ................................................ ........................42
Identification of Tolerance to Flood Stress ............................... ............... 42
R ationale and O objectives ......... ................................................. .... ........... 45
M materials and M methods ....................................................................... ..................4 5
Leaf A rea and Plant H eight .................................................. ...................46
Statistical A analysis ............................................. .... .. ...... ........ 47
R results and D discussion ... ........... .............................. .......... .... ..... ....... ....48
Stem Dry Weight, Dry Matter Partitioning, and Height ...................................48
Leaf Expansion, Dry Matter Accumulation, and Partitioning.............................52
T otal Plant D ry W eight ............................................... ............................ 59
R oot: Shoot R atio .................................................. .. .......... ..... .......... .... 62
Correlations of Above Ground Plant Growth to Flood Tolerance ....................63
Contrast Between Flood Tolerance Groups ................................ ............... 66
C o n clu sio n s ..................................................... ................ 6 7

4 RESEARCH PERSPECTIVES ............................................................................ 69

APPENDIX

A D A TA FR OM EX PERIM EN T 2..................................................... .....................72

B LEAF NUTRIENT AND SPAD DATA FROM EXPERIMENTS 1 AND 2............84

L IST O F R E F E R E N C E S ...................... .. .. ............. ....................................................89

B IO G R A PH IC A L SK E TCH ..................................................................... ..................93
















LIST OF TABLES


Table page

2-1 Experimental designs used for soybean flooding experiments.............................15

2-2 Preliminary Mehlich-I soil test results and fertilizer recommendations ..............17

2-3 Weekly average temperature and precipitation data for Belle Glade EREC,
M ay-August 2004. ................................... ... ... ... .... ............ 22

2-4 Primary root dry weight, dry matter partitioning, and relative dry weight............23

2-5 Genotype comparisons for significant dry weight genotype x flood interaction
term s ...................................... ...................................................... 2 5

2-6 Genotype comparisons for significant dry matter partitioning genotype x flood
interaction term s................................................ ................ 2 7

2-7 Primary root length, average diameter, surface area, and volume 49 days after
sow ing ..............................................................................29

2-8 Adventitious root dry weights and dry matter partitioning ..............................31

2-9 Genotype comparisons for significant adventitious root dry weight genotype x
flo o d interaction term ......................................... .............................................32

2-10 Adventitious root length, average diameter, surface area, and volume 49 days
after sow in g ............. ......... .. .. .......... ..... .... ...... ............................... 3 3

2-11 Nodule dry weight and dry matter partitioning..................... ............... 35

2-12 Spearman's rank correlation coefficients for selected rhizosphere plant
components under flooded conditions as correlated to tolerance rank.................36

2-13 Rhizosphere dry weight contrast for flood "tolerant" vs. "sensitive" genotypes
as defined by C ornelious.............................................. .............................. 40

2-14 Primary and adventitious root length, surface area, diameter, and volume,
contrast value for flood "tolerant" vs. flood "sensitive genotypes as defined
by C ornelious. ........................................................................40









3-1 Genotype yield reduction and flood injury scored for most tolerant and most
sensitive A rkansas R IL 's. ........................................................... .....................46

3-2 Regression equations and R2 values for leaf area by genotype used to
determine non-destructive leaf area values.......... .......................................47

3-3 Stem dry weight and dry matter partitioning (Experiment 1).............................49

3-4 Genotype comparison for all significant dry matter partitioning flood x
genotype interaction term s ............................................................................... ...51

3-5 Plant heights.................................................... ...................... ..... ........ 53

3-6 Leaf dry weights and dry matter partitioning. ...................................................... 54

3-7 Increm ental leaf area expansion....................................... .......................... 56

3-8 L eaf area ...................................................................................................... .......60

3-9 T otal plant dry w eight. ............................................................................. .... .... 6 1

3-10 R oot: Shoot ratios ......................................................................... ......... ............62

3-11 Tolerance of all genotypes to early-season flooding. .........................................64

3-12 Stem dry weight means for flooded treatments and ranks used for correlations...64

3-13 Correlations between selected plant measures and flood tolerance.....................65

3-14 Stem dry matter partitioning means for flooded treatments and ranks used for
correlations ............. .... .......... ................... ............. ... ...... 65

3-15 Leaf dry weight means for flooded treatments and ranks used for correlations....66

3-16 Aboveground dry weight contrasts between "tolerant" vs. "sensitive"
genotypes as defined by Cornelious (2003) across 2- and 4-week flood
treatm ents ............................................. ............................ 67

3-17 Plant height, incremental leaf area, and leaf area values, contrasts between
"tolerant" vs. "sensitive" genotypes as defined by Cornelious (2003) across 2-
and 4-w eek flood treatm ents ........................................ ............................ 67

A -l Prim ary root dry w eights. ........................................................... .....................73

A-2 Primary root length, diameter, surface area, and volume. ....................................74

A-3 Adventitious root dry weight and dry matter partitioning. ...................................75

A-4 Adventitious root length, diameter, surface area, and volume. ..........................76









A-5 Nodule dry weight and dry matter partitioning............................ ..............77

A-6 Stem dry weight and dry matter partitioning. .............................. ................ 78

A -7 P lant height. ...................................................... ................. 79

A-8 Leaf Dry weights and dry matter partitioning......................................................80

A -9 L eaf area v alu es. .................. .... ........ ...................... .. ........ .......... .. .... 1

A -10 T total plant dry w eight ............................................................................. ..... 82

A -11 Root:shoot ratios .................. ........................................... ... ......... 83

A -12 T tolerance at 49 D A S .............................................................................. .... ........83

B-l Leaf nutrient analysis (Experiment 1).........................................................85

B-2 Leaf nutrient analysis (Experiment 2)................ .......................................86

B-3 SPAD readings (Experiment 1). ........................................ ....................... 87

B-4 SPAD readings (Experiment 2). ........................................ ....................... 88
















LIST OF FIGURES


Figure page

2-1 Rainfall at EREC Belle Glade over the duration of Experiments 1 and 2. Day 1
for Experiment 1 = May 25, 2004. Day 1 for Experiment 2 = June 29, 2004. .......22

2-2 Graph showing lack of linear fit in the relationship between adventitious root
length and dry w eight ....................... ........ .......... ............... ......... 38

2-3 Graph showing lack of linear fit in the relationship between adventitious root
surface area and dry w eight ...................... .... .......... ..................... ............... 38

3-1 Incremental leaf area expansion for all genotypes across flood treatments ............57

3-2 Incremental leaf area expansion for "most sensitive" genotypes...........................57

3-3 Incremental leaf area expansion for "most tolerant" genotypes.............................. 58

3-4 Incremental leaf area expansion for control genotype Hinson ...............................58

3-5 Linear regression of tolerance ranks from Arkansas and Belle Glade ....................68















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MORPHOLOGICAL ADAPTATIONS OF SOYBEAN IN RESPONSE TO EARLY
SEASON FLOOD STRESS

By

Thomas L. Henshaw

August 2005

Chair: Robert A. Gilbert
Major Department: Interdisciplinary Ecology

Soybean has been considered for some time as a crop with potential for flood

tolerance because of its highly adaptive nature under flood stress. The designation of

genetic markers for flood tolerance in southern varieties has kindled interest in further

defining the physiological properties associated with acclimation to flooding. The

objective of this research was to identify distinguishable morphological changes in

response to early season flooding (V2 growth stage), in an attempt to isolate a

characteristic or groups of characteristics that can be used for identification of potentially

flood tolerant varieties. A lysimeter trial was carried out at the University of Florida

Everglades Research and Education Center, Belle Glade, Florida, from May to August

2004. Eleven genotypes (ten numbered Recombinant Inbred Lines (RIL) and Hinson)

were flooded at the V2 growth stage. Plants were subjected to one of three flood regimes

(well watered, 2 weeks of flooding with 2-week recovery, or 4 weeks of flooding).

Consistent patterns of adaptation developed with additional flood duration including









decrease in leaf area, total biomass, and nodulation accumulation, and an increase in

adventitious root length. Genotypic response to flooding was significantly different for

all plant tissue types, while differentiation between varieties based on flood tolerance was

inconsistent. Growth characteristics including primary root dry weight, length, surface

area, and adventitious root dry weight have been identified as positively correlated to

flood tolerance under these experimental conditions.














CHAPTER 1
INTRODUCTION

Overview

This chapter provides an overview of the rationale, approach, and hypotheses

underlying this study pertaining to the morphological response of soybean to early-season

flood stress. Subsequent chapters include comprehensive literature reviews, materials

and methods, results, discussion and conclusion sections. Chapter 2 outlines the effect of

flooding treatments on primary roots, adventitious roots, and nodules. Chapter 3

describes the effects of the same treatments on stem, leaf, and whole plant biomass as

well as the relationship between root and shoot growth. Chapter 4 contains my

perspectives on the research process based on my experience during my M.S. This

division of chapters allows for the independent analysis of plant components that are

interactive in nature, as well as a comprehensive analysis of the plant as an entire entity.

Introduction

Background

The Everglades Agricultural Area (EAA) is located on the southeastern shore of

lake Okeechobee, primarily in Hendry and Palm Beach Counties, Florida. With a

sugarcane production area of approximately 180,000 ha (Schueneman, 2002), this region

is home to more than 50% of the United States sugar cane production. Established in the

1920's, the sugarcane industry boomed in the 1960's with the exclusion of Cuban access

to US markets. Agricultural production in the area was made possible primarily by two

technological developments, the introduction of varieties adapted to Florida's climate and









the development of a regional drainage structure including canals that allowed for control

of water levels in this often-flooded area (Alvarez and Polopolus, 2002).

The soils primarily used for sugarcane production are Histosols characterized by

high organic matter content, deep organic matter profile, and low clay mineral content

(Rice et al., 2005). While fertilization recommendations vary dramatically depending on

soil location and properties, these soils are generally recognized as highly fertile.

Conversely, approximately 20% of the sugarcane grown in Florida is grown on sandy

mineral soils adjacent to the muck soils of the EAA. Soils in this region are fine to

coarse textured sands with varying organic matter content predominantly classified as

Spodosols, but Entisols and Mollisols occur in transitional areas. The majority of these

soils are subject to flooding because of the close proximity of bedrock and hard pans

resulting in seasonally high water tables. High variability in soil characteristics,

inherently low nutrient content, and drainage concerns combine to create a need for

intensive crop management in this region (Gilbert et al., 2002).

Crop rotations with sugarcane on mineral soils

Sugarcane is a perennial crop, generally harvested at yearly intervals between

October and April. The following year's crop (ratoon) is the result of re-growth from the

rhizomes and stubble left after harvest. A field will remain in production until yields

have fallen below an acceptable threshold. The yearly reduction in yield is more

accentuated on mineral soils with their low organic matter content and limited inherent

soil fertility (Muchovej, 2002). While sugarcane fields may stay in production for

several years, once a field is removed from production, there may be an extended period

of fallow between final harvest and replanting. It is well documented that the use of a

leguminous cover crop can add both soil organic matter and offer a substantial source (up









to 50-200 kg ha-1) of organic nitrogen (Powers and McSorley, 2000). Considering the

soil fertility limitations of mineral soils for sugarcane production, use of a legume-based

rotation during fallow appears to be desirable.

Environmental and agronomic limitations make selection of an appropriate

rotational crop critical to its potential success in this region. Ideally, the rotation crop

would provide substantial amounts of biomass and provide a high rate of nitrogen

fixation. When selecting a leguminous rotation crop the following considerations are

important: 1) It must fit into the agronomic calendar used by sugarcane growers, that is

to say, there is a window of approximately six months in which the rotation crop should

reach its full beneficial potential; 2) It must not interfere with future cane production,

germination, or growth; 3) Its seed must be readily available; 4) It should provide

significant increases in cane production; 5) It must be financially appropriate from a

cost/benefit perspective; and 6) It must be adapted to the climatic conditions of the

region, including high average temperature, periodic flooding, and high water tables

(Muchovej, 2002).

Soybean (Glycine max. L. Merrill) as a rotational crop

Soybean, possibly because of its value as a food staple, is mentioned less often than

other annual legumes when considering potential green manures. Muchovej (2002)

however, cites that soybean can fix as much as 112 kg ha-1 N, more than cowpea (Vigna

sinesis), peanuts (Arachis hypogaea), or snap beans (Phaseolus vulgaris). While soybean

is generally considered susceptible to flood stress (Sullivan et al., 2001), it has proven to

be more resistant to soil waterlogging than cowpea, a commonly grown crop in the EAA

(Andreeva et al., 1987). Considering the limitations placed on variety selection for green

manures in this growing environment, soybean appears to merit additional investigation.









Rationale

Soybean has not been grown commercially in South Florida. As such there is

limited information on the acclimation of this species to the specific production

environments that prevail in this region and its performance under local conditions.

Concurrently there is increased interest in identifying and generating flood tolerant

varieties that can allow for increased production in areas prone to waterlogging across the

United States. The opportunity to investigate soybean flood tolerance is both pertinent to

the potential use of soybean as a rotational crop in South Florida and potentially

enlightening in the development of new tolerant varieties. In addition the availability of

ten recombinant inbred lines (RIL's) bred and tested for flood tolerance at the University

of Arkansas (Comelious, 2003) offered a unique opportunity to combine documented

tolerance research with additional studies on plant growth.

Selection of genetic material

All experiments included in this study drew on the pool often RIL's generated and

previously tested for flood tolerance at the University of Arkansas (Cornelious, 2003).

Each genotype had been rated based on visual "flood injury" and yield potential. The ten

RIL's included in this study represent the five "most tolerant" and five "most sensitive"

lines based on the Arkansas grading methods. Genotypes available included: most

sensitive, 91209-119, 91209-220, 91209-269, 91209-143, 91209-297 and most tolerant,

91209-126, 91209-142, 91209-151, 91210-316, and 91210-350. The trial also included

Hinson, a recommended variety for soybean maturity group VIII in Florida, as a

commercial check.









Flood tolerance

The available genetic material had been rated for tolerance based on response to a

10-14 day flood treatment at the R2 growth stage (Fehr and Caviness, 1977). A 0-9

visual flood injury scale was used with 0 = no injury and 9 =severe injury. In addition,

soybean grain yield response was measured as a percent decrease in yield for flooded

treatments compared with non-flooded treatments (1-[grain yield flood / grain yield

control])* 100% (Cornelious, 2003). For the purpose of this study "tolerance" is defined

as the mean total plant dry weight of flooded treatments expressed as a percentage of the

total plant dry weight of the non-flooded treatment (plant dry weight flood / plant dry

weight non-flood). Using this approach, the relationship between flooded and non-

flooded treatments is maintained. The expression of tolerance is however, the inverse of

that used by Cornelious, resulting in higher tolerance rating ratios for materials with

higher tolerances.

Approach

Following the logic of Bacanamwo and Purcell (1999a, 1999b) and emphasizing

the key role of morphological adaptations in the avoidance of flood injury, the intent of

these experiments was to determine if a link exists between identifiable morphological

traits and flood "tolerance." Flooding at an early season growth stage (V2) was selected

in an attempt to determine the possibility of reducing the time and expense associated

with genotypic flood tolerance identification. V2 had been identified as an early season

soybean growth stage susceptible to flood stress (Linkemer et al., 1998), thus flooding at

this stage would highlight any genotypic differences. Morphological changes have been

used for early identification of growth traits in other legume species (Sultana et al.,

2002). Morphological changes of interest based on the current literature on the









adaptation of plants to flood stress include: Root growth parameters including dry

weights of primary and adventitious roots, primary and adventitious root length and area,

and nodule development. Shoot growth parameters of interest included dry weights of

leaves and stems, SPAD leaf greenness readings, and plant height.

Null Hypotheses

1. HoI: No significant soybean morphological differences will occur between soybean
genotypes subjected to various flooding treatments (Chapters 2 and 3).

2. HoII: No significant soybean morphological differences will be observed between
flood treatments (Chapters 2 and 3).

3. Ho0II: There are no significant flood x genotype interactions for soybean
morphological traits (Chapters 2 and 3).

4. HoIv: There are no significant differences in flood "tolerance" among genotypes
when subjected to various flood treatments (Chapter 3).

5. Hov: There is no significant relationship between flood tolerance and soybean
morphological traits (Chapters 2 and 3).

6. HovI: There is no significant difference between flood tolerance rankings reported
in this study and flood tolerance rankings reported by the University of Arkansas
(Chapter 3).















CHAPTER 2
THE EFFECT OF FLOODING AND GENOTYPE ON THE SOYBEAN (GLYCINE
MAX L. MERRILL) RHIZOSPHERE

Review of Literature

Identification of Tolerance to Flood Stress

Flood tolerance may vary greatly between plant species. Variation in maximum

allowable flood duration ranging from a few hours to weeks can be attributed to two

primary factors: the increased availability of oxygen to the roots provided by

morphological adaptations in tolerant plants, and different biochemical responses to

anaerobic conditions (Kramer and Boyer, 1995). Intra-species variation in flood

tolerance depends greatly on the plant organs directly affected by flooding, stage of plant

development at which the flood is imposed, and external conditions such as temperature

(Vartapetian and Jackson, 1997).

While a net reduction in growth is to be expected under anaerobic conditions, many

plants exhibit a number of adaptive morphological responses that result in increased

growth or extension of certain tissues (Jackson and Drew, 1984). The primary adaptation

of the rhizosphere is the development of adventitious roots. Adventitious roots are

defined as non-primary roots emerging from hypocotyl tissue (Zobel, 1991) and are

commonly viewed as a partial replacement for the incapacitated original root system

(Jackson and Drew, 1984).

Various methods have been used to identify flood tolerance. It is generally

accepted that yield of high quality marketable seeds is the ultimate judging factor (Van









Toai et al., 1994; Roiselle and Hamblin, 1981). Van Toai et al. (1994) rated flood

tolerance of 84 varieties by total grain yield under flooded and non-flooded conditions,

allowing for a comparative ranking of tolerance based on yield. Roiselle and Hamblin

(1981) used a comparative yield analysis defined as the average of flooded and non-

flooded yields. This approach selects the variety promising highest yield return

regardless of stress conditions. Van Toai and Nurijani (1996) flooded soybeans for the

length of the growing season (approximately 12 weeks) and measured average seed yield

of flooded plants and SPAD leaf color reading for each variety. These values were

divided by the values for the control. The resulting ratio represented a measure of

tolerance based on relative yield within varieties rather than total yield. Bacanamwo and

Purcell (1999a, 1999b) took a different approach to determining tolerance. Emphasizing

the necessity of morphological adaptations for avoidance of flooding injury, they

suggested that selecting varieties for morphological adaptations to flooding may be the

best way to select for flooding tolerance. This study uses an approach similar to that of

Van Toai and Nurijani (1996) while incorporating the morphological emphasis of

Bacanamwo and Purcell (1999a, 1999b). Using a flooded to control ratio allows for the

elimination of bias given to genotypes accumulating greater quantities of biomass under

all treatments.

While soybean is generally considered susceptible to flooding (Bacanamwo and

Purcell, 1999a, 1999b; Sullivan et al., 2001) showing reductions in growth and yield

when flooded for 7 days (Oosterhuis et al., 1990; Sallam and Scott, 1987), there is

evidence that in comparison to other legumes, soybean has the ability to adapt to soil

waterlogging. Andreeva et al. (1987) reported soybean to be more flood tolerant than









cowpea. Boru et al. (2003) showed no negative effects on survival or leaf greenness of

soybean plants grown in nitrogen gas with no detectable oxygen for 14 days, suggesting

that soybean is more tolerant to increased levels of water and decreased oxygen than

previously thought.

The effects of waterlogging on the soybean rhizosphere are substantial and

generally negative. A decrease in nitrogen accumulation for flooded soybean plants

(Pankhurst and Sprent, 1975; Sugimoto and Satou, 1990) has been identified as the

limiting factor to growth (Bacanamwo and Purcell, 1999a). Flooding for one week was

sufficient to reduce leaf nitrogen concentration levels below deficiency at early

vegetative stages (Sullivan et al., 2001). Reduction in nitrogen has been attributed to

decreased nodulation (Sprent, 1972; Sallam and Scott, 1987; Sung; 1993), increased

levels of ethylene (Sprent and Gallacher, 1976), and decreased nitrogenase activity

(Bennett and Albrecht, 1984). However, after 4 days of flooding there was no

destruction of cellular mitochondria in roots (Andreeva et al., 1987). Maintaining

mitochondrial integrity is essential to continued nitrogenase activity. After an initial

depression in nitrogenase activity flooded soybean plants actually recovered a level of

activity comparable to that of the control (Bennett and Albrecht, 1984). Though a

reduction in all root growth parameters was observed (Sallam and Scott, 1987), the

development of new nodules at the soil surface and on newly developed adventitious

roots offset the loss of original nodule function (Bacanamwo and Purcell, 1999b; Bennett

and Albrecht, 1984) in flooded soybean. Plant nitrogen fixation returned to near normal

levels within 15 days after removal of flood treatments of up to 14 days (Sugimoto and

Satou, 1990; Bennett and Albrecht, 1984). The recovery of nodule function coinciding









with the rebound in plant growth after the removal of flood treatments provides evidence

that active root nodules are necessary for flood tolerance (Sprent, 1972; Sugimoto and

Satou, 1990).

Short-term acclimation to flooding through biochemical responses may allow for

avoidance of certain injurious factors (Vartapetian and Jackson, 1997). These

biochemical changes have been shown, in the case of wheat, to be temporary (Good and

Muench, 1993). Long-term flood tolerance will require morphological adaptations in the

plant allowing for increased aeration of the roots (Vartapetian and Jackson, 1997;

Bacanamwo and Purcell, 1999b).

"Adventitious rooting is a mechanism for replacing existing roots that have been

killed or whose function is impaired by anoxia at depth" (Vartapetian and Jackson, 1997).

Extensive adventitious root development has been reported to enhance oxygen transport

from the stem to the roots (Visser et al., 1996) and reduce flooding injury in soybeans

(Lee et al., 2003). Adventitious root fresh weight as a percentage of total root weight was

greatly increased by flooding. Twenty-one days of flooding at the V4-V6 growth stage

resulted in adventitious root percentages of 33-41% total root fresh weight (Bacanamwo

and Purcell, 1999b). Pires et al. (2002) reported similar results for a 14-day flood at the

V2 growth stage.

Aerenchyma is spongy modified tissue containing large connected pores that

facilitates gas exchange (Evans, 2003). Flooding in soybean plants greatly increased the

incidence of aerenchyma in the cortex of roots. Aerenchyma development was primarily

evident in new adventitious roots, whereas primary roots of flooded plants exhibited

tightly packed cortical cells (Bacanamwo and Purcell, 1999b). Cortical aerenchyma









formation especially in adventitious roots was apparent after 21 days of flooding from the

V2 to V5 growth stages (Pires et al., 2002). Inhibition of adventitious root and

aerenchyma development through the application of 5 uM Ag, which counters ethylene

reduction, increased the flooding susceptibility of soybeans. An additional loss of 25%

biomass and leaf nitrogen accumulation over simple flooded treatments was witnessed

with the application of Ag. This indicates that adventitious root and aerenchyma

formation are important for acclimation to flooding (Bacanamwo and Purcell, 1999b).

Selecting Soybean Genotypes for Flood Tolerance

Though the ultimate criterion for judging flood tolerance is seed yield, early-season

screening techniques for flood tolerance would decrease the time and resources required

for plant breeding programs. Several traits have been used to make determinations about

flood tolerance in soybean including: leaf color, plant height, chlorophyll content, and

biomass of roots and shoots. Attempts to use these characteristics to develop a flood

resistant line have yet to be fully successful (Van Toai et al., 2001). Flood tolerance in

soybean plants is considered to be a developed adaptation specific to particular genotypes

rather than an inherent property (Van Toai and Nurijani, 1996), therefore there is genetic

variability in tolerance among cultivars (Van Toai et al., 1994; Van Toai and Nurijani,

1996).

Van Toai et al. (2001) used 122 recombinant inbred lines (RIL) of an Archer x

Minsoy cross and 86 RIL's of an Archer x Noir cross, noting that Archer is more flood

tolerant than either of the other two varieties. Flooding response was measured by a

comparison of plant height and seed yield. A single quantitative trait locus (QTL) was

identified for both increased height and increased yield. Plants containing the Archer

Sat_064 allele yielded 95% more and were 16% taller than control plants. The effect of a









single marker on both height and yield suggests that plants that grew better under

waterlogged conditions also yielded more (Van Toai et al., 2001)

A second trial performed in Costa Rica, flooding plants for 10-14 days until mild

chlorosis was noted, showed a very different result (Reyna et al., 2003). Plants

containing the Archer Sat_064 marker showed no difference in yield between flooded

and non-flooded treatments. Plants containing the southern (A5403) Sat_064 allele did

show significant yield advantages over plants not containing that marker (Reyna et al.,

2003).

While both trials do show genotypic variation in flood tolerance and the potential

for identification of pertinent genetic markers, the inconsistent demonstration of flood

tolerance in these strains highlights the current limitations of the flood tolerance

identification process in soybean. There is a critical need for further investigation of both

genetic and morphological flood tolerance indicators. Positive correlations between early-

season flood adaptations, and flood tolerance as defined by grain yield, could greatly

reduce the time and resources needed to identify potentially flood tolerant varieties for

breeding purposes.

This chapter highlights the morphological adaptations of the rhizosphere portion of

the soybean plant to flooding in an attempt to isolate specific physiological characteristics

that may help identify flood tolerant germplasm. Reporting the growth patterns of

isolated plant components and their correlation to tolerance allows us to more closely

examine the immediate effects of flood treatments on these components. The rhizosphere

fraction is most immediately affected by flooding due to its proximity to the source of

stress (Kramer and Boyer, 1995). Our objectives were to examine the effects of flooding









soybean on 1) growth of the primary rooting system, 2) the development and expansion

of adventitious roots, 3) nodule growth, and also to 4) determine the relationship of

rhizosphere morphological changes to overall flood tolerance.

Materials and Methods

Two experiments were conducted at the Everglades Research and Education Center

(EREC, 260 39' N, 800 38' W), in Belle Glade, Florida. Twenty-four separate concrete

lysimeters were used in two separate experiments. The lysimeters used for Experiment 1

had an inside measurement of 3.5 m x 0.97 m and a depth of 0.91 m, while those used for

the Experiment 2 measured 2.29 m x 1.07 m also at a depth of 0.91 m. Each lysimeter

was equipped with a gravel leach bed approximately 15 cm in depth and a pvc release

valve extending from one end (short side) of the container. Inserting or removing a

stopper from the release valve allowed free drainage by gravity of water accumulated in

the leach bed. Flood treatments were maintained at constant height through the use of a

float valve set to add water whenever the level fell below 3 cm above the soil surface.

Each lysimeter was filled to approximately 5 cm below the container lip with a topsoil

mix (Odum's Inc., West Palm Beach, Fl) consisting of 90% coarse sand and 10 %

organic soil. This topsoil mix was chosen as it most closely approximated the mineral

soils in the Everglades Agricultural Area. Planting for Experiment 1 took place on May

25, 2004 and planting for Experiment 2 was performed on June 29, 2004. Both

experiments had a total duration of 49 days.

Selection of Genetic Material

Experiment 1 included ten Recombinant Inbred Lines (RIL's) (provided by Dr.

Pengyin Chen, University of Arkansas) generated and previously tested for flood

tolerance at the University of Arkansas (Cornelious, 2003). Each genotype had been









rated based on visual "flood injury" and yield. The ten genotypes included in this study

represented the five "most tolerant" and five "least tolerant" lines based on the Arkansas

grading methods. The most tolerant genotypes included were 91209-126, 91209-142,

91209-151, 91210-316, and 91210-350. The most sensitive genotypes were 91209-119,

91209-143, 91209-220, 91209-269, and 91209-297. The trial also included Hinson, a

commercial variety recommended for soybean maturity group VIII in Florida. Flood

tolerance rankings remained unknown to the researchers until the conclusion of the trials.

Due to space limitations, six of the eleven genotypes used in Experiment 1 of the

lysimeter trial were selected for Experiment 2. Selection of these six genotypes was

based on the comparison of dry weight data from a 21-day preliminary trial. The ratios

of plant dry weights from the 21-day flood treatment to plant dry weights of a non-

flooded treatment were calculated for each genotype. The three genotypes showing the

greatest flood/dry ratio and the two varieties having the lowest flood/dry ratio were

selected for the second experiment. Hinson was also included to continue as a

commercial check. The genotypes selected were: 91209-126, 91209-142, 91209-220,

91209-269, 91210-350, and Hinson.

Experimental Design

Experiment 1 consisted of a 3 x 11 factorial experiment arranged in a split-plot

design with 4 replicates (2.1). Each of the twelve lysimeters represented one of three

flood treatments or main plot effects; no flood, 2 week flood duration followed by 2

weeks without flood, or 4 week flood duration. The main plots (lysimeters) where then

divided into eleven sub-plots representing each of the eleven selected soybean genotypes.

Flood treatments were randomly assigned to the three lysimeters within each replication

and genotypes were randomly assigned plot position within the lysimeters. This process










was repeated independently for each of the four replicates. The design of Experiment 2

was similar to Experiment 1, however only the six selected genotypes were used.

Table 2-1 Experimental designs used for soybean flooding experiments.
Experiment 1 3 x 11 factorial design Experiment 2 3 x 6 factorial design
Main Plots Sub-plots Replications Main Plots Sub-plots Replications
Flood Treatment Genotype 4 Flood Treatment Genotype 4
Non-flooded 91209-119 Non-flooded 91209-126
2 Week Flood 91209-126 2 Week Flood 91209-142
4 Week Flood 91209-142 4 Week Flood 91209-220
91209-143 91209-269
91209-151 91210-350
91209-220 Hinson
91209-269
91209-297
91210-316
91210-350
Hinson

Crop Management

Seeds were inoculated with soybean-specific rhizobium to enhance initial

nodulation. Plant spacing was 10 cm within rows and 28 cm between rows. Sowing was

designed to minimize border effects. A row of Hinson seed was added to each end of the

lysimeter and each sub plot row was bordered with a Hinson plant. Each lysimeter in

Experiment 1 was sown with seven plants per genotype, while eight plants per genotype

were sown in Experiment 2. Any seeding gaps were re-sown after germination to

eliminate the "gap effect" and maintain the integrity of the sampling pattern. These late-

sown plants were omitted during sampling.

Flood treatments were imposed when 50% of the plants reached the V2 growth

stage across sub-plots genotypess), main-plots (flood treatments), and replicates. V2 is

defined as the full extension of the second trifoliolate leaf beyond the unifoliate node

(Fehr and Caviness, 1977) and was identified as an early season growth stage susceptible

to flooding stress (Linkemer et al., 1998). Plants were maintained as "well-watered" up









to the initiation of the flood treatments as determined by soil volumetric water content

(VWC) measure of 0.15 as taken by a Field Scout TDR 100 (Spectrum Technologies

Inc., Plainfield, IL). Flood treatments imposed at the V2 growth stage consisted of

raising the lysimeter water level to approximately 3 cm above the soil surface. Flooding

depth was maintained at this level using a float valve positioned to allow automatic refill

when water height dropped below 3 cm. Each lysimeter within a replicate was assigned a

flood treatment of: no flood, 2-week duration, or 4-week duration. Treatments of 2

weeks and 4 weeks were initiated at the same time allowing the 2-week flooded plants a

recovery period of 2 weeks. The non-flooded treatment was maintained at near field

capacity throughout the trial. The 2-week duration treatment was maintained near field

capacity for the final two weeks of the trial, after the imposed flood was removed. Flood

treatments were identical for both experiments.

