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THESIS

3009

This is to certify that the

thesis entitled

PHOSPHORUS, PHYTIC ACID AND ZINC CONTENT IN SEEDS OF
DRY BEANS (PHASEOLUS VULGARIS, L.): THEIR INTER;
RELATIONSHIPS, INHERITANCE AND RANGE IN VARIABILITY
AMONG GENOTYPES

presented by

Karen Ann Cichy

has been accepted towards fulfillment
of the requirements for

”.5. degree in Plant BY‘EEdIng
and Genetics

Major pr fe or

Date W0 A

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6/01 c10lRC/DateDuo.p6&p.15

PHOSPHORUS, PHYTIC ACID, AND ZINC CONTENT IN SEEDS
OF DRY BEANS (PHASEOLUS VULGARIS L.): THEIR
INTERRELATIONSHIPS, INHERITANCE, AND RANGE IN
VARIABILITY AMONG GENOTYPES

By

Karen Ann Cichy

A THESIS
Submitted to
Michigan State University

In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE

Plant Breeding and Genetics Program

2002

ABSTRACT

PHOSPHORUS, PHYTIC ACID, AND ZINC CONTENT IN SEEDS OF DRY
BEANS (PHASEOLUS VULGARIS L.): THEIR INTERRELATIONSHIPS,

INHERITANCE, AND RANGE IN VARIABILITY AMONG GENOTYPES

By

Karen Ann Cichy

Phytic acid, the major form of phosphorus in bean seeds, is considered an
antinutrient in humans because it reduces the bioavailability of zinc. Plant breeding
for increased seed zinc and reduced phytic acid is one strategy to improve the zinc
nutritional value of the bean. Accordingly, experiments were conducted with two
navy bean cultivars that differed in seed zinc content. ‘Voyager’ (high seed Zn) and
‘Albion’ (low seed Zn) were grown under different combinations of P and Zn
fertilizer. An inheritance study was also conducted with the same cultivars. Parents
were intermated to produce Pl, F2, and backcross generations. The seed of the
fertilizer and inheritance studies was harvested and screened for phytic acid, P, and
Zn levels. The results of the fertilizer study indicated that seed Zn levels were higher
in Voyager than Albion at 2 and 4 mg g'1 soil Zn in combination with all P fertilizer
treatments. The results of the inheritance study indicated variability for seed zinc
levels. The trait also showed high heritability. An additional 48 bean genotypes were
also screened for phytic acid and Zn levels. There was limited variability in seed

phytic acid levels, but substantial variability in seed Zn levels among the genotypes.

ACKNOWLEDGEMENTS

I am grateful to the members of my guidance committee: Dr. James Kelly, Dr.
Bernard Knezek, and Dr. Maurice Bennink, for their advice, suggestions, interest,
and criticism.

I would also like to thank my major advisor, Dr. George Hosfield, for his support
and guidance. He gave me the opportunity to conduct the research in an area of plant
genetics of particular interest to me.

I am very grateful to Ken Grafton and John Moraghan at North Dakota State
University. They provided me plant material and very valuable assistance in
understanding the nature of the trait and guidance in reviewing the literature.

I would like to thank also the many people in the USDA, including Shawna Bushey,
Robyn Engleright, Missy Stiefel, and Dr. Robert Olien, who provided technical
assistance in many aspects of my work and who also made coming to work each day
enjoyable.

I would like to thank my parents and my brothers and sister for their support and
understanding. I am also thankful to my husband to be, David Mota-Sanchez for his
faith in me. I would also like to thank the many friends I made while at Michigan

State University who created a wonderful environment in which to work and live.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ vi

LIST OF FIGURES ........................................................................... viii

INTRODUCTION AND LITERATURE REVIEW ....................................... 1
Introduction .................................................................................. 1
Literature Review ........................................................................... 2

Phytic acid, its Function in Seeds, Structure, Biosynthesis, Breakdown,

and Physiological role .............................................................. 2
Role of Phytic Acid in Human Nutrition ....................................... IO
Phytic Acid and Zinc Bioavailability .................................. 10
Phytic Acid and Protein Interaction .................................... l4
Phytic Acid and the Environment ............................................... 14
Ways to Reduce Phytic Acid in Foods ......................................... 15
Seed Preparation ............................. .. ........................... 16
Plant Growing Conditions and Genetic Intervention ............... 17

CHAPTER I: PHYTIC ACID, PHOSPHORUS, AND ZINC ACCUMULATION
AND THEIR INTERACTIONS IN THE SEED OF A ZINC EFFICIENT AND
NON EFFICIENT NAVY BEAN CULTIV AR ............................................ 21

Introduction ................................................................................. 21

iv

Materials and Methods .................................................................... 24
Results and Discussion .................................................................... 27
Conclusions ................................................................................. 40

CHAPTER 2: INHERITANCE OF SEED PHYTIC ACH), PHOSPHORUS AND

ZINC LEVELS IN THE SEED OF NAVY BEAN ....................................... 43
Introduction ................................................................................. 43
Materials and Methods ..................................................................... 45
Results and Discussion .................................................................... 51

Seed Phytic Acid Phosphorus Concentration ................................. 51

Seed Non Phytic Acid Phosphorus Concentration ............................ 57

Seed Zinc Concentration ......................................................... 59
Conclusions ................................................................................. 60

CHAPTER 3: SCREENING OF PHASEOLUS VULGARIS L. GENOTYPES FOR

VARIABILITY IN PHYTIC ACID AND ZINC IN RAW AND THERMALLY

PROCESSED SEED ........................................................................... 62
Introduction ................................................................................. 62
Materials and Methods ..................................................................... 63
Results and Discussion .................................................................... 66
Conclusions ................................................................................. 73

APPENDIX ..................................................................................... 74

LITERATURE CITED ........................................................................ 84

LIST OF TABLES

Table 1. Mean phytic acid phosphorus concentration [PA-P], non-phytic acid
phosphorus concentration [non PA-P], and total phosphorus concentration [P] in
Voyager and Albion navy bean cultivars grown under three levels of P and five
levels of Zn fertilizer... ............................................................................................ .28

Table 2. Means for phytic acid phosphorus (PA-P) content per experimental unit
(seed per 3 plants), non-phytic acid phosphorus (non PA-P) content per experimental
unit and total phosphorus (P) content per experimental unit in Voyager and Albion
navy bean cultivars grown under three levels of P and five levels of Zn fertilizer

................................................................................................... 30
Table 3. Means for seed yield, seed zinc concentration [Zn] and zinc content per
experimental unit (Zn) (seed of three plants) in Voyager and Albion navy bean
cultivars grown under three levels of P and five levels of Zn
fertrlrzer36

Table 4. The percent of phosphorus (P) in the phytic acid (PA) form in seed of
Voyager and Albion navy bean cultivars grown under three levels of phosphorus and
five levels of zinc

fertilizer ....... . ...................................................................... i ........... 38

Table 5. The regression relationship (R2) between three phosphorus (P) treatments
added to the soil and the concentrations of phosphorus in phytic acid (PA-P) and
non-phytic acid (non PA-P) forms in seed of two navy bean cultivars. . . .39

Table 6. Multiple linear regression for the effect of cultivar and P and Zn added to
the soil on phytic acid phosphorus (Pa-P), non-phytic acid phosphorus (non Pa-P),
and zinc (Zn) in the seed oftwo navy bean cultivars......................................39

Table 7. Coefficients of gene effects in generation means analysis .................... 50

vi

Table 8. Mean levels of total seed phosphorus (P), the phytic acid (PA-P) and non—
phytic acid fractions of phosphorus (non PA-P), the percent of total seed phosphorus
in the phytic acid form (%PA-P) and seed zinc (Zn) in two navy bean cultivars and
four additional generations established by an initial cross between the cultivars... . .52

Table 9. Chi-square test for the adequacy of an additive/dominance generation
means model for the concentration of seed phytic acid phosphorus [PA-P], non-
phytic acid phosphorus [non PA-P], and zinc [Zn] in a cross between Voyager and
Albion navy bean cultivars and four additional generations established by an initial
cross between the cultivars .................................................................... 55

Table 10. Estimates and standard error of genetic effects in the generation means
analysis model for the concentration of seed phosphorus [P], phytic acid phosphorus
[PA-P], non phytic acid phosphorus [non PA-P], percent phytic acid phosphorus [%
PA-P] and zinc [Zn] in a cross between Voyager and Albion navy bean cultivars and
four additional generations established by an initial cross between the cultivars... . .56

Table 11. The variance components and heritability estimates of phytic acid
phosphorus [PA-P], non-phytic acid phosphorus [non PA-P], total phosphorus [P]
and zinc [Zn] concentration in the seed derived from a population developed fi’om a
cross between Albion and Voyager navy bean. ........................................... 56

Table 12. The commercial class, seed coat color, seed weight and yield of 48 dry
bean genotypes screened for the concentration of phytic acid phosphorus, total
phosphorus, and zinc in the seed ............................................................. 67

Table 13. The concentration of zinc [Zn], phosphorus [P], and the phytic acid form
of phosphorus [PA-P] in raw and thermally processed seed of 48 dry bean
genotypes ....................................................................................... 69

vii

LIST OF FIGURES

Figure l. The chemical structure of phytic acid as a chair conformation. . . . . . . . . .2
Figure 2. Phytic acid biosynthesis pathway ................................................. 6
Figure 3. Phytase action on phytate in seeds ................................................ 8

Figure 4. Comparison of the concentrations of phosphorus in phytic acid [PA-P] and
non- phytic acid [non PA-P] forms in seed of Voyager (Voy) and Albion (Alb) navy
bean cultivars grown under three levels of P (O, 60, and 120mg P kg") and five
levels of Zn (0, 0.5, 1, 2, and 4 mg Zn kg") fertilizer. Fig. 4A shows the responses
for the two cultivars for seed phosphorus in the phytic acid [PA-P] form. Fig. 4B
shows the responses for the two cultivars for seed P in the non phytic acid

[non PA-P] form ................................................................................ 29

Figure 5. Comparison of the content per experimental unit (seed of 3 plants) of
phosphorus in phytic acid (PA-P) and non-phytic acid (non PA-P) forms in seed of
Voyager (Voy) and Albion (Alb) navy bean cultivars grown under three levels of P
(O, 60, and 120mg P kg") and five levels onn (O, 0.5, I, 2, and 4 mg Zn kg")
fertilizer. Fig. 5A shows the responses for the two cultivars for seed phosphorus in
the phytic acid (PA-P). Fig. SB shows the responses for the two cultivars for seed
non PA-P ......................................................................................... 31

Figure 6. Seed yield per experimental unit (3 plants) of Voyager (Voy) and Albion
(Alb) navy bean cultivars grown under three levels of P (O, 60, and 120mg P kg")
and five levels onn (O, 0.5, 1, 2, and 4 mg Zn kg") fertilizer .......................... 33

Figure 7. Correlations between the concentration of zinc [Zn] and the concentration
of phosphorus in the phytic acid form [PA-P] in the seed of Albion and Voyager
navy beans grown under three levels of P (O, 60, and 120mg P kg") and five levels of
Zn (0, 0.5, 1, 2, and 4 mg Zn kg") fertilizer ................................................. 42

Figure 8. Frequency distribution of phosphorus concentration [P] in Albion and
Voyager navy bean seed and four generations derived from the intermating of Albion
and Voyager. The arrows indicate the mean [P] of each generation. . .53

viii

Figure 9. Frequency distribution of phytic acid phosphorus concentration [PA-P] in
Albion and Voyager navy bean seed and four generations derived from the
intermating of Albion and Voyager. The arrows indicate the mean [PA-P] of each
generation ....................................................................................... 54

Figure 10. Frequency distribution of the non-phytic acid phosphorus concentration
[non PA-P] in Albion and Voyager navy bean seed and four generations derived
from the intermating of Albion and Voyager. The arrows indicate the mean

[non PA-P] of each generation ............................................................... 58

Figure l 1. Frequency distribution of zinc concentration [Zn] in Albion and Voyager
navy bean seed and four generations derived from the intermating of Albion and
Voyager. The arrows indicate the mean [Zn] of each generation ........................ 61

ix

INTRODUCTION AND LITERATURE REVIEW

INTRODUCTION

Dry bean (Phaseolus vulgaris L.) is an important food source worldwide, and is
a staple for many people in Latin America and Africa and those on vegetarian diets
in developed countries. Beans are nutritionally valuable because they are high in
complex carbohydrates (~ 40%) and protein (19 to 33 %), low in fat, high in dietary
fiber and are a good source of iron, calcium, thiamine, folic acid, and niacin (Shellie
and Hosfield, 1991). Despite dry bean’s nutritional benefits, the crop is
underutilized because of several antinutrients that limit food quality (J affe, 1973).
Phytate, the main phosphorus storage form in legume seeds, is one such antinutrient
(Barampama and Simard, 1993; Oberlas, 1975). Phytate tightly binds divalent
cations and reduces their availability and utilization in humans and animals. Zinc is
the cation most tightly bound to phytate. Studies have shown that zinc
bioavailability in vivo is considerably less than the actual zinc content of the seed
(World Health Organization, 1996).