Fertilizer application amount was based on a soil analysis performed at the

Everglades Soil Testing Laboratory (2.2). Nitrogen tests are not considered to be

meaningful for Florida's sandy soils and therefore not included (Sartain, 2001). The

nitrogen application of 22 kg ha-1 was based on University of Florida Extension

recommendations for snap beans on similar soils within the region Nitrogen was

applied at 36 and 24 grams per lysimeter in Experiments 1 and 2 respectively using a urea

(21% N) formulation. Phosphorus was applied as triple super phosphate at 110 kg P205

ha-1 translating to an application rate of 82 grams and 56 grams of fertilizer per lysimeter

for Experiments 1 and 2 respectively. Potassium, also recommended at 110 kg K20 ha-1,

was applied as potassium chloride at 63 and 43 grams per lysimeter in Experiments 1 and


No N fertilizer recommendations for soybean in South Florida have been established.













Table 2-2 Preliminary Mehlich-I soil test results and fertilizer recommendations.
Soil Test Values (kg ha-1) Recommendations (kg ha-1)
Sample ID Soil Texture pH P K Ca Mg Si (mg kg-1) P205 K20 Mg
Lysimeter 1 sand 6.5 88 22 996 29 7 112 112 0
Lysimeter 2 sand 6.6 77 21 984 21 7 112 112 0
Lysimeter 3 sand 6.5 72 25 1049 29 7 112 112 0









2. Fertilizer was banded between rows and applied at sowing for both Experiments 1 and

2. An additional application of MicroMax (Lacebark Inc., Stillwater, OK) micronutrient

powder containing 15% S, 12% Fe, 2.5% Mn, 1% Zn, 0.5% Cu, 0.1% B, and 0.005% Mo

was applied to Experiment 1 at a rate 30 g lysimeter-1 at 14 and 21 days after planting.

For Experiment 2 the entire 60 g recommended dosage was applied 14 days after

planting.

Ridomil Gold (Sygenta Crop Protection, Inc.) fungicide was applied prior to

sowing in order to suppress root damage from Phytophthora root rot (at a rate of 11.9 and

7.2 g lysimeter-1) in Experiments 1 and 2 respectively.

Measurements

Volumetric water content (VWC) was measured every other day and used primarily

to determine "flooded" and "well watered" soil status. One sample was taken from each

end (short side) of the lysimeters for two total samples and averaged to give VWC of the

lysimeter as a whole. Each sample was taken halfway between planting rows in order to

minimize root disturbance. The exact sampling position was varied on each date to avoid

air pockets created by previous samples that would have affected VWC readings.

Flooded soils had VWC measurements > 0.45. Non-flooded treatments were maintained

at approximately 0.15 VWC using overhead watering.

Plant biomass sampling

Soybean biomass was sampled on three occasions over the course of each trial: prior to

the initiation of the flood treatments at 21 days after sowing (DAS), at the cessation of

the 2- week flood duration 35 DAS, and at final harvest 49 DAS. At each sampling date

a whole-plant sample was obtained including a 10 cm x 15 cm x 30 cm deep soil volume









centered around the stem of the plant to be harvested that contained the root portion of

that plant.

At 21 DAS one plant per sub-plot was harvested. Each plant was divided into root,

stem, and leaf portions. Roots were determined to begin at the soil surface. Leaf blades

were removed right above the petiole with petiole weight being included in the stem

fraction. The 35 DAS sampling also utilized one plant per sub-plot. Each plant was

divided into primary root, adventitious root, nodule, stem, and leaf components. Primary

and adventitious roots were segregated based on a visible gap between rooting layers

generally located just below the soil surface (Stoffella et al., 1979). No distinction was

made between nodules taken from adventitious and primary roots. The final harvest

sample consisted of three plants. Each of the samples was divided into plant components

using the same criteria employed at 35 DAS. Adventitious roots and primary roots from

one of the three plants were preserved separately for root scanning by wrapping them in a

paper towel, sealing them in a plastic freezer bag, and freezing (-20C).

All samples (with the exception of those frozen for subsequent root analysis) were

dried at 65C to a constant weight. Dry weights were recorded for each plant component

for all sampling dates, sub-plots, main-plots, and replicates. For the purpose of statistical

analysis the three samples taken at 49 DAS were averaged to calculate dry weight per

plant.

Root growth

Primary and adventitious root samples collected at harvest were scanned using

WinRhizoPro (Regent Instruments, Sainte-Foy, QC, Canada) and analyzed for total root

length, average diameter, projected surface area, and volume. Each sample was

suspended in water and scanned individually. After scanning all samples were dried at









65 C to a constant weight. The resulting dry weight was used to complete the 49 DAP

harvest dry-weight calculations.

Statistical Analysis

Analyses of variance were performed for all experimental measures using proc

GLM in SAS (Littell et al., 2002) with flood regimes being the main plot treatments and

genotypes the sub-plots. Significant differences among cultivars and flood treatments

were determined using Fisher's protected LSD test (P<0.05). Further analysis of

significant interaction terms was performed using the proc GLM 'Estimate' statement in

SAS using the flood x genotype means for genotype 91210-350 as the comparative base.

91210-350 was chosen for comparison based on its performance as the genotype "most

tolerant" to flooding in the Arkansas trials (Cornelious, 2003). The proc GLM 'Estimate'

statement was also used to contrast group response to flooding. The mean value for all

flood "tolerant" genotypes as defined by Cornelious (2003) across the 2-and 4-week

flood treatments was contrasted against the mean value for all flood "sensitive"

genotypes. Additionally, proc CORR in SAS was used to calculate Spearman's rank

correlation coefficients to determine significant correlations between morphological

measurements and genotype flood tolerance rankings.

For the purposes of this study flood tolerance was defined as the mean total plant

dry weight of flooded treatments divided by the total plant dry weight of the non-flooded

treatment (plant dry weight flood / plant dry weight non-flood). Using this method,

correlations were made between soybean morphological changes and early-season flood

tolerance, as well as flood tolerance defined by grain yield (Cornelious, 2003).









Results and Discussion

Results from Experiment 2 are presented in Appendix A. These results were

rendered ineffectual by the inability to establish an effective non-flooded control.

Discrepancies between Experiment 1 and 2 may be attributable to persistent precipitation

post-flood initiation for Experiment 2. During Experiment 2 twenty-seven days of rain

marked the period between flood initiation and final harvest including 10 consecutive

days immediately post-flooding (Figure 2-1). In contrast for Experiment 1 only 10 days

of precipitation occurred during the flood initiation to final harvest period (Figure 2-1).

Evaluation of weather data for this time period (2.3) points most strongly to precipitation

as the cause of Experiment 2 growth inconsistent with Experiment 1. Noting the

propensity for extended periods of precipitation during late summer in south Florida, two

recommendations can be made: 1) Soybean trials should be performed with earlier

sowing dates, preferably April-May in south Florida, and 2) In order to better guarantee

the quality of the control future experiments should be conducted under conditions

controlled more completely for external environmental influences.

Primary Root Dry Weight and Dry Matter Partitioning

No significant differences in the main (flood) effect were noted in primary root dry

weights based on samples taken 21 or 35 days after sowing (2.4). However, at final

harvest (49 DAS) the non-flooded treatment root weight was significantly greater (822

mg plant-1), than either 2-week (325 mg plant-) or 4-week (302 mg plant-) flooded

treatments (2.4).

Decrease in root biomass accumulation in flooded soybean has been noted by Pires

et al. (2002) and Sallam and Scott (1987). Dry weight losses are generally attributed to

dieback of the primary rooting system (Varpetian and Jackson, 1997,











Belle Glade EREC Rainfall


19 25 31 37


43 49


Days After Sowing

Figure 2-1 Rainfall at EREC Belle Glade over the duration of Experiments 1 and 2. Day
1 for Experiment 1 = May 25, 2004. Day 1 for Experiment 2 = June 29, 2004.


Table 2-3 Weekly average temperature and precipitation data for Belle Glade EREC,
May-August 2004.


Max Air Temp


Week Starting
May 23rd
May 30th
June 6th
June 13th
June 20th
June 27th
July 4th
July 11th
July 18th
July 25th
August 1st
August 8th
August 15th


32.4
33.7
31.6
32.4
33.7
32.5
33.7
33.2
31.4
31.9
32.0
33.5
32.7


Min Air Temp


18.5
21.1
21.5
22.5
22.6
22.0
21.9
21.4
22.0
21.9
23.0
23.0
22.9


Avg. Air Temp.


25.2
26.4
25.5
26.5
27.2
26.4
26.8
26.1
25.6
25.7
25.6
26.9
26.6


-Z Experiment
C Experiment 2





IM


Rainfall
mm


1.8
10.5
13.1
5.5
5.9

















Table 2-4 Primary root dry weight, dry matter partitioning, and relative dry weight.
Days After Sowing
21 35 49
Dry Weight Partitioning: Dry Weight Partitioning Dry Weight RDW* Partitioning
mg plant1 mg plant- mg plant-
Flood
2 Week 159 0.244 279 0.213 325 b 0.469 0.118
4 Week 168 0.249 259 0.189 302 b 0.431 0.138
Non-flooded 152 0.235 254 0.188 822 a 0.166
LSD 0.05 32 0.220 36 0.016 68 0.010 0.010
Significance ns ns ns ns ** ns ns

Genotype
Tolerant
91210-350 244 a 0.248 261 bc 0.169 c 472 bc 0.422 bcd 0.139 ab
91210-316 133 c 0.248 243 c 0.187 bc 528 ab 0.310 cd 0.124 b
91209-126 171 bc 0.256 267 bc 0.200 ab 482 bc 0.549 abc 0.127 b
91209-151 133 c 0.262 234 c 0.204 ab 506 abc 0.272 d 0.130 b
91209-142 133 c 0.241 213 c 0.198 abc 539 ab 0.405 bcd 0.131 b
Sensitive
91209-220 175 bc 0.231 270 bc 0.195 bc 458 bc 0.386 bcd 0.139 ab
91209-119 153 bct 0.263 317 ab 0.226 a 519 ab 0.599 ab 0.155 a
91209-269 143 c 0.236 223 c 0.211 ab 413 bc 0.643 ab 0.155 a
91209-297 131 c 0.215 253 bc 0.204 ab 386 c 0.302 cd 0.154 a
91209-143 207 ab 0.247 257 a 0.192 bc 624 a 0.328 cd 0.155 a
Control
Hinson 133 c 0.222 271 bc 0.182 bc 385 c 0.730 a 0.138 ab
LSD 0.05 61 0.041 68 0.0 131 0.030 0.019
Significance ** ns ** ** **

Flood x Genotype
Significance ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means in the same column followed by the same letter are not significantly different (P<0.05).
fDry matter partitioning is defined as primary root dry weight / total plant dry weight.
RDW = Relative dry weight is defined as primary root dry weight for flood treatment / primary root dry weight for non-flood treatment.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).









Jackson and Ricard, 2003) and a decrease in the amount of photo-assimilate available for

additional root extension (Trought and Drew, 1980). Delays in response to flooding have

also been reported by Sallam and Scott (1987). The delayed response of primary root dry

weight accumulation to 14 days of flooding starting at the V2 growth stage is most likely

attributed to the slow primary root growth rate of the soybean plant during the initial

growth phase (Sung-Guang et al., 1996). As the soybean plant reached the rapid growth

phase 35 DAS, flooding served to delay root elongation and thereby reduced dry weight

accumulation. However, based on the continual accumulation of additional biomass in

flooded roots it could be concluded that soybean plants produce new root material at a

greater rate than dieback of existing roots.

Genotypic differences in primary root dry weights persisted over all sampling dates

(2.4). Developmental differences in plant biomass based on soybean genotype were

documented by Bacanamwo and Purcell (1999a). In order to understand the contribution

of genotypic variation in relation to the main flood effect, it is necessary to separate

genotype responses by flood treatment. Differences in genotypic response to flood

treatment resulted in significance in the interaction term. Primary root dry weight

genotype x flood interaction was significant for the 35 DAS and 49 DAS sampling dates

(2.4). 2.5 presents the performance of each genotype under all flood treatments in

relation to a base value for 91210-350. Genotype 91209-143 had significantly greater

primary root dry weight in the non-flooded treatment, however in general primary root

dry weight of the germplasm tested was not significantly different from 91210-350 under

flooded conditions.













Table 2-5 Genotype comparisons for significant dry weight genotype x flood interaction terms. Values are differences between
genotype and 91210-350.
Primary Root 35 DAS Primary Root 49 DAS
non-flooded 2 week 4 week non-flooded 2 week 4 week
mg plant1
Genotype
Tolerant
91210-316 88 ns -140 -3 ns 204 ns -36 ns 1 ns
91209-126 50 ns -55 ns 23 ns -49 ns 88 ns -8 ns
91209-151 -23 ns -73 ns 15 ns 195 ns -38 ns -55 ns
91209-142 5 ns -85 ns -63 ns 121 ns 101 ns -19 ns
Sensitive
91209-220 -33 ns 35 ns 25 ns 12 ns 29 ns -84 ns
91209-119 143 88 ns -63 ns -16 ns 88 ns 71 ns
91209-269 -30 ns -38 ns -45 ns 212 ns 59 ns -24 ns
91209-297 43 ns -58 ns -8 ns -4 ns -51 ns -204 ns
91209-143 205 ** 33 ns 35 ns 360 ** 135 ns -39 ns
Control
Hinson -18 ns 30 ns 18 ns -251 59 ns -68 ns
91210-350t 215 303 265 789 286 341
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
tGenotype 91210-350 has been identified as the "most tolerant" genotype (Cornelious, 2003).
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).









Expressed as the ratio of primary root dry weight to total plant dry weight,

differences in partitioning of dry matter to primary roots due to flooding was not

significant (2.4). A steady decline, however, in the fraction of total biomass allocated to

roots was witnessed across sampling dates. As with dry weight, dry matter partitioning to

roots was significantly different among genotypes at 35 and 49 DAS. However,

partitioning was relatively constant ranging from 0.124 to 0.155 among all genotypes.

The genotype x flood interaction term was significant at 49 days after sowing. Genotype

91209-151 had a significantly greater allocation of biomass to primary roots than

genotype 91210-350 in the non-flooded treatment (+.039) and significantly lower fraction

allocated for the 4-week flood treatment (-.067) (2.6). Genotype 91210-350 was notable

for its high partitioning to roots under the 4-week flood treatment.

Relative dry weight measured at 49 days after sowing for Experiment 1 showed no

significant difference between the 2 and 4-week flood treatments (2.4). Significant

genotype differences were noted (2.4). No significant interaction existed between

genotype and flood effects.

The range in relative dry weights of primary roots demonstrates a strong genotypic

variation in ability to continue primary root growth under flooded conditions, with the

highest fraction maintenance for Hinson at 0.73 and the lowest for 91209-151 at 0.27. It

is important to note that accumulation of primary root biomass was reduced for all

genotypes under flooded relative to non-flooded treatments, this is again consistent with

the findings ofPires et al. (2002) and Sallam and Scott (1987).










Table 2-6 Genotype comparisons for significant dry matter partitioning genotype x flood
interaction terms. Values are differences between genotype and 91210-350.
Primary Root Dry Matter Partitioning: 49 DAS
Non-flooded 2 week 4 week
Genotype
Tolerant
91210-316 0.015 ns -0.019 ns -0.043 *
91209-126 0.012 ns -0.007 ns -0.042 *
91209-151 0.040 -0.002 ns -0.067 **
91209-142 0.033 ns -0.010 ns -0.049 **
Sensitive
91209-220 0.014 ns 0.005 ns -0.018 ns
91209-119 0.065 ** 0.003 ns -0.021 ns
91209-269 0.003 ns 0.029 ns -0.014 ns
91209-297 0.028 ns 0.022 ns -0.083 ns
91209-143 0.037 0.003 ns 0.062 ns
Control
Hinson 0.020 ns -0.014 ns -0.039 *
91210-350t 0.139 0.115 0.165
S,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.

tDry matter partitioning is defined as tap root dry weight / total plant dry weight.
tGenotype 91210-350 has been identified as the "most tolerant" genotype (Cornelious, 2003).
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).









Primary Root Length, Average Diameter, Surface Area, and Volume

Morphological characteristics of the primary roots followed the same pattern as root dry

weight. Plants in non-flooded treatments had significantly greater primary root length,

average diameter, surface area, and volume than flooded plants (2.7). No significant

differences were observed between genotypes, nor were any interaction terms significant.

Sallam and Scott (1987) describe the relationship between primary root area, extension

and dry weight as linear, while recording a noted decrease in the addition of root length

and area with the imposition of flood treatments. The linear nature of this relationship

implies that the same factors working to suppress dry weight accumulation are inhibiting

primary root extension and area. As with root dry weight, reduction in assimilates

directed to flooded roots (Trought and Drew, 1980) and dieback of primary roots

(Vartapetian and Jackson, 1997; Jackson and Ricard, 2003) serve to limit additional

growth.

Adventitious Root Dry Weight and Dry Matter Partitioning

As the primary rhizosphere morphological response to flooding in soybean, adventitious

root development is of particular interest in evaluating potentially tolerant genotypes.

Since adventitious roots developed only on flooded samples, analyses for these samples

were limited to dry weight, partitioning percentage, and root morphology.

Adventitious root dry weight was significantly affected by flood duration at 35 and

49 days after sowing (2.8), with the 4-week flood treatment biomass being greater than

the 2-week flood treatment. Plants in both flood treatments continued to add biomass to

the adventitious roots between 35 and 49 days after sowing, but statistical trends were

maintained. Significant differences among genotypes developed only at the










Table 2-7 Primary root length, average diameter, surface area, and volume 49 days after
sowing.
Length Avg. Diameter Surface Area Volume
cm plant-1 mm plant1 cm2 plant1 cm3 plant1
Flood
2 Week 1115 bt 0.266 b 83 b 0.498 b
4 Week 918 b 0.305 b 81 b 0.579 b
Non-flooded 1917 a 0.494 a 168 a 1.210 a
LSD 0.05 251 0.057 20 0.138
Significance ** ** *

Genotype
Tolerant
91210-350 1127 0.337 99 0.713
91210-316 1570 0.486 137 0.965
91209-126 1418 0.360 119 0.803
91209-151 1291 0.352 108 0.736
91209-142 1304 0.373 111 0.768
Sensitive
91209-220 1206 0.305 100 0.668
91209-119 1337 0.297 108 0.721
91209-269 1446 0.345 117 0.779
91209-297 1089 0.353 96 0.698
91209-143 1444 0.391 129 0.932
Control
Hinson 1158 0.299 91 0.587
LSD 0.05 481 0.109 38 0.256
Significance ns ns ns ns

Flood x Genotype
Significance ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).


49 DAS sampling date, and the genotype x flood interaction was also significant at this

date (2.8). Analysis of the significant interaction term revealed five genotypes with

significantly greater adventitious root weight than 91210-350 after 4 weeks of flooding.

Genotype 91209-151 produced 98 mg plant- more adventitious roots than 91210-350

(2.9). Significant increases in adventitious root development are cited as a key

morphological response to flooding in soybeans (Pires et al., 2002; Sallam and Scott,

1987, Bacanamwo and Purcell, 1999a). It should be noted that adventitious root biomass









(generally associated with flooding) continued to increase in the 2-week treatment after

the removal of flood treatments. This demonstrated that adventitious roots might have

assumed responsibility for primary root function in flooded plants; this is consistent with

the role defined for adventitious roots by Jackson and Drew (1984).

Adventitious root dry weight partitioning was significantly different between flood

treatments at 49 days after sowing (2.8). As with dry weight, the 4-week flood treatment

had a greater fraction of total biomass allocated to adventitious roots than did the 2-week

treatment. Significant genotype differences appeared at 35 days after sowing and

continued at 49 day after sowing.

Adventitious Root Length, Average Diameter, Surface Area, and Volume

Adventitious root length, average diameter, surface area, and volume were

significantly greater in the 4-week compared to 2-week flood treatments (2.10). Total

elongation was more than double (1235 cm) for the 4-week versus the 2-week treatment

(603 cm). Greater continued elongation of adventitious roots in the 4-week flooded

treatment may indicate a continuing dependence of plants in this treatment on

adventitious roots for plant aeration. Conversely, slower adventitious root elongation










Table 2-8 Adventitious root dry weights and dry matter partitioning.
Days After Sowing
35 49
Dry Weight Partitioning4 Dry Weight Partitioning
mg plant1 mg plant1
Flood
2 Week 18 bt 0.012 73 b 0.026 b
4 Week 26 a 0.019 94 a 0.038 a
Non-flooded -
LSD 0.05 7 0.004 13 0.003
Significance ** ns **

Genotype
Tolerant
91210-350 13 0.005 b 32 cd 0.014 d
91210-316 14 0.010 ab 65 ab 0.024 ab
91209-126 19 0.013 ab 69 ab 0.022 ab
91209-151 9 0.006 b 68 ab 0.027 a
91209-142 12 0.011 ab 74 a 0.021 bc
Sensitive
91209-220 16 0.009 ab 52 abc 0.022 ab
91209-119 14 0.012 ab 72 ab 0.024 ab
91209-269 12 0.013 ab 49 bc 0.022 ab
91209-297 15 0.011 ab 19 d 0.015 cd
91209-143 22 0.014 a 53 abc 0.020 bcd
Control
Hinson 14 0.008 ab 59 ab 0.022 ab
LSD 0.05 14 0.008 25 0.006
Significance ns ** **

Flood x Genotype
Significance ns ns ** ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
tDry matter partitioning is defined as adventitious root dry weight / total plant dry weight.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table 2-9 Genotype comparisons for significant adventitious root dry weight genotype x
flood interaction term. Values are differences between genotype and 91210-
350.
Adventitious Root 49 DAS
2 week 4 week
Genotype mg plant 1
Tolerant
91210-316 16 ns 84 **
91209-126 56 54 *
91209-151 10 ns 98***
91209-142 45 83 **
Sensitive
91209-220 33 ns 23 ns
91209-119 38 ns 83 **
91209-269 19 ns 33 ns
91209-297 -16 ns -23 ns
91209-143 39 ns 22 ns
Control
Hinson 18 ns 62 **
91210-350t 49 46
S,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.

t91210-350 has been identified as the "most tolerant" variety (Comelious, 2003).
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table 2-10 Adventitious root length, average diameter, surface area, and volume 49 days
after sowing.
Length Average Diameter Surface Area Volume
cm plant- mm plant cm2 plant' cm3 plant'
Flood
2 Week 603 bt 0.213 b 40.3 b 0.216 b
4 Week 1235 a 0.282 a 87.3 a 0.499 a
Non-flooded
LSD 0.05 245 0.038 18.8 0.123
Significance *

Genotype
Tolerant
91210-350 868 0.247 64.1 0.394
91210-316 1100 0.297 83.6 0.512
91209-126 899 0.220 62.0 0.343
91209-151 1007 0.242 67.8 0.366
91209-142 938 0.234 69.7 0.422
Sensitive
91209-220 660 0.220 46.7 0.265
91209-119 1478 0.301 94.0 0.478
91209-269 920 0.246 64.7 0.368
91209-297 371 0.209 24.3 0.127
91209-143 765 0.245 51.6 0.278
Control
Hinson 1103 0.262 72.8 0.387
LSD 0.05 574 0.089 44.1 0.288
Significance ns ns ns ns

Flood x Genotype
Significance ns ns ns ns
*,***** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

post-flood in the 2-week flooded treatment suggests that sufficient primary root function

remains after 2 weeks of flooding to support plant growth once reallocation of

photosynthate to primary root development is initiated. Genotypic differences were not

significant, nor were the genotype x flood interactions.

Nodule Dry Weights and Dry Matter Partitioning

Nodule dry weights at 35 days after sowing were significantly different, with non-

flooded soybeans accumulating three times the biomass of 2- and 4-week flooded

treatments (2.11). At 49 DAS, the 4-week flood treatment had significantly lower nodule









biomass (65 mg plant-) than the 2-week flood (147 mg plant-1), which was significantly

less than the non-flooded control (235 mg plant-'). Genotype effects on nodule weights

were significant at 35 and 49 DAS (2.11). The interaction term between genotype and

flood was not significant. The effect of 14 days of flooding on nodulation in our

experiment is consistent with the findings of Sallam and Scott (1987), including the

increase in nodule dry weight of the 2-week flood treatment after the removal of

flooding. Bennett and Albrecht (1984) suggested that soybean plants retain the ability to

recuperate nodule function when aerobic conditions are restored, a conclusion supported

by the findings of this study. Prolonged flood duration resulting in decreased nodulation

is also consistent with other research findings (Sprent, 1972; Sung, 1993). Sallam and

Scott (1987) found that nodule function in plants flooded at the V4 growth stage returned

to normal levels, however, the same study revealed that nodules of plants with flood

imposed at the VI growth stage never recovered function.

The effect of flood on partitioning to nodules was significant only at the 35 DAS

sampling date (2.11). At that date, the non-flooded treatment had significantly greater

dry matter partitioning to nodules compared with flooded treatments. Genotype effects

were significant at 35 and 49 days after sowing (2.11) This included a significant

interaction effect at 35 DAS (2.11).

Correlations of morphological characters to flood tolerance

Significant Spearman's rank correlation coefficients for flood tolerance (as defined in this

study) and primary root dry weight at 49 days after sowing (2.12) underscore the










Table 2-11 Nodule dry weight and dry matter partitioning.
Days After Sowing
35 49
Dry Weight Partitioning: Dry Weight Partitioning
mg plant'1 mg plant'1
Flood
2 Week 12 bt 0.009 b 147 b 0.046
4 Week 13 b 0.010 b 65 c 0.025
Non-flooded 41 a 0.026 a 235 a 0.041
LSD 0.05 13 0.006 43 0.006
Significance ns

Genotype
Tolerant
91210-350 16 cd 0.010 c 138 bd 0.035 c
91210-316 35 abc 0.023 ab 206 ab 0.052 ab
91209-126 41 ab 0.029 a 213 ab 0.053 ab
91209-151 26 bcd 0.026 ab 186 ab 0.049 ab
91209-142 21 bcd 0.015 bc 247 a 0.058 a
Sensitive
91209-220 15 cd 0.011 c 136 bcd 0.037 c
91209-119 7 d 0.006 c 142 bc 0.038 c
91209-269 8 d 0.008 c 59 de 0.022 d
91209-297 9 d 0.007 c 71 cde 0.019 d
91209-143 56 a 0.026 ab 191 ab 0.042 bc
Control
Hinson 6 d 0.004 c 46 e 0.013 d
LSD 0.05 24 0.012 81 0.011
Significance ** ***

Flood x Genotype
Significance ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
tDry matter partitioning is defined as nodule dry weight / total plant dry weight.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table 2-12 Spearman's rank correlation coefficients for selected rhizosphere plant
components under flooded conditions as correlated to tolerance rank.
Tolerance
Root parameter Spearman's Rank Correlation Coefficient
Primary root dry weight 49 DAS 0.618 *
Primary root length 0.818 **
Primary root area 0.727 *
Primary root surface area 0.727 *
Primary root diameter 0.218 ns
Primary root volume 0.418 ns
Primary root partitioning 49 DAS -0.155 ns
Adventitious root dry weight 49 DAS 0.645 *
Adventitious root length 0.500 ns
Adventitious root area 0.418 ns
Adventitious root surface area 0.418 ns
Adventitous root diameter 0.155 ns
Adventitous root volume 0.255 ns
Nodule dry weight 0.391 ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively


importance of continued root development in maintaining plant integrity under flooded

conditions. The dieback of the primary rooting system and replacement of function by

adventitious roots is cited as a central response of soybeans to flooding (Bacanamwo and

Purcell, 1999a; Pires et al., 2002). Root damage due to hypoxia may also lead to

insufficient transfer of water, inorganic minerals, and hormones resulting in shoot

damage (Vartapetian and Jackson, 1997; Jackson and Ricard, 2003). Maintaining

primary root function is clearly essential to continued plant growth. Plant flood tolerance

was likewise strongly correlated to primary root length and correlated to primary root

surface area (2.12). The findings of this study in describing significant correlations

between primary root length, surface area, dry weight and tolerance are unique in their

attempt to relate measured root parameters to a quantitative analysis of plant

performance. The relationships between primary root length, surface area and dry weight

are consistent with the findings of Sallam and Scott (1987). The consistency of the

correlations between these growth parameters and early-season flood tolerance supports









the hypothesis that maintenance of primary root extension and accumulation of root

biomass may serve as an indicator of soybean flood tolerance.

The Spearman's rank correlation coefficient analysis showed a positive correlation

between adventitious root dry weight and flood tolerance at 49 days after sowing (2.12),

however correlations between flood tolerance and adventitious root length and surface

area at the same sample date were not significant. The positive correlation between

adventitious root dry weight and tolerance is also consistent with the concept that root

function under flooded conditions in enhanced by the aeration provided by aerenchyma in

adventitious roots (Jackson and Drew, 1984). Lee et al. (2003) stated that forced early

development of adventitious roots in soybean served to significantly mitigate crop

susceptibility to waterlogging. This study is unique in that it provides a detailed analysis

of adventitious root development in flooded soybeans. This analysis has led to two

unanticipated results. The lack of significant correlation between adventitious root

length, average diameter, surface area, or volume and tolerance is contradictory to the

concept that adventitious roots provide a necessary means of adaptation to flood stress,

assuming that increased adventitious root development leads to increased root aeration.

The significant correlation between adventitious root dry weight and tolerance and lack

of correlation between length and tolerance may be an indication that the linear

relationship between root length, area, and dry weight that exists for primary roots is not

present with adventitious roots. Figures 2.2 and 2.3 chart this relationship across












Adventitious root length x dry weight


3

U,

S2
U,
0

0


e-
0,


0 -
0.00


0.05


0.10


0.15


0.20


0.25


Dry Weight 49 DAS (g plant')

Figure 2-2 Graph showing lack of linear fit in the relationship between adventitious root
length and dry weight.



Adventitious root surface area x dry weight


0.05


0.10 0.15
Dry Weight 49 DAS (g plant1)


0.20


0.25


Graph showing lack of linear fit in the relationship between adventitious root
surface area and dry weight.


y =0.410 + 5.10x
R2 = 0.221


0 0
0


o Oo O
0 0 Ooo<00 ^
o


180
,-160
E 140
120
2100
80
^ 60
40
I) 20
0
0


Figure


Sy = 27.9 + 356x
0 o R2= 0.195
O


0 0



o 0
o 0
0 0 "6 00


.00


2-3


)









genotypes and flood treatments. This may point to a greater relative importance in

adaptation to flooding associated with adventitious root dry weight than length, surface

area, average diameter, or volume.

Contrast Between Flood Tolerance Groups

In order to better judge the performance of flood tolerance groups in relation to

their original flood tolerance rankings (Comelious, 2003), group means were compared

across 2- and 4-week flood treatments. Prior to the imposition of flood treatments no

significant differences were noted between flood tolerance groups (Tables 2.13 and 2.14).

Strong early growth in the primary roots of flood tolerant genotypes led to significantly

greater accumulation of root biomass by the 35 DAS sampling date (2.13). The

difference was not significant, however, by the 49 DAS sampling date. Adventitious root

and nodule development both showed significantly greater accumulation of dry matter in

the sensitive group than in the tolerant group by the 49 DAS sampling date (2.13).

Noting the significant correlations between primary root dry matter accumulation,

adventitious root dry matter accumulation, and early season tolerance, (2.12) this is

further evidence that in this study the "sensitive" genotypes as defined by Comelious

(2003) proved to have a greater resistance to flooding than those previously defined as

"tolerant."

Conclusions

The existence of significant correlations between rhizosphere morphology and

flood tolerance as defined in this study substantiates the assertion that morphological

adaptations may serve as an indicator of flood tolerance. The strength and consistency of

correlations between primary root dry weight, length, surface area and tolerance support

the conclusion that the measure most worth pursuing in prediction of tolerance is










Table 2-13 Rhizosphere dry weight contrast for flood "tolerant" vs. "sensitive"
genotypes as defined by Cornelious (2003).