Reduction of seed phytate in dry bean may be possible through genetic
manipulation or by altering the growing conditions or processing methods of seeds.
Accordingly, a study was conducted to:'(1) Determine the effects of phosphorus and
zinc fertilizer treatments on total seed phosphorus, phytic acid, and zinc in a zinc
efficient and a zinc inefficient navy bean cultivar. (2) Ascertain the inheritance of
seed phosphorus, phytate, and zinc in a zinc efficient and inefficient cultivar using
generation means analysis. (3) Screen a select sample of dry bean cultivars and

breeding lines to identify the range in variability in phytic acid, phosphorus, and zinc

content in raw and cooked bean seed for plant breeding purposes. (4) Propose a
plant breeding strategy to increase seed zinc while simultaneously reducing phytate

levels in bean seed.
LITERATURE REVIEW

Phytic Acid, its Function in Seeds, Structure, Biosynthesis, Breakdown, and
Physiological Role:

Phytate (myo-inositol 1,2,3,4,5,6 hexakisphosphate) is a storage form of
phosphorus found in all plant seeds; it is most abundant in cereal and legume seeds
(Oberlas, 1983). Sixty to eighty percent of the total phosphorus in legume seeds is
stored as phytate. Phytate has a strong negative charge that is maintained over a
wide pH range. The molecule has 6 phosphate groups (Fig.1) that can covalently
bind to cations, especially KI, Ca2+, Mg2+, Fe”, and Zn“ (Tsao, 1997). These
cations, along with phosphorus and myo-inositol form a pool used by seedlings

during the early stages of development.

 

Figure 1. The chemical structure of phytic acid (chair conformation)

The types of cations stored as part of a phytate complex differ among species
and differ within diverse cells of the same species (Lott et al., 1993). A study
conducted by Reddy and Pierson (1987) found that in great northern bean (P.
vulgaris) calcium, magnesium, and potassium were most frequently stored with
phytate (6.4mg/g, 20.5mg/ g and 2.6mg/ g respectively). Phytate complexes
containing calcium are water insoluble. In great northern bean, 40% of the total
phytate was water-insoluble and the other 60% was in a water-soluble form (Reddy
et al., 1988). Iron, zinc, and sodium are also stored as part of the complex, but in
much lower concentrations (528ug/g, 133 ug, and 157ug g" respectively) as
expressed on a dry weight basis (Reddy et al., 1988).

In the seed of common bean, the majority of phytate is located in the cotyledons,
typically within protein bodies, which are membrane bound vacuoles 1.5 to 8 um in
diameter (Lott et al., 1993). In great northern bean, protein bodies contained 34.3%
protein, 30% carbohydrates, and 26.6% phytic acid (Reddy and Pierson, 1987).
Inside the protein bodies, phytate makes up electron dense particles called globoid
crystals (Lott and Buttrose, 1977). Globoid accumulation occurs during the
maturation phase of seed development, which is the phase of rapid seed reserve
synthesis and accumulation (Lott et al., 1993). In rice (Oryza sativa L.), globoid
accumulation has been shown to occur as early as four days after anthesis, which is
early for a seed reserve storage molecule. This fact suggests phytic acid may serve a
role during seed development, in addition to its role in providing nutrients to the

growing seedling (Yoshida et al., 1999).

Biosynthesis of phytic acid is believed to take place in the same tissue where it
is stored. Organ et a1. (1988) demonstrated that phytate synthesis in castor bean
(Ricinus communis) occurred in embryonic tissue in the presence of exogenous
carbon and phosphorus. Under such conditions, phytate synthesis was not a
temporary phenomenon, but occurred throughout seed development in response to
added phosphate, even during post germination growth. Greenwood and Bewley
(1984) suggested that in castor bean synthesis occurs in the cytoplasm outside the
developing protein bodies on cistemal endoplasmic reticulum, and following
synthesis, phytate is transported to protein bodies via endoplasmic reticulum
vesicles. The conclusions drawn by these authors were based on light microscopy
and energy dispersive x-ray analysis data. A study in rice (0. sativa) also indicated
phytate biosynthesis occurs in the embryo soon after anthesis. The enzyme that
catalyzes the production of myo-inositol 1 phosphate from glucose-6-phosphate,
myo-inositol 1 phosphate synthase, appears to direct phytate biosynthesis (Yoshida
etaL,l999)

There is a growing literature regarding how phytate biosynthesis occurs. The
pathway was first elucidated in the cellular slime mold, Dictyostelium (Stephens and
Irvine, 1990). Phytic acid biosynthesis occurs via stepwise phosphorylation of myo-
inositol (3) P. (abbrev. Ins (3) P). Stephens and Irvine (1990) found the biosynthesis
to proceed as follows: Ins (3) P -> Ins (3,6) P2 -* Ins (3,4,6) P; —> Ins (1,3,4,6) P4
-* Ins (l ,3,4,5,6) P5 -* InsP6. By 1996, the pathway was elucidated in the plant
kingdom. In the organism Spirodela polyrhiza, Brearly and Hanke (1996a,b) found

the pathway for phytate biosynthesis to be: Ins (3) P -* Ins (3,4) P2 -* Ins (3,4,6) P3

-’ Ins (3,4,5,6) P4 -’ Ins (l,3,4,5,6) Ps-e InsP6, which was different than the pathway
in Dictyostelium. Interestingly, lower inositol phosphates, InsP. through InsP4
apparently have a role as intracellular messengers in plants and animals (Urbano et
al., 2000), but in the phytic acid biosynthesis pathways elucidated in Dictyostelium
and S. polyrhiza, there was no obvious direct link for the intermediates to function as
signal molecules (Brearley and Hanke, 1996b). This indicates that the lower inositol
phosphates involved in signaling may be phosphorylated at different positions of the
inositol ring than are the intermediates of phytic acid.

The branch of the pathway prior to the stepwise phosphorylation of Ins (3) P
is the cyclization of D-glucose-6-phosphate to Ins (3) P, catalyzed by D-myo-inositol
(3) Plsynthase (MIPS) (Figure 2.). This step is not unique to phytic acid
biosynthesis (Loewus, 1990). In fact, most of the synthesized Ins (3) P is
dephosphorylated to free myo-inositol. Myo-inositol is an important sugar in plants
and is a precursor to compounds with a diverse array of functions including signal
transduction, stress response, cell wall structure, and hormonal homeostasis (Loewus
and Murthy, 2000). Despite its role in numerous cellular processes, characterization
and cloning of the MIPS enzyme gene has been helpful in understanding the role of
phytic acid in the plant. The MIPS gene was cloned in a number of plant species,
including Arabidopsis thaliana (Johnson 1994), Citrus paradisii (Abu-Abied and
Holland, 1994), Mesembryamhemum crystallinum (Ishitani et al., 1996), Nicotiana
tabacum (Hara et al., 2000), P. vulgaris (Wang and Johnson, 1995), and Glycine
max (Hegeman et al., 2001). The availability of the sequence has made possible

experiments that demonstrate the role of the MIPS enzyme in myo-inositol synthesis,

0
0H 5 O O“ P- —i—L—0H
H
4 OH H 1 (I)H
H 3 2 H
H OH
glucose-s-phosphate
D-myo-inositol(3)Plsynthase (MIPS)
H O—P
OH OH inositol monophosphatase
___¥
‘—
inositol kinase
H H
OH H

 

inositol (3) phosphate,

 

 

 

H
OH
H
O—P H inositol (1 ,3,4,5,6) phosphate5
inositol (3,4,6) phosphate3 /
ins (l,3,4,5,6) 5 phosphate 2-kinase
H O—P
o/P O—P
O—P H
H 0"”
H H
O—P H

phytic acid (inositol phosphates)

Figure 2. Phytic acid biosynthesis pathway

and the central role of myo-inositol in the plant. Keller et al. (1998) used antisense
technology to develop transgenic potato (Solanum tuberosum L.) lines with the
MIPS gene knocked out. These transgenic lines had a 93% reduction in leaf inositol
levels compared to the wild type. Raffinose and galactinol levels were reduced by
90%. Additional altered phenotypes were also observed in the transgenic plants,
including altered leaf morphology, reduced apical dominance, and early leaf
senescence (Keller et al., 1998).

Phytate breakdown occurs in germinating seeds. Phytate is hydrolyzed into
inorganic P, lower P esters of myo-inositol, free myo-inositol, and cations by a class
of nonspecific phosphomonoesterases called phytases (Reddy et al., 1989). Phytases
catalyze the stepwise removal of the inorganic orthophosphate from phytate. Data
obtained from monocot seed phytases of spelt (T riticum spelta L.), rye (Secale
cereale L.), barley (Hordeum vulgare L.), and oat (Avena sativa L.) presents a
generalized scheme for the action of phytases (Fig. 3) (Greiner and Alminger, 2000).
The lower inositol phosphates produced by the action of plant, microbial, and fungal
phytases are different from inositol phosphate intermediates of the phytic acid
biosynthetic pathway (Brearley and Hanke, 1996b). The 3-phytases appear to be
characteristic of microorganisms, they act by hydrolyzing InsP6 to Ins (1,2,4,5,6) P5,
with the exception of Paramecium and Escherichia coli, which both have 6-phytases
(Greiner and Alminger, 2000). In the seeds of higher plants, 4-phytases or 6-
phytases act on phytic acid (Cosgrove, 1980). Most plant phytases, including 4-

phytases and 6-phytases, have optimal activity at low pH.

Ins(1,2,3,4,5,6)P6 (Phytic acid)

Phytase

Ins (1,2,3,5,6) P5
j Phytase

Ins (l,2,5,6) P4

¢ Phytase

Ins (1,2,6) P,

l Phytase

Ins (1,2) P2

¢ Phytase

Ins (2)P

Myoinositol monophosphatase

Free myoinositol

Figure 3. Phytase action on phytate in seeds (Greiner and Alminger, 2001)

Barrientos et al. (1994) characterized a 5-phytase in lily (Lilium longiflorum L.)
pollen with a pH 8 optimum. Unlike acid phytates, this alkaline phytase could not
dephosphorylate beyond InsP3. The InsP4, InsP3, and Inst isomers produced by the
alkaline phytase also differ from those of the acid phytates, and to this date, the
physiological significance of these intermediates has not been established.

There is a small amount of enzymatic activity in non-germinated seeds, but
phytases are most active during germination. Lolas and Markakis (1977), showed
that the phytase activity of navy bean (P. vulgaris) increased slightly during the first
two days of germination, followed by a 7-fold increase in activity from day 2 to day
6 of germination. A gradual decrease in activity after day 6 was noted (Lolas and
Markakis, 1977). The optimal temperature for phytase activity in navy bean was
50°C. Seeds may contain pre-existing phytase, but additional phytase is synthesized
during germination (Cosgrove, 1980). Phytase induction and synthesis appeared to
V be activated, in part, by low orthophosphate levels in germinating seedlings (Gibson
and Ullah, 1990), Similarly, synthesis and activity of phytase was inhibited by high
levels of orthophosphate (Kikunaga et al., 1991).

In addition to its importance as a cation storage molecule, phytic acid has
recently been shown in potato (S. tuberosum) to be a component of a Ca2+
dependent signaling pathway of stomata guard cells that inhibits K+ inward flux
(Lemtiri-Chlieh at al., 2000). Additional undiscovered functions of phytic acid may

exist in plants.

Role of Phytic Acid in Human Nutrition

Much of the interest in phytate has traditionally been based on its role in human
and animal nutrition. Under human physiological conditions, some phytate
complexes are insoluble, whereas others are soluble during digestion. The liberated
phytic acid that was part of the soluble complexes forms new complexes with
minerals that are present in the gut (Schlemmer et al., 1995). This process decreases
the bioavailabity of phosphorus and cations, especially iron and zinc, stored as a part
of the complex.

Phytic acid and Zinc Bioavailability:

Numerous studies, using a variety of experimental techniques, have shown a
correlation between high phytate diets and limited zinc absorbance in the
gastrointestinal tract of humans and animals (Saha et al., 1994; House et a1, 1982;
Tumlund et al., 1984; Sandstrom et a1, 1988; Lonnerdaleta1., 1988; Hunt et al.,
1998; Zhou et al., 1992). Zinc is a micronutrient required in the human diet. The
recommended daily allowance established for people of the United States is 12 to 15
mg for adults and 10 mg for children. However, this amount of zinc may be too low
for people who consume phytate rich vegetarian diets. Zinc deficiency in humans is
widespread, and is due not only to the zinc content of the diet, but also to its
bioavailability. Although extreme zinc deficiency is rare, marginal deficiency has
been documented in North and South America, and parts of Europe, Asia, and Africa
(Welch, 1993). Based on national food balance data collected by the FAO, it is
estimated that 49% of the human population is at risk for inadequate zinc in their diet

(International Zinc Association, 2000). Marginal zinc deficiency in humans can

10

cause dermatitis, a weakened immune system, prolonged wound healing, taste
dysfunction, impaired dark vision, diarrhea, anorexia, decreased learning capacity,
and depression. Zinc deficiency can also cause abnormal fetal development,
premature births, and reduced growth rates of infants and children (Hambidge,
2000).