Days After Sowing
21 35 49
mg
Dry weight:
Primary root -6 tns -193 1 ns
Adv. root --18 ns 95 **
Nodule 18 ns 266 *


,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Negative values denote a greater mean value for "tolerant" treatments.


Table 2-14 Primary and adventitious root length, surface area, diameter, and volume,
contrast value for flood "tolerant" vs. flood "sensitive genotypes as defined
by Cornelious (2003).
49 Days After Sowing
Length Surface area Diameter Volume
cm cm2 mm cm3
Primary root: -122.1 t ns 12.4 ns 0.031 ns 0.192 ns
Adv. root: 617.7 ns 66.1 ns 0.019 ns 0.519 ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Negative values denote a greater mean value for "tolerant" treatments.

continued primary root elongation and primary root biomass accumulation. However, as

highlighted in Chapter 3, the fact that tolerance rankings based on dry weights at 49 DAS

did not correspond with rankings based on yield and visual flood injury as outlined in

Cornelious (2003) limit the application of this methodology. It is impossible to discern

whether these differences in rank arise from differences in response between plants

flooded at the V2 growth stage versus those flooded at the R2 stage, or from climatic

differences between experiments.

In order to better understand the possibility of using these significant correlations

between morphological traits and flood tolerance as early season indicators of flood

tolerant genotypes, it will be necessary to further investigate the response of the selected

genotypes to flooding at the V2 growth stage. Rankings, similar to those presented in

Cornelious (2003), but based on flood imposition at the V2 rather than R2 growth stage






41


would allow for extrapolation of early season morphological data to year end grain yield.

Based on the strength of multiple significant correlations, there is reason to believe that

links between grain yield and early season morphological response can be established,

greatly reducing the time need to identify potentially tolerant genotypes.














CHAPTER 3
THE EFFECT OF EARLY-SEASON FLOODING AND GENOTYPE ON SOYBEAN
(GLYCINE MAX L. MERRILL) ABOVEGROUND GROWTH AND TOTAL PLANT
BIOMASS

Review of Literature

Identification of Tolerance to Flood Stress

An overall decrease in soybean shoot growth under flooded conditions results from

the inability of the root system to maintain normal function in relation to the transport of

water, nutrients, hormones, and assimilates (Jackson and Drew, 1984). The first

symptom to appear in shoots is a wilting of the leaves based on increased resistance to

water flow through the root (Kramer, 1951). Some dispute as to the origin of reduced

leaf expansion remains (Jackson and Drew, 1984). However, there is solid evidence that

extended periods of root anoxia will result in a decline in leaf area accumulation. Sojka

et al. (1975) reported a reduction of greater than seventy percent in leaf area of wheat

after twenty-five days of root flooding as compared to non-flooded controls. Prolonged

flooding will eventually lead to epinasty, leaf chlorosis, and plant death (Kramer and

Boyer, 1995).

Net CO2 assimilation per unit of leaf area is depressed with root waterlogging

(Trought and Drew, 1980) primarily due to stomatal closure reducing Rubisco production

(Jackson and Drew, 1984). Decreases in assimilation will eventually also reduce dry

weight accumulation (Trought and Drew 1980; Sojka et al., 1975). This may, however,

be proceeded by a period of increased accumulation of assimilates in leaves resulting

from decreased export of photosynthate to the roots (Jackson and Drew, 1984).









Coinciding with the extension of adventitious roots from the hypocotyl,

hypertrophy, the swelling of the stem base or hypocotyl, is another common response to

flooding. Resulting from a swelling of cells in the cortex, hypertrophy is often

accompanied by a collapse of cells to form gas filled spaces (Kawase, 1981).

The effects of waterlogging on soybean shoot growth and aboveground biomass

accumulation are substantial and generally negative. Linkemer et al. (1998) reported a

significant decline in pod number, pods per node, branch number, and seed size after 7

days of flooding at various vegetative and reproductive growth stages. Oosterhuis et al.

(1990) confirmed an overall reduction in dry weight production in soybean. Seven days

of flood created a 14-day lag in biomass accumulation at the V4 growth stage, and a

greater than 14 day lag at the R2 stage. Both studies added, however, that biomass

accumulation returned to a similar rate with the control 7 days after removal of the flood

treatment. Reduction in pod number was confirmed by Sullivan et al. (2001). They also

reported a reduction in height for 3, 5, and 7-day floods at early vegetative growth stages.

Flooding reduced leaf number (Sallam and Scott, 1987), and prolonged effects on canopy

height and reduced dry weight were still noticeable at maturity (Scott et al., 1989).

Yield reductions in soybean ranged from 20-93% for 2 and 6-day floods

respectively (Sullivan et al., 2001). Singh and Singh (1995), Oosterhuis et al. (1990), and

Scott et al. (1989) all reported yield reductions for all flooded treatments ranging in

duration from 24 hours to 14 days. Decreases in yield can be attributed to reduced pod

growth, seed size, and number of seeds per pod (Linkemer et al., 1998) brought about by

nitrogen stress and reduced photosynthesis (Oosterhuis et al., 1990).









Stem hypertrophy is an adaptation to flooding that has been reported to serve as a

primary pathway for conducting atmospheric air needed for adequate root aeration

(Shimamura et al., 2003). Changes in stem diameter have also been shown to closely

correlate to flood tolerance in soybean (Pires et al., 2002). As such it is likely that the

relationship between stem development and flooding tolerance may be useful in selecting

flood tolerant soybean genotypes. Similarly, the generic responses in plant leaf growth

attributed to flooding including: wilting (Kramer, 1951), reduction in leaf area

accumulation (Sojka et al., 1975) and leaf chlorosis (Kramer and Boyer, 1995), have been

confirmed for soybean by Sallam and Scott (1987) who reported a reduction in leaf

number after 7 days of flooding, and Bacanamwo and Purcell (1999a) who observed a

general reduction in leaf area for all treatments and genotypes as compared to controls.

The consistency of these results highlights the potential value of leaf measures in

assessing flood tolerance.

The ultimate factor for judgment of plant tolerance to flooding is the yield of high-

quality marketable seeds (Van Toai et al., 1994; Roiselle and Hamblin, 1981). Within

this definition varying methods have been used to establish tolerance rankings. Van Toai

et al. (1994) rated the tolerance of 84 tested varieties by ranking total yield under flooded

conditions and comparing these to corresponding rankings under non-flooded conditions.

Roiselle and Hamblin (1981) promoted the use of a comparative yield analysis taking the

average of flooded and non-flooded yields. This selects genotypes broadly adapted to

both stress and non-stress conditions. Van Toai and Nurijani (1996) identified tolerant

genotypes by taking the average seed yield and average SPAD leaf greenness reading of

flooded plants and dividing these values by the values for the non-flooded control. The









resulting ratio demonstrated the ability of plants to maintain growth under flooded

conditions. Bacanamwo and Purcell (1999a, 1999b) took a different approach in

determining tolerance; emphasizing the necessity of morphological adaptations for

avoidance of flooding injury, they suggested that selecting varieties for morphological

adaptations to flooding may be the best way to select for flooding tolerance.

Rationale and Objectives

Identification of soybean flood tolerance in the vegetative stage could greatly

reduce the time frame required to identify potentially flood tolerant varieties for breeding

purposes. In addition insight may be gained as to the relationship between early and late

season morphological responses to flooding. Comparing early-season flood adaptations,

with late-season flood tolerance (3.1; Comelious, 2003) may provide a link between

early-season morphological flood responses and end of season tolerance. The objectives

of this study were 1) to monitor changes in soybean stem and leaf growth in response to

early-season flooding, 2) compare early-season morphological adaptations to early-

season flood tolerance, and 3) determine the relationship between early-season and late-

season flood tolerance

Materials and Methods

Detailed general materials and methods can be found in Chapter 2. Included here is

the development of methods specific to aboveground growth and total plant biomass.









Table 3-1 Genotype yield reduction and flood injury scored for most tolerant and most
sensitive Arkansas RIL's.

Most tolerant Most sensitive
Genotype Yield reduction Injury Genotype Yield reduction Injury
% 0-9 % 0-9
91210-350 32.2 2.8 91209-220 67.9 5.8
91210-316 33.0 3.0 91209-119 65.2 6.2
91209-126 37.2 3.7 91209-269 63.6 5.9
91209-151 43.1 4.0 91209-297 69.8 6.4
91209-142 49.1 4.0 91209-143 69.8 6.4
t Cornelious (2003)

Leaf Area and Plant Height

Determination of total leaf area per plant was established as described by

Wiseman and Bailey (1975). Thirty trifoliolate leaves from each of the eleven genotypes

to be studied were removed from plants in a pre-trial run. A range of trifoliolate leaf

sizes (small, medium, large) was taken from both flooded and non-flooded plants. The

length and maximum width of the center leaflet of each trifoliolate was measured

manually. Each of the three trifoliolate leaflets was passed through a LI-3000 portable

leaf area meter (LI-COR Inc., Lincoln, NE) and actual total leaf area for the trifoliolate

leaf was recorded. A linear regression equation was developed that expressed leaf area as

a function of the product of the length and width of the center leaflet. Genotype specific

regression equations for each of the eleven genotypes had an R2 value > 0.92 (3.2) and

these equations were used to predict leaf area based on length x width measurements of

individual trifoliolate leaves. Leaf area measurements were taken from two

predetermined plants in each sub-plot at 3.5 and 7-day intervals for Experiments 1 and 2,

respectively. Incremental leaf area expansion is defined as the addition of leaf area

between day "x" and day "y", where the value used for analysis is (value day y value

day x).










Plant height was measured twice weekly from the soil surface to the uppermost

growth point of the stem. Three measurements were taken from each sub-plot across all

Table 3-2 Regression equations and R2 values for leaf area by genotype used to
determine non-destructive leaf area values.
Genotype Regression Equations R
Tolerant
91210-350 y= 1.70x + 2.13 0.976
91210-316 y= 1.99x-0.135 0.977
91209-126 y = 1.80x + 1.29 0.921
91209-151 y= 1.81x+ 1.39 0.968
91209-142 y= 1.81x+ 1.09 0.932
Sensitive
91209-220 y = 1.83x + 1.80 0.977
91209-119 y= 1.74x + 2.94 0.959
91209-269 y = 1.92x + 2.31 0.922
91209-297 y = 2.03x 0.811 0.982
91209-143 y = 2.12x 0.222 0.985
Control
Hinson y = 1.66x + 4.36 0.951
tTolerant and Sensitive determination based on descending ranking (Cornelious, 2003).
4 y=trifioliolate area, x=length x width of center leaflet.

main-plots and replicates. The average of individual plant heights was used to represent

plot height for statistical analysis. The same three plants were measured at each sampling

date.

Statistical Analysis

Analyses of variance were performed for all experimental measurements using proc

GLM in SAS (Littell et al., 2002). Data was analyzed using a factorial experiment with

a split-plot design with flood regimes being the main plot treatments and genotypes the

sub-plots. Significant differences among cultivars and flood treatments were determined

using Fisher's protected LSD test (P<0.05). Further analysis of significant interaction

terms was performed using the proc GLM 'Estimate' statement in SAS using the flood x

genotype means for genotype 91210-350 as the comparative base. Genotype 91210-350

was chosen for comparison as it was reported to be the genotype "most tolerant" to









flooding in the Arkansas trials (Cornelious, 2003). The proc GLM 'Estimate' statement

was also used to contrast group response to flooding. The mean value for all "tolerant"

genotypes as defined by Cornelious (2003) across the 2- and 4-week flood treatments was

contrasted against the mean value for all "sensitive" genotypes. Additionally proc CORR

in SAS was used to calculate Spearman's rank correlation coefficient to determine

significant correlations between morphological measurements and tolerance rankings.

Each morphological measure was ranked, based on numerical values and then correlated

to numerical tolerance rankings based on whole plant dry weights. For the purposes of

this study, flood tolerance was defined as the mean total plant dry weight of flooded

treatments divided by the total plant dry weight of the non-flooded treatment (plant dry

weight flood / plant dry weight non-flood).

Results and Discussion

While results from Experiments 1 and 2 indicate consistent patterns of adaptation

to flooding for aerial components of soybean, in relation to stem dry matter

accumulation, partitioning, and plant height; as stated in Chapter 2, repeated rainfall

events interfered with flooding treatments in Experiment 2. The inability to establish an

effective control makes the presentation of this data inconsistent. Thus all Experiment 2

data can be found in Appendix A The consistency between quantifiable effects of

flooding on aerial components in Experiments 1 and 2 may indicate that aerial plant

growth parameters are more highly sensitive to the effects of imposed flooding than

rhizosphere components.

Stem Dry Weight, Dry Matter Partitioning, and Height

Flooding did not cause significant differences in stem dry weight at the 35 DAS

sampling date. However, stem dry weight in the non-flooded treatment was significantly














Table 3-3 Stem dry weight and dry matter partitioning (Experiment 1).
Days After Sowing
21 35 49
Dry Weight Partitioning$ Dry Weight Partitioning Dry Weight Partitioning
mg plant mg plant mg plant-1
Flood
2 Week 167 0.255 448 0.323 b 1006 b 0.385 b
4 Week 162 0.249 502 0.343 a 980 b 0.431 a
Non-flooded 165 0.247 390 0.291 c 1770 a 0.345 c
LSD 0.05 17 0.012 69 0.0 21 0.0
Significance ns ns ns ** **
Genotype
Tolerant
91210-350 243 a 0.275 a 590 a 0.367 a 1380 ab 0.427 ab
91210-316 125 e 0.235 d 414 bc 0.302 c 1460 ab 0.366 def
91209-126 160 d 0.236 d 406 bc 0.300 c 1360 ab 0.360 ef
91209-151 126 e 0.247 bcd 376 bc 0.320 bc 1330 abc 0.389 cd
91209-142 143 de 0.251 bcd 393 bc 0.344 ab 1540 a 0.381 cde
Sensitive
91209-220 195 bc 0.256 abcd 448 c 0.300 c 1170 abcd 0.375 def
91209-119 140 det 0.234 d 458 abc 0.314 bc 1280 abcd 0.379 de
91209-269 158 de 0.261 abc 340 c 0.305 c 906 d 0.354 f
91209-297 165 cd 0.265 ab 408 bc 0.325 bc 937 cd 0.432 a
91209-143 212 ab 0.251 bcd 588 a 0.321 bc 1510 ab 0.386 cd
Control
Hinson 145 de 0.290 cd 497 ab 0.310 c 1114 bcd 0.405 bc
LSD 0.05 32 0.023 133 0.031 400 0.031
Significance *

Flood x Genotype
Significance ns ns ns ns ns*
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
$ Dry matter partitioning is defined as stem dry weight / total plant dry weight.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










greater than either the 2 or 4-week flood treatment at 49 DAS (3.3). Significant

differences among genotypes were noted at all sampling dates. There was no significant

flood x genotype interaction on stem dry weight at any sampling date.

The observed pattern in stem dry weight accumulation was consistent with other plant

components. Little effect on the overall growth of the plant was recorded at 14 days after

flooding. This may be attributed to the natural delay in plant growth (Hanway and

Weber, 1971; Sun-Guang et al., 1996) with the flooding treatment resulting in a delay in

the rapid growth phase beyond 35 DAS. While measurements of stem dry weight at 35

DAS were not significantly different, it is apparent that damage to the soybean plant has

occurred during these first 14 days of flooding. This is indicated by the continued slow

biomass accumulation of plants under the 2-week flood treatment after the removal of the

flood. Dry weight for non-flood and 2-week flood treatments was not significantly

different at the time of removal of the 2-week flood treatment (3.3). Yet, 14 days later at

the 49 DAS sampling date, non-flood plants had a significantly greater stem dry weight

than 2-week flooded plants. Genotype 91210-350 had consistently high stem weights

(3.3).

The amount of dry weight partitioned to the stem was significantly different

between flood treatments at 35 and 49 DAS (3.3). A significant difference among

genotypes was noted in stem partitioning at all sampling dates (3.3). Significant

interactions between flood and genotype occurred at the 49 DAS sampling date (3.3).

Further analyses of the interaction terms show 91210-350 consistently partitioned more

of its total biomass to the stem compared to other genotypes (3.4). With the prolonging

of the flood treatments this trend became more pronounced.










Table 3-4 Genotype comparison for all significant dry matter partitioning flood x


Dry Matter Partitioning:
Stem 49 DAS
non-flooded 2 week 4 week
Genotype
Tolerant
91210-316 -0.019 ns -0.059 ** -0.107***
91209-126 -0.032 ns -0.067** -0.104***
91209-151 -0.038 ns -0.049 -0.028 ns
91209-142 -0.018 ns -0.046 -0.076 **
Sensitive
91209-220 -0.051 -0.047 -0.061 **
91209-119 -0.031 ns -0.042 ns -0.075 **
91209-269 -0.062 ** -0.063 ** -0.096 ***
91209-297 -0.043 0.022 ns -0.032 ns
91209-143 -0.029 ns -0.063 ** -0.033 ns
Control
Hinson -0.032 ns -0.012 ns -0.016 ns
91210-350t 0.377 0.424 0.482


,**,*** Significant at the 0.05, 0.01,


and <0.001


levels of probab


ility respectively.


t Genotype 91210-350 was identified as the "most tolerant" genotype (Cornelious, 2003).
$ Dry matter partitioning is defined as plant part dry weight / total plant dry weight.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).
genotype interaction terms. Values represent difference between genotype and 91210-


350.









Significant differences in plant height between flood treatments occurred only at 25

and 28 DAS (Tables 3.5). Significant differences among genotypes were noted almost

uniformly across experiments with the only exception occurring at 36 DAS (3.5). There

was no significant interaction between flood and genotype effects at any sampling date

Significantly lower soybean plant heights at harvest as a result of flooding have

been reported by: Sallam and Scott (1987), Scott et al. (1989), and Linkemer et al.

(1998). Pires et al. (2002) on the other hand did not find any significant difference in

plant height after 21 days of flooding initiated at the V2 growth stage. The consistency

of our findings with that of Pires et al. (2002) can most likely be attributed to similarities

between the two studies in relation to plant growth stage at the time of flood imposition

(V2) and final plant harvest before maturity. Significant differences in plant height were

reported at final harvest (Linkemer et al., 1998; Sallam and Scott, 1987; Scott et al.,

1989) implying that flood initiation at an early growth stage had a lasting effect on stem

elongation. Early sampling for morphological changes in our experiment may not have

allowed enough time post-flood for significant height differences to develop.

Leaf Expansion, Dry Matter Accumulation, and Partitioning

Leaf dry weight showed significant flood treatments effects at 49 DAS (3.6). Non-

flood treatments accumulated more biomass in the leaves (2220 mg plant-) compared to

flooded treatments, while the 2-week treatment had a significantly greater dry weight

(1190 mg plant-) than the 4-week treatment (919 mg plant-1). Significant differences in

leaf dry weight were noted among genotypes at all sampling dates (3.6). Interactions

between flood and genotype treatments were not significant.













Table 3-5 Plant heights.
Days After Sowing
21 25 28 32 36 39 44 47
cm plant1
Flood
2 Week 9.38 11.5 a 12.4 a 12.9 16.5 17.4 20.0 21.9
4 Week 9.45 11.2 b 11.9 b 12.5 15.2 16.9 20.9 23.0
Non-flood 9.12 10.2 c 10.9 c 11.5 14.2 16.4 19.9 22.0
LSD 0.05 0.21 0.3 0.3 0.4 1.7 0.6 0.9 1.0
Significance ns ns ns ns ns ns

Genotype
Tolerant:
91210-350 11.10 a 12.8 a 13.9 a 14.6 a 17.5 19.7 a 23.0 a 25.4 a
91210-316 8.82 ef 10.8 c 11.6 cd 12.5 c 15.0 17.1 cd 21.1 bc 23.5 bc
91209-126 8.42 fg 9.94 de 10.6 e 11.4 ef 13.9 16.1 de 20.1 cd 22.2 cd
91209-151 8.93 e 10.0 cd 11.4 cd 11.9 cde 18.3 16.3 de 20.1 cd 22.4 cd
91209-142 9.00 e 10.8 c 11.5 cd 11.9 cde 14.9 17.2 cd 21.1 bc 23.8 abc
Sensitive
91209-220 10.60 b 12.2 a 13.2 b 13.7 b 16.5 18.8 ab 22.1 ab 24.4 ab
91209-119 8.89 et 10.4 cdce 11.1 de 11.5 de 14.1 16.1 de 20.1 cd 22.1 cd
91209-269 8.29 g 9.85 e 10.5 e 10.6 f 13.0 14.0 f 17.1 e 18.4 e
91209-297 10.00 c 11.6 b 12.6 b 13.3 b 15.7 17.8 bc 20.2 cd 21.4 d
91209-143 9.56 d 10.9 c 11.9 c 12.2 cd 14.6 16.5 de 19.5 cd 21.5 d
Control
Hinson 8.93 e 10.6 c 11.5 cd 11.9 cde 14.3 15.7 e 18.6 de 20.6 d
LSD 0.05 0.41 0.5 0.6 0.7 3.3 1.2 1.8 1.9
Significance *** *** *** *** ns *

Flood x Genotype
Significance ns ns ns ns ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
$ Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).















Table 3-6 Leaf dry weights and dry matter partitioning.
Days After Sowing
21 35 49
Dry Weight Partitioningf Dry Weight Partitioning Dry Weight Partitioning
mg plant'1 mg plant'1 mg plant-1
Flood
2 Week 328 0.501 621 0.443 b 1190 b 0.423 b
4 Week 325 0.503 646 0.438 b 919 c 0.369 c
Non-flooded 338 0.518 668 0.495 a 2220 a 0.448 a
LSD 0.05 33 0.017 98 0.015 210 0.016
Significance ns ns ns ** *

Genotype
Tolerant
91210-350 425 a 0.477 728 ab 0.449 cde 1390 abcd 0.383 e
91210-316 280 de 0.517 674 abcd 0.478 abc 1730 a 0.434 abc
91209-126 343 bc 0.508 633 bcd 0.458 bcde 1620 abc 0.438 ab
91209-151 248 e 0.491 512 cd 0.443 de 1470 abc 0.404 cde
91209-142 287 cde 0.508 495 d 0.432 e 1660 ab 0.409 bcde
Sensitive
91209-220 389 ab 0.513 690 abc 0.486 ab 1440 abc 0.426 abcd
91209-119 297 cdet 0.502 646 bcd 0.441 de 1370 abcd 0.405 cde
91209-269 308 cde 0.504 519 cd 0.463 bcd 1220 cd 0.447 a
91209-297 320 cd 0.519 588 bcd 0.453 cde 1010 d 0.379 e
91209-143 421 a 0.502 845 a 0.447 de 1690 ab 0.397 de
Control
Hinson 320 cd 0.539 764 ab 0.495 a 1290 bcd 0.421 abcd
LSD 0.05 63 0.034 187 0.029 410 0.030
Significance *** ns

Flood x Genotype
Significance ns ns ns ns ns **
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
f Dry matter partitioning is defined as leaf dry weight / total plant dry weight.
Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).









Our findings that leaf dry weight for flooded treatments is reduced in comparison to

non-flooded treatments and that two-week post-flood recovery is sufficient to observe a

significant recuperation of leaf biomass are consistent with the generic plant response to

flooding, but to our knowledge have not been previously reported for soybean. In

soybean, reductions in leaf area of flooded plants (Bacanamwo and Purcell, 1999a) as

well as reduction in leaf number (Sallam and Scott, 1987) have been reported which

logically should result in a reduction in leaf dry weight such as we have documented.

Differences in total leaf biomass accumulation between 35 and 49 DAS show continued

leaf growth for all treatments. Significant differences in leaf dry weight between the 2

and 4-week flood treatments at 49 DAS demonstrates an enhanced ability of the 2-week

treatment to accumulate biomass in the leaves after removal of flood. This is consistent

with increases in plant biomass accumulation after removal of flood reported by

Bacanamwo and Purcell (1999b) and Oosterhuis et al. (1990).

While patterns in total leaf biomass accumulation and leaf expansion mirror one

another, an analysis of incremental leaf expansion showed greater genotype sensitivity to

flooding than leaf biomass, resulting in a significant genotype x flood interaction term at

36 DAS (3.7). Figures 3.1-3.4 show the range of genotypic incremental leaf area

expansion response to flood treatments. Noting that leaf expansion is one of the first

plant parameters affected by flooding (Kramer, 1951), trends in the response of leaf area

expansion to flood treatments may serve as a more sensitive indicator of plant

performance than that of total leaf area accumulation.

The fraction of total biomass partitioned to leaves was significantly different for the

flood treatments at 35 and 49 DAS (3.6). At 49 DAS the non-flooded treatment










Table 3-7 Incremental leaf area expansion.
Days After Sowing
14 22 28 36 43
cm2
Flood
2 Week 20.6 51.8 48.9 57 b 66 b
4 Week 19.4 51.5 55.5 49 b 49 b
Non-flooded 17.9 53.5 63.9 153 a 247 a
LSD 0.05 1.9 5.4 7.7 17 32
Significance ns ns ns ** **

Genotype
Tolerant:
91210-350 24.3 bct 57.8 bc 59.5 cde 89 83 cd
91210-316 16.2 d 46.0 de 60.5 bcd 105 166 a
91209-126 14.6 de 47.0 de 44.9 ef 81 149 ab
91209-151 11.7 e 40.6 e 41.5 f 89 134 abcd
91209-142 13.2 de 40.6 e 40.2 f 92 143 abc
Sensitive
91209-220 21.5 c 54.6 bcd 53.5 cdef 79 102 bcd
91209-119 15.1 de 49.2 cde 52.9 cdef 76 129 abcd
91209-269 15.8 d 46.9 de 48.4 def 70 84 cd
91209-297 29.0 a 70.7 a 74.8 ab 66 76 d
91209-143 26.4 ab 63.5 ab 76.9 a 108 168 a
Control
Hinson 24.2 bc 58.1 bc 64.1 abc 94 95 bcd
LSD 0.05 3.5 10.4 14.8 33.1 61
Significance *** *** ns

Contrast T vs. S

Flood x Genotype
Significance ns ns ns ns
,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
4Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

















500
E 400
S300
= 200
* o 10 0
0 0
S-100
-200


Incremental Leaf Area Expansion Across
Genotypes


i


_-2 week
0 week
- --non-flooded


7 19 24 32 40
Days after sowing


Figure 3-1 Incremental leaf area expansion for all genotypes across flood treatments.


Incremental Leaf Area Expansion 91209-220


500
400
300
c 200
0
'V 100
a o0
'i -100
-200


7 19 24
Days after sowing


32 40


Incremental Leaf Area Expansion 91209-269

500 -


C 400
u 300
S200
V 100
0
L -100
-200


7 19 24 32 40
c Days after sowing



Incremental Leaf Area Expansion 91209-143


500
S400-
E
S300
- 200-
0-
*I 100-
C
1 0-
. o
L -100-
-200


02 week
0 week
- non-flooded

-----------------" ----------




7 19 24 32 40
Days after sowing


Incremental Leaf Area Expansion 91209-119

500
500 -02 week
400 0 week
300 -- non-flooded
200
100
0
100
-200
7 19 24 32 40
Days after sowing



Incremental Leaf Area Expansion 91209-297

500
-0-2 week
400 0 week
300 frnon-flooded
200
100 n
0
100
-200
7 19 24 32 40
Days after sowing


Figure 3-2 Incremental leaf area expansion for "most sensitive" genotypes (Cornelious,

2003).


S2 week
0 week
-non-flooded


S2 week
0 week
--non-flooded

















Incremental Leaf Area Expansion 91209-350

500
0 2 week
400 week
300 non-flooded


Incremental Leaf Area Expansion 91210-316

0
- I ,------------
0 2 week
0 30 week
0 non-flooded


7 19 24

Days after sowing


32 40


Incremental Leaf Area Expansion 91209-126


_ 0 2 week
0 week
non-flooded



-----.---- ---4 --





7 19 24 32 40
Days after sowing



Incremental Leaf Area Expansion 91209-142

02 week
week
A non-flooded


: ^ ^ ^91


a





500

400

4 300

200

' 100

w 0

-100

-200
c






500
400
E300

0200


100
"' n


w -

-100
-200 ----------------------


7 19 24
Days after sowing


b





500
400

E 300
S200

100
0
-100

-200
d


7 19 24
Days after sowing


32 40


Incremental Leaf Area Expansion 91209-151

S -2 week
O week
non-flooded



__"


7 19 24
Days after sowing


32 40


32 40


Figures 3-3 Incremental leaf area expansion for "most tolerant" genotypes (Cornelious,

2003).


500 Incremental Leaf Area Expansion Hinson


400

1300
U


-100



-00
100

200-


7 19 24
Days after sowing


32 40


Figure 3-4 Incremental leaf area expansion for control genotype Hinson.


- ---2 week
0 week
--non-flooded
T


a
.2

X
W









had a significantly greater allocation of biomass to the leaves, and the 4-week flooded

treatment had the lowest. Significant genotypic differences were observed at the 35 and

49 DAS sampling dates. Flood by genotype interactions were significant at 49 DAS

(3.6). The decline in fraction of biomass partitioned to leaves with flood durationis

again a natural consequence of the inability of the plant to maintain plant processes and

production on a whole plant level under flooded conditions.

Flooded treatments recorded significantly lower leaf areas at later sampling dates

(36-43 DAS) (3.8). Genotype effects were significant at all but the final sampling date

(43 DAS). No significant interaction between flood and genotype effects was recorded at

any sampling date. The relative decline in leaf area due to flooding ranged from 54-57%

from non-flooded treatments at 43 DAS. Our results are consistent with the findings of

Bacanamwo and Purcell (1999a) who recorded a reduction in leaf area between 49-60%

after 21 days of flooding with respect to non-flooded controls.

Total Plant Dry Weight

Total plant dry weight was significantly less for flooded treatments at 49 DAP

(Tables 3.9) accumulating only 44 53% the total biomass of non-flooded controls. No

significant difference between flooded treatments was recorded at final harvest.

Significant differences among genotypes were noted at all sample dates, however no

significant interaction between flood and genotype was recorded for any sample date.

Trends in accumulation of soybean plant biomass when flooded were in agreement with

previous studies; Oosterhuis et al. (1990) cited a general reduction in plant biomass with

flooding at the V4 growth stage, while Scott et al. (1989) reported a reduction of 41-56%

in total biomass as compared to non-flooded controls with 14 days of flooding at the V4










Table 3-8 Leaf area.
Days After Sowing
14 22 28 36 43
cm2
Flood
2 Week 20.6 72.5 121 178 b 245 b
4 Week 19.4 70.9 126 176 b 225 b
Non-flooded 17.9 71.3 135 288 a 535 a
LSD 0.05 1.8 6.1 11 25.0 52
Significance ns ns ns *

Genotype
Tolerant:
91210-350 24.3 bc 82.0 bc 142 cd 231 abc 314
91210-316 16.2 d 62.3 ef 123 def 228 bcd 394
91209-126 14.6 de 61.6 ef 106 fg 188 cd 337
91209-151 11.7 e 52.3 f 93.8 g 182 cd 316
91209-142 13.2 de 53.8 ef 94.1 g 186 cd 330
Sensitive
91209-220 21.5 c 76.1 cd 130 cde 209 bcd 311
91209-119 15.1 det 64.3 de 117 ef 193 bcd 322
91209-269 15.8 d 62.7 ef 111 efg 181 d 265
91209-297 29.0 a 99.7 a 175 a 240 ab 317
91209-143 26.4 ab 89.9 ab 167 ab 275 a 443
Control
Hinson 24.2 bc 82.3 bc 146 bc 240 ab 336
LSD 0.05 3.5 11.9 21 48 101
Significance *** *** *** ** ns

Flood x Genotype
Significance ns ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
:Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).