Dialysis is one method used to estimate Zn bioavailability by simulating human
digestion in vitro. In addition to in vitro studies, in vivo studies have been conducted
to determine the effect of phytate on zinc bioavailability in laboratory animals,
typically rats. In vivo experiments involved feeding intrinsically labeled 65Zn to rats
as part of controlled diets, followed by measuring the radioactivity to determine Zn
absorption. Similar zinc radioisotope experiments can be safely conducted in
humans. Radionuclide labeled 65 Zn can be used with whole body counting to
measure post-prandial Zn absorption (Wise, 1995). Stable isotope 67Zn can be safely
used in humans. In addition, in animals and humans, nutritional balance studies
have been conducted to estimate absorption by feeding a know amount of zinc in the
diet and measuring the amount in the feces (Wisker et al., 1991). Numerous zinc
absorbance studies in humans and animals have indicated that the phytic acid/zinc
molar ratio (moles phytate/moles zinc) is useful to predict possible zinc deficiency
based on composition of the diet (Torre, 1991) (Oberlas and Harland, 1981).
Typically, total diet phytate/zinc molar ratios of greater than 12 to 15 indicate
chemical zinc deficiency (Reddy et al., 1989).

The majority of experimental evidence for the inhibitory effect of phytic acid on

zinc absorbance has been conduced in laboratory animals. House et al, (1982)

11

combined 65 Zn tracing and nutritional balance studies in rats to examine how the
addition of 2% sodium phytate to a diet affected zinc bioavailability. These authors
(House et al., 1982) observed a decrease in true zinc absorption from 46.8% in the
basal diet to 32% in the phytate diet. Zhou et al., (1992) found that reducing the
amount of endogenous phytate in soybean by limiting phosphorus to the growing
plant or by altering processing methods increased zinc bioavailability in weanling
rats. The results were based on the measurement of the tibia zinc content of the rats
(Zhou et al., 1992). Saha et al. (1994) studied Zn bioavailability in rats with
intrinsically labeled whole-wheat flour. These authors (Saha et al., 1994) also
measured endogenous phytate instead of sodium phytate. Diets that were 0.19%
phytate had higher zinc absorption than diets that contained 1.64% phytate, whereas
there was no difference in zinc absorbance between diets that contained 1.64% and
1.85% phytic acid.

Since phytic acid (InsP6) is just one of the inositol phosphates found in grain and
legumes seed, Lonnerdal et al (1989) looked specifically at how phytic acid and each
of the lower inositol phosphates affected 65Zn absorption in rats. Phytic acid (InsP6)
was found to strongly inhibit zinc absorption; InsPs exhibited decreased inhibition
compared to InsPé, whereas InsP, and InsP4 showed no inhibition, indicating reduced
dephosphorylation of phytic acid can positively affect zinc absorption.

Although laboratory animals are used to understand the relationship between
phytate and zinc bioavailability in humans, animal diets are different from those of
humans and extrapolations should be made with caution (Wise, 1995). Studies on

human subjects have been conducted to a lesser extent than for animals. Tumlund et

12

al. (1984) used stable isotope 67Zn to determine zinc absorbance in four adult human
males fed controlled diets for 63 days that varied over time in sodium phytate and or-
cellulose levels. Average apparent zinc absorbance was 34% in the basal diet with a
range of 21.9% to 51.3%. With the addition of 2.34g of phytate to the diet, an
amount equivalent to a phytate/zinc molar ratio of 15, the apparent mean zinc
absorbance fell to 17.5% with a range of 14.2% to 24.9%. Sandstrom et al. (1989)
used 65Zn in humans to study the effect of various animal and plant protein sources
on zinc absorbance. Sandstrom et al. (1989) found that after the addition of fish or
chicken as a protein source to a bean (P. vulgaris) meal, the zinc absorbance
increased 50 to 70% while the total zinc content remained constant. Hunt et al.
(1997) compared zinc absorbance with extrinsically labeled “Zn from human
subjects who maintained lacto-ovo vegetarian and omnivorous diets over an eight-
week period. The subjects who maintained the non-vegetarian diet had molar ratios
of phytate to zinc of less than five while subjects who maintained the lacto-ovo
vegetarian diet had phytate to zinc molar ratios of 5 to 15. The zinc content of
subjects maintaining the typical lacto-ovo vegetarian diet was estimated to be 10-
30% less than subjects maintaining a non-vegetarian diet and subjects maintaining
the lacto-ovo vegetarian diet absorbed 35% less zinc. The results of these studies
(Sandstrom et al., 1989, and Hunt et al., 1997) indicate zinc deficiency would likely
be greater in people who maintain vegetarian diets with a phytate to zinc molar ratio
greater than 15 than non-vegetarians. Molar ratios greater than 15 are often found in
diets with limited animal protein and protein from grain and legumes (Hunt et al.,

1997)

13

Phytic Acid and Protein Interaction:

Although phytic acid is best known for its role in reducing micronutrient
bioavailability, phytic acid has been implicated (Cheryan, 1980) in reducing protein
utilization as well. Two factors are involved in reduced protein utilization due to
phytic acid: First, phytic acid can form complexes with proteins, which may
decrease their solubility. Interactions between phytic acid and protein are most
prevalent at low pH (Cheryan, 1980). Second, phytic acid may reduce activity of
protein digestive enzymes. In vitro studies showed that phytic acid reduced the
activity of the proteases pepsin and trypsin (Knuckles et al., 1989), (V aintraub and
Buhnaga, 1991). However, in vivo studies showed no effect of phytic acid on
protein digestive enzymes; the involvement of phytic acid in protein bioavailability
is inconclusive. Vaintraub and Bulmaga (1991) speculated that the discrepancy
between the in vitro and in vivo studies may have been due to differences in the
formation of phytate, enzyme, protein, or substrate complexes in each of these
systems, and most likely were affected by pH changes.

Phytic Acid and the Environment:

One side effect of the limited digestibility of phytate by monogastric animals is
that the majority of phosphorus in feed made from cereal or legume seeds is
excreted. For example, 80% of the P in a typical corn diet fed to swine and poultry
is in the form of phytate. The P is unavailable to these animals because they lack the
phytases necessary to hydrolyze phytate (Ertl et al., 1998). Animal waste is the
principle source of phosphorus pollution in agricultural systems (Ertl et al., 1998).

The average P in animal manures was estimated as follows: beef -5.6 g kg"; dairy-

14

11.7 g kg"; poultry layers-20.8 g kg"; broilers-16.9 g kg"; sheep-10.3 g kg"; swine-
17.6 g kg"; and turkeys-16.5 g kg". Phosphorus pollutes watersheds via runoff and
erosion of agricultural lands and causes eutrophication of streams, rivers, and lakes.
This form of pollution reduces value and use of surface water for fisheries,
recreation, industry and drinking (Sharpley et al., 1994). Lott et al. (2000) estimate
that 4.1 billion metric tons of field crops, fruits, and seeds are produced annually,
which contain 35 million metric tons of phytic acid - 9.9 million tons of which are
phosphorus. Lott et al. (2000) used these estimates in conjunction with the FAO
fertilizer yearbook (1995), and calculated that about 65% of the P fertilizer sold in
the world ends up as phytic acid phosphorus. This figure represents a significant loss
of P from soils, crops, and animals. Ertl et a1. (1998) studied the effects of feeding
swine and poultry diets consisting of low phytic acid corn. These authors (Ertl et al.,
1998) found that com with normal levels of phytic acid contained 3.8 g kg" total P
with 84% in the form of phytic acid, while the low phytic acid corn contained 3.9 g
kg" total P and only 33% as phytic acid. The animals fed the low phytic acid corn
diet had a 9 to 40% reduction in fecal P. Low phytate crop varieties may hold the
answer to effectively reducing the P pollution in the environment. This type of work
is in the preliminary stages and in the instance of Ertl et al’s work (1998), the low
phytic acid cultivars also had a significant yield reduction.
Ways to Reduce Phytic Acid in Foods:
One way to ameliorate the problems caused by phytate in human and animal

nutrition and the environment it to reduce its levels in food. Scientists have

15

examined genetic variability, plant production conditions and post harvest seed
handling as possible means to reduce seed phytic acid.
Seed Preparation:

Preparation, cooking, and processing methods all influence the amount of
phytate present in foods. Schlemmer et al. (1995) found that soaking soya bean
(Soja hispida Max.), mung bean (Phaseolus aureus Roxb.), brown bean and white
bean (P. vulgaris), green pea and yellow pea (Pisum sativum L.), or lentils (Lens
esculenta Moench) for 8, 16, or 24 hours at 22°C had no affect on phytic acid levels
in the seed. A number of other studies (Reddy et al., 1989; Greiner and Konietzny,
1998) have also shown that soaking does not change seed phytic acid levels in
legumes. The effect of standard cooking in boiling water for two to three hours has
been more variable. Schlemmer et al. (1995) found that cooking caused the
hydrolysis of a minimal amount of the phytic acid to InsPs. On the other hand,
Greiner and Konietzny (1998) found that cooking reduced the phytic acid content in
beans 16 to 24% with a corresponding increase in InsPs, InsP4, and InsP3. Thermal
processing (canning) of foodstuffs has also been examined as a means to reduce
phytic acid, but results were conflicting in regard to effect. For example, Schlemmer
et al. (1995) found canning had no effect, whereas Tabekhia and Luh (1980) showed
a 70 to 75% reduction in phytic acid with canning in beans (P. vulgaris).

The processing methods that have lead to the sharpest decline in phytic acid
are those that activate phytase. For example, a 10-hour, 60°C water bath heat
treatment (optimal temperature of phytase) of navy bean caused a 90% drop in seed

phytic acid (Chang et al., 1977). Fermentation also activates endogenous phytases of

16

yeast and other microorganisms that significantly reduce phytic acid. Kannan et al.
(2001) found fermented black bean (P. vulgaris) had 10% less phytate than the non-
ferrnented control. Conversely, a number of compounds sometimes used in the
fermentation process, especially calcium and magnesium salts have been shown to
inhibit phytate hydrolysis, thereby preventing a significant reduction in seed phytic
acid (Reddy et al., 1989). One way to circumvent the inactivation of phytase during
fermentation is to add exogenous phytase to food. Phytase is a common additive of
animal feed used to improve the diet and limit phosphorus excretion in manure (Ertl
etaL,1998)

Plant Growing Conditions and Genetic Intervention:

The principal environmental factor that affects the content of seed phytic acid is
available soil P. A strong linear relationship between P fertilizer levels and seed
phytic acid concentration has been shown in the seed of a number of crops including,
soybean (G. max) (Lolas et al., 1976) wheat (Asada et al., 1969) and'oats (0. sativa)
(Miller et al., 1980). The reduction of P fertilizer is not a practical way to reduce
phytic acid levels because phosphorus is required for plant growth and seed yield
(Raboy, 1985).

Genetic factors that influence P uptake and translocation also influence seed
phytic acid content (Raboy and Dickinson, 1993). Screening of dry bean (P.
vulgaris) genotypes from North America, Central America, South America, Europe,
and Africa has revealed significant variability in seed phytic acid levels (19.6 to 27.5
umol g") (Welch et al., 2000). In order to reduce seed phytic acid without also

reducing seed total P, seed phytic acid specific genetic factors must be identified.

17

When one only examines the variability in phytic acid among cultivars of a species,
it is impossible to determine if the differences are due to factors influencing P uptake
or phytic acid synthesis (Raboy and Dickinson, 1993).

It has been possible to separate genetic factors controlling total seed P from
phytic acid P. Mutation breeding has been used to develop low phytic acid lines in
maize (Zea. mays) (Larson and Raboy, 1999), barley (Hordeum vulgare L.) (Larson
et al., 1998), rice (0. sativum) (Larson et al., 2000), and soybean (G. max) (Wilcox
et al., 2000). Raboy et al. (1990) studied a series of mutations in maize that affected
germ and aleurone tissue. These authors found that certain germ mutations had low
levels of phytic acid and abnormally high concentrations of inorganic phosphorus,

. suggesting tissue specificity for phytic acid synthesis in maize. Two non-allelic low
phytic acid mutants were found in maize and barley, and they were classified as lpal
and lpa2 (Larson et al., 1998; Raboy et al., 2000). The lpal mutant showed a 66%
reduction in phytic acid phosphorus and a corresponding increase in inorganic P.
The lpa2 mutants similarly had a 50% reduction in phytic acid P and a corresponding
increase in Pi, but unlike the lpal mutants, the lpa2 mutants also showed an increase
in lower inositol phosphates compared to the wild type. Larson and Raboy (1999)
hypothesize that the lpal mutant interrupts an early step in the phytic acid
biosynthesis pathway, possibly the MIPS gene or a MIPS regulatory gene. The lpa2
mutant, on the other hand, appeared to affect a step later in the pathway. A genetic
mapping experiment showed that one lpal mutant of maize mapped to same region
of chromosome 18 of maize; thus providing additional evidence that the mutation

affected the MIPS gene. In barley, the lpal mutant did not map to the same region of

18

the genome as MIPS, suggesting this mutant may be a trans-acting factor (Larson
and Raboy, 1999). Raboy et al. (2001) tested low phytic acid maize mutants to
determine their agronomic performance. Fourteen near-isogenic maize hybrid pairs
were created with an lpal mutant or its non-mutated counterpart serving as a parent
in each of the pairs. The mutants had no effect on germination, stalk strength,
flowering date, or grain moisture at harvest, but each of the mutant classes showed a
4% to 23% reduction in seed yield as compared to the wild type. An animal feeding
study with the grain showed that the low phytic acid mutants had 50% more
available P that resulted in a 50% reduction in P waste (Spencer et al., 2000).

The best strategy to reduce seed phytate levels may be by finding and using seed
specific promoters to the MIPS or other genes in the phytic acid pathway. There is
evidence in soybean for the existence of a family of MIPS genes important in
different plant parts. Hageman et al. (2001) found four DNA sequences that
hybridized to the MIPS clone. One of the sequences, GmMIPSl, was expressed in
immature cotyledons and was developmentally regulated indicated by its reduced
expression levels as the seed matured.

Low phytic acid mutants have been identified in many major grain and legume
crops, including maize, rice, barley, and soybean. These crops are staples in the diet
of humans and animals around the world. Dry bean is a valuable, protein rich
legume missing from this list. The identification of a low phytic acid genotype in
this crop would be very useful for the development of bean cultivars with improved
micronutrient bioavailability. The detection and selection of beans with increased

density of zinc is also an important strategy to improve the micronutrient value of the

19

bean seed. Preliminary studies are necessary to understand the interaction of phytic
acid and zinc in beans under different environmental conditions, the inheritance of
zinc and phytic acid levels in the bean seed, and the variability of these seed
components in existing gerrnplasm. Once these essential steps are conducted, the
best suited procedure to develop a bean with increased zinc density and decreased

phytic acid density should be straightforward to identify.

20

CHAPTER 1: PHYTIC ACID, PHOSPHORUS, AND ZINC
ACCUMULATION AND THEIR INTERACTIONS IN THE SEED
OF A ZINC EFFICIENT AND N ON-EFFICIENT NAVY BEAN

C ULTIVAR

INTRODUCTION

Improving the zinc nutritional value of dry bean (Phaseolus vulgaris L.) for

human consumption requires an understanding of the relationship among phosphorus

 

(P), phytic acid (PA) and zinc (Zn) in the seed. Phytic acid is a storage form of
phosphorus found in all plant seeds; it is most abundant in cereal and legume seeds
(Oberlas, 1983). PA is a polyanion at physiological pH and an effective chelator of
nutritionally important mineral cations such as calcium, zinc, and iron (Raboy et al.,
2001). The binding of Zn by PA in beans can contribute to zinc deficiency in human
populations (Hambidge, 2000).

The relationship between P and Zn has been studied in the soil, and in roots and
shoots of numerous plant species, including dry bean. However, neither the P and
Zn relationship, nor the PA, P, and Zn relationship has been studied in dry bean
seeds. Although, genetic differences for plant response to low zinc soils have been
identified in bean, the responses to P and Zn fertilizers have not been characterized
in the seed of this crop.

Uptake of Zn by plant roots occurs predominately through diffusion of the
divalent cation (Znfl) from the soil solution. Soil type, pH, temperature and P level

(Moraghan and Mascagni, 1991) influence zinc availability in the root zone.

21

Genotypic differences in susceptibility to deficient soil zinc of P. vulgaris were
initially identified in studies with the navy bean cultivars Sanilac and Saginaw
(Ambler and Brown, 1969). In low Zn soils, plants of both cultivars developed
visual symptoms of Zn deficiency (necrotic and discolored leaves) although
‘Sanilac’ had the more severe symptoms. ‘Sanilac’ also had lower yield and reduced
shoot Zn content compared to ‘Saginaw’, but at normal soil Zn levels, the two
cultivars had comparable yields (Ambler and Brown, 1969). In addition, when
grown in Zn deficient soils, ‘Sanilac’ had more P in the shoots than ‘Saginaw’, but

‘Saginaw’ had more P in the roots than ‘Sanilac’.

Zinc efficiency is the term used to describe a genotype that has a higher yield
than a standard (‘check’) genotype when grown in soils limiting in Zn for the check.
‘Saginaw.’ is an example of a Zn efficient genotype and ‘Sanilac’ is an example of a
Zn inefficient one. The Zn inefficiency trait in navy bean is believed to have
originated in ‘Sanilac’. This cultivar originated as an X-ray mutant and was the first
navy bean cultivar with a determinant growth habit (Judy et al., 1965). ‘Sanilac’ was
then used as a parent to transfer the determinacy trait into many other navy bean
cultivars. Many of the early determinant navy bean cultivars are in the pedigrees of
current day navy bean cultivars. ‘Sanilac’ was zinc inefficient and supposedly this

trait was transferred to other navy beans along with ‘Sanilac’s’ determinacy trait

(Kelly, 2000).

Zinc efficient navy bean cultivars also accumulate higher levels of seed Zn than
Zn inefficient ones. Moraghan and Grafton (1999) showed that ‘Norstar’ and

‘Voyager’- both Zn efficient- and ‘Albion’ and ‘Avanti’- both zinc inefficient- had

22

similar seed Zn concentrations in the absence of added Zn, but the efficient cultivars
had 15 to 20% higher seed Zn than the inefficient cultivars when Zn was added to
the soil. The response of ‘Voyager’ and ‘Albion’ seed [Zn] when Zn was added to
the soil was observed under both greenhouse and field conditions. Under field

conditions, with no added Zn, ‘Albion’ had the highest seed P of the four cultivars.

_b_"- 'l'Zg‘.- “I.

This may indicate that the differences in Zn efficiency noted among the cultivars
may be based on a plant’s ability to limit excessive P uptake when Zn in the soil is
limiting.

A high level of soil phosphorus is an environmental factor that can influence

 

 

plant zinc deficiency in P. vulgaris, especially where available soil Zn is low. This
phenomenon, known as P induced Zn deficiency is believed to be caused by a
number of factors. The principle factor is a dilution effect such that the increased
plant tissue grth due to the presence of added P causes a dilution of Zn
concentration in the whole plant (Moraghan, 1980). Additional proposed
mechanisms include: 1) P and Zn interact in the soil and limit zinc availability
(Brown et al., 1970). 2) Phosphorus inhibits the translocation of Zn from roots to
shoots (Singh et al., 1988). 3) Zinc deficiency promotes the increased transport of P
from the roots to the leaves resulting in P toxicity symptoms (Loneragan et al., 1979
and Loneragan et al., 1982). 4) High P soils inhibit mycorrizal root infection, which
decreases Zn uptake (Singh et al., 1986).

Detailed studies ofP induced Zn deficiency in P. vulgaris have indicated
possible mechanisms of the interaction. Singh et al. (1988) found the shoot

concentration of Zn to decrease sharply as the amount of P added to the soil

23

increased, but the addition of P beyond 40 mg P kg" did not greatly influence Zn
concentration further. McKenzie and Soper (1983) found that the presence of added
Zn (8 mg Zn kg" soil) prevented a P induced reduction of Zn concentration in plant
tissue. Zinc deficiency appeared to be caused by two factors. First, zinc
concentration was diluted in plant tissue because added P fertilizer caused an
increase in plant mass. Second, in the presence of high levels of P, there was
restricted translocation of Zn from the roots to aboveground plant parts in low soil
Zn in certain genotypes.

Since the Zn efficiency trait affects seed Zn concentration, it may also influence
seed P and PA content, especially considering that increased P fertilizer causes a
decrease in the concentration of Zn [Zn] in the seed. This study was undertaken
because of the lack of information on how soil P and Zn affect [Zn] in the seed of P.
vulgaris. In addition, Zn is an essential micronutrient for human nutrition whose
bioavailability is reduced by phytic acid, and understanding the relationship between
PA and Zn in the seed may be helpful for improving the Zn nutritional value of the
seed. The objective of the study was to ascertain the relationship among seed P, PA,
and Zn in a Zn efficient and Zn inefficient navy bean cultivars grown under different

treatments of P and Zn fertilizers.
MATERIALS AND METHODS

‘Voyager’ and ‘Albion’ were the experimental material used in this study and
are known to be Zn efficient and non-efficient cultivars, respectively. ‘Voyager’ and
‘Albion’ were grown in pots in a greenhouse at North Dakota State University. Five

rates of Zn (0, 0.5, 1.0, 2.0, and 4.0 mg ZnSO4-Zn kg") and three rates of P (0,

24

60,120 Ca (H2P04)2-P kg") fertilizer were applied in a factorial combination to each
of the cultivars, for a total of 30 treatments. Each treatment was replicated four
times, and the experiment was arranged in a randomized complete block design with
pots as the experimental unit. The soil used in each pot was a Wheatville loam
(coarse-silty over clayey, mixed over smectitic, superactive, frigid Aerie
Calciaquolls) that had a pH of 8.2 and contained 12.2 g inorganic C kg", 8.0 mg
NaHCO3-extractable P kg", 0.35 mg DTPA-extractable Zn kg" , 4.5 mg DTPA-
extractable Fe kg", and 25 mg NO3-N kg". A basal dressing of 70 mg NH4NO3-N
kg", 90 mg KZSO4-K kg" was mixed with the soil before addition to the pots, and 1
mg Na2B4Oy-B kg" and 2 mg Fe EDDHA-F e kg" were applied in liquid form to
each pot. Eight seeds were planted per pot, and thinned to 3 plants per pot 7 days

after emergence. Pots were watered as needed with distilled, deionized water.

Pods from each experimental unit (i.e., 3-plants per pot) were harvested when
80% of them had changed from green to pale yellow or brown (mature color). At the
time of the final pod harvest, pod walls, stern tissue, and leaves were separated and
oven dried at 50°C for 72 hours, weighed, ground in a stainless steel mill, and
analyzed for Zn and P. Two subsamples of seed from the 3-p1ants of each pot were
washed with distilled water, oven dried at 70°C for 24 hours, finely ground to fit
through a sieve with 0.25 min openings. Total P was measured by a molybdenum
blue procedure and Zn was measured by atomic adsorption. For each analysis, 0.5 g
of seed was ashed in a muffle furnace at 500°C for 6 hours. Next, 25ml of 3N
HNO3 was added to the seeds. Afier one hour the mixture was filtered to remove

undissolved particles. These samples were directly analyzed for Zn concentration by

25

 

atomic adsorption spectroscopy. For P analysis, the samples were diluted 10:1 v:v in
0.3N NaOH. Following the initial dilution, they were diluted again 15:1 v:v with
2.5N sulfuric acid containing 0.006% ammonium molybdate, 0.0002% antimony
potassium tartrate, and 0.005% l-ascorbic acid. The sample absorbance was read on

a Brinkmann dipping probe with a 880nm filter.

An additional subsarnple of seed was freeze dried, ground and analyzed for PA.
Sample preparation for the analysis of phytic acid concentration [PA] in bean seed
was conducted following the procedure of Lehrfeld (1980). Phytic acid was
extracted from the samples by the addition of 3ml 0.5M HCl (trace element grade) to
0.100 g’of sample. The acidified samples were mechanically agitated for 2 hours at
21°C. Samples were centrifuged at 12,000g for 15 minutes. The supemant was
collected and diluted 1:5 (v/v) with distilled, deionized water. The diluted sample
(15 ml total) was passed through a Bond Elut strong anion exchange column (Varian,
Walnut Creek, CA) for purification. The column was washed once with 10 ml 0.05
M HCl, and PA was eluted with 3m] 2M HCl. The eluted fraction containing PA
was air dried and dissolved in 5mM sodium acetate. The dissolved sample was
filtered through a 2 pm filter. Phytic acid levels in each sample were quantified by
high performance liquid chromatography with a refractive index detector. The
column used for analysis was a Waters Symmetry C18 column (3.9mm x 150mm)
(Waters, Milford, MA) heated to a temperature of 40°C. Sodium acetate (5mM) was
used as the solvent at a flow rate of 1.4 ml min". Phytic acid dodecasodium salt

from corn (Z. mays L.) (Sigma, St. Louis, MO) was used as a standard to determine

26

PA concentration. The PA values were divided by 3.55 to obtain P stored as PA

(PA-P) values (Lott et al., 2000).

All statistical analyses were conducted using SAS software (SAS Institute, Cary,
NC, 1985). A paired t-test was used to determine differences between ‘Albion’ and
‘Voyager’ at different fertilizer treatments for seed P, PA-P, non PA-P, P as PA-P,

Zn, and seed yield.

RESULTS AND DISCUSSION

Analyses of Variance, (Table A1) indicated that P and Zn fertilizer treatments
and P x Zn interactions influenced the concentration of PA-P in the seed, but the
cultivar effect for this character was not significant. The general trend noted for
‘Albion’ and ‘Voyager’ was that as P fertilizer increased, seed [PA-P] also increased
and as Zn fertilizer increased, seed [PA-P] decreased except at the lowest level of P
(Figure 4A & Table I). The concentrations of seed P not stored in PA [non PA-P]
and total seed P [P] were significantly affected by P and Zn fertilizer treatments and
cultivar. The [P] and [non PA-P] were significantly affected by P x Zn, cultivar x
Zn, cultivar x P, and cultivar x P x Zn interactions (Table A1). The [non PA-P]
tended to increase as P fertilizer increased and decrease as Zn fertilizer increased
(Table 1; Fig 4B). The striking difference between seed [non PA-P] and [PA-P] was
that [PA-P] increased almost identically in ‘Albion’ and ‘Voyager’ as added P was
increased but the [non PA-P] in the seed was cultivar dependent (Fig 4B). These
results are clearly shown in Figure 4A and B at the 60 mg kg" soil and 120 mg kg"

soil P fertilizer treatments.