Table 3-9 Total plant dry weight.
Days After Sowing
21 35 49

mg plant-1
Flood
2 Week 655 1370 2810 b
4 Week 655 1450 2360 b
Non-flooded 655 1350 5050 a
LSD 0.05 66 200 520
Significance ns ns **

Genotype
Tolerant:
91210-350 913 a 1610 ab 3420 abc
91210-316 538 e 1380 bc 3990 a
91209-126 675 cd 1370 bc 3740 ab
91209-151 507 e 1160 c 3560 abc
91209-142 563 de 1140 c 4070 a
Sensitive
91209-220 759 bc 1440 bc 3260 abcd
91209-119 590 de 1440 bc 3380 abcd
91209-269 609 de 1110 c 2640 cd
91209-297 616 de 1270 bc 2420 d
91209-143 839 ab 1860 a 4070 a
Control
Hinson 598 de 1550 ab 2930 bcd
LSD 0.05 126 380 991
Significance *** ** *

Flood x Genotype
Significance ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
tTolerant and Sensitive determined based on descending ranking (Cornelious, 2003).

growth stage. From our results, it is apparent that sufficient damage occurred to the 2-

week flooded plants to retard overall growth for at least 14 days following the removal of

flooding. Oosterhuis et al. (1990) showed increases in biomass accumulation to near-

normal rates 14 days after the removal of a 7-day flood treatment. In our experiment, 14

days was not a long enough period to see significant recovery from the 2-week flood

treatment.










Root:Shoot Ratio

Flooding had a significant effect on root:shoot ratios at 49 DAP (3.10). Genotype

effects were also significant at 49 DAP with 91209-119 and 91209-143 having the

highest values. The interaction between flood and genotype was not significant.

Table 3-10 Root:Shoot ratios
Days After Planting
21 35 49

Flood
2 Week 0.327 0.310 0.239 b
4 Week 0.349 0.289 0.254 ab
Non-flooded 0.310 0.275 0.263 a
LSD 0.05 0.057 0.037 0.019
Significance ns ns *

Genotype
Tolerant:
91210-350 0.385 0.228 0.234 bc
91210-316 0.321 0.286 0.251 ab
91209-126 0.347 0.323 0.254 ab
91209-151 0.358 0.313 0.263 ab
91209-142 0.319 0.294 0.268 ab
Sensitive
91209-119 0.366 0.332 0.278 a
91209-220 0.303 0.277 0.249 abc
91209-269 0.312 0.319 0.249 abc
91209-297 0.277 0.287 0.234 bc
91209-143 0.329 0.304 0.279 a
Control
Hinson 0.289 0.243 0.212 c
LSD 0.05 0.109 0.070 0.038
Significance ns ns *

Flood x Genotype
Significance ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively
t Treatment means followed by the same letter are not significantly different (P<0.05).
:Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003).

Consistent with the findings of Sallam and Scott (1987), root:shoot ratios decreased

for all treatments with time. This can be accounted for by the continual accumulation of

aboveground biomass regardless of treatment. Flooding at the V2 growth stage did not

appear to prevent the accumulation of aboveground biomass, rather slow the initiation of









the rapid growth phase. The decreases in root:shoot ratio for the 2-wk flood (3.10) are

not consistent with the findings of Bacanamwo and Purcell (1999a) and Sallam and Scott

(1987). In our experiment, the relative loss of aboveground biomass in leaf and stem

tissues was not as pronounced as the loss of root dry weight. The development of

adventitious roots, while present, was not sufficient to offset the loss of primary root dry

weight.

Correlations of Above Ground Plant Growth to Flood Tolerance

Genotypes were ranked based on flood tolerance (3.11) and stem dry weight (3.12).

Spearman's rank correlation coefficients were significant for stem dry weight and flood

tolerance (3.13). The positive correlation reported by Pires et al. (2002) between stem

diameter and flood tolerance was attributed to increased hypertrophy in the more tolerant

variety. Our results, to our knowledge not previously reported in the scientific literature,

indicate a similar correlation may exist between stem dry weight and flood tolerance.

This relationship is consistent with the findings of Pires et al. (2002), assuming that

increased hypertrophy coincides with greater stem dry weight.

A negative correlation between stem dry matter partitioning and tolerance existed

at 49 DAS (3.13) based on genotype tolerance rankings (3.11) and stem dry matter

partitioning rankings across flooded treatments (3.14). The percentage of total biomass

associated with the stem is a logical inverse measure of plant performance under

flooding. While stem dry weight is positively correlated with tolerance, necrosis and

dieback of leaves and roots in non-tolerant genotypes may leave a greater percentage of

total biomass in the stems. Plants that are least able to support near-normal leaf and root

growth and develop fewer adventitious roots would have the greatest percentage of










biomass associated with the stem. Based on the negative correlation between stem dry

weight partitioning and tolerance it may be desirable to use partitioning percentage for

Table 3-11 Tolerance of all genotypes to early-season flooding.
Tolerance Ratio Rank
91209-119 0.779 at 1
91209-126 0.745 a 2
91209-269 0.739 ab 3
91209-142 0.671 abc 4
Hinson 0.635 abcd 5
91209-220 0.469 abcd 6
91209-151 0.462 abcd 7
91209-143 0.441 bcd 8
91210-316 0.466 cd 9
91210-350 0.410 cd 10
91209-297 0.335 d 11
t Treatment means followed by the same letter are not significantly different (P<0.05)
$ Tolerance is defined as total mean plant dry weight flooded treatments / total dry weight
non-flooded treatment. Based on dry weights at 49 DAS sampling.

Table 3-12 Stem dry weight means for flooded treatments and ranks used for correlations.
Days After Sowing
21 35 49
Genotype (mg plant1) (mg plant') (mg plant ) rank
91209-142 153 423 1350 1
91209-119 140 470 1190 2
91209-126 158 378 1160 3
Hinson 150 560 1120 4
91209-143 198 606 1070 5
91209-151 121 428 1040 6
91210-316 120 405 1020 7
91210-350 224 643 1010 8
91209-220 205 529 880 9
91209-269 168 390 830 10
91209-297 174 396 610 11










Table 3-13 Correlations between selected plant measures and flood tolerance.
Experiment 1 Tolerance
Plant measure Spearman's Rank Correlation Coefficient
Stem dry weight 49 DAS 0.600 *
Stem partitioning 35 DAS -0.209 ns
Stem partitioning 49 DAS -0.618 *
Leaf dry weight 49 DAS 0.627 *
Leaf partitioning 35 DAS -0.245 ns
Leaf partitioning 49 DAS 0.545 ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively



Table 3-14 Stem dry matter partitioning means for flooded treatments and ranks used for
correlations.
Days After Sowing
21 35 49
Genotype rank
91209-297 0.273 0.327 0.481 1
91210-350 0.269 0.371 0.453 2
Hinson 0.248 0.317 0.435 3
91209-151 0.249 0.346 0.415 4
91209-143 0.243 0.350 0.405 5
91209-220 0.264 0.314 0.399 6
91209-119 0.224 0.329 0.395 7
91209-142 0.262 0.365 0.391 8
91209-269 0.270 0.329 0.374 9
91210-316 0.229 0.317 0.370 10
91209-126 0.238 0.299 0.368 11

flooded treatments as a measure of tolerance. A significant positive correlation was

noted between leaf dry weight and flood tolerance at 49 DAS (3.13) when genotype

tolerance ranks (3.12) were correlated to leaf dry weight ranks across flood treatments

(3.15). The correlation between leaf dry weight and flood tolerance is most likely due to

the enhanced ability of the most tolerant plants to continue transport of essential water

and nutrients from the roots to the leaves allowing for continued photosynthesis and leaf

expansion.










Table 3-15 Leaf dry weight means for flooded treatments and ranks used for
Days After Planting
21 35 49
Genotype (mg plant1) (mg plant') (mg plant') rank
91209-142 289 483 1420 1
91209-126 339 595 1350 2
91209-119 315 596 1220 3
91210-316 279 593 1020 4
91209-143 409 756 1130 5
Hinson 325 816 1070 6
91209-151 236 526 1020 7
91209-269 309 530 1000 8
91209-220 391 751 940 9
91210-350 386 765 860 10
91209-297 318 555 450 11
correlations.

Contrast Between Flood Tolerance Groups

The difference between flood tolerance groups was consistently highlighted by

strong early growth in the "tolerant" grouping and better performance under flooded

conditions for the "sensitive" grouping (Tables 3.16 and 3.17). Significant differences in

pre-flood values for stem dry weight, leaf dry weight, plant height, incremental leaf area

accumulation, and total leaf area (3.16 and 3.17) are all based on the greater total value of

the "tolerant" genotype grouping. In each instance the, flood duration led to gains by the

"sensitive" genotype grouping, resulting in either no significant difference between

groups (stem dry weight, incremental leaf area accumulation, and total leaf area) or a

significant difference based on greater value for the "sensitive" grouping (leaf dry weight

and plant height) (Tables 3.16 and 3.17). This is further evidence of the lack of

correspondence between tolerance rankings as defined in this study and tolerance

rankings as defined by Cornelious (2003).










Table 3-16 Aboveground dry weight contrasts between "tolerant" vs. "sensitive"
genotypes as defined by Cornelious (2003) across 2- and 4-week flood
treatments.
Days After Sowing
21 35 49
mg
Dry weight:
Stem -109 t* -116 ns 994 ns
Leaf -213 -228 ns 1121 *
Total -328 ns -536 ns 2502 ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Negative values denote a greater mean value for "tolerant" treatments.

Table 3-17 Plant height, incremental leaf area, and leaf area values, contrasts between
"tolerant" vs. "sensitive" genotypes as defined by Cornelious (2003) across 2-
and 4-week flood treatments.
Days After Planting
21 25 28 32 36 39 44 47
cm
Plant height -1.58 t** -0.6 ns -1.13 ns -0.12 ns 5.31 ns 0.77 ns 3.31 ns 6.25 *


14 22 28 36 43
Incremental Leaf Area -32.6 *** -55.4 ** -67.4 ** 67.9 ns 118.4 ns

Leaf Area -32.6 *** -88.0 *** -155.4 *** -87.4 ns 31.0 ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Negative values denote a greater mean value for "tolerant" treatments.

Conclusions

Significant positive correlations were found between soybean stem dry weight

and leaf dry weight and flood tolerance. However, soybean stem partitioning percentage

was negatively correlated with flood tolerance. The importance of stem development

both as a positive measure of adaptation and a negative reflection of plant performance is

highlighted in its relationship to tolerance. Significant correlations between plant growth

parameters and tolerance as defined in this study solidify the link between morphological

adaptations and the ability of soybean to grow under flooded conditions. The







68


dissimilarity between tolerance ranks for this study, with flooding at the V2 growth stage,


and tolerance rankings as established by Cornelious (2003) for flooding at the R2 growth


stage (Figure 3-5) may reflect variation in plant response to flooding at these stages. In



Linear Regression of Flood Tolerance Rankings

12
91209-297
10 91210-350
0 91210-316
8 0 91209-143
"_ 91209-151
S 6 91209-220
91209-142
4 0 91209-269 y = 5 93 0 078xI
91209-126 R2 = 0006
2 0 91209-119 < All Genotypes by Rank

0
0 2 4 6 8 10 12
Arkansas (Cornelious, 2003)




Figure 3-5 Linear regression of tolerance ranks from Arkansas (Cornelious, 2003) and
Belle Glade.

order to better understand the relationship between early season morphological response


and end of season tolerance it will be necessary to further investigate the effects of V2


flooding on soybean yield for these genotypes. By establishing rankings through


comparable means to those presented in Cornelious (2003), but based on early season


flood imposition, it would be possible to judge the effectiveness of early season tolerance


identification. Insight may also be gained as to the consistency of genotype tolerance


rankings with regards to timing of flood imposition. The data presented in this Chapter


warrants further investigation as it notes significant relationships between plant


morphology and flood tolerance.














CHAPTER 4
RESEARCH PERSPECTIVES

The process of developing and executing an academic experiment has been

enlightening on a professional, academic, and personal level. What I may have

considered to be an exercise in strict repetition before beginning has turned out to be a

challenging and creative process that pushed my abilities to learn.

From an academic perspective the experiments reported in this thesis offer, in my

opinion, an important contribution to increased understanding of the processes involved

in soybean adaptation to flood stress. Assuming that to be the intent of performing the

experiments, then this project has to be considered a success. The success we obtained

was not however the success we anticipated. It appears that by answering one question

the possibility to ask five new questions has arisen. That in itself may be the beauty of

coming to a greater understanding of any topic. Only through obtaining different results

than expected have I better understood how I might have gone about this project.

Without rehashing the contents of the previous three chapters, I do believe there is

validity to the concept that morphological response serves as a good indicator of flood

tolerance. I also believe that there is a strong possibility that early identification of flood

tolerant varieties is possible, at least in relation to early season flood stress. That being

said, additional evidence is needed and should be sought. Recommendations for future

projects in this area would include: 1) Morphology of flooding response at the R2

growth stage (this being the stage at which initial flood tolerance ranking were

determined) 2) Effects of flooding at the V2 stage on yield. This would allow early









season tolerance rankings based on biomass and yield based tolerance rankings to be

compared therefore establishing the predictive ability of those early season rankings.

From a professional perspective, my time at the University of Florida has been very

productive. The first question asked of me while inquiring about the School of Natural

Resources and Environment was "what are your career goals?" While I still may not be

able to answer that question specifically, I do know that I came in with little

understanding of the functioning of plants and their relation to the greater environment

and I am leaving with more information and thought dedicated to this topic than I could

have hoped for. Acquiring this information was my primary goal, but the conversations,

classes, and atmosphere associated with its acquisition have been of greater benefit than

the raw information ever will be. My recommendation to anyone pursuing a degree at

this level is, understand what you want to know before you begin looking for it.

Anything else you find along the way becomes a bonus to knowing where you are going.

Personal lessons learned have proven to be the greatest body of knowledge

obtained in this process. First, consistent work pays off. Hard work is good, but too

much hard work at any one time becomes stress. Consistent work allows for continual

meeting of goals and the appearance of progress. Second, be ready for the unexpected.

You cannot know what the outcome of the experiment will be before it is performed, if

you do then is it really worth doing? When the unexpected hits you, run with it,

sometimes it is much more interesting than the expected. Likewise, for the sake of the

experiment and science, do everything you can to avoid the unexpected. Try to anticipate

what could go wrong and avoid the possibility. In the meantime, make sure you can

document what is going on just in case you did not anticipate every possibility. Finally,






71


take control of knowing the subject. It is your project, you degree and while the process

must be and always will be a collaborative effort, on a very basic level it is your

responsibility to make sure it goes well.

This chapter is for the benefit of the students that may take the time to pick up and read

portions of this thesis. Understand that hindsight is always 20-20 (to be exceptionally

cliched). The strength and weaknesses of the previous three chapters are the experiences

represented in the fourth















APPENDIX A
DATA FROM EXPERIMENT 2













Table A-i Primary root dry weights.
Days After Sowing
21 35 49
Dry Weight Partitioning: Dry Weight Partitioning Dry Weight RDW* Partitioning
mg plant-1 mg plant1 mg plant'
Flood
2 Week 151 0.213 497 0.218 375 0.675 0.147
4 Week 164 0.227 342 0.165 447 0.813 0.163
Non-flooded 179 0.290 468 0.191 633 0.143
LSD 0.05 39 0.037 66 0.015 109 0.200 0.015
Significance ns ns ns ns ns ns ns

Genotype
Tolerant
91210-350 182 0.220 422 0.164 d 488 0.565 0.143 bc
91209-126 164 0.234 529 0.214 abt 571 0.775 0.159 ab
91209-142 147 0.237 394 0.221 a 440 0.719 0.169 a
Sensitive
91209-220 174 0.225 443 0.179 cd 413 0.976 0.149 abc
91209-269 189 0.259 424 0.197 bc 547 0.674 0.154 abc
Control
Hinson 134 0.183 405 0.175 d 451 0.759 0.133 c
LSD 0.05 56 0.053 923 0.021 154 0.400 0.021
Significance ns ns ns *** ns ns *

Flood x Genotype
Significance ns ns ns ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means in the same column followed by the same letter are not significantly different (P<0.05).
$ Dry matter partitioning is defined as primary root dry weight / total plant dry weight.
RDW = Relative dry weight is defined as primary root dw for flood treatment / primary root dw for non-flood treatment.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table A-2 Primary root length, diameter, surface area, and volume.
49 Days After Planting
Length Avg.Diameter Surface Area Volume
cm plant-1 mm plant' cm2 plant 1 cm3 plant
Flood
2 Week 1718 0.439 128 0.793
4 Week 2740 0.677 205 1.28
Non-flooded 1481 0.506 134 1.04
LSD 0.05 693 0.140 46 0.3
Significance ns ns ns ns

Genotype
Tolerant
91210-350 1782 0.521 147 1.04
91209-126 2198 0.634 178 1.22
91209-142 1796 0.515 142 0.958
Sensitive
91209-220 1740 0.474 134 0.876
91209-269 1945 0.524 158 1.07
Control
Hinson 2418 0.576 175 1.06
LSD 0.05 979 0.198 65 0.384
Significance ns ns ns ns

Flood x Genotype
Significance ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
tTolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table A-3 Adventitious root dry weight and dry matter partitioning.
Days After Planting
35 49
Dry Weight Partitioningt Dry Weight Partitioning
mg plant' mg plant'
Flood
2 Week 14 1.09 26 1.10
4 Week 39 1.28 35 1.26
Non-flooded -
LSD 0.05 11 0.40 11 0.28
Significance ns ns ns ns

Genotype
Tolerant:
91210-350 14 1.020 21 0.884
91209-126 17 0.865 23 0.830
91209-142 12 0.988 21 0.928
Sensitive
91209-220 21 0.440 15 0.591
91209-269 18 0.793 28 0.811
Control
Hinson 22 0.619 16 0.676
LSD 0.05 15 0.561 15 0.401
Significance ns *** ns ns

Flood x Genotype
Significance ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
tDry matter partitioning is defined as adventitious root dry weight / total plant dry weight.
:Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table A-4 Adventitious root length, diameter, surface area, and volume.
Length Average Diameter Surface Area Volume
cm plant'1 mm plant cm2 plant1 cm3 plant-
Flood
2 Week 231 0.247 16.9 0.104
4 Week 560 0.293 38.4 0.211
Non-flooded -
LSD 0.05 195 0.038 14.1 0.823
Significance ns ns ns ns

Genotype
Tolerant
91210-350 278 0.244 19.9 0.115
91209-126 469 0.275 33.1 0.193
91209-142 376 0.267 27.2 0.157
Sensitive
91209-220 422 0.296 29.8 0.169
91209-269 408 0.289 30.2 0.179
Control
Hinson 420 0.247 25.8 0.129
LSD 0.05 338 0.066 24.4 0.143
Significance ns ns ns ns

Flood x Genotype
Significance ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
tTolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table A-5 Nodule dry weight and dry matter partitioning.
Days After Planting
35 49
Dry Weight Partitioning: Dry Weight Partitioning
mg plant' % mg plant' %
Flood
2 Week 4 0.127 57 2.42
4 Week 15 0.643 68 2.41
Non-flooded 34 1.57 85 1.67
LSD 0.05 16 0.734 39 0.95
Significance ns ns ns ns

Genotype
Tolerant
91210-350 35 1.53 61 bc 1.75 b
91209-126 17 1.12 82 bt 2.24 b
91209-142 5 0.215 58 bc 2.23 b
Sensitive
91209-220 6 0.282 25 c 1.11 b
91209-269 21 1.00 155 a 4.29 a
Control
Hinson 16 0.537 41 bc 1.38 b
LSD 0.05 23 1.04 56 1.34
Significance ns ns ** **

Flood x Genotype
Significance ns ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
tDry matter partitioning is defined as nodule dry weight / total plant dry weight.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).













Table A-6 Stem dry weight and dry matter partitioning.
Days After Sowing
21 35 49
Dry Weight Partitioning: Dry Weight Partitioning Dry Weight Partitioning
mg plant- mg plant 1mg plant
Flood
2 Week 193 0.268 780 0.337 949 b 0.394
4 Week 190 0.261 773 0.359 1114 b 0.423
Non-flooded 186 0.256 774 0.312 1670 a 0.369
LSD 0.05 31 0.018 112 0.014 284 0.015
Significance ns ns ns ns ** ns

Genotype
Tolerant
91210-350 228 0.273 997 a 0.368 a 1410 0.442 a
91209-126 197 0.255 753 bct 0.309 d 1320 0.378 c
91209-142 168 0.271 617 c 0.349 ab 990 0.389 c
Sensitive
91209-220 199 0.256 816 ab 0.338 bc 1090 0.394 bc
91209-269 173 0.245 710 bc 0.319 cd 1310 0.356 d
Control
Hinson 191 0.271 793 b 0.334 bc 1380 0.414 b
LSD 0.05 43 0.026 158 0.209 400 0.022
Significance ns ns ** *** ns ***

Flood x Genotype
Significance ns ns ns ns ns*
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
$ Dry matter partitioning is defined as stem dry weight / total plant dry weight.
Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).










Table A-7 Plant height.
Days After Sowing
21 30 38 43
cm
Flood
2 Week 9.06 13.8 19.6 23.4
4 Week 8.89 13.7 19.1 23.4
Non-flooded 9.40 12.7 18.7 23.4
LSD 0.05 0.60 0.7 1.2 1.5
Significance ns ns ns ns

Genotype
Tolerant:
91210-350 9.29 ab 14.5 b 20.8 b 25.8 a
91209-126 9.25 abc 12.9 c 18.6 c 23.3 b
91209-142 9.17 abc 12.4 cd 17.7 cd 21.7 bc
Sensitive
91209-220 9.85 a 15.8 a 22.9 a 27.7 a
91209-269 8.44 c 11.8 d 16.5 d 20 c
Control
Hinson 8.70 bc 13.1 c 18.1 c 21.9 bc
LSD 0.05 0.84 0.9 1.6 2.2
Significance ** *

Flood x Genotype
Significance ns ns ns ns


*,**,*** Significant at the 0.05, 0.01, and <0.001


levels of probability respectively.


t Treatment means followed by the same letter are not significantly different (P<0.05).
:Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).













Table A-8 Leaf Dry weights and dry matter partitioning.
Days After Sowing
21 35 49
Dry Weight Partitioning: Dry Weight Partitioning Dry Weight Partitioning
mg plant1 mg plant1 mg plant
Flood
2 Week 369 0.520 1001 0.438 c 1040 b 0.424 b
4 Week 377 0.511 960 0.451 b 1040 b 0.373 c
Non-flooded 374 0.505 1180 0.480 a 2040 a 0.471 a
LSD 0.05 59 0.028 130 0.008 240 0.016
Significance ns ns ns ** ** **

Genotype
Tolerant
91210-350 422 0.507 1160 a 0.448 d 1310 0.389 c
91209-126 362 0.511 1110 at 0.457 cd 1530 0.432 ab
91209-142 308 0.492 750 b 0.421 e 1070 0.411 bc
Sensitive
91209-220 399 0.519 1140 a 0.471 ab 1290 0.441 a
91209-269 356 0.497 1020 a 0.465 bc 1590 0.439 a
Control
Hinson 396 0.546 1130 a 0.477 a 1470 0.433 ab
LSD 0.05 84 0.040 180 0.011 350 0.023
Significance ns ns ** *** ns **

Flood x Genotype
Significance ns ns ns ns*
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
$ Dry matter partitioning is defined as leaf dry weight / total plant dry weight.
Tolerant and Sensitive determined based on descending rankings (Cornelioius, 2003).










Table A-9 Leaf area values.
Days After Sowing
16 22 29 37 44
cm2 plant-
Flood
2 Week 48.9 at 113 169 228 b 245 b
4 Week 44.7 ab 104 159 225 b 254 b
Non-flooded 40.6 b 105 179 333 a 492 a
LSD 0.05 6.6 13 23 38 69
Significance ns ns **

Genotype
Tolerant:
91210-350 53.4 a 118 a 183 ab 277 ab 342
91209-126 38.4 c 99 bc 164 b 269 b 363
91209-142 34.6 c 80 c 128 c 187 c 252
Sensitive
91209-220 48.5 ab 117 ab 182 ab 266 b 286
91209-269 40.4 bc 97 c 154 bc 246 b 353
Control
Hinson 52.8 a 132 a 203 a 238 a 385
LSD 0.05 9.3 18 32 54 98
Significance ** *** ** ** ns

Flood x Genotype
Significance ns ns ns ns ns
**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
4Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).










Table A-10 Total plant dry weight
Days After Planting
21 35 49
g plant1
Flood
2 Week 0.713 2.30 2.45 b
4 Week 0.731 2.13 2.73 b
Non-flooded 0.739 2.46 4.43 a
LSD 0.05 0.107 0.29 0.65
Significance ns ns **

Genotype
Tolerant:
91210-350 0.831 2.59 a 3.29
91209-126 0.705 2.42 a 3.53
91209-142 0.623 1.78 b 2.58
Sensitive
91209-220 0.773 2.42 a 2.83
91209-269 0.718 2.19 a 3.62
Control
Hinson 0.721 2.37 a 3.63
LSD 0.05 0.151 0.42
Significance ns ** ns

Flood x Genotype
Significance ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
:Tolerant and Sensitive determined based descending ranking (Cornelious, 2003).










Table A-11 Root:shoot ratios
Days After Planting
21 35 49

Flood
2 Week 0.273 0.292 a 0.227 ab
4 Week 0.296 0.237 b 0.256 a
Non-flooded 0.360 0.263 b 0.192 b
LSD 0.05 0.129 0.027 0.041
Significance ns **

Genotype
Tolerant:
91210-350 0.283 0.228 b 0.206
91209-126 0.309 0.307 a 0.238
91209-142 0.311 0.301 a 0.255
Sensitive
91209-220 0.291 0.237 b 0.204
91209-269 0.439 0.277 a 0.262
Control
Hinson 0.224 0.235 b 0.184
LSD 0.05 0.183 0.037 0.058
Significance ns *ns

Flood x Genotype
Significance ns ns ns
*,*",* Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
:Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003).


Table A-12 Tolerance at 49 DAS
Tolerance Rank
91209-220 0.723 1
91209-142 0.714 2
91209-126 0.664 3
91209-269 0.635 4
91210-350 0.555 5
Hinson 0.552 6















APPENDIX B
LEAF NUTRIENT AND SPAD DATA FROM EXPERIMENTS 1 AND 2













Table B-l Leaf nutrient analysis (Experiment 1)
N P K Mg S Ca Mo Mn Fe
Treatment g kg'1 mg kg1
Flood
2 Week 3.05 0.64 b 1.39 0.44 a 0.264 1.98 a 155 38.1 73.5 ab
4 Week 2.59 0.87 a 1.43 0.32 b 0.22 1.56 c 94.3 25.9 85.8 a
Non-flood 2.86 0.43 c 1.23 0.456 a 0.25 1.82 b 117 25.9 68.6 b
LSD 0.05 0.24 0.11 0.12 0.027 0.21 0.09 17.3 4.4 13.7
Significance ns ** ns ** ns ** ns ns *

Genotype
Tolerant
91210-350 3.03 abc 0.76 abc 1.39 0.41 0.25 1.91 a 130 bc 37.9 a 77.6
91210-316 3.02 abc 0.55 cd 1.31 0.41 0.24 1.67 bc 84.9 e 24.7 c 67.4
91209-126 2.69 cd 0.57 cd 1.29 0.36 0.22 1.76 abc 92.3 de 31.5 abc 72.7
91209-151 2.71 cd 0.46 d 1.19 0.42 0.23 1.91 a 96.9 de 32.5 abc 70.4
91209-142 3.22 ab 0.44 d 1.28 0.4 0.24 1.79 ab 99.3 cde 30.6 abc 83.1
Sensitive
91209-220 2.84 bc 0.65 abcd 1.37 0.37 0.24 1.67 bc 89.1 de 26.3 c 82.6
91209-119 2.89 abct 0.620 bcd 1.34 0.43 0.27 1.91 a 121 cd 33.1 abc 73.7
91209-269 2.29 d 0.73 abc 1.39 0.45 0.26 1.81 ab 155 b 27.3 bc 67.9
91209-297 2.85 abc 0.84 a 1.54 0.41 0.25 1.61 c 108 cde 24.9 c 61.9
91209-143 3.32 a 0.65 abcd 1.29 0.4 0.24 1.92 a 96.4 de 25.6 c 86.3
Control
Hinson 2.32 d 0.83 ab 1.49 0.42 0.26 1.69 bc 269 a 35.4 ab 92.3
LSD 0.05 0.45 0.21 0.23 0.05 0.04 0.18 33.0 8.4 26.2
Significance ** ** ns ns ns ** *** ns

Flood x Genotype
Significance ns ns ns ns ns ns ns


*,**,*** Significant at the 0.05, 0.01, and <0.001


levels of probability respectively.


t Treatment means followed by the same letter are not significantly different (P<0.05).
4Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003).













Table B-2 Leaf nutrient analysis (Experiment 2)
N P K Mg S Ca Mo Mn Fe
Treatment g kg'1 mg kg1
Flood
2 Week 2.87 at 0.543 1.27 a 0.413 b 0.273 a 2.34 a 228 a 48.4 a 133
4 Week 2.26 b 0.632 1.15 b 0.296 c 0.202 b 1.68 b 120 c 37.9 b 120
Non-flood 2.43 b 0.525 1.04 c 0.571 a 0.289 a 2.28 a 202 b 36.9 b 117
LSD 0.05 0.21 0.059 0.08 0.023 0.016 0.080 17 4.5 18
Significance ** ns *** ** *** ** ns

Genotype
Tolerant:
91210-350 2.63 ab 0.616 a 1.13 0.452 a 0.261 2.29 a 199 b 43.2 ab 150 a
91209-126 2.57 ab 0.513 b 1.14 0.399 b 0.245 2.12 b 136 d 46.3 a 106 c
91209-142 2.56 ab 0.513 b 1.09 0.450 a 0.253 2.21 ab 171 c 43.3 ab 110 bc
Sensitive
91209-220 2.11 c 0.657 a 1.09 0.417 b 0.258 2.13 b 153 cd 39.4 bc 125 abc
91209-269 2.86 a 0.513 b 1.25 0.416 b 0.265 1.95 c 167 c 35.4 c 113 bc
Control
Hinson 2.41 bc 0.590 ab 1.23 0.425 ab 0.246 1.89 c 273 a 39.1 bc 137 ab
LSD 0.05 0.3 0.084 0.12 0.033 0.023 0.11 24 6.4 26
Significance ** ** ns ns ** *

Flood x Genotype
Significance ns ns ns ns ns ** ** ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
4Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).














Table B-3 SPAD readings (Experiment 1).
Days After Sowing
21 25 28 32 36 39 44 47
Flood
2 Week 21.2 17.9 18.4 16.7 17.4 b 18.7 b 24.4 a 23.9 a
4 Week 21.0 17.9 17.7 16.0 17.4 b 17.9 b 23.6 b 19.2 b
Non-flooded 22.2 18.3 17.9 17.6 19.9 a 21.1 a 19.3 a 22.9 a
LSD 0.05 1.2 0.9 0.8 0.7 0.9 1.0 1.3 1.3
Significance ns ns ns ns ** **

Genotype
Tolerant$
91210-350 21.3 18.1 abc 18.5 ab 17.7 19.9 ab 21.3 a 22.7 b 21.8 bcde
91210-316 22.2 19.4 a 19.3 a 16.7 18.5 abc 21.1 a 23.8 ab 24.1 ab
91209-126 21.6 17.6 bc 17.5 bc 16.5 18.4 abc 19.6 abc 23.4 ab 23.1 abc
91209-151 22.4 19.5 a 18.5 ab 17.1 20.2 a 20.6 ab 24.1 ab 23.2 abc
91209-142 21.1 18.3 ab 17.6 bc 16.9 20.0 ab 21.5 a 25.6 a 24.4 a
Sensitive
91209-220 21.2 17.6 bc 17.4 bc 16.0 16.4 de 17.9 de 22.3 b 21.5 cde
91209-119 21.4 18.0 abct 17.9 abc 16.2 18.3 bcd 18.7 abcd 22.7 b 22.3 abc
91209-269 21.1 17.9 abc 17.7 abc 17.1 17.2 cd 18.6 cde 19.7 c 21.1 cde
91209-297 23.1 18.4 ab 19.2 a 17.5 15.2 e 14.8 f 19.4 c 19.4 e
91209-143 20.3 16.9 bc 17.5 bc 16.8 18.6 abc 19.8 abc 23.0 b 21.9 bcd
Control
Hinson 20.2 16.5 c 16.7 c 16.3 17.4 cd 17.6 e 19.4 c 19.7 de
LSD 0.05 2.3 1.6 1.6 1.4 1.9 1.9 2.5 2.5
Significance ns ns *** *** *** **

Flood x Genotype
Significance ns ns ns ns ns ns
,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means in the same column followed by the same letter are not significantly different (P<0.05).
4Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).













Table B-4 SPAD readings (Experiment 2).
Days After Sowing
21 30 38 43

Flood
2 Week 31.3 at 23.8 a 20.8 b 22.3 b
4 Week 28.7 b 22.4 b 21.4 b 23.5 a
Non-flooded 30.2 ab 24.4 a 23.3 a 22.7 ab
LSD 0.05 1.8 0.9 1.3 1.2
Significance ns

Genotype
Tolerant:
91210-350 30.4 26.7 a 23.9 a 22.3 ab
91209-126 29.5 22.1 cd 22.4 a 23.8 a
91209-142 30.3 24.9 b 22.7 a 24.2 a
Sensitive
91209-220 31.0 21.2 d 18.8 b 19.8 c
91209-269 29.3 23.4 c 22.7 a 24.7 a
Control
Hinson 29.8 22.9 c 20.6 b 21.5 bc
LSD 0.05 2.5 1.3 1.8 1.8
Significance ns *

Flood x Genotype
Significance ns ns ns
*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.
t Treatment means followed by the same letter are not significantly different (P<0.05).
:Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).

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MORPHOLOGICAL ADAPTATIONS OF SOYBEAN IN RESPONSE TO EARLY SEASON FLOOD STRESS By THOMAS L. HENSHAW A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Thomas L. Henshaw

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This thesis is dedicated to my family for getti ng me here and to Erin for keeping me here. We may be young, but we are on our way.

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ACKNOWLEDGMENTS I would like to thank my committee, Drs. Robert Gilbert, Johannes Scholberg, and Tom Sinclair, for their availability, patience, and help. I thank all the people in Belle Glade that made this project possible Ron Gosa, Lee Liang, and Pepe Gonzalez. Special appreciation goes to Dr. Chen and the University of Arkansas soybean breading program for providing germplasm and background information. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.............................................................................................................x ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 Overview.......................................................................................................................1 Introduction...................................................................................................................1 Background............................................................................................................1 Crop rotations with sugarcane on mineral soils.............................................2 Soybean (Glycine max. L. Merrill) as a rotational crop.................................3 Rationale................................................................................................................4 Selection of genetic material..........................................................................4 Flood tolerance...............................................................................................5 Approach.......................................................................................................................5 Null Hypotheses............................................................................................................6 2 THE EFFECT OF FLOODING AND GENOTYPE ON THE SOYBEAN (GLYCINE MAX L. MERRILL) RHIZOSPHERE....................................................7 Review of Literature.....................................................................................................7 Identification of Tolerance to Flood Stress...........................................................7 Selecting Soybean Genotypes for Flood Tolerance............................................11 Materials and Methods...............................................................................................13 Selection of Genetic Material..............................................................................13 Experimental Design...........................................................................................14 Crop Management...............................................................................................15 Measurements...............................................................................................18 Plant biomass sampling................................................................................18 Root growth..................................................................................................19 Statistical Analysis..............................................................................................20 v

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Results and Discussion...............................................................................................21 Primary Root Dry Weight and Dry Matter Partitioning......................................21 Primary Root Length, Average Diameter, Surface Area, and Volume...............28 Adventitious Root Dry Weight and Dry Matter Partitioning..............................28 Adventitious Root Length, Average Diameter, Surface Area, and Volume.......30 Nodule Dry Weights and Dry Matter Partitioning..............................................33 Correlations of morphological characters to flood tolerance..............................34 Contrast Between Flood Tolerance Groups........................................................39 Conclusions.........................................................................................................39 3 THE EFFECT OF EARLY-SEASON FLOODING AND GENOTYPE ON SOYBEAN (GLYCINE MAX L. MERRILL) ABOVEGROUND GROWTH AND TOTAL PLANT BIOMASS.......................................................................................42 Review of Literature...................................................................................................42 Identification of Tolerance to Flood Stress.........................................................42 Rationale and Objectives.....................................................................................45 Materials and Methods...............................................................................................45 Leaf Area and Plant Height.................................................................................46 Statistical Analysis..............................................................................................47 Results and Discussion...............................................................................................48 Stem Dry Weight, Dry Matter Partitioning, and Height.....................................48 Leaf Expansion, Dry Matter Accumulation, and Partitioning.............................52 Total Plant Dry Weight.......................................................................................59 Root:Shoot Ratio.................................................................................................62 Correlations of Above Ground Plant Growth to Flood Tolerance......................63 Contrast Between Flood Tolerance Groups........................................................66 Conclusions.........................................................................................................67 4 RESEARCH PERSPECTIVES..................................................................................69 APPENDIX A DATA FROM EXPERIMENT 2................................................................................72 B LEAF NUTRIENT AND SPAD DATA FROM EXPERIMENTS 1 AND 2............84 LIST OF REFERENCES...................................................................................................89 BIOGRAPHICAL SKETCH.............................................................................................93 vi

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LIST OF TABLES Table page 2-1 Experimental designs used for soybean flooding experiments..............................15 2-2 Preliminary Mehlich-I soil test results and fertilizer recommendations................17 2-3 Weekly average temperature and precipitation data for Belle Glade EREC, May-August 2004..................................................................................................22 2-4 Primary root dry weight, dry matter partitioning, and relative dry weight............23 2-5 Genotype comparisons for significant dry weight genotype x flood interaction terms.......................................................................................................................25 2-6 Genotype comparisons for significant dry matter partitioning genotype x flood interaction terms.....................................................................................................27 2-7 Primary root length, average diameter, surface area, and volume 49 days after sowing....................................................................................................................29 2-8 Adventitious root dry weights and dry matter partitioning....................................31 2-9 Genotype comparisons for significant adventitious root dry weight genotype x flood interaction term............................................................................................32 2-10 Adventitious root length, average diameter, surface area, and volume 49 days after sowing............................................................................................................33 2-11 Nodule dry weight and dry matter partitioning......................................................35 2-12 Spearmans rank correlation coefficients for selected rhizosphere plant components under flooded conditions as correlated to tolerance rank..................36 2-13 Rhizosphere dry weight contrast for flood tolerant vs. sensitive genotypes as defined by Cornelious........................................................................................40 2-14 Primary and adventitious root length, surface area, diameter, and volume, contrast value for flood tolerant vs. flood sensitive genotypes as defined by Cornelious.........................................................................................................40 vii

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3-1 Genotype yield reduction and flood injury score for most tolerant and most sensitive Arkansas RILs.......................................................................................46 3-2 Regression equations and R2 values for leaf area by genotype used to determine non-destructive leaf area values............................................................47 3-3 Stem dry weight and dry matter partitioning (Experiment 1)................................49 3-4 Genotype comparison for all significant dry matter partitioning flood x genotype interaction terms.....................................................................................51 3-5 Plant heights...........................................................................................................53 3-6 Leaf dry weights and dry matter partitioning........................................................54 3-7 Incremental leaf area expansion.............................................................................56 3-8 Leaf area.................................................................................................................60 3-9 Total plant dry weight............................................................................................61 3-10 Root:Shoot ratios...................................................................................................62 3-11 Tolerance of all genotypes to early-season flooding.............................................64 3-12 Stem dry weight means for flooded treatments and ranks used for correlations...64 3-13 Correlations between selected plant measures and flood tolerance.......................65 3-14 Stem dry matter partitioning means for flooded treatments and ranks used for correlations.............................................................................................................65 3-15 Leaf dry weight means for flooded treatments and ranks used for correlations....66 3-16 Aboveground dry weight contrasts between tolerant vs. sensitive genotypes as defined by Cornelious (2003) across 2and 4-week flood treatments...............................................................................................................67 3-17 Plant height, incremental leaf area, and leaf area values, contrasts between tolerant vs. sensitive genotypes as defined by Cornelious (2003) across 2and 4-week flood treatments..................................................................................67 A-1 Primary root dry weights.......................................................................................73 A-2 Primary root length, diameter, surface area, and volume......................................74 A-3 Adventitious root dry weight and dry matter partitioning.....................................75 A-4 Adventitious root length, diameter, surface area, and volume..............................76 viii

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A-5 Nodule dry weight and dry matter partitioning......................................................77 A-6 Stem dry weight and dry matter partitioning.........................................................78 A-7 Plant height............................................................................................................79 A-8 Leaf Dry weights and dry matter partitioning........................................................80 A-9 Leaf area values.....................................................................................................81 A-10 Total plant dry weight............................................................................................82 A-11 Root:shoot ratios....................................................................................................83 A-12 Tolerance at 49 DAS..............................................................................................83 B-1 Leaf nutrient analysis (Experiment 1)....................................................................85 B-2 Leaf nutrient analysis (Experiment 2)....................................................................86 B-3 SPAD readings (Experiment 1).............................................................................87 B-4 SPAD readings (Experiment 2).............................................................................88 ix

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LIST OF FIGURES Figure page 2-1 Rainfall at EREC Belle Glade over the duration of Experiments 1 and 2. Day 1 for Experiment 1 = May 25, 2004. Day 1 for Experiment 2 = June 29, 2004........22 2-2 Graph showing lack of linear fit in the relationship between adventitious root length and dry weight...............................................................................................38 2-3 Graph showing lack of linear fit in the relationship between adventitious root surface area and dry weight......................................................................................38 3-1 Incremental leaf area expansion for all genotypes across flood treatments.............57 3-2 Incremental leaf area expansion for most sensitive genotypes.............................57 3-3 Incremental leaf area expansion for most tolerant genotypes...............................58 3-4 Incremental leaf area expansion for control genotype Hinson.................................58 3-5 Linear regression of tolerance ranks from Arkansas and Belle Glade.....................68 x

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MORPHOLOGICAL ADAPTATIONS OF SOYBEAN IN RESPONSE TO EARLY SEASON FLOOD STRESS By Thomas L. Henshaw August 2005 Chair: Robert A. Gilbert Major Department: Interdisciplinary Ecology Soybean has been considered for some time as a crop with potential for flood tolerance because of its highly adaptive nature under flood stress. The designation of genetic markers for flood tolerance in southern varieties has kindled interest in further defining the physiological properties associated with acclimation to flooding. The objective of this research was to identify distinguishable morphological changes in response to early season flooding (V2 growth stage), in an attempt to isolate a characteristic or groups of characteristics that can be used for identification of potentially flood tolerant varieties. A lysimeter trial was carried out at the University of Florida Everglades Research and Education Center, Belle Glade, Florida, from May to August 2004. Eleven genotypes (ten numbered Recombinant Inbred Lines (RIL) and Hinson) were flooded at the V2 growth stage. Plants were subjected to one of three flood regimes (well watered, 2 weeks of flooding with 2-week recovery, or 4 weeks of flooding). Consistent patterns of adaptation developed with additional flood duration including xi

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decrease in leaf area, total biomass, and nodulation accumulation, and an increase in adventitious root length. Genotypic response to flooding was significantly different for all plant tissue types, while differentiation between varieties based on flood tolerance was inconsistent. Growth characteristics including primary root dry weight, length, surface area, and adventitious root dry weight have been identified as positively correlated to flood tolerance under these experimental conditions. xii

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CHAPTER 1 INTRODUCTION Overview This chapter provides an overview of the rationale, approach, and hypotheses underlying this study pertaining to the morphological response of soybean to early-season flood stress. Subsequent chapters include comprehensive literature reviews, materials and methods, results, discussion and conclusion sections. Chapter 2 outlines the effect of flooding treatments on primary roots, adventitious roots, and nodules. Chapter 3 describes the effects of the same treatments on stem, leaf, and whole plant biomass as well as the relationship between root and shoot growth. Chapter 4 contains my perspectives on the research process based on my experience during my M.S. This division of chapters allows for the independent analysis of plant components that are interactive in nature, as well as a comprehensive analysis of the plant as an entire entity. Introduction Background The Everglades Agricultural Area (EAA) is located on the southeastern shore of lake Okeechobee, primarily in Hendry and Palm Beach Counties, Florida. With a sugarcane production area of approximately 180,000 ha (Schueneman, 2002), this region is home to more than 50% of the United States sugar cane production. Established in the 1920s, the sugarcane industry boomed in the 1960s with the exclusion of Cuban access to US markets. Agricultural production in the area was made possible primarily by two technological developments, the introduction of varieties adapted to Floridas climate and 1

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2 the development of a regional drainage structure including canals that allowed for control of water levels in this often-flooded area (Alvarez and Polopolus, 2002). The soils primarily used for sugarcane production are Histosols characterized by high organic matter content, deep organic matter profile, and low clay mineral content (Rice et al., 2005). While fertilization recommendations vary dramatically depending on soil location and properties, these soils are generally recognized as highly fertile. Conversely, approximately 20% of the sugarcane grown in Florida is grown on sandy mineral soils adjacent to the muck soils of the EAA. Soils in this region are fine to coarse textured sands with varying organic matter content predominantly classified as Spodosols, but Entisols and Mollisols occur in transitional areas. The majority of these soils are subject to flooding because of the close proximity of bedrock and hard pans resulting in seasonally high water tables. High variability in soil characteristics, inherently low nutrient content, and drainage concerns combine to create a need for intensive crop management in this region (Gilbert et al., 2002). Crop rotations with sugarcane on mineral soils Sugarcane is a perennial crop, generally harvested at yearly intervals between October and April. The following years crop (ratoon) is the result of re-growth from the rhizomes and stubble left after harvest. A field will remain in production until yields have fallen below an acceptable threshold. The yearly reduction in yield is more accentuated on mineral soils with their low organic matter content and limited inherent soil fertility (Muchovej, 2002). While sugarcane fields may stay in production for several years, once a field is removed from production, there may be an extended period of fallow between final harvest and replanting. It is well documented that the use of a leguminous cover crop can add both soil organic matter and offer a substantial source (up

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3 to 50-200 kg ha-1) of organic nitrogen (Powers and McSorley, 2000). Considering the soil fertility limitations of mineral soils for sugarcane production, use of a legume-based rotation during fallow appears to be desirable. Environmental and agronomic limitations make selection of an appropriate rotational crop critical to its potential success in this region. Ideally, the rotation crop would provide substantial amounts of biomass and provide a high rate of nitrogen fixation. When selecting a leguminous rotation crop the following considerations are important: 1) It must fit into the agronomic calendar used by sugarcane growers, that is to say, there is a window of approximately six months in which the rotation crop should reach its full beneficial potential; 2) It must not interfere with future cane production, germination, or growth; 3) Its seed must be readily available; 4) It should provide significant increases in cane production; 5) It must be financially appropriate from a cost/benefit perspective; and 6) It must be adapted to the climatic conditions of the region, including high average temperature, periodic flooding, and high water tables (Muchovej, 2002). Soybean (Glycine max. L. Merrill) as a rotational crop Soybean, possibly because of its value as a food staple, is mentioned less often than other annual legumes when considering potential green manures. Muchovej (2002) however, cites that soybean can fix as much as 112 kg ha-1 N, more than cowpea (Vigna sinesis), peanuts (Arachis hypogaea), or snap beans (Phaseolus vulgaris). While soybean is generally considered susceptible to flood stress (Sullivan et al., 2001), it has proven to be more resistant to soil waterlogging than cowpea, a commonly grown crop in the EAA (Andreeva et al., 1987). Considering the limitations placed on variety selection for green manures in this growing environment, soybean appears to merit additional investigation.

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4 Rationale Soybean has not been grown commercially in South Florida. As such there is limited information on the acclimation of this species to the specific production environments that prevail in this region and its performance under local conditions. Concurrently there is increased interest in identifying and generating flood tolerant varieties that can allow for increased production in areas prone to waterlogging across the United States. The opportunity to investigate soybean flood tolerance is both pertinent to the potential use of soybean as a rotational crop in South Florida and potentially enlightening in the development of new tolerant varieties. In addition the availability of ten recombinant inbred lines (RILs) bred and tested for flood tolerance at the University of Arkansas (Cornelious, 2003) offered a unique opportunity to combine documented tolerance research with additional studies on plant growth. Selection of genetic material All experiments included in this study drew on the pool of ten RILs generated and previously tested for flood tolerance at the University of Arkansas (Cornelious, 2003). Each genotype had been rated based on visual flood injury and yield potential. The ten RILs included in this study represent the five most tolerant and five most sensitive lines based on the Arkansas grading methods. Genotypes available included: most sensitive, 91209-119, 91209-220, 91209-269, 91209-143, 91209-297 and most tolerant, 91209-126, 91209-142, 91209-151, 91210-316, and 91210-350. The trial also included Hinson, a recommended variety for soybean maturity group VIII in Florida, as a commercial check.

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5 Flood tolerance The available genetic material had been rated for tolerance based on response to a 10-14 day flood treatment at the R2 growth stage (Fehr and Caviness, 1977). A 0-9 visual flood injury scale was used with 0 = no injury and 9 =severe injury. In addition, soybean grain yield response was measured as a percent decrease in yield for flooded treatments compared with non-flooded treatments (1-[grain yield flood / grain yield control])*100% (Cornelious, 2003). For the purpose of this study tolerance is defined as the mean total plant dry weight of flooded treatments expressed as a percentage of the total plant dry weight of the non-flooded treatment (plant dry weight flood / plant dry weight non-flood). Using this approach, the relationship between flooded and non-flooded treatments is maintained. The expression of tolerance is however, the inverse of that used by Cornelious, resulting in higher tolerance rating ratios for materials with higher tolerances. Approach Following the logic of Bacanamwo and Purcell (1999a, 1999b) and emphasizing the key role of morphological adaptations in the avoidance of flood injury, the intent of these experiments was to determine if a link exists between identifiable morphological traits and flood tolerance. Flooding at an early season growth stage (V2) was selected in an attempt to determine the possibility of reducing the time and expense associated with genotypic flood tolerance identification. V2 had been identified as an early season soybean growth stage susceptible to flood stress (Linkemer et al., 1998), thus flooding at this stage would highlight any genotypic differences. Morphological changes have been used for early identification of growth traits in other legume species (Sultana et al., 2002). Morphological changes of interest based on the current literature on the

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6 adaptation of plants to flood stress include: Root growth parameters including dry weights of primary and adventitious roots, primary and adventitious root length and area, and nodule development. Shoot growth parameters of interest included dry weights of leaves and stems, SPAD leaf greenness readings, and plant height. Null Hypotheses 1. H0I: No significant soybean morphological differences will occur between soybean genotypes subjected to various flooding treatments (Chapters 2 and 3). 2. H0II: No significant soybean morphological differences will be observed between flood treatments (Chapters 2 and 3). 3. H0III: There are no significant flood x genotype interactions for soybean morphological traits (Chapters 2 and 3). 4. H0IV: There are no significant differences in flood tolerance among genotypes when subjected to various flood treatments (Chapter 3). 5. H0V: There is no significant relationship between flood tolerance and soybean morphological traits (Chapters 2 and 3). 6. H0VI: There is no significant difference between flood tolerance rankings reported in this study and flood tolerance rankings reported by the University of Arkansas (Chapter 3).

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CHAPTER 2 THE EFFECT OF FLOODING AND GENOTYPE ON THE SOYBEAN (GLYCINE MAX L. MERRILL) RHIZOSPHERE Review of Literature Identification of Tolerance to Flood Stress Flood tolerance may vary greatly between plant species. Variation in maximum allowable flood duration ranging from a few hours to weeks can be attributed to two primary factors: the increased availability of oxygen to the roots provided by morphological adaptations in tolerant plants, and different biochemical responses to anaerobic conditions (Kramer and Boyer, 1995). Intra-species variation in flood tolerance depends greatly on the plant organs directly affected by flooding, stage of plant development at which the flood is imposed, and external conditions such as temperature (Vartapetian and Jackson, 1997). While a net reduction in growth is to be expected under anaerobic conditions, many plants exhibit a number of adaptive morphological responses that result in increased growth or extension of certain tissues (Jackson and Drew, 1984). The primary adaptation of the rhizosphere is the development of adventitious roots. Adventitious roots are defined as non-primary roots emerging from hypocotyl tissue (Zobel, 1991) and are commonly viewed as a partial replacement for the incapacitated original root system (Jackson and Drew, 1984). Various methods have been used to identify flood tolerance. It is generally accepted that yield of high quality marketable seeds is the ultimate judging factor (Van 7

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8 Toai et al., 1994; Roiselle and Hamblin, 1981). Van Toai et al. (1994) rated flood tolerance of 84 varieties by total grain yield under flooded and non-flooded conditions, allowing for a comparative ranking of tolerance based on yield. Roiselle and Hamblin (1981) used a comparative yield analysis defined as the average of flooded and non-flooded yields. This approach selects the variety promising highest yield return regardless of stress conditions. Van Toai and Nurijani (1996) flooded soybeans for the length of the growing season (approximately 12 weeks) and measured average seed yield of flooded plants and SPAD leaf color reading for each variety. These values were divided by the values for the control. The resulting ratio represented a measure of tolerance based on relative yield within varieties rather than total yield. Bacanamwo and Purcell (1999a, 1999b) took a different approach to determining tolerance. Emphasizing the necessity of morphological adaptations for avoidance of flooding injury, they suggested that selecting varieties for morphological adaptations to flooding may be the best way to select for flooding tolerance. This study uses an approach similar to that of Van Toai and Nurijani (1996) while incorporating the morphological emphasis of Bacanamwo and Purcell (1999a, 1999b). Using a flooded to control ratio allows for the elimination of bias given to genotypes accumulating greater quantities of biomass under all treatments. While soybean is generally considered susceptible to flooding (Bacanamwo and Purcell, 1999a, 1999b; Sullivan et al., 2001) showing reductions in growth and yield when flooded for 7 days (Oosterhuis et al., 1990; Sallam and Scott, 1987), there is evidence that in comparison to other legumes, soybean has the ability to adapt to soil waterlogging. Andreeva et al. (1987) reported soybean to be more flood tolerant than

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9 cowpea. Boru et al. (2003) showed no negative effects on survival or leaf greenness of soybean plants grown in nitrogen gas with no detectable oxygen for 14 days, suggesting that soybean is more tolerant to increased levels of water and decreased oxygen than previously thought. The effects of waterlogging on the soybean rhizosphere are substantial and generally negative. A decrease in nitrogen accumulation for flooded soybean plants (Pankhurst and Sprent, 1975; Sugimoto and Satou, 1990) has been identified as the limiting factor to growth (Bacanamwo and Purcell, 1999a). Flooding for one week was sufficient to reduce leaf nitrogen concentration levels below deficiency at early vegetative stages (Sullivan et al., 2001). Reduction in nitrogen has been attributed to decreased nodulation (Sprent, 1972; Sallam and Scott, 1987; Sung; 1993), increased levels of ethylene (Sprent and Gallacher, 1976), and decreased nitrogenase activity (Bennett and Albrecht, 1984). However, after 4 days of flooding there was no destruction of cellular mitochondria in roots (Andreeva et al., 1987). Maintaining mitochondrial integrity is essential to continued nitrogenase activity. After an initial depression in nitrogenase activity flooded soybean plants actually recovered a level of activity comparable to that of the control (Bennett and Albrecht, 1984). Though a reduction in all root growth parameters was observed (Sallam and Scott, 1987), the development of new nodules at the soil surface and on newly developed adventitious roots offset the loss of original nodule function (Bacanamwo and Purcell, 1999b; Bennett and Albrecht, 1984) in flooded soybean. Plant nitrogen fixation returned to near normal levels within 15 days after removal of flood treatments of up to 14 days (Sugimoto and Satou, 1990; Bennett and Albrecht, 1984). The recovery of nodule function coinciding

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10 with the rebound in plant growth after the removal of flood treatments provides evidence that active root nodules are necessary for flood tolerance (Sprent, 1972; Sugimoto and Satou, 1990). Short-term acclimation to flooding through biochemical responses may allow for avoidance of certain injurious factors (Vartapetian and Jackson, 1997). These biochemical changes have been shown, in the case of wheat, to be temporary (Good and Muench, 1993). Long-term flood tolerance will require morphological adaptations in the plant allowing for increased aeration of the roots (Vartapetian and Jackson, 1997; Bacanamwo and Purcell, 1999b). Adventitious rooting is a mechanism for replacing existing roots that have been killed or whose function is impaired by anoxia at depth (Vartapetian and Jackson, 1997). Extensive adventitious root development has been reported to enhance oxygen transport from the stem to the roots (Visser et al., 1996) and reduce flooding injury in soybeans (Lee et al., 2003). Adventitious root fresh weight as a percentage of total root weight was greatly increased by flooding. Twenty-one days of flooding at the V4-V6 growth stage resulted in adventitious root percentages of 33-41% total root fresh weight (Bacanamwo and Purcell, 1999b). Pires et al. (2002) reported similar results for a 14-day flood at the V2 growth stage. Aerenchyma is spongy modified tissue containing large connected pores that facilitates gas exchange (Evans, 2003). Flooding in soybean plants greatly increased the incidence of aerenchyma in the cortex of roots. Aerenchyma development was primarily evident in new adventitious roots, whereas primary roots of flooded plants exhibited tightly packed cortical cells (Bacanamwo and Purcell, 1999b). Cortical aerenchyma

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11 formation especially in adventitious roots was apparent after 21 days of flooding from the V2 to V5 growth stages (Pires et al., 2002). Inhibition of adventitious root and aerenchyma development through the application of 5 M Ag, which counters ethylene reduction, increased the flooding susceptibility of soybeans. An additional loss of 25% biomass and leaf nitrogen accumulation over simple flooded treatments was witnessed with the application of Ag. This indicates that adventitious root and aerenchyma formation are important for acclimation to flooding (Bacanamwo and Purcell, 1999b). Selecting Soybean Genotypes for Flood Tolerance Though the ultimate criterion for judging flood tolerance is seed yield, early-season screening techniques for flood tolerance would decrease the time and resources required for plant breeding programs. Several traits have been used to make determinations about flood tolerance in soybean including: leaf color, plant height, chlorophyll content, and biomass of roots and shoots. Attempts to use these characteristics to develop a flood resistant line have yet to be fully successful (Van Toai et al., 2001). Flood tolerance in soybean plants is considered to be a developed adaptation specific to particular genotypes rather than an inherent property (Van Toai and Nurijani, 1996), therefore there is genetic variability in tolerance among cultivars (Van Toai et al., 1994; Van Toai and Nurijani, 1996). Van Toai et al. (2001) used 122 recombinant inbred lines (RIL) of an Archer x Minsoy cross and 86 RILs of an Archer x Noir cross, noting that Archer is more flood tolerant than either of the other two varieties. Flooding response was measured by a comparison of plant height and seed yield. A single quantitative trait locus (QTL) was identified for both increased height and increased yield. Plants containing the Archer Sat_064 allele yielded 95% more and were 16% taller than control plants. The effect of a

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12 single marker on both height and yield suggests that plants that grew better under waterlogged conditions also yielded more (Van Toai et al., 2001) A second trial performed in Costa Rica, flooding plants for 10-14 days until mild chlorosis was noted, showed a very different result (Reyna et al., 2003). Plants containing the Archer Sat_064 marker showed no difference in yield between flooded and non-flooded treatments. Plants containing the southern (A5403) Sat_064 allele did show significant yield advantages over plants not containing that marker (Reyna et al., 2003). While both trials do show genotypic variation in flood tolerance and the potential for identification of pertinent genetic markers, the inconsistent demonstration of flood tolerance in these strains highlights the current limitations of the flood tolerance identification process in soybean. There is a critical need for further investigation of both genetic and morphological flood tolerance indicators. Positive correlations between early-season flood adaptations, and flood tolerance as defined by grain yield, could greatly reduce the time and resources needed to identify potentially flood tolerant varieties for breeding purposes. This chapter highlights the morphological adaptations of the rhizosphere portion of the soybean plant to flooding in an attempt to isolate specific physiological characteristics that may help identify flood tolerant germplasm. Reporting the growth patterns of isolated plant components and their correlation to tolerance allows us to more closely examine the immediate effects of flood treatments on these components. The rhizosphere fraction is most immediately affected by flooding due to its proximity to the source of stress (Kramer and Boyer, 1995). Our objectives were to examine the effects of flooding

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13 soybean on 1) growth of the primary rooting system, 2) the development and expansion of adventitious roots, 3) nodule growth, and also to 4) determine the relationship of rhizosphere morphological changes to overall flood tolerance. Materials and Methods Two experiments were conducted at the Everglades Research and Education Center (EREC, 26o 39' N, 80o 38' W), in Belle Glade, Florida. Twenty-four separate concrete lysimeters were used in two separate experiments. The lysimeters used for Experiment 1 had an inside measurement of 3.5 m x 0.97 m and a depth of 0.91 m, while those used for the Experiment 2 measured 2.29 m x 1.07 m also at a depth of 0.91 m. Each lysimeter was equipped with a gravel leach bed approximately 15 cm in depth and a pvc release valve extending from one end (short side) of the container. Inserting or removing a stopper from the release valve allowed free drainage by gravity of water accumulated in the leach bed. Flood treatments were maintained at constant height through the use of a float valve set to add water whenever the level fell below 3 cm above the soil surface. Each lysimeter was filled to approximately 5 cm below the container lip with a topsoil mix (Odums Inc., West Palm Beach, Fl) consisting of 90% coarse sand and 10 % organic soil. This topsoil mix was chosen as it most closely approximated the mineral soils in the Everglades Agricultural Area. Planting for Experiment 1 took place on May 25, 2004 and planting for Experiment 2 was performed on June 29, 2004. Both experiments had a total duration of 49 days. Selection of Genetic Material Experiment 1 included ten Recombinant Inbred Lines (RILs) (provided by Dr. Pengyin Chen, University of Arkansas) generated and previously tested for flood tolerance at the University of Arkansas (Cornelious, 2003). Each genotype had been

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14 rated based on visual flood injury and yield. The ten genotypes included in this study represented the five most tolerant and five least tolerant lines based on the Arkansas grading methods. The most tolerant genotypes included were 91209-126, 91209-142, 91209-151, 91210-316, and 91210-350. The most sensitive genotypes were 91209-119, 91209-143, 91209-220, 91209-269, and 91209-297. The trial also included Hinson, a commercial variety recommended for soybean maturity group VIII in Florida. Flood tolerance rankings remained unknown to the researchers until the conclusion of the trials. Due to space limitations, six of the eleven genotypes used in Experiment 1 of the lysimeter trial were selected for Experiment 2. Selection of these six genotypes was based on the comparison of dry weight data from a 21-day preliminary trial. The ratios of plant dry weights from the 21-day flood treatment to plant dry weights of a non-flooded treatment were calculated for each genotype. The three genotypes showing the greatest flood/dry ratio and the two varieties having the lowest flood/dry ratio were selected for the second experiment. Hinson was also included to continue as a commercial check. The genotypes selected were: 91209-126, 91209-142, 91209-220, 91209-269, 91210-350, and Hinson. Experimental Design Experiment 1 consisted of a 3 x 11 factorial experiment arranged in a split-plot design with 4 replicates (2.1). Each of the twelve lysimeters represented one of three flood treatments or main plot effects; no flood, 2 week flood duration followed by 2 weeks without flood, or 4 week flood duration. The main plots (lysimeters) where then divided into eleven sub-plots representing each of the eleven selected soybean genotypes. Flood treatments were randomly assigned to the three lysimeters within each replication and genotypes were randomly assigned plot position within the lysimeters. This process

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15 was repeated independently for each of the four replicates. The design of Experiment 2 was similar to Experiment 1, however only the six selected genotypes were used. Table 2-1 Experimental designs used for soybean flooding experiments. rop Management Seeds were inoculated with soybean-specific rhizobium to enhance initial nodulation. Plant spacing was 10 cm within rows and 28 cm between rows. Sowing was designed to minimize border effects. A row of Hinson seed was added to each end of the lysimeter and each sub plot row was bordered with a Hinson plant. Each lysimeter in Experiment 1 was sown with seven plants per genotype, while eight plants per genotype were sown in Experiment 2. Any seeding gaps were re-sown after germination to eliminate the gap effect and maintain the integrity of the sampling pattern. These late-sown plants were omitted during sampling. Flood treatments were imposed when 50% of the plants reached the V2 growth stage across sub-plots (genotypes), main-plots (flood treatments), and replicates. V2 is defined as the full extension of the second trifoliolate leaf beyond the unifoliate node (Fehr and Caviness, 1977) and was identified as an early season growth stage susceptible to flooding stress (Linkemer et al., 1998). Plants were maintained as well-watered up Experiment 13 x 11 factorial designExperiment 23 x 6 factorial designMain PlotsSub-plotsReplicationsMain PlotsSub-plotsReplicationsFlood TreatmentGenotype4Flood TreatmentGenotype4Non-flooded91209-119Non-flooded91209-1262 Week Flood91209-1262 Week Flood91209-1424 Week Flood91209-1424 Week Flood91209-22091209-14391209-26991209-15191210-35091209-220Hinson91209-26991209-29791210-31691210-350Hinson C

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16 to the initiation of the flood treatments as determined by soil volumetric water content (VWC) measure of 0.15 as taken by a Field Scout TDR 100 (Spectrum Technologies Inc., Plainfield, IL). Flood treatments imposed at the V2 growth stage consisted of raising the lysimeter water level to approxi mately 3 cm above the soil surface. Flooding depth was maintained at this level using a float valve positioned to allow automatic refill when water height dropped below 3 cm. Each lysimeter within a replicate was assigned a flood treatment of: no flood, 2-week duration, or 4-week duration. Treatments of 2 weeks and 4 weeks were initiated at the sa me time allowing the 2-week flooded plants a recovery period of 2 weeks. The non-flooded treatment was maintained at near field capacity throughout the trial. The 2-week duration treatment was maintained near field capacity for the final two week s of the trial, af ter the imposed flood was removed. Flood treatments were identical for both experiments. Fertilizer application am ount was based on a soil analysis performed at the Everglades Soil Testing Laborat ory (2.2). Nitrogen tests are not considered to be meaningful for Floridas sandy soils and th erefore not included (Sartain, 2001). The nitrogen application of 22 kg ha-1 was based on University of Florida Extension recommendations for snap beans on similar soils within the region*. Nitrogen was applied at 36 and 24 grams per lysimeter in Experiments 1 and 2 respectively using a urea (21% N) formulation. Phosphorus was appl ied as triple supe r phosphate at 110 kg P2O5 ha-1 translating to an application rate of 82 grams and 56 grams of fertilizer per lysimeter for Experiments 1 and 2 respectively. Potassium, also recommended at 110 kg K2O ha-1, was applied as potassium chloride at 63 and 43 grams per lysimeter in Experiments 1 and No N fertilizer recommendations for soybean in South Florida have been established.