27

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29

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Mn .8 _. d4

 

 

Seed PA-P and Non PA-P content (Table 2 and Fig. 5) which is the amount
of each of the components per experimental unit (seed from 3 plants), showed
similar patterns of accumulation as compared to PA-P and non PA-P concentration,
respectively (Table l and Figure 4). One notable exception was the response by
‘Albion’ at 60 and 120 mg kg'l P fertilizer treatments to the addition of 0.5 mg kg"
Zn fertilizer treatment. The large increase in PA-P and Non PA-P content resulted f
from a corresponding increase in seed yield (Fig. 6) at these P levels with the
addition of 0.5 mg kg" soil Zn. The concentration of seed PA-P and Non PA-P did

not increase because of the dilution effect caused by the increased seed yield.

 

The various concentration responses noted in the seed when P and Zn fertilizer
levels were increased provided a basis for making inferences from the data. First,
the levels of seed [Non PA-P] were more tightly controlled than seed [PA-P] such
that when excess P was added to the soil, the P most likely was translocated into seed
PA rather than in some other form of P. This was especially true when the levels of
P fertilizer were above 60 mg kg" soil. The data upon which this assumption is
based is limited to measurements of seed P. Previous studies in common bean where
the effects of P fertilizer on P in other plant parts were investigated also support this
assumption. For example, Lynch ct al., (1991) indicated that as P fertilizer increased
leaf [P] also increased. As P available to the roots increased so did the amount of P
transiocatcd to the shoots (ranging from 20 to 80% of P in roots). On the other hand,
remobilization of Zn and P to the seed from shoots and leaves remained constant
under low or high P fertilizer conditions at 60 to 67% of the total Zn and P in the

shoots and stems (Snapp and Lynch, 1996). The results of the current study

32

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33

suggested that, in general, as P fertilizer increases a higher proportion is expected to
accumulate in the seeds because of the increased translocation from the roots to the
shoots. The work of Snapp and Lynch (1996) support this suggestion. The
differences in seed [PA-P] and [Non PA-P] noted at each treatment level could also
be due to ‘Voyager’ and ‘Albion’ sharing common alleles for the genes that control
the regulation of PA synthesis in the seed. However, the levels of at least one of the

forms of non PA-P in the seed may be regulated differently in ‘Voyager’ as

liar.- i Bar: “Hill

compared to ‘Albion’. Non PA-P is found by subtracting PA-P from total P and

includes components of molecules such as DNA, RNA, sugars, phospholipids,

[lmdp‘t‘i- .4 153th r S

phosphate esters (other than phytic acid) and free inorganic phosphate.

Yield differences between ‘Albion’ and ‘Voyager’ in response to Zn fertilizer
treatments are shown in Figure 6. At the fertilizer treatments 0 mg kg" soil Zn, 120
mg kg’lsoil P and 0 mg kg"soil Zn, 60 mg kg’lsoil P, ‘Albion’ showed a markedly
reduced seed yield compared to ‘Voyager’ at the same fertilizer levels. The yield of
‘Albion’ was significantly increased by added Zn fertilizer, and although ‘Voyager’
also showed a yield increase in response to added Zn, it was less dramatic than the
response of ‘Albion’. For example, at the 120 mg kg'lsoil P, 0 mg kg"soil Zn
treatment, the seed yield of ‘Albion’ was 50% lower than at the 60 P mg kg'lsoil, 0
mg kg"soil Zn treatment. The seed yield for ‘Albion’ at 0 mg kg"soil P, 0 mg kg'
lsoil Zn was two to three times higher than at the 60 mg kg'lsoil P, 0 mg kg'lsoil Zn
and 120mg kg"soil P, 0 mg kg " soil Zn treatments, respectively. These data
indicated that a plant response to phosphorus was a key factor in differentiating

‘Voyager’ (Zn efficient) from ‘Albion’ (Zn inefficient).

34

In addition to differences in yield, ‘Albion’ and ‘Voyager’ also showed
differences in levels of total P accumulation. ‘Albion’ had significantly higher levels
of seed P than ‘Voyager’ at the 60 mg kg'lsoil P, under all levels of Zn fertilizer, and
at 120 mg kg'lsoil P under 0, 0.5, and 1 mg kg'lsoil Zn treatments (Table l).
Moraghan and Grafton (1999) saw similar higher levels of P in the seed of Zn
inefficient navy bean cultivars as compared to efficient cultivars at low levels of soil
Zn. However, Moraghan and Grafton (1999) did not look at the effect of different P
fertilizer levels on the concentration of P in the seed of the cultivars. For the current
study, there was different P fertilizer treatments in addition to different Zn fertilizer
treatments. Based on these data, phosphorus induced Zn deficiency may be the
cause of the Zn inefficiency characteristic of ‘Albion’. One may speculate that the
gene(s) that control Zn efficiency in navy bean also may be involved in P regulation
in the seed or the mechanism of Zn efficiency may be related to the plant’s ability to
exclude P from the shoots and seeds.

Zn efficiency in bean not only influenced seed yield in low Zn soils, but also
seed [Zn] (Table 3). At 0 mg kg’1 soil P, at all levels of Zn fertilizer, ‘Voyager’ had,
on average, 17% higher seed [Zn] than ‘Albion’. Also, at 60 mg kg" soil P and 120
mg kg" soil P at 2, and 4ug kg"Zn fertilizer, seed [Zn] in ‘Voyager’ exceeded the
seed [Zn] of ‘Albion’ by an average of 23%. In this study, ‘Voyager’ generally had
higher seed [Zn] than ‘Albion’ at low soil P levels and at intermediate to high soil P
levels when Zn levels in the soil were high (Table 3).

The results of this study may provide insight as to whether navy beans with a

high concentration of seed zinc have an improved Zn nutritional value over beans

35

 

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with a low concentration of Zn. Before this question can be answered studies should
be conducted to ascertain whether the results of greenhouse pot studies on seed [Zn]
correlate highly with field experiments. Second, culinary studies are necessary to
establish whether Zn levels of Zn efficient and inefficient cultivars are significantly
different afier beans are prepared for consumption. Finally, Zn feeding studies
involving Zn and PA are needed to determine what levels of seed [Zn] in navy bean
can improve human nutrition.

In dry bean, one must give consideration to [PA] in attempts to improve the

micronutrient density of this crop. Once consumed in human foods PA binds to

 

minerals in the intestinal tract to form mixed salts that are largely excreted (Raboy et :4
al., 2001). Hence, Zn bioavailability in humans whose diets consist mainly of
cereals and legumes is dependant not only on Zn intake but also the intake of PA.

In the present study, ‘Albion’ and ‘Voyager’ did not differ in their amount of
PA-P, but they differed in the amount of non PA-P and total P (Table A1). Hence,
one can conclude that ‘Albion’ and ‘Voyager’ differed significantly in the % of total
P stored as phytic acid at the 60 and 120 mg kg " P fertilizer treatment level (Table
4). ‘Voyager’ had a higher % of total P stored as PA than ‘Albion’ because of the
differences in the non PA-P fractions. The differences between ‘Voyager’ and
‘Albion’ noted in this study for PA-P and Non PA-P suggests that selecting plants
with low phytic acid or low % of total P stored as PA per se may not be the most
effective way to reduce seed phytic acid. Raboy et al. (1990) suggested the use of
increased inorganic P levels in the seed as a selection criterion because total P and

PA-P are strongly correlated. By selecting for reduced PA-P one may also decrease

37

Table 4. The percent of phosphorus (P) in the phytic acid (PA) form in seed of
Voyager and Albion navy bean cultivars grown under three levels of
phosphorus and five levels of zinc fertilizer.

 

 

 

 

Soil treatment P in PA form Sig. of
P Zn Albion(a) Voyager(b) a—b at
__ mg kg" % P 9051‘,"
0 O 44.8 50.3 NS
0.5 47.0 50.8 NS
1 50.5 48.2 NS
2 49.6 46.6 NS
4 50.0 53.2 NS
60 0 51.3 67.6 **
0.5 51.6 60.2 **
1 52.5 60.4 **
2 52.3 59.1 **
4 49.4 56.4 **
120 0 52.6 63.4 **
0.5 55.8 62.4 **
1 58.5 60.1 NS
2 55.0 62.2 **
4 56.1 61.5 NS
ISE 2.24
§CV(%) 8

 

** significant at P $0.05, TNS: not significant at P $0.05
I SE = standard error of the mean
§CV = coefficient of variation

38

Table 5. The regression relationship (R2) between three phosphorus (P)
treatments added to the soil and the concentrations of phosphorus in phytic acid
(PA-P) and non-phytic acid (non PA-P) forms in seed of two navy bean
cultivars.

 

P added to soil (me P kg'l)

 

Cultivar 0 60 120 R2
------PA-P (mg g“)------

Albion 1.39 2.62 3.1 1 0.74

Voyager 1.35 2.67 3.16 0.75
----- Non PA-P (mg g")------

Albion 1.49 2.48 2.5 0.47

Voyager 1.36 1.69 1.94 0.57

 

Table 6. Multiple linear regression for the effect of cultivar and P and Zn
added to the soil on phytic acid phosphorus (Pa-P), non-phytic acid phosphorus
(non Pa-P), and zinc (Zn) in the seed of two navy bean cultivars.

 

 

 

 

Pa-P (Y1) Non Pa-P (Y2) Zn (Y3)

Overall R2 0.81 0.64 0.85
Correlation lrl p value [rl p value [r[ p value
Cultivar (X1) 0.01 NS -0.46 <.0001 0.24 0.008
P fertilizer

(X2) 0.86 <.0001 0.60 <.0001 -0.79 <.0001
Zn fertilizer

(X3) 026 0.0045 -0.26 0.0042 0.43 <.0001

 

39

the viability of the seed along with impairing its nutritional value. Seedling
emergence and dry weight accumulation are greater in P. vulgaris seed with high
levels of P as compared to seed with low levels of P (Teixeira et al., 1999). In the
current study, there was a strong linear relationship between seed [PA-P] and P
fertilizer level (r= 0.86) (Table 5 and Table 6) therefore by selecting for reduced
levels of PA, one would also be selecting for reduced total seed P. Phosphorus
fertilizer level was also correlated with seed [non PA-P], but the correlation was not
as strong as the correlation between P fertilizer level and seed [PA-P]. Based on the
results of the current study, the screening of germplasm based on Pi may prove
fruitful to lower [PA] in dry bean because a common phenotype seen in low phytic
acid mutants in other crops is an increase in Pi corresponding to the decrease in [PA-
P] (Raboy, 2001). Selecting germplasm for increased seed [Zn] appears to be a
promising method to increase the zinc density of the seed, which may improve zinc
bioavailability in terms of human nutrition. Not only was significant genotypic
variability found for seed [Zn], but seed [Zn] and [PA-P] were negatively correlated.
Figures 7, A1 and A2 show that seed [PA-P] and [Zn] are negatively correlated,
which indicates selection based solely on seed [Zn] will not cause an increase in seed
[PA-P].
CONCLUSIONS

The differences in seed yield, [Zn], and [P] in ‘Voyager’ and ‘Albion’ indicated
that the zinc inefficiency trait may be caused by P induced Zn deficiency and that the
signs of P induced Zn deficiency documented in roots and shoots of certain bean

cultivars is also seen in the seed of Albion.

40

‘Voyager’ (Zn efficient) had higher seed [Zn] than ‘Albion’ (Zn inefficient) at
various P and Zn fertilizer levels. Intrinsic differences between the cultivars for seed
zinc status may be useful to improve the zinc density of the seed to make more zinc
available in diets. ‘Voyagcr’ and ‘Albion’ did not differ in their amount of PA-P, but
seed [Zn] and [PA-P] were negatively correlated, indicating that a breeding strategy

to increase seed Zn should not cause a simultaneous increase in seed PA-P.

41

4.5 -

 

 

 

I
40 - " I r = -0.81**
‘ I I I
3.5-1
3.0- ‘ I'-
:8 . fi\ 5'-
' t
3 2'54 ' "l\ ' '-
‘ ‘§
I
5 2.0- I n "
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é . I . I I I
1.5- I
m d . II’. I I‘ I 3
H - I I 2:
1.0-
‘ I
05-
T I I I I I I I I I '

Zn (ug g")

Figure 7. Correlations between the concentration of zinc [Zn] and the
concentration of phosphorus in the phytic acid form [PA-P] in the seed of
Albion and Voyager navy beans grown under three levels of P (0, 60, and 120mg
P kg") and five levels of Zn (0, 0.5, 1, 2, and 4 mg Zn kg") fertilizer.

** significant at P $0.05

42

CHAPTER 2: INHERITANCE OF SEED PHYTIC ACID
PHOSPHORUS AND ZINC LEVELS IN THE SEED OF NAVY

BEAN

INTRODUCTION

Improving yield over a range of environments is generally the major goal of dry
bean breeding programs. However, bean breeders should not neglect food quality
improvements. Food quality in dry bean includes characteristics that have a direct
impact on human nutrition and those related to preparation for eating.

Breeding for improved nutritional quality is an endeavor that requires much
consideration. Enhancement of vitamins and minerals and reduction of antinutrients
in edible plant parts will not likely improve yield, and may actually decrease it
(Bliss, 1999). Except for economically important plant parts with enhanced carotene
or lycopene content, the appearance of nutritionally enhanced plants generally are
not noticeable to breeders, growers, or consumers. The benefits from improved
nutritional quality in a crop will only be realized by a population that has a
deficiency in the diet for the given vitamin or mineral. Do human populations exist
that can benefit from plant modification for increased nutritional quality? If one
considers the zinc status in humans, an estimated 49% of the world p0pulation is at
risk for low zinc intake (Brown et al., 2001). The areas of the world where people
are most at risk are South and Southeast Asia, Sub-Saharan Africa, and Latin
America and the Caribbean. In these regions, only 15 to 25% of the zinc is from

animal sources as compared to developed nations, where more than half the zinc

43

comes from animal sources. In many developing countries cereals and legumes are
staples in the diet. Phytic acid is an important antinutrient in these crops. The high
phytate: zinc molar ratios of plant based sources of zinc such as grains and legumes
as compared to animal sources of zinc is a major factor in determining the risk of
zinc deficiency (Frossard et al., 2000).

Zinc deficiency symptoms in humans include impairment of physical growth,
diarrhea, increased incidence of pneumonia and other upper respiratory infections,
decreased neurophysical performance, and impaired immune function (Hambidge,
2000). The diverse symptoms of zinc deficiency can be attributed to its central role
in many physiological processes. Zinc is essential for over 300 enzymes, hormones,
and structural proteins (Penny, 1998). Studies in Vietnam, Mexico, Guatemala,
India, Jamaica, Indonesia, Papua New Guinea, and Peru have demonstrated the
reduction of diarrhea and pneumonia associated with giving children daily zinc
supplementation (Penny, 1998). Supplementation and fortification are traditional
solutions that have been applied to nutrient deficiencies, and may also work to
decrease zinc deficiency in target populations (Ruel and Bouis, 1998). But, these are
not sustainable strategies and require a change in consumer behavior (Rengel et al.,
1999). On the other hand, increasing the bioavailable zinc in the diet through plant
breeding, offers a sustainable strategy that does not require a change in eating habits.

Plant breeding to meet consumer recommended dietary allowances (RDA) for a
given nutrient is a time consuming and costly process. Therefore a survey should be
conducted to determine the need, impact, and acceptability of a nutritionally

enhanced crop (Bliss and Quebedeaux, 1988). To maximize time and resources, the

44

breeder must possess some knowledge of the range of variability and the nature of
gene action of the trait.

Before a plant-breeding program is initiated to improve a particular trait, the
breeder must have knowledge of gene action involved in trait expression and the
degree to which genes are influenced by environmental fluctuation. Knowledge of
the type of gene action for a trait aids the breeder in ascertaining which breeding
procedure will efficiently improve the performance of a trait. Accordingly, a study
was conducted with three objectives: I) ascertain the inheritance of zinc and phytic
acid levels in navy bean. 2) determine the broad and narrow sense heritability of
phytic acid and zinc levels, and 3) discuss the results in terms of breeding strategies

to improve zinc content in dry bean seeds.
MATERIALS AND METHODS

‘Voyager’ a zinc efficient navy bean and ‘Albion’ a zinc inefficient navy bean

. were chosen for the study based on their differences in seed zinc concentration. A _
zinc efficient genotype has normal seed yield under low soil zinc cenditions, whereas
a zinc inefficient genotype has a reduced yield under low zinc soil levels that is
increased by the addition of zinc fertilizer. In 1998, seed of ‘Voyager’ and ‘Albion’
was planted in a greenhouse in a soil-less mixture (Sunshine Mix, Fison’s Inc.)
fertilized with 12-12-12 time-release fertilizer and watered daily. Upon flowering,
‘Voyager’ and ‘Albion’ were cross-fertilized to produce F1 seed. In 1999, F1 plants
were hybridized to each parent to produce backcross generations and F1 plants were
allowed to self-pollinate to produce F2 seed. Seed was harvested from each plant

individually and seed of each F1 plant was kept separate from F2 seed of other plants.

45

.V‘f'a'fl

 

l.‘.'_‘—“““:‘
. «n. I

In 1999 seed of the following generations: parent 1 (‘Albion’), parent 2
(‘Voyager’), F1, F2, BCIPI, and BCIP2 were hand-sown at Erie, ND. The soil type
was an Eckman (coarse-silty, mixed, superactive, frigid Calcic Hapludolls) with pH
of 5.8 and 2.4 mg kg'l DTPA-Zn, 84 mg kg'1 DTPA-Fe and 21 mg kg‘1 NaHCOyP.
Thirty seeds were distributed uniformly in 1.5-m rows, spaced 0.76 m apart. One
row was planted for each parent and the F1, BC 1P1, and BC1P2 , and eight rows of F2
seed were planted. Population size evaluated was based on seed availability.
Fertilizer and herbicide applications and common cultural practices followed
recommended practices for dry bean production in the northern Great Plains. Plots
were hand weeded as necessary. Plants were harvested individually by hand and
kept separate by placing each plant in a paper bag. Pods were removed from plants
and seed threshed from pods in the greenhouse by hand to avoid any contamination
with Zn containing appliances.

The pods were threshed by hand and a sub-sample of seed from each plant was
analyzed for total phosphorus, phytic acid, and Zn. Total P was measured by a
molybdenum blue procedure and zinc was measured by atomic absorption. For each
analysis, 0.5 g of seed was ashed in a muffle furnace at 500°C for 6 hours. Next,
25ml of 3N HNO3 was added to the seeds. After one hour the mixture was filtered
with paper filters to remove undissolved particles. These samples were directly
analyzed for zinc concentration by atomic adsorption spectroscopy. For P analysis,
the samples were diluted 10:1 v:v in 0.3N NaOH. Following the dilution, they were

diluted 15;1 v:v with 2.5N sulfuric acid containing 0.006% ammonium molybdate,

46

0.0002% antimony potassium tartrate, and 0.005% l-ascorbic acid. The sample

absorbance was read on a Brinkmann dipping probe with an 880nm filter.

An additional subsample of seed was freeze dried, ground, and analyzed for
phytic acid. Sample preparation for phytic acid [PA] analysis in bean seed was
conducted following the procedure of Lehrfeld (1980). Phytic acid was extracted
from the samples by the addition of 3m] 0.5M HCl (trace element grade) to 0.100 g
of sample. The acidified samples were mechanically agitated for 2 hours at 21°C.
Samples were centrifuged at 12,000g for 15 minutes. The supemant was collected
and diluted 1:5 (v/v) with distilled, deionized water. The diluted sample (15 ml
total) was passed through a Bond Elut strong anion exchange column (Varian,
Walnut Creek, CA) for purification. The column was washed once with 10 ml 0.05
M HCl, and phytic acid was eluted with 3m12M HCl. The eluted fraction containing
PA wasair dried and dissolved in 5mM sodium acetate. The dissolved sample was
filtered through a 2pm filter. Phytic acid levels in each sample were quantified by
high performance liquid chromatography with a refractive index detector. The
column used for analysis was a Waters Symmetry C18 column (3.9mm x 150mm)
(Waters, Milford, MA) heated to a temperature of 40°C. Sodium acetate (5mM) was
used as the solvent at a flow rate of 1.4 ml min". Phytic acid dodecasodium salt
from corn (Zea. mays L.) (Sigma, St. Louis, MO) was used as a standard to
determine PA concentration.

Based on these data, the following characters were analyzed for variability: total
phosphorus (P), phytic acid-phosphorus (PA-P), non-phytic acid-phosphorus (non

PA-P), percent phosphorus as phytic acid (%PA-P), and zinc (Zn). PA-P was

47

determined by dividing the concentration of phytic acid by 3.55 (Lott at al., 2000)
and non PA-P was determined by subtracting PA-P from total P.

Chi-square analyses were conducted for frequency distributions of seed Znin
the F2 and BCIP. _2 generations to test the goodness of fit of the data to hypothesized
genetic ratios. Seed samples were classified into two groups, low zinc and high zinc.
Discriminant analysis was the statistical procedure used to classify each plant in the
F2 and backcross generations into either high or low seed [Zn]. In this procedure, the
pooled standard deviation of Voyager and Albion was estimated. The cutoff value
between the high and low Zn classes was determined by subtracting the mean of
Albion from the mean [Zn] of Voyager. That number was divided by the pooled
variance. The resulting number was multiplied by the mean of Albion plus the mean
of Voyager and divided by two. Any value above the resulting number was
categorized as high seed [Zn], and any sample with a value below the number was
categorized as low seed [Zn].

A mixed effects model (PROC MIXED in SAS) was used to determine
significant variation among means of the six generations for each of the five seed
characteristics (seed total P, PA-P, non PA-P, %P as PA, and Zn) of interest. In this
model, replications were considered random effects, and generations were
considered fixed effects. Variance was partitioned in a generation means analysis
model according to the model: Y = m + (a) [d] + (B)[h] + (a2)[i] + (2043)[j] + (Bz)[l]
where Y was the observed mean of a generation (Holthaus et al., 1996).

Weighted least squares regression was used to fit six generations to six variables

beginning with the midparent. Each generation was weighted by the inverse of the

48

variance of its mean (Mather and Jinks, 1971). The six variables used in the model to
describe the phenotype were: midparent value (m), additive effects [(1], dominance
effects [h], additive x additive interactions [i], additive x dominant interactions [j],
and dominance x dominance interactions [1]. The model parameters were estimated
from the means of each generation and genetic coefficients (Table 7). Relationships
among the six generations used to establish gene effect estimates for generation

means analysis (Gamble, 1962) were:

rn F2

[d] = PlFl - Pth
[h] = - I/.z P1 - V2 P2 ‘i' F1 - 4F2 + 2P1F1 'i' 2P2F|
[I] = - 4F; + 2P|F1 + 2P2F|
U] = - V2 PI + 1/2 P2 + PlFl - P2Fl
[I] = P] + P2 + 2F1 + 4F; - 4P|F| - 4P2F1

From the above estimates, expected means were found and a chi square test was used
to determine if the data fit an additive/ dominance model which only included m, [d],
and [h] or an epistasis model which takes all six gene effects into consideration. T-
tests were used to determine which of the genetic estimates were significant. These
analyses were conducted with the aid of a spreadsheet developed by Ng (1990).
Estimates of broad sense and narrow sense heritability were calculated for seed
zinc concentration by using the variance of the parent, F1, F2, and backcross
generations to estimate phenotypic (Vp), environmental (V5), total genetic (Vo),

additive genetic (VA), and dominance genetic variances (VD). Where:

VP 2 VF2
VB: V4 (Vm) + % (V92) + V2 (VF!)
VG = Vrz - VE

VA = 2(VF2) — VBClPl — VBClPZ
VD: VBClPl + VBCIPZ - VF: - VT;

49

Table 7. Coefficients of gene effects in generation means analysis.

 

Gene effects

 

 

Generation m [d] [h] [i] [j] [l]
Pl 1 l 0 1 0 0

P2 1 -l 0 l 0 0

F1 1 0 l 0 0 1
F2 1 0 V2 0 0 ‘A
BCIPI 1 '/z ‘/2 'A ‘A ‘A
BC 1P2 l -‘/2 ‘/2 'A -‘/4 ‘A

 

m: midparent, [d]: additive ['n]: dominance [i]: additive x additive
[j]: additive x dominance [l]: dominance x dominance (Holthaus et al., 1996).

50

Broad sense heritability = hzb = (VA + VD)NF2 where VA + VD represent the genetic
variance of F2 (Allard, 1960). Narrow sense heritability = h‘n = VA/VF2 (Warner,

1952).

RESULTS AND DISCUSSION

Except for BCP2, there were no differences among generations for total seed
phosphorus (Table 8 and Fig 8). The six generations showed various differences
among generations for phytic acid phosphorus, non-phytic acid phosphorus, and %
of total phosphorus as phytic acid (Table 8). The lack of variability for total seed P
precluded performing a generation means analysis in the ‘Albion’ x ‘Voyager’
derived population.

Seed Phytic Acid Phosphorus Concentration:

The mean PA-P concentration of Albion and Voyager was 1.7 mg g '1 and
2.0 mg g " respectively (Table 8). Since these values were not significantly different
- from each other, the F2 and BC generation data were not able to be categorized into
classes of high and low phytic acid phosphorus. However, generation means
analysis provided data to make a few genetic inferences. First, based on linear
contrasts, the mean of the F; generation (2.19) deviated significantly from the mid-
parent value (Figure 9). A significant deviation of the F1 hybrid from the mid-parent
indicates that dominance was a property of the genetic system influencing phytic
acid phosphorus content in seeds. The broad sense heritability for phytic acid
phosphorus was 0.17 (Table 11). This low value indicated that environmental effects

greatly overshadowed genetic effects for this trait, thus selecting for low PA-P will

51

Table 8. Mean levels of total seed phosphorus (P), the phytic acid (PA-P) and
non-phytic acid fractions of phosphorus (non PA-P), the percent of total seed
phosphorus in the phytic acid form (%PA—P) and seed zinc (Zn) in two navy
bean cultivars and four additional generations established by an initial cross

between the cultivars.

 

 

 

Percent
total P
Phytic Non phytic as
acid P acid P phytic
Phosphorus (mg/ g) (mg/g) acid Zinc
Generation (mg/ g) [PA-P] [non PA-P] (%) (pg/g)
PIT 4.9 01 1.7 a 3.2 a 35 a 21.7 0
P21" 4.9 a 2.0 a,b 2.9 a,b,d 42 a,b 31.2 b
F; 5.0 a 2.2 b 2.8 a,e,f 45 b 28.5c,d,e
F2 4.9 a 2.5 c 2.5 b,e,g 52 c 28.5 c
BCp. 4.9 a,c 2.5 c 2.5 dfg 52 c 26.5 e
BCp2 4.3 be 2.8 d 1.7 c 64 d 30.7 b,d
F Value 1.40 20.65 5.63 12.53 19.12
P 0.26 <.0001 <.0005 <.0001 <.0001

 

1' P1 and P2 are Albion and Voyager, respectively

IMeans the do not share a letter are significantly different from each other

(Tukey’s test, P < 0.05).