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17 Table 2-2 Preliminary Mehlich-I soil test results and fertilizer recommendations. Soil Test Values (kg ha-1) Recommendations (kg ha-1) Sample ID Soil Texture pH P K Ca Mg Si (mg kg-1) K P2O5 2O Mg Lysimeter 1 sand 6.5 88 22 996 29 7 112 112 0 Lysimeter 2 sand 6.6 77 21 984 21 7 112 112 0 Lysimeter 3 sand 6.5 72 25 1049 29 7 112 112 0

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18 2. Fertilizer was banded between rows and applied at sowing for both Experiments 1 and 2. An additional application of MicroMax (Lacebark Inc., Stillwater, OK) micronutrient powder containing 15% S, 12% Fe, 2.5% Mn, 1% Zn, 0.5% Cu, 0.1% B, and 0.005% Mo was applied to Experiment 1 at a rate 30 g lysimeter-1 at 14 and 21 days after planting. For Experiment 2 the entire 60 g recommended dosage was applied 14 days after planting. Ridomil Gold (Sygenta Crop Protection, Inc.) fungicide was applied prior to sowing in order to suppress root damage from Phytophthora root rot (at a rate of 11.9 and 7.2 g lysimeter-1) in Experiments 1 and 2 respectively. Measurements Volumetric water content (VWC) was measured every other day and used primarily to determine flooded and well watered soil status. One sample was taken from each end (short side) of the lysimeters for two total samples and averaged to give VWC of the lysimeter as a whole. Each sample was taken halfway between planting rows in order to minimize root disturbance. The exact sampling position was varied on each date to avoid air pockets created by previous samples that would have affected VWC readings. Flooded soils had VWC measurements > 0.45. Non-flooded treatments were maintained at approximately 0.15 VWC using overhead watering. Plant biomass sampling Soybean biomass was sampled on three occasions over the course of each trial: prior to the initiation of the flood treatments at 21 days after sowing (DAS), at the cessation of the 2week flood duration 35 DAS, and at final harvest 49 DAS. At each sampling date a whole-plant sample was obtained including a 10 cm x 15 cm x 30 cm deep soil volume

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19 centered around the stem of the plant to be harvested that contained the root portion of that plant. At 21 DAS one plant per sub-plot was harvested. Each plant was divided into root, stem, and leaf portions. Roots were determined to begin at the soil surface. Leaf blades were removed right above the petiole with petiole weight being included in the stem fraction. The 35 DAS sampling also utilized one plant per sub-plot. Each plant was divided into primary root, adventitious root, nodule, stem, and leaf components. Primary and adventitious roots were segregated based on a visible gap between rooting layers generally located just below the soil surface (Stoffella et al., 1979). No distinction was made between nodules taken from adventitious and primary roots. The final harvest sample consisted of three plants. Each of the samples was divided into plant components using the same criteria employed at 35 DAS. Adventitious roots and primary roots from one of the three plants were preserved separately for root scanning by wrapping them in a paper towel, sealing them in a plastic freezer bag, and freezing (-20oC). All samples (with the exception of those frozen for subsequent root analysis) were dried at 65oC to a constant weight. Dry weights were recorded for each plant component for all sampling dates, sub-plots, main-plots, and replicates. For the purpose of statistical analysis the three samples taken at 49 DAS were averaged to calculate dry weight per plant. Root growth Primary and adventitious root samples collected at harvest were scanned using WinRhizoPro (Regent Instruments, Sainte-Foy, QC, Canada) and analyzed for total root length, average diameter, projected surface area, and volume. Each sample was suspended in water and scanned individually. After scanning all samples were dried at

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20 65 oC to a constant weight. The resulting dry weight was used to complete the 49 DAP harvest dry-weight calculations. Statistical Analysis Analyses of variance were performed for all experimental measures using proc GLM in SAS (Littell et al., 2002) with flood regimes being the main plot treatments and genotypes the sub-plots. Significant differences among cultivars and flood treatments were determined using Fishers protected LSD test (P<0.05). Further analysis of significant interaction terms was performed using the proc GLM Estimate statement in SAS using the flood x genotype means for genotype 91210-350 as the comparative base. 91210-350 was chosen for comparison based on its performance as the genotype most tolerant to flooding in the Arkansas trials (Cornelious, 2003). The proc GLM Estimate statement was also used to contrast group response to flooding. The mean value for all flood tolerant genotypes as defined by Cornelious (2003) across the 2-and 4-week flood treatments was contrasted against the mean value for all flood sensitive genotypes. Additionally, proc CORR in SAS was used to calculate Spearmans rank correlation coefficients to determine significant correlations between morphological measurements and genotype flood tolerance rankings. For the purposes of this study flood tolerance was defined as the mean total plant dry weight of flooded treatments divided by the total plant dry weight of the non-flooded treatment (plant dry weight flood / plant dry weight non-flood). Using this method, correlations were made between soybean morphological changes and early-season flood tolerance, as well as flood tolerance defined by grain yield (Cornelious, 2003).

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21 Results and Discussion Results from Experiment 2 are presented in Appendix A. These results were rendered ineffectual by the inability to establish an effective non-flooded control. Discrepancies between Experiment 1 and 2 may be attributable to persistent precipitation post-flood initiation for Experiment 2. During Experiment 2 twenty-seven days of rain marked the period between flood initiation and final harvest including 10 consecutive days immediately post-flooding (Figure 2-1). In contrast for Experiment 1 only 10 days of precipitation occurred during the flood initiation to final harvest period (Figure 2-1). Evaluation of weather data for this time period (2.3) points most strongly to precipitation as the cause of Experiment 2 growth inconsistent with Experiment 1. Noting the propensity for extended periods of precipitation during late summer in south Florida, two recommendations can be made: 1) Soybean trials should be performed with earlier sowing dates, preferably April-May in south Florida, and 2) In order to better guarantee the quality of the control future experiments should be conducted under conditions controlled more completely for external environmental influences. Primary Root Dry Weight and Dry Matter Partitioning No significant differences in the main (flood) effect were noted in primary root dry weights based on samples taken 21 or 35 days after sowing (2.4). However, at final harvest (49 DAS) the non-flooded treatment root weight was significantly greater (822 mg plant-1), than either 2-week (325 mg plant-1) or 4-week (302 mg plant-1) flooded treatments (2.4). Decrease in root biomass accumulation in flooded soybean has been noted by Pires et al. (2002) and Sallam and Scott (1987). Dry weight losses are generally attributed to dieback of the primary rooting system (Varpetian and Jackson, 1997,

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22 Table 2-3 Weekly average temperature and precipitation data for Belle Glade EREC, May-August 2004. Max Air TempMin Air TempAvg. Air Temp.RainfalloCmmWeek StartingMay 23rd32.418.525.20.0May 30th33.721.126.46.4June 6th31.621.525.58.7June 13th32.422.526.513.1June 20th33.722.627.20.5June 27th32.522.026.41.5July 4th33.721.926.87.9July 11th33.221.426.14.7July 18th31.422.025.61.8July 25th31.921.925.710.5August 1st32.023.025.613.1August 8th33.523.026.95.5August 15th32.722.926.65.9 Belle Glade EREC Rainfall40506031374349 Sowing 010203017131925Days Aftermm Experiment1 Experiment 2 Figure 2-1 Rainfall at EREC Belle Glade over the duration of Experiments 1 and 2. Day 1 for Experiment 1 = May 25, 2004. Day 1 for Experiment 2 = June 29, 2004.

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23 Table 2-4 Primary root dry weight, dry matter partitioning, and relative dry weight. Days After Sowing 213549Dry WeightPartitioningDry Weight PartitioningDry WeightRDW*Partitioningmg plant-1mg plant-1mg plant-1Flood2 Week1590.2442790.213325b0.4690.1184 Week1680.2492590.189302b0.4310.138Non-flooded1520.2352540.188822a-0.166LSD 0.05320.220360.016680.0100.010Significancensnsnsns**nsnsGenotypeTolerant91210-350244a0.248261bc0.169c472bc0.422bcd0.139ab91210-316 133c0.248243c0.187bc528ab0.310cd0.124b91209-126171bc0.256267bc0.200ab482bc0.549abc0.127b91209-151133c0.262234c0.204ab506ab c 0.272d0.130b91209-142133c0.241213c0.198abc539ab0.405bcd0.131bSensitive91209-220 175bc0.231270bc0.195bc458bc0.386bcd0.139ab91209-119153bc0.263317ab0.226a519ab0.599ab0.155a91209-269 143c0.236223c0.211ab413bc0.643ab0.155a91209-297 131c0.215253bc0.204ab386c0.302cd0.154a91209-143207ab0.247257a0.192bc624a0.328cd0.155aControlHinson 133c0.222271bc0.182bc385c0.730a0.138abLSD 0.05610.041680.01310.0300.019Significance**ns******** Fl oo d x G eno t ype Significancensns*ns*ns**,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means in the same column followed by the same letter are not significantly different (P<0.05).Dry matter partitioning is defined as primary root dry weight / total plant dry weight.* RDW = Relative dry weight is defined as primary root dry weight for flood treatment / primary root dry weight for non-flood treatment. Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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24 Jackson and Ricard, 2003) and a decrease in th e amount of photo-assi milate available for additional root extension (Tr ought and Drew, 1980). Delays in response to flooding have also been reported by Sallam and Scott (1987). The delayed response of primary root dry weight accumulation to 14 days of flooding starting at the V2 growth stage is most likely attributed to the slow primary root growth rate of the soybean pl ant during the initial growth phase (Sung-Guang et al., 1996). As the soybean plant reached the rapid growth phase 35 DAS, flooding served to delay root elongation and thereby reduced dry weight accumulation. However, based on the continual accumulation of additional biomass in flooded roots it could be concl uded that soybean plants produ ce new root material at a greater rate than dieb ack of existing roots. Genotypic differences in primary root dry weights persisted over all sampling dates (2.4). Developmental differences in plant biomass based on soybean genotype were documented by Bacanamwo and Purcell (1999a). In order to understand the contribution of genotypic variation in relati on to the main flood effect, it is necessary to separate genotype responses by flood treatment. Differences in genotypic response to flood treatment resulted in significance in the in teraction term. Primary root dry weight genotype x flood interaction was significan t for the 35 DAS and 49 DAS sampling dates (2.4). 2.5 presents the performance of each genotype under all flood treatments in relation to a base value for 91210-350. Genotype 91209-143 had significantly greater primary root dry weight in the non-flooded treatment, however in general primary root dry weight of the germplasm tested was not significantly different from 91210-350 under flooded conditions.

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25 Table 2-5 Genotype comparisons for significant dry weight genotype x flood interaction terms. Values are differences between genotype and 91210-350. Primary Root 35 DASPrimary Root 49 DASnon-flooded2 week4 week non-flooded2 week 4 weekmg plant-1GenotypeTolerant91210-31688ns-140*-3ns204ns-36ns1ns91209-126 50ns-55ns23ns-49ns88ns-8ns91209-151 -23ns-73ns15ns195ns-38ns-55ns91209-142 5ns-85ns-63ns121ns101ns-19nsSensitive91209-220 -33ns35ns25ns12ns29ns-84ns91209-119 143*88ns-63ns-16ns88ns71ns91209-269 -30ns-38ns-45ns212ns59ns-24ns91209-297 43ns-58ns-8ns-4ns-51ns-204ns91209-143 205**33ns35ns360**135ns-39nsControlHinson -18ns30ns18ns-251*59ns-68ns91210-350215303265789286341*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.Genotype 91210-350 has been identified as the "most tolerant" genotype (Cornelious, 2003).Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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26 Expressed as the ratio of primary root dry weight to total plant dry weight, differences in partitioning of dry matter to primary roots due to flooding was not significant (2.4). A steady decline, however, in the fraction of total biomass allocated to roots was witnessed across sampling dates. As with dry weight, dry matter partitioning to roots was significantly different among genotypes at 35 and 49 DAS. However, partitioning was relatively constant ranging from 0.124 to 0.155 among all genotypes. The genotype x flood interaction term was significant at 49 days after sowing. Genotype 91209-151 had a significantly greater allocation of biomass to primary roots than genotype 91210-350 in the non-flooded treatment (+.039) and significantly lower fraction allocated for the 4-week flood treatment (-.067) (2.6). Genotype 91210-350 was notable for its high partitioning to roots under the 4-week flood treatment. Relative dry weight measured at 49 days after sowing for Experiment 1 showed no significant difference between the 2 and 4-week flood treatments (2.4). Significant genotype differences were noted (2.4). No significant interaction existed between genotype and flood effects. The range in relative dry weights of primary roots demonstrates a strong genotypic variation in ability to continue primary root growth under flooded conditions, with the highest fraction maintenance for Hinson at 0.73 and the lowest for 91209-151 at 0.27. It is important to note that accumulation of primary root biomass was reduced for all genotypes under flooded relative to non-flooded treatments, this is again consistent with the findings of Pires et al. (2002) and Sallam and Scott (1987).

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27 Table 2-6 Genotype comparisons for significant dry matter partitioning genotype x flood interaction terms. Values are differences between genotype and 91210-350. Primary Root Dry Matter Partitioning 49 DASNon-flooded2 week 4 weekGenotypeTolerant91210-3160.015ns-0.019ns-0.043*91209-126 0.012ns-0.007ns-0.042*91209-1510.040*-0.002ns-0.067**91209-1420.033ns-0.010ns-0.049**Sensitive91209-2200.014ns0.005ns-0.018ns91209-1190.065**0.003ns-0.021ns91209-269 0.003ns0.029ns-0.014ns91209-2970.028ns0.022ns-0.083ns91209-1430.037*0.003ns0.062nsControlHinson0.020ns-0.014ns-0.039*91210-350 0.1390.1150.165*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.Dry matter partitioning is defined as tap root dry weight / total plant dry weight. Genotype 91210-350 has been identified as the "most tolerant" genotype (Cornelious, 2003).Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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28 Primary Root Length, Average Diameter, Surface Area, and Volume Morphological characteristics of the primary roots followed the same pattern as root dry weight. Plants in non-flooded treatments had significantly greater primary root length, average diameter, surface area, and volume than flooded plants (2.7). No significant differences were observed between genotypes, nor were any interaction terms significant. Sallam and Scott (1987) describe the relationship between primary root area, extension and dry weight as linear, while recording a noted decrease in the addition of root length and area with the imposition of flood treatments. The linear nature of this relationship implies that the same factors working to suppress dry weight accumulation are inhibiting primary root extension and area. As with root dry weight, reduction in assimilates directed to flooded roots (Trought and Drew, 1980) and dieback of primary roots (Vartapetian and Jackson, 1997; Jackson and Ricard, 2003) serve to limit additional growth. Adventitious Root Dry Weight and Dry Matter Partitioning As the primary rhizosphere morphological response to flooding in soybean, adventitious root development is of particular interest in evaluating potentially tolerant genotypes. Since adventitious roots developed only on flooded samples, analyses for these samples were limited to dry weight, partitioning percentage, and root morphology. Adventitious root dry weight was significantly affected by flood duration at 35 and 49 days after sowing (2.8), with the 4-week flood treatment biomass being greater than the 2-week flood treatment. Plants in both flood treatments continued to add biomass to the adventitious roots between 35 and 49 days after sowing, but statistical trends were maintained. Significant differences among genotypes developed only at the

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29 Table 2-7 Primary root length, average diameter, surface area, and volume 49 days after sowing. 49 DAS sampling date, and the genotype x flood interaction was also significant at this date (2.8). Analysis of the significant interaction term revealed five genotypes with significantly greater adventitious root weight than 91210-350 after 4 weeks of flooding. Genotype 91209-151 produced 98 mg plant more adventitious roots than 91210-350 (2.9). Significant increases in adventitious root development are cited as a key morphological response to flooding in soybeans (Pires et al., 2002; Sallam and Scott, 1987, Bacanamwo and Purcell, 1999a). It should be noted that adventitious root biomass LengthAvg. DiameterSurface AreaVolumecm plant-1mm plant-1cm2 plant-1cm3 plant-1Flood2 Week1115b0.266b83b0.498b4 Week918b0.305b81b0.579bNon-flooded1917a0.494a168a1.210aLSD 0.052510.057200.138Significance********GenotypeTolerant91210-35011270.337990.71391210-31615700.4861370.96591209-12614180.3601190.80391209-15112910.3521080.73691209-14213040.3731110.768Sensitive91209-22012060.3051000.66891209-119 13370.2971080.72191209-269 14460.3451170.77991209-29710890.353960.69891209-14314440.3911290.932ControlHinson11580.299910.587LSD 0.054810.109380.256SignificancensnsnsnsFlood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003). -1

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30 (generally associated with flooding) continued to increase in the 2-week treatment after the removal of flood treatments. This demonstrated that adventitious roots might have assumed responsibility for primary root function in flooded plants; this is consistent witthe role defined for adventitious roots by Jackson and Drew (1984). Adventitious root dry weight partitioning was significantly diffe h rent between flood treatmge Diameter, Surface Area, and Volume e signifotal t gation ents at 49 days after sowing (2.8). As with dry weight, the 4-week flood treatment had a greater fraction of total biomass allocated to adventitious roots than did the 2-week treatment. Significant genotype differences appeared at 35 days after sowing and continued at 49 day after sowing. Adventitious Root Length, Avera Adventitious root length, average diameter, surface area, and volume wer icantly greater in the 4-week compared to 2-week flood treatments (2.10). Telongation was more than double (1235 cm) for the 4-week versus the 2-week treatmen(603 cm). Greater continued elongation of adventitious roots in the 4-week flooded treatment may indicate a continuing dependence of plants in this treatment on adventitious roots for plant aeration. Conversely, slower adventitious root elon

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31 Table 2-8 Adventitious root dry weights and dry matter partitioning. Days After Sowing3549Dry WeightPartitioningDry WeightPartitioningmg plant-1mg plant-1Flood2 Week18b0.01273b0.026b4 Week26a0.01994a0.038aNon-flooded----LSD 0.0570.004130.003Significance**ns*****GenotypeTolerant91210-350130.005b32cd0.014d91210-316140.010ab65ab0.024ab91209-126 190.013ab69ab0.022ab91209-15190.006b68ab0.027a91209-142 120.011ab74a0.021bcSensitive91209-220 160.009ab52abc0.022ab91209-119 140.012ab72ab0.024ab91209-269 120.013ab49bc0.022ab91209-297 150.011ab19d0.015cd91209-143 220.014a53abc0.020bcdControlHinson 140.008ab59ab0.022abLSD 0.05140.008250.006Significancens*****Flood x Genotype Significancensns**ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Dry matter partitioning is defined as adventitious root dry weight / total plant dry weight.Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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32 Table 2-9 Genotype comparisons for significant adventitious root dry weight genotype x flood interaction term. Values are differences between genotype and 91210350. Adventitious Root 49 DAS2 week 4 weekGenotypemg plant -1Tolerant91210-31616ns84**91209-12656*54*91209-15110ns98***91209-14245*83**Sensitive91209-220 33ns23ns91209-11938ns83**91209-26919ns33ns91209-297-16ns-23ns91209-14339ns22nsControlHinson18ns62**91210-350 4946*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.91210-350 has been identified as the "most tolerant" variety (Cornelious, 2003).Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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33 Table 2-10 Adventitious root length, average diameter, surface area, and volume 49 days after sowing. LengthAverage DiameterSurface AreaVolumecm plant-1mm plant-1cm2 plant-1cm3 plant-1Flood2 Week603b0.213b40.3b0.216b4 Week1235a0.282a87.3a0.499aNon-flooded----LSD 0.052450.03818.80.123Significance****GenotypeTolerant91210-350 8680.24764.10.39491210-316 11000.29783.60.51291209-126 8990.22062.00.34391209-151 10070.24267.80.36691209-142 9380.23469.70.422Sensitive91209-220 6600.22046.70.26591209-119 14780.30194.00.47891209-269 9200.24664.70.36891209-297 3710.20924.30.12791209-143 7650.24551.60.278ControlHinson 11030.26272.80.387LSD 0.055740.08944.10.288SignificancensnsnsnsFlood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003). post-flood in the 2-week flooded treatment suggests that sufficient primary root function remains after 2 weeks of flooding to support plant growth once reallocation of photosynthate to primary root development is initiated. Genotypic differences were not significant, nor were the genotype x flood interactions. Nodule Dry Weights and Dry Matter Partitioning Nodule dry weights at 35 days after sowing were significantly different, with non-flooded soybeans accumulating three times the biomass of 2and 4-week flooded treatments (2.11). At 49 DAS, the 4-week flood treatment had significantly lower nodule

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34 biomass (65 mg plant-1) than the 2-week flood (147 mg plant-1), which was significantly less than the non-flooded control (235 mg plant-1). Genotype effects on nodule weights were significant at 35 and 49 DAS (2.11). The interaction term between genotype and flood was not significant. The effect of 14 days of flooding on nodulation in our experiment is consistent with the findings of Sallam and Scott (1987), including the increase in nodule dry weight of the 2-week flood treatment after the removal of flooding. Bennett and Albrecht (1984) suggested that soybean plants retain the ability to recuperate nodule function when aerobic conditions are restored, a conclusion supported by the findings of this study. Prolonged flood duration resulting in decreased nodulation is also consistent with other research findings (Sprent, 1972; Sung, 1993). Sallam and Scott (1987) found that nodule function in plants flooded at the V4 growth stage returned to normal levels, however, the same study revealed that nodules of plants with flood imposed at the V1 growth stage never recovered function. The effect of flood on partitioning to nodules was significant only at the 35 DAS sampling date (2.11). At that date, the non-flooded treatment had significantly greater dry matter partitioning to nodules compared with flooded treatments. Genotype effects were significant at 35 and 49 days after sowing (2.11) This included a significant interaction effect at 35 DAS (2.11). Correlations of morphological characters to flood tolerance Significant Spearmans rank correlation coefficients for flood tolerance (as defined in this study) and primary root dry weight at 49 days after sowing (2.12) underscore the

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35 Table 2-11 Nodule dry weight and dry matter partitioning. Days After Sowing3549Dry WeightPartitioningDry WeightPartitioningmg plant-1mg plant-1Flood2 Week12b0.009b147b0.0464 Week13b0.010b65c0.025Non-flooded41a0.026a235a0.041LSD 0.05130.006430.006Significance***nsGenotypeTolerant91210-350 16cd0.010c138bd0.035c91210-316 35abc0.023ab206ab0.052ab91209-126 41ab0.029a213ab0.053ab91209-15126bcd0.026ab186ab0.049ab91209-142 21bcd0.015bc247a0.058aSensitive91209-220 15cd0.011c136bcd0.037c91209-119 7d0.006c142bc0.038c91209-269 8d0.008c59de0.022d91209-297 9d0.007c71cde0.019d91209-143 56a0.026ab191ab0.042bcControlHinson 6d0.004c46e0.013dLSD 0.05240.012810.011Significance***********Flood x Genotype Significancens*nsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Dry matter partitioning is defined as nodule dry weight / total plant dry weight.Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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36 Table 2-12 Spearmans rank correlation coefficients for selected rhizosphere plant components under flooded conditions as correlated to tolerance rank. importance of continued root development in maintaining plant integrity under flooded conditions. The dieback of the primary rooting system and replacement of function by adventitious roots is cited as a central response of soybeans to flooding (Bacanamwo and Purcell, 1999a; Pires et al., 2002). Root damage due to hypoxia may also lead to insufficient transfer of water, inorganic minerals, and hormones resulting in shoot damage (Vartapetian and Jackson, 1997; Jackson and Ricard, 2003). Maintaining primary root function is clearly essential to continued plant growth. Plant flood tolerance was likewise strongly correlated to primary root length and correlated to primary root surface area (2.12). The findings of this study in describing significant correlations between primary root length, surface area, dry weight and tolerance are unique in their attempt to relate measured root parameters to a quantitative analysis of plant performance. The relationships between primary root length, surface area and dry weight are consistent with the findings of Sallam and Scott (1987). The consistency of the correlations between these growth parameters and early-season flood tolerance supports ToleranceRoot parameterSpearman's Rank Correlation CoefficientPrimary root dry weight 49 DAS0.618*Primary root length0.818**Primary root area0.727*Primary root surface area0.727*Primary root diameter 0.218nsPrimary root volume0.418nsPrimary root partitioning 49 DAS-0.155nsAdventitious root dry weight 49 DAS0.645*Adventitious root length0.500nsAdventitious root area0.418nsAdventitious root surface area0.418nsAdventitous root diameter0.155nsAdventitous root volume0.255nsNodule dry weight0.391ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively

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37 the hypothesis that maintenance of primary root extension and accumulation of root biomass may serve as an indicator of soybean flood tolerance. The Spearmans rank correlation coefficient analysis show ed a positive correlation betwet in alysis e ts is not en adventitious root dry weight and flood tolerance at 49 days after sowing (2.12), however correlations between flood tolerance and adventitious root length and surface area at the same sample date were not significant. The positive correlation between adventitious root dry weight and tolerance is also consistent with the concept that roofunction under flooded conditions in enhanced by the aeration provided by aerenchymaadventitious roots (Jackson and Drew, 1984). Lee et al. (2003) stated that forced early development of adventitious roots in soybean served to significantly mitigate crop susceptibility to waterlogging. This study is unique in that it provides a detailed anof adventitious root development in flooded soybeans. This analysis has led to two unanticipated results. The lack of significant correlation between adventitious root length, average diameter, surface area, or volume and tolerance is contradictory to thconcept that adventitious roots provide a necessary means of adaptation to flood stress,assuming that increased adventitious root development leads to increased root aeration. The significant correlation between adventitious root dry weight and tolerance and lack of correlation between length and tolerance may be an indication that the linear relationship between root length, area, and dry weight that exists for primary roopresent with adventitious roots. Figures 2.2 and 2.3 chart this relationship across

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38 Adventitious root length x dry weight y =0.410 + 5.10xR2 = 0.22101230.000.050.100.150.200.25Dry Weight 49 DAS (g plant-1)Length (thousands cm) Figure 2-2 Graph showing lack of linear fit in the relationship between adventitious root length and dry weight. Adventitious root surface area x dry weight y = 27.9 + 356xR2 = 0.1950204060801001201401601800.000.050.100.150.200.25Dry Weight 49 DAS (g plant-1)Surface area (cm2) Figure 2-3 Graph showing lack of linear fit in the relationship between adventitious root surface area and dry weight.

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39 genotypes and flood treatments. This may point to a greater relative importance in adaptation to flooding associated with adventitious root dry weight than length, surface area, average diameter, or volume. Contrast Between Flood Tolerance Groups In order to better judge the performance of flood tolerance groups in relation to their original flood tolerance rankings (Cornelious, 2003), group means were compared across 2and 4-week flood treatments. Prior to the imposition of flood treatments no significant differences were noted between flood tolerance groups (Tables 2.13 and 2.14). Strong early growth in the primary roots of flood tolerant genotypes led to significantly greater accumulation of root biomass by the 35 DAS sampling date (2.13). The difference was not significant, however, by the 49 DAS sampling date. Adventitious root and nodule development both showed significantly greater accumulation of dry matter in the sensitive group than in the tolerant group by the 49 DAS sampling date (2.13). Noting the significant correlations between primary root dry matter accumulation, adventitious root dry matter accumulation, and early season tolerance, (2.12) this is further evidence that in this study the sensitive genotypes as defined by Cornelious (2003) proved to have a greater resistance to flooding than those previously defined as tolerant. Conclusions The existence of significant correlations between rhizosphere morphology and flood tolerance as defined in this study substantiates the assertion that morphological adaptations may serve as an indicator of flood tolerance. The strength and consistency of correlations between primary root dry weight, length, surface area and tolerance support the conclusion that the measure most worth pursuing in prediction of tolerance is

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40 Table 2-13 Rhizosphere dry weight contrast for flood tolerant vs. sensitive genotypes as defined by Cornelious (2003). Table 2-14 Primary and adventitious root length, surface area, diameter, and volume, d continued p and primary root biomass accumulation. However, as the possibility of using these significant correlations betweted Days After Sowing213549mgDry weight:Primary root-6ns-193*1nsAdv. root--18ns95**Nodule-18ns266**,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Negative values denote a greater mean value for "tolerant" treatments. contrast value for flood tolerant vs. flood sensitive genotypes as defineby Cornelious (2003). rimary root elongation 49 Days After SowingLengthSurface areaDiameterVolumecmcm2mmcm3Primary root:-122.1 ns12.4ns0.031ns0.192nsAdv. root:617.7ns66.1ns0.019ns0.519ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Negative values denote a greater mean value for "tolerant" treatments. highlighted in Chapter 3, the fact that tolerance rankings based on dry weights at 49 DAS did not correspond with rankings based on yield and visual flood injury as outlined in Cornelious (2003) limit the application of this methodology. It is impossible to discernwhether these differences in rank arise from differences in response between plants flooded at the V2 growth stage versus those flooded at the R2 stage, or from climaticdifferences between experiments. In order to better understand en morphological traits and flood tolerance as early season indicators of flood tolerant genotypes, it will be necessary to further investigate the response of the selecgenotypes to flooding at the V2 growth stage. Rankings, similar to those presented in Cornelious (2003), but based on flood imposition at the V2 rather than R2 growth stage

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41 would allow for extrapolation of early season morphological data to year end grain yield.Based on the strength of multiple significant correlations, there is reason to believe that links between grain yield and early season morphological response can be established, greatly reducing the time need to identify potentially tolerant genotypes.

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CHAPTER 3 THE EFFECT OF EARLY-SEASON FLOODING AND GENOTYPE ON SOYBEAN (GLYCINE MAX L. MERRILL) ABOVEGROUND GROWTH AND TOTAL PLANT BIOMASS Review of Literature Identification of Tolerance to Flood Stress An overall decrease in soybean shoot growth under flooded conditions results from the inability of the root system to maintain normal function in relation to the transport of water, nutrients, hormones, and assimilates (Jackson and Drew, 1984). The first symptom to appear in shoots is a wilting of the leaves based on increased resistance to water flow through the root (Kramer, 1951). Some dispute as to the origin of reduced leaf expansion remains (Jackson and Drew, 1984). However, there is solid evidence that extended periods of root anoxia will result in a decline in leaf area accumulation. Sojka et al. (1975) reported a reduction of greater than seventy percent in leaf area of wheat after twenty-five days of root flooding as compared to non-flooded controls. Prolonged flooding will eventually lead to epinasty, leaf chlorosis, and plant death (Kramer and Boyer, 1995). Net CO2 assimilation per unit of leaf area is depressed with root waterlogging (Trought and Drew, 1980) primarily due to stomatal closure reducing Rubisco production (Jackson and Drew, 1984). Decreases in assimilation will eventually also reduce dry weight accumulation (Trought and Drew 1980; Sojka et al., 1975). This may, however, be preceeded by a period of increased accumulation of assimilates in leaves resulting from decreased export of photosynthate to the roots (Jackson and Drew, 1984). 42

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43 Coinciding with the extension of adventitious roots from the hypocotyl, hypertrophy, the swelling of the stem base or hypocotyl, is another common response to flooding. Resulting from a swelling of cells in the cortex, hypertrophy is often accompanied by a collapse of cells to form gas filled spaces (Kawase, 1981). The effects of waterlogging on soybean shoot growth and aboveground biomass accumulation are substantial and generally negative. Linkemer et al. (1998) reported a significant decline in pod number, pods per node, branch number, and seed size after 7 days of flooding at various vegetative and reproductive growth stages. Oosterhuis et al. (1990) confirmed an overall reduction in dry weight production in soybean. Seven days of flood created a 14-day lag in biomass accumulation at the V4 growth stage, and a greater than 14 day lag at the R2 stage. Both studies added, however, that biomass accumulation returned to a similar rate with the control 7 days after removal of the flood treatment. Reduction in pod number was confirmed by Sullivan et al. (2001). They also reported a reduction in height for 3, 5, and 7-day floods at early vegetative growth stages. Flooding reduced leaf number (Sallam and Scott, 1987), and prolonged effects on canopy height and reduced dry weight were still noticeable at maturity (Scott et al., 1989). Yield reductions in soybean ranged from 20-93% for 2 and 6-day floods respectively (Sullivan et al., 2001). Singh and Singh (1995), Oosterhuis et al. (1990), and Scott et al. (1989) all reported yield reductions for all flooded treatments ranging in duration from 24 hours to 14 days. Decreases in yield can be attributed to reduced pod growth, seed size, and number of seeds per pod (Linkemer et al., 1998) brought about by nitrogen stress and reduced photosynthesis (Oosterhuis et al., 1990).

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44 Stem hypertrophy is an adaptation to flooding that has been reported to serve as a primary pathway for conducting atmospheric air needed for adequate root aeration (Shimamura et al., 2003). Changes in stem diameter have also been shown to closely correlate to flood tolerance in soybean (Pires et al., 2002). As such it is likely that the relationship between stem development and flooding tolerance may be useful in selecting flood tolerant soybean genotypes. Similarly, the generic responses in plant leaf growth attributed to flooding including: wilting (Kramer, 1951), reduction in leaf area accumulation (Sojka et al., 1975) and leaf chlorosis (Kramer and Boyer, 1995), have been confirmed for soybean by Sallam and Scott (1987) who reported a reduction in leaf number after 7 days of flooding, and Bacanamwo and Purcell (1999a) who observed a general reduction in leaf area for all treatments and genotypes as compared to controls. The consistency of these results highlights the potential value of leaf measures in assessing flood tolerance. The ultimate factor for judgment of plant tolerance to flooding is the yield of high-quality marketable seeds (Van Toai et al., 1994; Roiselle and Hamblin, 1981). Within this definition varying methods have been used to establish tolerance rankings. Van Toai et al. (1994) rated the tolerance of 84 tested varieties by ranking total yield under flooded conditions and comparing these to corresponding rankings under non-flooded conditions. Roiselle and Hamblin (1981) promoted the use of a comparative yield analysis taking the average of flooded and non-flooded yields. This selects genotypes broadly adapted to both stress and non-stress conditions. Van Toai and Nurijani (1996) identified tolerant genotypes by taking the average seed yield and average SPAD leaf greenness reading of flooded plants and dividing these values by the values for the non-flooded control. The

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45 resulting ratio demonstrated the ability of plants to maintain growth under flooded conditions. Bacanamwo and Purcell (1999a, 1999b) took a different approach in determining tolerance; emphasizing the necessity of morphological adaptations for avoidance of flooding injury, they suggested that selecting varieties for morphological adaptations to flooding may be the best way to select for flooding tolerance. Rationale and Objectives Identification of soybean flood tolerance in the vegetative stage could greatly reduce the time frame required to identify potentially flood tolerant varieties for breeding purposes. In addition insight may be gained as to the relationship between early and late season morphological responses to flooding. Comparing early-season flood adaptations, with late-season flood tolerance (3.1; Cornelious, 2003) may provide a link between early-season morphological flood responses and end of season tolerance. The objectives of this study were 1) to monitor changes in soybean stem and leaf growth in response to early-season flooding, 2) compare early-season morphological adaptations to early-season flood tolerance, and 3) determine the relationship between early-season and late-season flood tolerance Materials and Methods Detailed general materials and methods can be found in Chapter 2. Included here is the development of methods specific to aboveground growth and total plant biomass.

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46 Table 3-1 Genotype yield reduction and flood injury score for most tolerant and most sensitive Arkansas RILs. Most tolerantMost sensitiveGenotypeYield reduction Injury GenotypeYield reduction Injury%0-9%0-991210-35032.22.891209-22067.95.891210-31633.03.091209-11965.26.291209-12637.23.791209-26963.65.991209-15143.14.091209-29769.86.491209-14249.14.091209-14369.86.4 Cornelious (2003) Leaf Area and Plant Height Determination of total leaf area per plant was established as described by Wiseman and Bailey (1975). Thirty trifoliolate leaves from each of the eleven genotypes to be studied were removed from plants in a pre-trial run. A range of trifoliolate leaf sizes (small, medium, large) was taken from both flooded and non-flooded plants. The length and maximum width of the center leaflet of each trifoliolate was measured manually. Each of the three trifoliolate leaflets was passed through a LI-3000 portable leaf area meter (LI-COR Inc., Lincoln, NE) and actual total leaf area for the trifoliolate leaf was recorded. A linear regression equation was developed that expressed leaf area as a function of the product of the length and width of the center leaflet. Genotype specific regression equations for each of the eleven genotypes had an R2 value > 0.92 (3.2) and these equations were used to predict leaf area based on length x width measurements of individual trifoliolate leaves. Leaf area measurements were taken from two predetermined plants in each sub-plot at 3.5 and 7-day intervals for Experiments 1 and 2, respectively. Incremental leaf area expansion is defined as the addition of leaf area between day x and day y, where the value used for analysis is (value day y value day x).

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47 Plant height was measured twice weekly from the soil surface to the uppermost growth point of the stem. Three measurements were taken from each sub-plot across all Table 3-2 Regression equations and R2 values for leaf area by genotype used to determine non-destructive leaf area values. ain-plots and replicates. The average of individual plant heights was used to represent plot height for statistical analysis. The same three plants were measured at each sampling date. Statistical Analysis Analyses of variance were performed for all experimental measurements using proc GLM in SAS (Littell et al., 2002). Data was analyzed using a factorial experiment with a split-plot design with flood regimes being the main plot treatments and genotypes the sub-plots. Significant differences among cultivars and flood treatments were determined using Fishers protected LSD test (P<0.05). Further analysis of significant interaction terms was performed using the proc GLM Estimate statement in SAS using the flood x genotype means for genotype 91210-350 as the comparative base. Genotype 91210-350 was chosen for comparison as it was reported to be the genotype most tolerant to GenotypeRegression EquationR2Tolerant91210-350y = 1.70x + 2.130.97691210-316y = 1.99x 0.1350.97791209-126y = 1.80x + 1.290.92191209-151y = 1.81x + 1.390.96891209-142y = 1.81x + 1.090.932Sensitive91209-220y = 1.83x + 1.800.97791209-119y = 1.74x + 2.940.95991209-269y = 1.92x + 2.310.92291209-297y = 2.03x 0.8110.98291209-143y = 2.12x 0.2220.985ControlHinsony = 1.66x + 4.360.951Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003). y=trifioliolate area, x=length x width of center leaflet. m

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48 flooding in the Arkansas trials (Cornelious, 2003). The proc GLM Estimate statement was also used to contrast group response to flooding. The mean value for all tolerant genotypes as defined by Cornelious (2003) across the 2and 4-week flood treatments was contrasted against the mean value for all sensitive genotypes. Additionally proc CORR in SAS was used to calculate Spearmans ra nk correlation coefficient to determine significant correlations between morphological measurements and tolerance rankings. Each morphological measure was ranked, based on numerical values and then correlated to numerical tolerance rankings based on whole plant dry weights. For the purposes of this study, flood tolerance was defined as th e mean total plant dry weight of flooded treatments divided by the tota l plant dry weight of the nonflooded treatment (plant dry weight flood / plant dry weight non-flood). Results and Discussion While results from Experiments 1 and 2 i ndicate consistent patterns of adaptation to flooding for aerial components of soyb ean, in relation to stem dry matter accumulation, partitioning, and plant height; as stated in Chapter 2, repeated rainfall events interfered with flooding treatments in Experiment 2. The inability to establish an effective control makes the presentation of this data inconsistent. Thus all Experiment 2 data can be found in Appendix A The cons istency between quantifiable effects of flooding on aerial components in Experiments 1 and 2 may indicate that aerial plant growth parameters are more highly sensitiv e to the effects of imposed flooding than rhizosphere components. Stem Dry Weight, Dry Matter Partitioning, and Height Flooding did not cause signifi cant differences in stem dry weight at the 35 DAS sampling date. However, stem dry weight in the non-flooded treatment was significantly

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49 Table 3-3 Stem dry weight and dry matter partitioning (Experiment 1). Days After So wing213549Dry Weight PartitioningDry Weight PartitioningDry Weight Partitioningmg plant-1mg plant-1mg plant-1Flood2 Week1670.2554480.323b1006b0.385b4 Week1620.2495020.343a980b0.431aNon-flooded1650.2473900.291c1770a0.345cLSD 0.05170.012690.0210.0Significancensnsns*****GenotypeTolerant91210-350 243a0.275a590a0.367a1380ab0.427ab91210-316 125e0.235d414bc0.302c1460ab0.366def91209-126 160d0.236d406bc0.300c1360ab0.360ef91209-151 126e0.247bcd376bc0.320bc1330abc0.389cd91209-142 143de0.251bcd393bc0.344ab1540a0.381cdeSensitive91209-220 195bc0.256abcd448c0.300c1170abcd0.375def91209-119 140de0.234d458abc0.314bc1280abcd0.379de91209-269 158de0.261abc340c0.305c906d0.354f91209-297 165cd0.265ab408bc0.325bc937cd0.432a91209-143212ab0.251bcd588a0.321bc1510ab0.386cdControlHinson 145de0.290cd497ab0.310c1114bcd0.405bcLSD 0.05320.0231330.0314000.031Significance*************Flood x Genotype Significancensnsnsnsns***,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05). Dry matter partitioning is defined as stem dry weight / total plant dry weight. Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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50 greater than either the 2 or 4-week flood treatment at 49 DAS (3.3). Significant differences among genotypes were noted at all sampling dates. There was no significant flood x genotype interaction on stem dry weight at any sampling date. The observed pattern in stem dry weight accumulation was consistent with other plant components. Little effect on the overall growth of the plant was recorded at 14 days after flooding. This may be attributed to the natural delay in plant growth (Hanway and Weber, 1971; Sun-Guang et al., 1996) with the flooding treatment resulting in a delay in the rapid growth phase beyond 35 DAS. While measurements of stem dry weight at 35 DAS were not significantly different, it is apparent that damage to the soybean plant has occurred during these first 14 days of flooding. This is indicated by the continued slow biomass accumulation of plants under the 2-week flood treatment after the removal of the flood. Dry weight for non-flood and 2-week flood treatments was not significantly different at the time of removal of the 2-week flood treatment (3.3). Yet, 14 days later at the 49 DAS sampling date, non-flood plants had a significantly greater stem dry weight than 2-week flooded plants. Genotype 91210-350 had consistently high stem weights (3.3). The amount of dry weight partitioned to the stem was significantly different between flood treatments at 35 and 49 DAS (3.3). A significant difference among genotypes was noted in stem partitioning at all sampling dates (3.3). Significant interactions between flood and genotype occurred at the 49 DAS sampling date (3.3). Further analyses of the interaction terms show 91210-350 consistently partitioned more of its total biomass to the stem compared to other genotypes (3.4). With the prolonging of the flood treatments this trend became more pronounced.

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51 Table 3-4 Genotype comparison for all significant dry matter partitioning flood x genotype interaction terms. Values represent difference between genotype and 91210-350. Dry Matter Partitioning Stem 49 DASnon-flooded2 week 4 weekGenotypeTolerant91210-316 -0.019ns-0.059**-0.107***91209-126 -0.032ns-0.067**-0.104***91209-151 -0.038ns-0.049*-0.028ns91209-142 -0.018ns-0.046*-0.076**Sensitive91209-220 -0.051*-0.047*-0.061**91209-119 -0.031ns-0.042ns-0.075**91209-269 -0.062**-0.063**-0.096***91209-297 -0.043*0.022ns-0.032ns91209-143 -0.029ns-0.063**-0.033nsControlHinson -0.032ns-0.012ns-0.016ns91210-3500.3770.4240.482*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Genotype 91210-350 was identified as the "most tolerant" genotype (Cornelious, 2003). Dry matter partitioning is defined as plant part dry weight / total plant dry weight. Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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52 Significant differences in plant height be tween flood treatments occurred only at 25 and 28 DAS (Tables 3.5). Significant differe nces among genotypes were noted almost uniformly across experiments with the only ex ception occurring at 36 DAS (3.5). There was no significant interaction between flood and genotype effects at any sampling date Significantly lower soybean plant heights at harvest as a result of flooding have been reported by: Sallam and Scott (1987), Scott et al (1989), and Linkemer et al. (1998). Pires et al. (2002) on the other ha nd did not find any signi ficant difference in plant height after 21 days of flooding initiated at the V2 growth stage. The consistency of our findings with that of Pi res et al. (2002) can most likely be attributed to similarities between the two studies in re lation to plant growth stage at the time of flood imposition (V2) and final plant harvest before maturity. Significant differences in plant height were reported at final harvest (Linkemer et al., 1998; Sallam and Scott, 1987; Scott et al., 1989) implying that flood initiation at an early growth stage had a lasting effect on stem elongation. Early sampling for morphological ch anges in our experiment may not have allowed enough time post-flood for significan t height differences to develop. Leaf Expansion, Dry Matter Accumulation, and Partitioning Leaf dry weight showed significant flood treatments effects at 49 DAS (3.6). Nonflood treatments accumulated more biom ass in the leaves (2220 mg plant-1) compared to flooded treatments, while the 2-week treatm ent had a significantly greater dry weight (1190 mg plant-1) than the 4-week treatment (919 mg plant-1). Significant differences in leaf dry weight were noted among genotypes at all sampling dates (3.6). Interactions between flood and genotype treat ments were not significant.

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Table 3-5 Plant heights. Days After Sowing2125283236394447cm plant-1Flood2 Week9.3811.5a12.4a12.916.517.420.021.94 Week9.4511.2b11.9b12.515.216.920.923.0Non-flood9.1210.2c10.9c11.514.216.419.922.0LSD 0.050.210.30.30.41.70.60.91.0Significancens**nsnsnsnsnsGenotypeTolerant91210-350 11.10a12.8a13.9a14.6a17.519.7a23.0a25.4a91210-316 8.82ef10.8c11.6cd12.5c15.017.1cd21.1bc23.5bc91209-126 8.42fg9.94de10.6e11.4ef13.916.1de20.1cd22.2cd91209-151 8.93e10.0cd11.4cd11.9cde18.316.3de20.1cd22.4cd91209-142 9.00e10.8c11.5cd11.9cde14.917.2cd21.1bc23.8abcSensitive91209-220 10.60b12.2a13.2b13.7b16.518.8ab22.1ab24.4ab91209-119 8.89e10.4cdce11.1de11.5de14.116.1de20.1cd22.1cd91209-269 8.29g9.85e10.5e10.6f13.014.0f17.1e18.4e91209-297 10.00c11.6b12.6b13.3b15.717.8bc20.2cd21.4d91209-143 9.56d10.9c11.9c12.2cd14.616.5de19.5cd21.5dControlHinson 8.93e10.6c11.5cd11.9cde14.315.7e18.6de20.6dLSD 0.050.410.50.60.73.31.21.81.9Significance************ns*********Flood x Genotype Significancensnsnsnsnsnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05). Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003). 53

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54 Table 3-6 Leaf dry weights and dry matter partitioning. Days After So wing213549Dry WeightPartitioningDry WeightPartitioningDry WeightPartitioningmg plant-1mg plant-1mg plant-1Flood2 Week3280.5016210.443b1190b0.423b4 Week3250.5036460.438b919c0.369cNon-flooded3380.5186680.495a2220a0.448aLSD 0.05330.017980.0152100.016Significancensnsns****GenotypeTolerant91210-350 425a0.477728ab0.449cde1390abcd0.383e91210-316 280de0.517674abcd0.478abc1730a0.434abc91209-126 343bc0.508633bcd0.458bcde1620abc0.438ab91209-151 248e0.491512cd0.443de1470abc0.404cde91209-142 287cde0.508495d0.432e1660ab0.409bcdeSensitive91209-220 389ab0.513690abc0.486ab1440abc0.426abcd91209-119 297cde0.502646bcd0.441de1370abcd0.405cde91209-269 308cde0.504519cd0.463bcd1220cd0.447a91209-297 320cd0.519588bcd0.453cde1010d0.379e91209-143 421a0.502845a0.447de1690ab0.397deControlHinson 320cd0.539764ab0.495a1290bcd0.421abcdLSD 0.05630.0341870.0294100.030Significance***ns*******Flood x Genotype Significancensnsnsnsns***,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05). Dry matter partitioning is defined as leaf dry weight / total plant dry weight.Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).

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55 Our findings that leaf dry weight for flooded treatments is reduced in comparison to non-flooded treatments and that two-week post-flood recovery is sufficient to observe a significant recuperation of leaf biomass are consistent with the generic plant response to flooding, but to our knowledge have not been previously reported for soybean. In soybean, reductions in leaf area of flooded plants (Bacanamwo and Purcell, 1999a) as well as reduction in leaf number (Sallam and Scott, 1987) have been reported which logically should result in a reduction in leaf dry weight such as we have documented. Differences in total leaf biomass accumulation between 35 and 49 DAS show continued leaf growth for all treatments. Significant differences in leaf dry weight between the 2 and 4-week flood treatments at 49 DAS demonstrates an enhanced ability of the 2-week treatment to accumulate biomass in the leaves after removal of flood. This is consistent with increases in plant biomass accumulation after removal of flood reported by Bacanamwo and Purcell (1999b) and Oosterhuis et al. (1990). While patterns in total leaf biomass accumulation and leaf expansion mirror one another, an analysis of incremental leaf expansion showed greater genotype sensitivity to flooding than leaf biomass, resulting in a significant genotype x flood interaction term at 36 DAS (3.7). Figures 3.1-3.4 show the range of genotypic incremental leaf area expansion response to flood treatments. Noting that leaf expansion is one of the first plant parameters affected by flooding (Kramer, 1951), trends in the response of leaf area expansion to flood treatments may serve as a more sensitive indicator of plant performance than that of total leaf area accumulation. The fraction of total biomass partitioned to leaves was significantly different for the flood treatments at 35 and 49 DAS (3.6). At 49 DAS the non-flooded treatment

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56 Table 3-7 Incremental leaf area expansion. Days After Sowing1422283643cm2 Flood2 Week20.651.848.957b66b4 Week19.451.555.549b49bNon-flooded17.953.563.9153a247aLSD 0.051.95.47.71732Significancensnsns****GenotypeTolerant91210-35024.3bc57.8bc59.5cde8983cd91210-31616.2d46.0de60.5bcd105166a91209-12614.6de47.0de44.9ef81149ab91209-15111.7e40.6e41.5f89134abcd91209-14213.2de40.6e40.2f92143abcSensitive91209-22021.5c54.6bcd53.5cdef79102bcd91209-11915.1de49.2cde52.9cdef76129abcd91209-26915.8d46.9de48.4def7084cd91209-29729.0a70.7a74.8ab6676d91209-14326.4ab63.5ab76.9a108168aControlHinson24.2bc58.1bc64.1abc9495bcdLSD 0.053.510.414.833.161Significance*******ns*Contrast T vs. SFlood x Genotype Significancensnsns*ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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57 Incremental Leaf Area Expansion Across Genotypes-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Figure 3-1 Incremental leaf area expansion for all genotypes across flood treatments. Incremental Leaf Area Expansion 91209-119-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-143-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-220-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-269-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-297-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded a b c d e Figure 3-2 Incremental leaf area expansion for most sensitive genotypes (Cornelious, 2003).

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58 Incremental Leaf Area Expansion 91209-126-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-142-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-151-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91210-316-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Incremental Leaf Area Expansion 91209-350-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded a b c d e Figures 3-3 Incremental leaf area expansion for most tolerant genotypes (Cornelious, 2003). Incremental Leaf Area Expansion Hinson-200-1000100200300400500719243240Days after sowingExpansion (cm2) 2 week 4week non-flooded Figure 3-4 Incremental leaf area expansion for control genotype Hinson.

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59 had a significantly greater allocation of biomass to the leaves, and the 4-week flooded treatment had the lowest. Significant genotypic differences were observed at the 35 and 49 DAS sampling dates. Flood by genotype interactions were significant at 49 DAS (3.6). The decline in fraction of biomass partitioned to leaves with flood durationis again a natural consequence of the inability of the plant to maintain plant processes and production on a whole plant level under flooded conditions. Flooded treatments recorded significantly lower leaf areas at later sampling dates (36-43 DAS) (3.8). Genotype effects were significant at all but the final sampling date (43 DAS). No significant interaction between flood and genotype effects was recorded at any sampling date. The relative decline in leaf area due to flooding ranged from 54-57% from non-flooded treatments at 43 DAS. Our results are consistent with the findings of Bacanamwo and Purcell (1999a) who recorded a reduction in leaf area between 49-60% after 21 days of flooding with respect to non-flooded controls. Total Plant Dry Weight Total plant dry weight was significantly less for flooded treatments at 49 DAP (Tables 3.9) accumulating only 44 53% the total biomass of non-flooded controls. No significant difference between flooded treatments was recorded at final harvest. Significant differences among genotypes were noted at all sample dates, however no significant interaction between flood and genotype was recorded for any sample date. Trends in accumulation of soybean plant biomass when flooded were in agreement with previous studies; Oosterhuis et al. (1990) cited a general reduction in plant biomass with flooding at the V4 growth stage, while Scott et al. (1989) reported a reduction of 41-56% in total biomass as compared to non-flooded controls with 14 days of flooding at the V4

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60 Table 3-8 Leaf area. s Days After Sowing1422283643cm2Flood2 Week20.672.5121178b245b4 Week19.470.9126176b225bNon-flooded17.971.3135288a535aLSD 0.051.86.11125.052Significancensnsns**GenotypeTolerant91210-350 24.3bc82.0bc142cd231abc31491210-316 16.2d62.3ef123def228bcd39491209-126 14.6de61.6ef106fg188cd33791209-151 11.7e52.3f93.8g182cd31691209-142 13.2de53.8ef94.1g186cd330Sensitive91209-220 21.5c76.1cd130cde209bcd31191209-119 15.1de64.3de117ef193bcd32291209-269 15.8d62.7ef111efg181d26591209-297 29.0a99.7a175a240ab31791209-143 26.4ab89.9ab167ab275a443ControlHinson 24.2bc82.3bc146bc240ab336LSD 0.053.511.92148101Significance***********nsFlood x Genotype Significancensnsnsnsn*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).

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61 Table 3-9 Total plant dry weight. rowth stage. From our results, it is apparent that sufficient damage occurred to the 2-week flooded plants to retard overall growth for at least 14 days following the removal of flooding. Oosterhuis et al. (1990) showed increases in biomass accumulation to near-normal rates 14 days after the removal of a 7-day flood treatment. In our experiment, 14 days was not a long enough period to see significant recovery from the 2-week flood treatment. Days After Sowing213549mg plant-1Flood2 Week65513702810b4 Week65514502360bNon-flooded65513505050aLSD 0.0566200520Significancensns**GenotypeTolerant91210-350 913a1610ab3420abc91210-316 538e1380bc3990a91209-126 675cd1370bc3740ab91209-151 507e1160c3560abc91209-142 563de1140c4070aSensitive91209-220 759bc1440bc3260abcd91209-119 590de1440bc3380abcd91209-269 609de1110c2640cd91209-297 616de1270bc2420d91209-143 839ab1860a4070aControlHinson 598de1550ab2930bcdLSD 0.05126380991Significance******Flood x Genotype Significancensnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003). g

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62 Root:Shoot Ratio Flooding had a significant effect on root:shoot ratios at 49 DAP (3.10). Genotype effects were also significant at 49 DAP with 91209-119 and 91209-143 having the highest values. The interaction between flood and genotype was not significant. Table 3-10 Root:Shoot ratios Consistent with the findings of Sallam and Scott (1987), root:shoot ratios decreased for all treatments with time. This can be accounted for by the continual accumulation of aboveground biomass regardless of treatment. Flooding at the V2 growth stage did not appear to prevent the accumulation of aboveground biomass, rather slow the initiation of Days After Planting213549Flood2 Week0.3270.3100.239b4 Week0.3490.2890.254abNon-flooded0.3100.2750.263aLSD 0.050.0570.0370.019Significancensns*GenotypeTolerant91210-350 0.3850.2280.234bc91210-316 0.3210.2860.251ab91209-126 0.3470.3230.254ab91209-151 0.3580.3130.263ab91209-142 0.3190.2940.268abSensitive91209-119 0.3660.3320.278a91209-220 0.3030.2770.249abc91209-269 0.3120.3190.249abc91209-297 0.2770.2870.234bc91209-143 0.3290.3040.279aControlHinson 0.2890.2430.212cLSD 0.050.1090.0700.038Significancensns*Flood x Genotype Significancensnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003).

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63 the rapid growth phase. The decreases in root:shoot ratio for the 2-wk flood (3.10) are not consistent with the findings of Bacanamwo and Purcell (1999a) and Sallam and Sco(1987). In our experiment, the relative loss of aboveground biomass in leaf and stem tissues was not as pronounced as the loss of root dry weight. The development of adventitious roots, while present, was not sufficient to offset the loss of primary roweight. Correlati tt ot dry ons of Above Ground Plant Growth to Flood Tolerance dry weight (3.12). Speart and tolerance existed at 49 ass and f Genotypes were ranked based on flood tolerance (3.11) and stem mans rank correlation coefficients were significant for stem dry weight and flood tolerance (3.13). The positive correlation reported by Pires et al. (2002) between stem diameter and flood tolerance was attributed to increased hypertrophy in the more toleranvariety. Our results, to our knowledge not previously reported in the scientific literature, indicate a similar correlation may exist between stem dry weight and flood tolerance. This relationship is consistent with the findings of Pires et al. (2002), assuming that increased hypertrophy coincides with greater stem dry weight. A negative correlation between stem dry matter partitioning DAS (3.13) based on genotype tolerance rankings (3.11) and stem dry matter partitioning rankings across flooded treatments (3.14). The percentage of total biomassociated with the stem is a logical inverse measure of plant performance under flooding. While stem dry weight is positively correlated with tolerance, necrosis dieback of leaves and roots in non-tolerant genotypes may leave a greater percentage ototal biomass in the stems. Plants that are least able to support near-normal leaf and rootgrowth and develop fewer adventitious roots would have the greatest percentage of

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64 biomass associated with the stem. Based on the negative correlation between stem dry weight partitioning and tolerance it may be desirable to use partitioning percentage for Table 3-11 Tolerance of all genotypes to early-season flooding. able 3-12 Stem dry weight means for flooded treatments and ranks used for correlations. ToleranceRatioRank91209-119 0.779a191209-126 0.745a291209-269 0.739ab391209-142 0.671abc4Hinson 0.635abcd591209-220 0.469abcd691209-151 0.462abcd791209-143 0.441bcd891210-316 0.466cd991210-350 0.410cd1091209-297 0.335d11 Treatment means followed by the same letter are not significantly different (P<0.05) Tolerance is defined as total mean plant dry weight flooded treatments / total dry weight non-flooded treatment. Based on dry weights at 49 DAS sampling. T Days After Sowing 213549Genotype(mg plant-1)(mg plant-1)(mg plant-1)rank91209-142 1534231350191209-119 1404701190291209-126 15837811603Hinson 1505601120491209-1431986061070591209-151 1214281040691210-316 1204051020791210-350 2246431010891209-220 205529880991209-269 1683908301091209-297 17439661011

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65 Table 3-13 Correlations between selected plant measures and flood tolerance. Table 3-14 Stem dry matter partitioning means for flooded treatments and ranks used for flooded treeasure of tolerance. A significant positive correlation was s o f Experiment 1TolerancePlant measureSpearman's Rank Correlation CoefficientStem dry weight 49 DAS0.600*Stem partitioning 35 DAS-0.209nsStem partitioning 49 DAS-0.618*Leaf dry weight 49 DAS0.627*Leaf partitioning 35 DAS-0.245nsLeaf partitioning 49 DAS0.545ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively correlations. atments as a m Days After Sowing213549Genotyperank91209-297 0.2730.3270.481191210-350 0.2690.3710.4532Hinson 0.2480.3170.435391209-151 0.2490.3460.415491209-143 0.2430.3500.405591209-220 0.2640.3140.399691209-1190.2240.3290.395791209-142 0.2620.3650.391891209-269 0.2700.3290.374991210-316 0.2290.3170.3701091209-126 0.2380.2990.36811 noted between leaf dry weight and flood tolerance at 49 DAS (3.13) when genotype tolerance ranks (3.12) were correlated to leaf dry weight ranks across flood treatment(3.15). The correlation between leaf dry weight and flood tolerance is most likely due tthe enhanced ability of the most tolerant plants to continue transport of essential water and nutrients from the roots to the leaves allowing for continued photosynthesis and leaexpansion.