53.3

a 1 .t'IC': Li.“ -a '7:-

 

Albion J Voyager

30
20:
1o: [

 

 

 

50- l

30~
20¢
10-

Frequency

 

 

 

 

 

 

60.
BC

50: Albion .. BCVoyager

40
30«

 

 

 

   

Seed Phosphorus (mg g'l)
Figure 8. Frequency distribution of phosphorus concentration in Albion and Voyager
navy bean seed and four generations derived from Albion x Voyager. The arrows and
number in parenthesis indicate the mean phosphorus concentration of each generation.

5.3

35; Albion ; Voyager
301 ;
251
201
15$
10.

214121;

 

 

 

 

b
O

N
U!
AL41¥1

.3
0|
+1

10-

Frequency

 

 

O

 

 

 

BC

Albion . BCVoyager

   

 

 

 

{.0 1.5 2.0 2.5 3.05 3:554:0'415 {.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Seed phytic acid phosphorus (mg g")

Figure 9. Frequency distribution of phytic acid phosphorus concentration [PA-
P] in Albion and Voyager navy bean seed and four generations derived from the
intermating of Albion and Voyager. The arrows indicate the mean [PA-P] of
each generation.

54

Table 9. Chi-square test for the adequacy of an additive/dominance generation
means model for the concentration of seed phytic acid phosphorus [PA-P], non-
phytic acid phosphorus [non PA-P], and zinc [Zn] in a cross between Voyager
and Albion navy bean cultivars and four additional generations established by
an initial cross between the cultivars.

 

 

Seed Trait df A}
PA-P 3 91.9"
Non PA-P 3 23.4M
Zn 3 4.2

 

*, ** Significance at the 0.05, 0.01 probability levels, respectively based on X2 tests
with 6-3 = 3 degrees of freedom. Significance indicated the inadequacy of an
additive/dominance model.

55

Table 10. Estimates and standard error of genetic effects in the generation
means analysis model for the concentration of seed phosphorus [P], phytic acid
phosphorus [PA-P], non phytic acid phosphorus [non PA-P], percent phytic
acid phosphorus [°/o PA-P] and zinc [Zn] in a cross between Voyager and
Albion navy bean cultivars and four additional generations established by an
initial cross between the cultivars.

 

 

 

Genetic Effects
Seed
Trait m [61 1h] 10 [11 [ll
PA-P 1.0" 0.16 4.6" 0.82" 0.26 -3.4"
Non 4.6" 0.16 -6.8" -1.6""" 1.2 4.9"
PA-P
Zn 26” 4.7“ 8.1 0.7 -1.2 -5.3

 

*, ** Significance at the 0.05, 0.01 probability levels, respectively based on t-tests
with n-l = 5 degrees of freedom

Table 11. The variance components and heritability estimates of phytic acid
phosphorus [PA-P], non-phytic acid phosphorus [non PA-P], total phosphorus
[P] and zinc [Zn] concentration in the seed derived from a population developed
from a cross between Albion and Voyager navy bean.

 

 

 

Seed Traits

Variance

Components Zn" PA-P Non PA-P Total P
Vp 31.2 0.131 1.5 1.58
V; 5.9 0.105 0.46 0.294
VG 25.3 0.026 1.04 1.29
VA 25.2 0.14 1.57 1.1
VD 0.1 0114 -0.517 0.196
112., 0.85 0.17 0.70 0.84
h’, 0.81 ----------------

 

56

be difficult, especially in segregating generations. Narrow sense heritability was
unable to be estimated because of the presence of epistasis that would confound the
estimate (Table 11). It should also be noted that the additive variance was found to
be higher than total genetic variance for [PA-P] and non [PA-P] (Table 11). This
discrepancy is due to differences is methods of calculation. Based on the generation
means analysis of the ‘Albion’ x ‘Voyager’ cross, breeding for decreased levels of
phytic acid phosphorus will be difficult because of the limited variability and the low
heritability. Interestingly, the percent of total P in the phytic acid form was higher in
‘Voyager’ (42%) than in ‘Albion’ (35%) but the non PA-P was lower in ‘Voyager’
(2.9 mg g") than ‘Albion’ (3.2 mg g") (Table 8) suggesting that PA-P and non PA-P
may be under separate genetic control in the seed.

Seed Non Phytic Acid Phosphorus Concentration:

Differences between ‘Albion’ and ‘Voyager’ for levels of non PA-P (Table 8)
were minimal. The mid-parent value (3.1 mg g") was significantly higher than the
F1 value (2.8 mg g'1 ) (Figure 10) indicating that low levels of non PA-P is dominant
to high levels of non PA-P. Generation means analysis revealed that dominance,
additive x additive, and dominance x dominance effects contributed significantly to
the inheritance of non phytic acid phosphorus content in this cross (Table 9). The
broad sense heritability 112(3) estimate for non PA-P was 0.7 (Table 11). The estimate
of hzw) may be meaningless for this trait in this cross because the F1, F2 and BC
generation means were lower than the mean of either parent. Moreover, the

difference between the two parents for non PA-P between the two parents was small,

Albion . Voyager

 

 

 

 

5 - ‘9"-
:11

4A

Frequency
N u

 

 

 

 

 

5°? BC... J

on BC
40 . . ’ Voyager
30 . ‘

 

 

i
i

 

 

 

Seed non-phytic acid phosphorus (mg g")

Figure 10. Frequency distribution of the non-phytic acid phosphorus
concentration [non PA-P] in Albion and Voyager navy bean seed and four
generations derived from the intermating of Albion and Voyager. The arrows
indicate the mean [non PA-P] of each generation.

58

3.2 mg g'1 and 2.9 mg g'1 for ‘Albion’ and ‘Voyager’, respectively (Table 8, Figure
10). Parents that differ little in respect to the character examined cannot generate
large additive or genetic variances in their progenies (Simmonds, 1979).

Seed Zinc Concentration:

The zinc inefficient navy bean parent, ‘Albion’ had a lower seed zinc
concentration than ‘Voyager’, the zinc efficient parent (Table 8). Seed from plants
of the F2 generation derived from Albion x Voyager were scored for high or low seed
zinc concentration. The F2 population consisted of 117 high zinc plants and 51 low
Zn plants, which fit a 3:1 ratio based on chi square analysis. The frequency
distribution of seed [Zn] in the F2 (Figure 11) was not normally distributed
(p = 0.076), which also supported a single dominant gene model for the seed [Zn]
trait. The BCP, consisted of 15 high Zn plants and 14 low Zn plants, which fits a 1:1
ratio based on chi square analysis. The 1:1 ratio in a backcross generation supports
the single gene model of inheritance. The BCP2 consisted of 20 high Zn plants and 1
low Zn plant. Since the F2 and BCPr (the inefficient parent) segregation patterns
showed a goodness of fit consistent with a 3:1 and 1:1 ratio respectively, and the
segregation of the backcross to Voyager showed all high zinc, one can conclude that
zinc efficiency in this navy bean cross is controlled by a single gene, and that zinc
efficiency is dominant over zinc inefficiency.

The midparent value of seed zinc concentration for ‘Albion’ and ‘Voyager’ was
25.6ug g", and the mean value ofthe F] population was 28.4ug g'l (Figure 11).
According to a linear contrast, the F1 value was significantly higher than that of the

midparent (p= 0.05), which suggested that the gene controlling zinc efficiency shows

59

partial dominance. However, data derived from the F2 and BC generations indicated
that additive gene action was responsible for the majority of the variation for this
trait (Table 10). The chi-square test for the adequacy of an additive/dominance
model for zinc concentration indicated that the model was not disturbed by epistatic
interactions (Table 9). Broad sense heritabilities for zinc, phytic acid phosphorus,
non-phytic acid phosphorus and total phosphorus in seeds were 0.85, 0.17, 0.70, and
0.84, respectively (Table 11). The narrow sense heritability estimated for seed zinc
concentration of 0.82 (Table 11) supports the conclusion that additive genetic
variance had a major influence on trait expression. These high value broad and
narrow sense heritability estimates for seed zinc concentration indicated the trait was
not strongly influenced by environment, thus selection for increasing the level of
seed zinc would be effective in early generations. A selection strategy that is
appropriate for traits with high heritability estimates will be useful to develop inbred

lines with increased levels of seed [Zn].

CONCLUSIONS

Based on the generation derived from ‘Albion’ x ‘Voyager’, breeding for higher
seed [Zn] should be possible. Selections made at the highest levels of [Zn] indicated
that seed [Zn] could be increased by nearly 50%. Such increases may improve the
quality of the seed for human consumption.

Breeding for low PA-P in bean seed will be challenging because of the lack of
variability for the trait combined with its low heritability. The use of targeted
mutational breeding to inhibit a gene in the phytic acid synthesis pathway may be an

approach to develOp a low PA-P bean.

60

25«
20-
15-
10-

 

o 01
J J

Frequency

00'!

 

 

Albion

 

CAlbion

Voyager

 

 

Voyager

 

35 ?0*35 {5 20 25 30 35 40 45

Seed Zinc (pg g")

Figure 11. Frequency distribution of zinc concentration [Zn] in Albion and
Voyager navy bean seed and four generations derived from the intermating of
Albion and Voyager. The arrows indicate the mean [Zn] of each generation.

61

CHAPTER 3: SCREENING OF PHASEOLUS VULGARIS L.
GENOTYPES FOR VARIABILITY IN PHYTIC ACID AND ZINC

IN RAW AND THERMALLY PROCESSED SEED

INTRODUCTION

Phytic acid, the major form of phosphorus in cereal and legume seeds, reduces the
bioavailability of zinc in humans (Tumlund etal., 1984). Marginal zinc deficiency is
a widespread problem, especially for people consumingdiets rich in cereals and
legumes (Torre, 1991). Breeding for reduced levels of phytic acid along with
increased zinc density in the seed may be a sustainable approach to increase the
amount of bioavailable zinc in the diets of people suffering from zinc deficiency.
The use of preparation procedures that reduce the amount of phytic acid by
activating endogenous seed phytase may be another means to increase the percentage
of bioavailable zinc in phytate rich meals.

The reports are conflicting on the effect of cooking and thermal processing
(canning) bean seeds on the amount of phytic acid in bean seeds. Schlemmer et al.
(1995) found that cooking beans in boiling water for two to three hours did not
significantly reduce the phytic acid content of the seed. Greiner and Konietzny
(1998) found cooking to reduce phytic acid levels 16 to 24% in bean. The same
variability in results has been seen in thermal processing experiments with beans.
Schlemmer et al. (1995) found canning had no effect, whereas Tabekhia and Luh

(1980) showed a 70 to 75% reduction in phytic acid with canning. One reason for

62

the conflicting reports on how phytic acid is affected by thermal processing may be
that there is genetic variability in bean seed for the conditions under which phytase is
activated. Accordingly, this study was conducted to screen raw and thermally
processed seed of 48 dry bean genotypes of diverse market classes for variability in
levels of zinc, phytic acid, and total phosphorus. The objective of the study was to
identify germplasm that may be valuable in a bean-breeding program to increase the
density of zinc and reduce the amount of phosphorus in the phytic acid form in bean

seeds.
MATERIALS AND METHODS

A nursery consisting of 48 dry bean genotypes representing several market
classes of Middle American gene pool and one genotype of the Andean gene pool
(Jacob’s Cattle) was screened. The breeding lines and cultivars were planted at the
Saginaw Valley Bean and Sugar Beet Research Farm near Saginaw, Michigan in
1999. Seed of the entries were precision-drilled with a tractor mounted air planter
into four row plots in a Mistequay silty clay [fine-illitic (calcareous) frigid typic
l-Iaplaquolls] soil. Rows were 5 m long and spaced 0.5 m apart. Within row spacing
was 0.12 m. The planting arrangement was a 7x7-balanced lattice with three
replications. At planting a 21-7-0 and 4% Mn and 1% Zn fertilizer was applied in
bands in each row at the rate of 224 kg ha". A preplant herbicide application was
made following recommendations by the Michigan Dry Bean Production Advisory
Board Agronomist for 1999. Mature plants of the 48 entries were harvested from 6m
of the middle two rows of each plot (6.1m2) and threshed with an Almaco (Allen

Machine Co., Nevada, IA) stationary plot thresher. The threshed seed was cleaned of

63

plant debris, broken and/or diseased grains, and soil particles and sized using 4 x
19mm slotted metal sieves. Plot yield and 100 seed weight was determined at 16%
seed moisture. Seed samples were stored at 22°C and 75% relative humidity
(Hosfield et al., 1999).