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66 Table 3-15 Leaf dry weight means for flooded treatments and ranks used for ontrast Between Flood Tolerance Groups The difference between flood tolerance groups was consistently highlighted by strong early growth in the tolerant grouping and better performance under flooded conditions for the sensitive grouping (Tables 3.16 and 3.17). Significant differences in pre-flood values for stem dry weight, leaf dry weight, plant height, incremental leaf area accumulation, and total leaf area (3.16 and 3.17) are all based on the greater total value of the tolerant genotype grouping. In each instance the, flood duration led to gains by the sensitive genotype grouping, resulting in either no significant difference between groups (stem dry weight, incremental leaf area accumulation, and total leaf area) or a significant difference based on greater value for the sensitive grouping (leaf dry weight and plant height) (Tables 3.16 and 3.17). This is further evidence of the lack of correspondence between tolerance rankings as defined in this study and tolerance rankings as defined by Cornelious (2003). Days After Planting213549Genotype(mg plant-1)(mg plant-1)(mg plant-1)rank91209-142 2894831420191209-126 3395951350291209-119 3155961220391210-316 2795931020491209-143 40975611305Hinson 3258161070691209-151 2365261020791209-269 3095301000891209-220 391751940991210-350 3867658601091209-297 31855545011 correlations. C

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67 Table 3-16 Aboveground dry weight contrasts between tolerant vs. sensitive genotypes as defined by Cornelious (2003) across 2and 4-week flood treatments. Days After Sowing213549mgDry weight:Stem-109*-116ns994nsLeaf-213*-228ns1121*Total-328ns-536ns2502ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Negative values denote a greater mean value for "tolerant" treatments. Table 3-17 Plant height, incremental leaf area, and leaf area values, contrasts between tolerant vs. sensitive genotypes as defined by Cornelious (2003) across 2and 4-week flood treatments. onclusions Significant positive correlations were found between soybean stem dry weight and leaf dry weight and flood tolerance. However, soybean stem partitioning percentage was negatively correlated with flood tolerance. The importance of stem development both as a positive measure of adaptation and a negative reflection of plant performance is highlighted in its relationship to tolerance. Significant correlations between plant growth parameters and tolerance as defined in this study solidify the link between morphological adaptations and the ability of soybean to grow under flooded conditions. The Days After Planting2125283236394447cmPlant height -1.58**-0.6ns-1.13ns-0.12ns5.31ns0.77ns3.31ns6.25*1422283643Incremental Leaf Area-32.6***-55.4**-67.4**67.9ns118.4nsLeaf Area-32.6***-88.0***-155.4***-87.4ns31.0ns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Negative values denote a greater mean value for "tolerant" treatments. C

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68 dissimilarity between tolerance ranks for this study, with flooding at the V2 growth stage, and tolerance rankings as established by Cornelious (2003) for flooding at the R2 growth stage (Figure 3-5) may reflect variation in plant response to flooding at these stages. In Linear Regression of Flood Tolerance Rankings y = 5.93 0.078xR2 = 0.006024681012024681012Arkansas (Cornelious, 2003)Belle Glade All Genotypes by Rank 91210-350 91210-316 91209-126 91209-151 91209-142 91209-220 91209-119 91209-269 91209-297 91209-143 Figure 3-5 Linear regression of tolerance ranks from Arkansas (Cornelious, 2003) and Belle Glade. order to better understand the relationship between early season morphological response and end of season tolerance it will be necessary to further investigate the effects of V2 flooding on soybean yield for these genotypes. By establishing rankings through comparable means to those presented in Cornelious (2003), but based on early season flood imposition, it would be possible to judge the effectiveness of early season tolerance identification. Insight may also be gained as to the consistency of genotype tolerance rankings with regards to timing of flood imposition. The data presented in this Chapter warrants further investigation as it notes significant relationships between plant morphology and flood tolerance.

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CHAPTER 4 RESEARCH PERSPECTIVES The process of developing and executing an academic experiment has been enlightening on a professional, academic, and personal level. What I may have considered to be an exercise in strict repetition before beginning has turned out to be a challenging and creative process that pushed my abilities to learn. From an academic perspective the experiments reported in this thesis offer, in my opinion, an important contribution to increased understanding of the processes involved in soybean adaptation to flood stress. Assuming that to be the intent of performing the experiments, then this project has to be considered a success. The success we obtained was not however the success we anticipated. It appears that by answering one question the possibility to ask five new questions has arisen. That in itself may be the beauty of coming to a greater understanding of any topic. Only through obtaining different results than expected have I better understood how I might have gone about this project. Without rehashing the contents of the previous three chapters, I do believe there is validity to the concept that morphological response serves as a good indicator of flood tolerance. I also believe that there is a strong possibility that early identification of flood tolerant varieties is possible, at least in relation to early season flood stress. That being said, additional evidence is needed and should be sought. Recommendations for future projects in this area would include: 1) Morphology of flooding response at the R2 growth stage (this being the stage at which initial flood tolerance ranking were determined) 2) Effects of flooding at the V2 stage on yield. This would allow early 69

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70 season tolerance rankings based on biomass and yield based tolerance rankings to be compared therefore establishing the predictive ability of those early season rankings. From a professional perspective, my time at the University of Florida has been very productive. The first question asked of me while inquiring about the School of Natural Resources and Environment was what are your career goals? While I still may not be able to answer that question specifically, I do know that I came in with little understanding of the functioning of plants and their relation to the greater environment and I am leaving with more information and thought dedicated to this topic than I could have hoped for. Acquiring this information was my primary goal, but the conversations, classes, and atmosphere associated with its acquisition have been of greater benefit than the raw information ever will be. My recommendation to anyone pursuing a degree at this level is, understand what you want to know before you begin looking for it. Anything else you find along the way becomes a bonus to knowing where you are going. Personal lessons learned have proven to be the greatest body of knowledge obtained in this process. First, consistent work pays off. Hard work is good, but too much hard work at any one time becomes stress. Consistent work allows for continual meeting of goals and the appearance of progress. Second, be ready for the unexpected. You cannot know what the outcome of the experiment will be before it is performed, if you do then is it really worth doing? When the unexpected hits you, run with it, sometimes it is much more interesting than the expected. Likewise, for the sake of the experiment and science, do everything you can to avoid the unexpected. Try to anticipate what could go wrong and avoid the possibility. In the meantime, make sure you can document what is going on just in case you did not anticipate every possibility. Finally,

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71 take control of knowing the subject. It is your project, you degree and while the process must be and always will be a collaborative effort, on a very basic level it is your responsibility to make sure it goes well. This chapter is for the benefit of the students that may take the time to pick up and read portions of this thesis. Understand that hindsight is always 20-20 (to be exceptionally clichd). The strength and weaknesses of the previous three chapters are the experiences represented in the fourth

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APPENDIX A DATA FROM EXPERIMENT 2

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73Table A-1 Primary root dry weights. Days After Sowing213549Dry WeightPartitioningDry WeightPartitioningDry WeightRDW*Partitioningmg plant-1mg plant-1mg plant-1Flood2 Week1510.2134970.2183750.6750.1474 Week1640.2273420.1654470.8130.163Non-flooded1790.2904680.191633-0.143LSD 0.05390.037660.0151090.2000.015SignificancensnsnsnsnsnsnsGenotypeTolerant91210-3501820.2204220.164d4880.5650.143bc91209-1261640.2345290.214ab5710.7750.159ab91209-1421470.2373940.221a4400.7190.169aSensitive91209-220 1740.2254430.179cd4130.9760.149abc91209-269 1890.2594240.197bc5470.6740.154abcControlHinson 1340.1834050.175d4510.7590.133cLSD 0.05560.0539230.0211540.4000.021Significancensnsns***nsns*Flood x Genotype Significancensnsnsnsnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means in the same column followed by the same letter are not significantly different (P<0.05). Dry matter partitioning is defined as primary root dry weight / total plant dry weight.* RDW = Relative dry weight is defined as primary root dw for flood treatment / primary root dw for non-flood treatment.Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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74 Table A-2 Primary root length, diameter, surface area, and volume. 49 Days After PlantingLengthAvg.DiameterSurface AreaVolumecm plant-1mm plant-1cm2 plant-1cm3 plant-1Flood2 Week17180.4391280.7934 Week27400.6772051.28Non-flooded14810.5061341.04LSD 0.056930.140460.3SignificancensnsnsnsGenotypeTolerant91210-35017820.5211471.0491209-12621980.6341781.2291209-14217960.5151420.958Sensitive91209-22017400.4741340.87691209-26919450.5241581.07ControlHinson24180.5761751.06LSD 0.059790.198650.384SignificancensnsnsnsFlood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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75 Table A-3 Adventitious root dry weight and dry matter partitioning. Days After Planting3549Dry WeightPartitioningDry WeightPartitioningmg plant-1mg plant-1Flood2 Week141.09261.104 Week391.28351.26Non-flooded----LSD 0.05110.40110.28SignificancensnsnsnsGenotypeTolerant91210-350141.020210.88491209-126170.865230.83091209-142120.988210.928Sensitive91209-220210.440150.59191209-269180.793280.811ControlHinson220.619160.676LSD 0.05150.561150.401Significancens***nsnsFlood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.Dry matter partitioning is defined as adventitious root dry weight / total plant dry weight.Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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76 Table A-4 Adventitious root length, diameter, surface area, and volume. LengthAverage DiameterSurface AreaVolumecm plant-1mm plant-1cm2 plant-1cm3 plant-1Flood2 Week2310.24716.90.1044 Week5600.29338.40.211Non-flooded----LSD 0.051950.03814.10.823SignificancensnsnsnsGenotypeTolerant91210-3502780.24419.90.11591209-1264690.27533.10.19391209-1423760.26727.20.157Sensitive91209-2204220.29629.80.16991209-2694080.28930.20.179ControlHinson4200.24725.80.129LSD 0.053380.06624.40.143SignificancensnsnsnsFlood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively.Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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77 Days After Planting3549Dry WeightPartitioningDry WeightPartitioningmg plant-1%mg plant-1%Flood2 Week40.127572.424 Week150.643682.41Non-flooded341.57851.67LSD 0.05160.734390.95SignificancensnsnsnsGenotypeTolerant91210-350351.5361bc1.75b91209-126171.1282b2.24b<0.05)., 2003). Table A-5 Nodule dry weight and dry matter partitioning. 91209-14250.21558bc2.23bSensitive91209-22060.28225c1.11b91209-269211.00155a4.29aControlHinson160.53741bc1.38bLSD 0.05231.04561.34Significancensns****Flood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (PDry matter partitioning is defined as nodule dry weight / total plant dry weight.Tolerant and Sensitive determination based on descending ranking (Cornelious

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78 Table A-6 Stem dry weight and dry matter partitioning. Days After Sowing213549Dry Weight PartitioningDry Weight PartitioningDry Weight Partitioningmg plant-1mg plant-1mg plant-1Flood2 Week1930.2687800.337949b0.3944 Week1900.2617730.3591114b0.423Non-flooded1860.2567740.3121670a0.369LSD 0.05310.0181120.0142840.015Significancensnsnsns**nsGenotypeTolerant91210-350 2280.273997a0.368a14100.442a91209-126 1970.255753bc0.309d13200.378c91209-142 1680.271617c0.349ab9900.389cSensitive91209-220 1990.256816ab0.338bc10900.394bc91209-269 1730.245710bc0.319cd13100.356dControlHinson 1910.271793b0.334bc13800.414bLSD 0.05430.0261580.2094000.022Significancensns*****ns***Flood x Genotype Significancensnsnsnsns***,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05). Dry matter partitioning is defined as stem dry weight / total plant dry weight. Tolerant and Sensitive determination based on descending ranking (Cornelious, 2003).

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79 Days After Sowing21303843cm Flood2 Week9.0613.819.623.44 Week8.8913.719.123.4Non-flooded9.4012.718.723.4LSD 0.050.600.71.21.5SignificancensnsnsnsGenotypeTolerant91210-350 9.29ab14.5b20.8b25.8a91209-126 9.25abc12.9c18.6c23.3b91209-142 9.17abc12.4cd17.7cd21.7bcSensitiveacbctively.ent (P<0.05).nelious, 2003). Table A-7 Plant height. 91209-220 9.85a15.8a22.9a27.791209-269 8.44c11.8d16.5d20ControlHinson 8.70bc13.1c18.1c21.9LSD 0.050.840.91.62.2Significance**********Flood x Genotype Significancensnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respec Treatment means followed by the same letter are not significantly differTolerant and Sensitive determined based on descending rankings (Cor

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80 Table A-8 Leaf Dry weights and dry matter partitioning. Days After Sowing213549Dry Weight PartitioningDry WeightPartitioningDry Weight Partitioningmg plant-1mg plant-1mg plant-1Flood2 Week3690.52010010.438c1040b0.424b4 Week3770.5119600.451b1040b0.373cNon-flooded3740.50511800.480a2040a0.471aLSD 0.05590.0281300.0082400.016Significancensnsns******GenotypeTolerant91210-3504220.5071160a0.448d13100.389c91209-1263620.5111110a0.457cd15300.432ab91209-1423080.492750b0.421e10700.411bcSensitive91209-2203990.5191140a0.471ab12900.441a91209-2693560.4971020a0.465bc15900.439aControlHinson3960.5461130a0.477a14700.433abLSD 0.05840.0401800.0113500.023Significancensns*****ns**Flood x Genotype Significancensnsns*ns**,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05). Dry matter partitioning is defined as leaf dry weight / total plant dry weight.Tolerant and Sensitive determined based on descending rankings (Cornelioius, 2003).

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81 Table A-9 Leaf area values. s Days After Sowing1622293744cm2 plant-1Flood2 Week48.9a113169228b245b4 Week44.7ab104159225b254bNon-flooded40.6b105179333a492aLSD 0.056.613233869Significance*nsns***GenotypeTolerant91210-35053.4a118a183ab277ab34291209-12638.4c99bc164b269b36391209-14234.6c80c128c187c252Sensitive91209-22048.5ab117ab182ab266b28691209-26940.4bc97c154bc246b353ControlHinson52.8a132a203a238a385LSD 0.059.318325498Significance*********nsFlood x Genotype Significancensnsnsnsn*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).

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82 Table A-10 Total plant dry weight Days After Planting213549g plant-1Flood2 Week0.7132.302.45b4 Week0.7312.132.73bNon-flooded0.7392.464.43aLSD 0.050.1070.290.65Significancensns**GenotypeTolerant91210-3500.8312.59a3.2991209-1260.7052.42a3.5391209-1420.6231.78b2.58Sensitive91209-2200.7732.42a2.8391209-2690.7182.19a3.62ControlHinson0.7212.37a3.63LSD 0.050.1510.42Significancens**nsFlood x Genotype Significancensnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based descending ranking (Cornelious, 2003).

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83 Table A-11 Root:shoot ratios Days After Planting213549Flood2 Week0.2730.292a0.227ab4 Week0.2960.237b0.256aNon-flooded0.3600.263b0.192bLSD 0.050.1290.0270.041Significancens****GenotypeTolerant91210-3500.2830.228b0.20691209-1260.3090.307a0.23891209-1420.3110.301a0.255Sensitive91209-2200.2910.237b0.20491209-2690.4390.277a0.262ControlHinson0.2240.235b0.184LSD 0.050.1830.0370.058Significancens***nsFlood x Genotype Significanc e nsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003). Table A-12 Tolerance at 49 DAS ToleranceRank91209-2200.723191209-1420.714291209-1260.664391209-2690.635491210-3500.5555Hinson0.5526

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APPENDIX B LEAF NUTRIENT AND SPAD DATA FROM EXPERIMENTS 1 AND 2

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Table B-1 Leaf nutrient analysis (Experiment 1) NPK MgSCaMoMnFeTreatmentg kg-1mg kg-1Flood2 Week3.050.64b1.390.44a0.2641.98a15538.173.5ab4 Week2.590.87a1.430.32b0.221.56c94.325.985.8aNon-flood2.860.43c1.230.456a0.251.82b11725.968.6bLSD 0.050.240.110.120.0270.210.0917.34.413.7Significancens**ns**ns**nsns*GenotypeTolerant91210-3503.03abc0.76abc1.390.410.251.91a130bc37.9a77.691210-3163.02abc0.55cd1.310.410.241.67bc84.9e24.7c67.491209-1262.69cd0.57cd1.290.360.221.76abc92.3de31.5abc72.791209-1512.71cd0.46d1.190.420.231.91a96.9de32.5abc70.491209-1423.22ab0.44d1.280.40.241.79ab99.3cde30.6abc83.1Sensitive91209-2202.84bc0.65abcd1.370.370.241.67bc89.1de26.3c82.691209-1192.89abc0.620bcd1.340.430.271.91a121cd33.1abc73.791209-2692.29d0.73abc1.390.450.261.81ab155b27.3bc67.991209-2972.85abc0.84a1.540.410.251.61c108cde24.9c61.991209-1433.32a0.65abcd1.290.40.241.92a96.4de25.6c86.3ControlHinson2.32d0.83ab1.490.420.261.69bc269a35.4ab92.3LSD 0.050.450.210.230.050.040.1833.08.426.2Significance****nsnsns******nsFlood x Genotype Significance*nsnsns*nsnsnsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending ranking (Cornelious, 2003). 85

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Table B-2 Leaf nutrient analysis (Experiment 2) NPK MgSCaMoMnFeTreatmentg kg-1mg kg-1Flood2 Week2.87a0.5431.27a0.413b0.273a2.34a228a48.4a1334 Week2.26b0.6321.15b0.296c0.202b1.68b120c37.9b120Non-flood2.43b0.5251.04c0.571a0.289a2.28a202b36.9b117LSD 0.050.210.0590.080.0230.0160.080174.518Significance**ns************nsGenotypeTolerant91210-3502.63ab0.616a1.130.452a0.2612.29a199b43.2ab150a91209-1262.57ab0.513b1.140.399b0.2452.12b136d46.3a106c91209-1422.56ab0.513b1.090.450a0.2532.21ab171c43.3ab110bcSensitive91209-2202.11c0.657a1.090.417b0.2582.13b153cd39.4bc125abc91209-2692.86a0.513b1.250.416b0.2651.95c167c35.4c113bcControlHinson2.41bc0.590ab1.230.425ab0.2461.89c273a39.1bc137abLSD 0.050.30.0840.120.0330.0230.11246.426Significance****ns*ns*********Flood x Genotype Significancensnsnsnsns****nsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003). 86

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Table B-3 SPAD readings (Experiment 1). Days After Sowing2125283236394447Flood2 Week21.217.918.416.717.4b18.7b24.4a23.9a4 Week21.017.917.716.017.4b17.9b23.6b19.2bNon-flooded22.218.317.917.619.9a21.1a19.3a22.9aLSD 0.051.20.90.80.70.91.01.31.3Significancensnsnsns******GenotypeTolerant91210-350 21.318.1abc18.5ab17.719.9ab21.3a22.7b21.8bcde91210-316 22.219.4a19.3a16.718.5abc21.1a23.8ab24.1ab91209-12621.617.6bc17.5bc16.518.4abc19.6abc23.4ab23.1abc91209-151 22.419.5a18.5ab17.120.2a20.6ab24.1ab23.2abc91209-142 21.118.3ab17.6bc16.920.0ab21.5a25.6a24.4aSensitive91209-220 21.217.6bc17.4bc16.016.4de17.9de22.3b21.5cde91209-11921.418.0abc17.9abc16.218.3bcd18.7abcd22.7b22.3abc91209-269 21.117.9abc17.7abc17.117.2cd18.6cde19.7c21.1cde91209-297 23.118.4ab19.2a17.515.2e14.8f19.4c19.4e91209-143 20.316.9bc17.5bc16.818.6abc19.8abc23.0b21.9bcdControlHinson 20.216.5c16.7c16.317.4cd17.6e19.4c19.7deLSD 0.052.31.61.61.41.91.92.52.5Significancens**ns***********Flood x Genotype Significancensns*nsns*nsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means in the same column followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003). 87

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88 Table B-4 SPAD readings (Experiment 2). Day s After Sowing21303843Flood2 Week31.3a 23.8a20.8b22.3b4 Week28.7b22.4b21.4b23.5aNon-flooded30.2ab24.4a23.3a22.7abLSD 0.051.80.91.31.2Significance***nsGenotypeTolerant91210-350 30.426.7a23.9a22.3ab91209-12629.522.1cd22.4a23.8a91209-142 30.324.9b22.7a24.2aSensitive91209-220 31.021.2d18.8b19.8c91209-269 29.323.4c22.7a24.7aControlHinson29.822.9c20.6b21.5bcLSD 0.052.51.31.81.8Significancens*********Flood x Genotype Significancens*nsns*,**,*** Significant at the 0.05, 0.01, and <0.001 levels of probability respectively. Treatment means followed by the same letter are not significantly different (P<0.05).Tolerant and Sensitive determined based on descending rankings (Cornelious, 2003).

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LIST OF REFERENCES Alvarez, J. and L.C. Polopolus. 2002. Florida sugar industry. EDIS publication SC-042, University of Florida. Retrieved from http://edis.ifas.ufl.edu/SC042 07/25/05. Andreeva, N., K. Swaraj, G.I. Kozlova, and L.A. Raikhman. 1987. Changes in the ultra-structure and nitrogen fixation activity of soybean nodules under the influence of flooding. Sov. Plant Physiol. 34:427-435. Bacanamwo, M. and L.C. Purcell. 1999a. Soybean dry matter and N accumulation responses to flooding stress, N sources and hypoxia. J. Exp. Bot. 50:689-696. Bacanamwo, M. and L.C. Purcell. 1999b. Soybean root morphological and anatomical traits associated with acclimation to flooding. Crop Sci. 39:143-149. Bennett, J.M. and S.L. Albrecht. 1984. Drought and flooding effects on N2 fixation, water relations, and diffusive water resistance of soybean. Agron. J. 76:735-740. Boru, G., T. Van Toai, J. Alves, D. Hua, and M. Knee. 2003. Responses of soybean to oxygen deficiency and elevated root-zone carbon dioxide concentration. Ann. Bot-London. 91:447-453. Cornelious, B. 2003. Phenotypic evaluation and molecular basis for waterlogging tolerance in southern soybean populations. PhD Dissertation, University of Arkansas. Evans, D. 2003. Aerenchyma formation. New Phytol. 161:35-49. Fehr, W.R. and C.E. Caviness. 1977. Stages of soybean development. Extension Bulletin 80. Iowa State University, Ames, Iowa. Gilbert, R.A., R.W. Rice, and R.S. Lentini. 2002. Characterization of selected mineral soils used for sugarcane production. EDIS publication SC-027, University of Florida. Retrieved from http://edis.ifas.ufl.edu/SC027 07/25/05. Good, A.G. and D.G. Muench. 1993. Long-term anaerobic metabolism in root tissue. Metabolic products of pyruvate metabolism. Plant Physiol. 101:1163-1168. Hanway, J.J. and C.R. Weber. 1971. Dry matter accumulation in soybean (Glycine max. (L) Merrill) plants as influenced by N, P, and K fertilization. Agron. J. 63:263-266. 89

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90 Jackson, M.B. and M.C. Drew. 1984. Effects of flooding on growth and metabolism of herbaceous plants. p. 47-128. In Kozlowski, T.T. ed. Flooding and Plant Growth. Academic Press, Orlando, FL. Jackson, M.B. and B. Ricard. 2003. Physiology, biochemistry and molecular biology of plant root systems subjected to flooding of the soil. p. 193-207. In de Kroon, H. and Visser, E.J.W. eds. Root Ecology. Springer-Verlag, Berlin. Kawase, M. 1981. Effects of ethylene on aerenchyma development. Am. J. Bot. 68:651-658. Kramer, P.J. 1951. Causes and injury to plants resulting from flooded soil. Plant Physiol. 26:722-736. Kramer, P.J. and J.S. Boyer. 1995. Water Relations of Plants and Soils. Academic Press, San Diego, CA. Lee, K.H., S.W. Park, and Y.W. Kwon. 2003. Enforced early development of adventitious roots increase flooding tolerance in soybean. Japn. J. Crop Sci. 72:82-88. Linkemer, G., J.E. Board, and M.E. Musgrave. 1998. Waterlogging effect on growth and yield of late planted soybean. Crop Sci. 38:1576-1584. Littell, R.C., W.W. Stroup, and R.J. Freund. 2002. SAS for linear models. Fourth edition. SAS Institute. Cary, NC. Muchovej, R.M. 2002. Rotational crops for sugarcane grown on mineral soils. EDIS publication SC-053, University of Florida. Retrieved from http://edis.ifas.ufl.edu/SC053 07/25/05. Oosterhuis, D.M., H.D. Scott, R.E. Hampton, and S.D. Wullschleger. 1990. Physiological responses of two soybean (Glycine max L. Merr.) cultivars to short term flooding. Environ. Exp. Bot. 30:85-92. Pankhurst, L.E. and J.I. Sprent. 1975. Effects of water, aeration and salt regime on nitrogen fixation in a nodulation legume definition of optimum root environment. J. Exp. Bot. 26:60-67. Pires, J.F., E. Soprano, and B. Cassol. 2002. Morphophysiological changes in soybean in flooded soils. Pesqui. Agropecu. Bras. 37:41-50. Powers, L.E. and R. McSorley. 2000. Ecological Principles of Agriculture. Delmar, Albany, NY. Reyna, N., B. Cornelious, J.G. Shannon, and C. H. Sneller. 2003. Evaluation of QTL for waterlogging tolerance in southern soybean germplasm. Crop Sci. 43:2077-2082.

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91 Rice, R.W., R.A. Gilbert, and S.H. Daroub. 2005. Application of the soil taxonomy key to the organic soils of the Everglades Agricultural Area. EDIS publication AG-151, University of Florida. Retrieved from http://edis.ifas.ufl.edu/AG151 07/25/05. Roiselle, A.A and J. Hamblin. 1981. Theoretical aspects of selection for yield in stress and non-stress environments. Crop Sci. 21:943-946. Sallam, A. and H.D. Scott. 1987. Effects of prolonged flooding on soybeans during vegetative growth. Soil Sci. 144:61-66. Sartain, J.B. 2001. Soil testing and interpretation for Florida turf grass. EDIS publication SL-317, University of Florida. Retrieved from http://edis.ifas.ufl.edu/SL317 07/25/05. Schueneman, T.J. 2002. An overview of Florida sugarcane. EDIS publication SC-032, University of Florida. Retrieved from http://edis.ifas.ufl.edu/SC032. Scott, H.D. J. DeAngulo, M.B. Daniels, and L.S. Wood. 1989. Flood duration effects on soybean growth and yield. Agron. J. 81:631-636. Shimamura, S., T. Mochizuki, Y. Nada, and M. Fukuyama. 2003. Formation and function of secondary aerenchyma in hypocotyl, roots, and nodules of soybean (Glycine max) under flooded conditions. Crop Sci. 72:351-359. Singh, K.D. and N.P. Singh. 1995. Effect of excess soil water and nitrogen on yield, quality, and N-uptake of soybean (Glycine max. (L) Merrill). Ann. Agr. Res. 16:151-155. Sojka, R.E., L.H. Stolzy, and M.R. Kaufmann. 1975. Wheat growth related to rhizosphere temperature and oxygen levels. Agron. J. 67:591-596. Sprent, J.I. 1972. The effects of water stress on nitrogen fixing root nodules. New Phytol. 71:603-611. Sprent, J.I. and A. Gallacher. 1976. Anaerobiosis in soybean root nodules under water stress. Soil Biol. Biochem. 8:317-320. Stofella, P.J., R.F. Sandsted, R.W. Zobel, and W.L. Hymes. 1979. Root characteristics of black beans. II. Morphological diferences among genotypes. Crop Sci. 19:826-830. Sugimoto, H. and T. Satou. 1990. Excess moisture injury of soybean cultivated in upland field converted from paddy. Japn. J. Crop Sci. 59:727-732. Sullivan, M., T.T. Van Toai, N. Fausey, J. Beuerlein, R. Parkinson, and A. Soboyejo. 2001. Evaluating on-farm flooding impacts on soybean. Crop Sci. 41:93-100.

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92 Sultana, N., Y. Ozaki, and H. Okubo. 2002. Early identification of determinate growth habit in lablab bean (Lablab purpureus (L.) Sweet). J. Fac. Agr., Kyushu Unv. 46:31-38. Sung, F.J.M. 1993. Waterlogging effect on nodule nitrogenase and leaf nitrate reduction activities in soybean. Field Crops Res. 35:183-189. Sung Guang, Y., H. Yong, H. Zheng-Rhang, and D.P. Zhang. 1996. Studies on the growth and activities of soybean roots. Soybean Sci. 15:317-321. Trought, M.C.T. and M.C. Drew. 1980. The development of waterlogging damage in wheat seedlings (Triticum aestivum L.) 1. Shoot and root growth in relation to concentrations of dissolved gasses and solutes in the soil solution. Plant Soil 54:77-94. Van Toai, T.T., J.E. Beuerlein, A.F. Schmitthenner, and S.K. St. Martin. 1994. Genetic variability for flooding tolerance in soybean. Crop Sci. 34:1112-1115. Van Toai, T.T. and N. Nurijani. 1996. Screening for flood tolerance in soybean. Soybean Genetics Newsletter 23:210-213. Van Toai, T.T., S.K. St. Martin, K. Chase, G. Boru, V. Schnipke, A.F. Schmitthenner, and K.G. Lark. 2001. Identification of a QTL associated with tolerance of soybean to soil waterlogging. Crop Sci. 41:1247-1252. Vartapetian, B.B. and M.B. Jackson. 1997. Plant adaptations to anaerobic stress. Ann. Bot.-London (Supplement A):3-20. Visser, E.J.W., J.D. Cohen, G.W.M. Barendse, C.W.P.M. Blom, and L.A.C.J Voesenek. 1996. An ethylene-mediated increase in sensitivity to auxin induces adventitious root formation in flooded (Rumex phlustris) Sm. Plant Physiol. 112:1687-1692. Wiseman, J.V. and T.B. Bailey. 1975. Estimation of leaflet, trifoliate, and total leaf areas of soybeans. Agron. J. 67:26-30. Zobel, R.W. 1991. Root growth and development. p. 61-71. In Keister, D.L. and Cregow, P.B. eds. The Rhizosphere and Plant Growth. Kluwer Academic Publishers, The Netherlands.

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BIOGRAPHICAL SKETCH Tom Henshaw was born May 20, 1976, in Chillicothe, OH. After attending DePauw University and receiving a BA in Spanish and international business he joined the Peace Corps as an agriculture extension volunteer. His time in Paraguay returned his focus to the agricultural setting of his youth and encouraged him to pursue a greater understanding of the natural and biological world. Throughout his time in Florida he has maintained a blossoming relationship with Erin Moore, and they will be married in August of 2005. 93