A 250g sample of seed from each entry was analyzed for seed moisture
percentage using a moisture meter. Duplicate samples from each plot with a fresh-
weight equivalent of 100g total solids (Hosfield and Uebersax, 1980) were placed in
nylon mesh and equilibrated to 16% moisture in a humidified storage room.
Remnant seed from the 250 g sample not placed in the nylon mesh bags was placed
in small Kraft paper bags and stored at room temperature. The seed in the mesh bags
was thermally processed according to the procedures of Hosfield and Uebersax
(1980) and Hosfield and Uebersax et a1. (1984 a,b). Thermal processing of the seed
samples began by soaking at 21°C for 30 min and blanching at 88°C for 30 min.
Soaking and blanching were done in distilled water adjusted to 0.025 mol L"
calcium ion (Hosfield and Uebersax, 1980; Uebersax and Bedford, 1980). The
soaking and blanching procedures produced well-hydrated beans with minimum
bean damage, similar to beans soaked and blanched in the high-temperature systems
common throughout the US. camring industry (Hosfield and Uebersax, 1980).

After bean samples were blanched, each was immersed in tap water (22°C) for 5
min to cool. Next samples were drained, weighed, and filled into number 303 (100 x
75 mm) tin cans and covered with boiling brine. The brine was prepared by adding
142.0g sucrose and 113.4g of NaCl to 9.1 kg of distilled water adjusted to 0.025

mol/L (100 ppm) calcium ion. The cans were exhausted at 90°C for 5 min in a water

filled exhaust box, sealed and cooked in a commercial retort without agitation for 45
min at 116°C and 10.4 x 10 Pa (15 Psi). After the beans were thermally processed,
the cans were removed from the retort, cooled under running tap water (22°C) for 15
min, and stored inverted for a minimum of two weeks before cans were opened for
evaluation.

The concentration of phosphorus, phytic acid, and zinc was measured on both
the thermally processed and raw seed. Raw seed was the remnant from the 250 g
initial sample from each entry. Prior to analyses, the raw seed was freeze dried in a
Virtis, Genesis® freeze dryer. Seed was then ground to a fine powder with a coffee
grinder. Thermally processed beans were blended into a paste in a blender, freeze-
dried and ground to a fine powder in a Wiley mill with a 60-mesh screen. From this
point the raw and thermally processed seed was handled similarly. For P and Zn
analysis, 0.5 g of seed was weighed and placed in a ceramic crucible. The samples
were ashed in a muffle filmace for 8 hours at 495°C. Samples were then acidified
with 25ml 3N HNO3_ Samples were analyzed for Zn by atomic adsorption (V arian
Spectra AA-20 plus) and for total P by the colorimetric method developed by
Murphy and Riley (1963) with a Brinkman dipping probe with an 880 nm filter
attached to the probe. Samples were analyzed for phytic acid (PA) by HPLC.
Sample preparation for PA analysis in bean seed was conducted following the
procedure of Lehrfeld (1980). PA was extracted from the samples by the addition of
3m] 0.5M HCl (trace element grade) to 0.100 g of sample. The acidified samples
were mechanically agitated for 2 hours at 21 °C. Samples were centrifuged at

12,000g for 15 minutes. The supemant was collected and diluted 1:5 (v/v) with

65

distilled, deionized water. The diluted sample (15 ml total) was passed through a
Bond Elut strong anion exchange column (Varian, Walnut Creek, CA) for
purification. The column was washed once with 10 ml 0.05 M HCl, and phytic acid
was eluted with 3ml 2M HCl. The eluted fraction containing PA was air dried and
dissolved in 5mM sodium acetate. The dissolved sample was filtered through a 2pm
filter. PA levels in each sample were quantified by high performance liquid
chromatography with a refractive index detector. The column used for analysis was
a Waters Symmetry C 18 column (3.9mm x 150mm) (Waters, Milford, MA) heated
to a temperature of 40°C. Sodium acetate (5mM) was used as the solvent at a flow
rate of 1.4 ml min". Phytic acid dodecasodium salt from corn (Z. mays L.) (Sigma,
St. Louis, MO) was used as a standard to determine PA concentration. The amount
of P in the form of PA (PA-P) was determined by dividing the concentration of
phytic acid by 3.55 (Lott at al., 2000)

Data was converted to concentration on a dry weight basis. Statistical analyses,

including least significant differences among cultivars, were conducted using SAS

(SAS Inst., Inc., 1997).

RESULTS AND DISCUSSION

The identification, commercial class designation, seed coat color, seed weight,
and yield of the 48 dry bean genotypes screened for seed Zn, P, and PA-P are
presented in Table 12. In the raw seed, PA-P values ranged from 1.11 mg g" to 2.14
mg g "1 with a mean of 1.65 mg g"(Table 13). The cultivar with the lowest seed PA-
P (Carioca) was not significantly different from the mean PA-P value of all the

cultivars. PA-P made up 30 to 50% of the total seed phosphorus. The PA-P values

66

Table 12. The commercial class, seed coat color, seed weight and yield of 48 dry
bean genotypes screened for the concentration of phytic acid phosphorus, total
phosphorus, and zinc in the seed.

 

 

[Seed
Commercial Seed coat weight ’Yield
Identification class color (8 ‘100 seed ") (kg'ha")
BlackTurtle Soup Tropical black Black 23.4 2498
800242 Small white White 20.2 2687
Seafarer Navy White 21.2 3012
Domino Tropical black Black 22.5 2628
Sanilac Navy White 18.6 2864
Tuscola Navy White 20.3 2408
8217-III-24 Navy White 21.0 2427
Jalpataqua Tropical black Black 25.6 2602
Nep-2 Small white White 20.1 2944
San Fernando Tropical black Black 20.4 2539
Bunsi Navy White 21.2 2637
ICA Pijao Tropical black Black 22.2 2861
Aurora Small white White 17.2 2894
P766 Undefined Black 21.9 2275
J arnapa Tropical black Black 22.7 3190
Protop-Pl Pinto Brown mottle 29.7 2143
FF4-13-MMMM Tropical black Black 23.0 2743
C-20 Navy White 20.1 2715
F leetwood Navy White 19.8 3142
Mexico 12-1 Undefined Brown 21.6 1915
Carioca Pinto Brown mottle 26.2 1347
Laker Navy White 19.6 2485
Swan Valley Navy White 19.5 2818
Midnight Tropical black Black 20.6 2235
BAT 41 Small red Red 19.8 1950
lS-R- 1 48 Small red Red (shiny) 20.4 1950
BAT 1507 Small red Red (shiny) 24.6 2734
BAT 1376 Small red Red 24.4 1937
BAC 95 Small red Red (shiny) 24.0 2950
Harblack(opaque) Tropical black Black 20.0 2577
Harblack(shiny) Tropical black Black(shiny) 21 .0 2763
Cumulus Navy White 22.9 2830
N84004 Navy White 20.2 2984
C-20 Mutant Navy White 20.4 2452
Jacob’s Cattle Heirloom Red/white 50.8 1330
Huron Navy White 22.0 1554
Mayflower Navy White 21.7 2915
Albion Navy White 19.4 2124
Newport Navy White 21.3 2899
N81099 Navy White 21.5 3344

 

67

Table 12 (cont’d)

 

 

 

Identification Commercial Seed coat jSeed weight [Yield
class color (8 '100 seed ") (kghf')
Huetar Small red Red 23.7 2151
Black Jack Tropical black Black 23.0 2572
Raven Tropical black Black 19.5 2336
T-39 Tropical black Black 22.1 2644
Shiny Crow Tropical black Black 26.0 3039
BunsixRaven Tropical black Black 20.7 2654
Garnet Small red Red 29.7 2035
Rufiis Small red Red 35.4 3024
% CV 7.2 15.9
LSD (0.05) 2.6 669

 

I Seed weight and yield are based on seed at 16% moisture. Plants were grown at the
Michigan State University Bean & Beet Research Farm in 1999
(Hosfield et al., 1999).

68

Table 13. The concentration of zinc [Zn], [P], and the phytic acid form of
phosphorus [PA-P] in raw and thermally processed seed of 48 bean genotypes.

 

 

 

 

Seed phytic acid

Seed zinc Seed phosphorus phosphorus

Identification (ug-g"L (mg-g‘ ) (mg-g")
Raw Processed Raw Processed Raw Processed

Black Turtle Soup 30.2 25.1 4.08 3.71 1.31 0.89
800242 30.2 23.9 4.28 4.16 2.11 1.33
Seafarer 30.1 21.1 4.83 3.58 1.71 1.28
Domino 27.1 28.0 4.29 4.25 1.55 1.15
Sanilac 26.4 23.7 4.74 4.29 1.97 1.46
Tuscola 43.3 26.1 4.51 4.21 1.86 1.67
8217—111-24 32.4 26.8 4.77 4.04 1.71 1.09
Jalpataqua 29.5 26.7 4.44 3.90 1.79 1.37
Nep-2 29.2 25.5 4.48 4.26 2.00 1.24
San Fernando 31.4 31.1 4.81 4.16 1.97 1.09
Bunsi 27.7 26.6 4.69 3.99 2.14 1.49
ICA Pijao 27.1 25.2 4.05 4.04 2.00 0.98
Aurora 29.5 24.7 4.45 3.90 1.83 1.23
P766 29.2 34.5 4.67 4.41 2.14 1.55
Jampa 34.0 25.6 4.02 3.65 1.62 1.07
Protop-Pl 33.9 26.9 5.26 4.34 1.77 1.19
FF4-13-MMMM 29.0 25.1 4.15 3.84 1.63 1.08
C-20 66.9 22.4 4.41 3.84 1.79 1.04
Fleetwood 39.7 20.7 4.47 3.86 1.34 1.21
Mexico 12-1 27.9 25.5 3.92 3.58 1.54 0.86
Carioca 31.4 21.9 3.92 3.29 1.11 0.94
Laker 26.5 26.7 4.82 4.32 1.71 1.22
Swan Valley 25.1 21.3 4.11 3.81 1.49 1.19
Midnight 43.3 26.5 4.56 4.04 ' 1.82 1.21
BAT 41 29.1 25.7 5.89 4.42 1.93 1.42
15-R-148 32.3 27.0 4.03 4.05 1.5 0.83
BAT 1507 24.6 29.1 4.02 3.86 1.34 1.11
BAT 1376 29.8 22.9 4.47 4.46 1.51 1.36
BAC 95 26.0 24.4 3.99 3.59 1.36 0.79
Harblack (opaque) 40.0 23.8 4.60 4.16 1.58 0.90
Harblack (shiny) 27.5 28.0 4.46 3.89 1.48 1.20
Cumulus 28.0 22.3 3.97 3.88 1.42 0.96
N84004 36.2 24.7 4.25 3.94 1.76 1.47
020 Mutant 27.1 23.5 4.28 3.77 1.68 1.11
Jacob’s Cattle 46.2 23.0 4.08 3.81 1.43 1.09
Huron 26.6 22.9 4.31 4.10 1.43 1.14
Mayflower 30.9 24.1 4.22 4.12 1.55 1.31
Albion 29.9 23.9 4.42 4.28 1.68 1.26

Newport 26.5 20.5 3.68 3.63 1.57 1.11

 

69

Table 13 (cont’d)

 

 

 

Identification Seed zinc Seed phosphorus Seed phytic acid
(88'8") (mg'g'L phyhows (mm
Raw Processed Raw Processed Raw Processed
N81099 39.4 23.0 4.22 4.32 1.69 1.57
Huetar 33.7 26.2 4.10 3.91 1.33 0.98
Black Jack 26.8 27.0 4.39 4.34 1.55 1.50
Raven 26.3 23.8 4.77 3.83 1.75 0.86
T-39 40.6 32.4 4.33 4.16 1.56 0.97
Shiny Crow 24.3 24.2 3.95 3.97 1.53 0.89
Bunsi x Raven 33.1 26.7 4.49 3.97 1.66 1.08
Garnet 25.9 22.9 4.00 3.85 1.57 1.06
Rufus 27.2 21.3 4.16 3.89 1.61 1.12
mean 31.1 25.1 4.37 3.99 1.65 1.17
SE 0.87 0.37 0.06 0.03 0.04 0.03
LSD (0.05) 12.1 5.1 0.78 0.42 0.54 0.41

 

70

for the raw seed were lower than the 60 to 80% that have been reported in the
literature (Reddy et al., 1989). One reason for the reduced levels of PA-P in this
study may be that the bean seed was harvested in October of 1999 but not screened
until 2002. Beans were stored where summertime temperatures may have reached
25° C. At room temperature storage for 1.5 years, white beans were shown to lose
10% of the PA (Ockenden et al., 1997). Therefore, one might expect some
breakdown of PA at storage temperatures above RT and especially if they were
stored for more than one year. The thermally processed seed had on average 1.17
mg g-l PA-P, and a range of 0.8 to 1.67 mg g-l PA. These data did not indicate
significant genetic variability for PA-P of thermally processed seed. There was on
average 29% reduction PA-P than the raw seed (Figure A3 and Table 13). A study
by Greiner and Konietzny (1998) indicated a similar reduction in seed phytic acid
following a 15 h soak in water and cooking in boiling water for 2 hours. They found
endogenous phytases to be responsible for the reduction in phytic acid. Such
reductions in PA-P many be beneficial to human nutrition... According to nutrition
studies where humans were fed diets consisting of low phytic acid maize (25% of
total seed P is PA~l'-‘) and normal maize (75% of total seed P is PA-P), the zinc
absorbance of the low PA maize was 30% while the absorbance was only 17% for

the normal maize (Adan-rs, 2000).

The genetic variability in raw seed [P] ranged from 3.7 mg g" to 5.9 mg g" with a
- mean of 4.4 mg g" (Figure A4 and Table 13). Thermally processed seed [P] ranged
from 3.3 mg g" to 4.5 mg g" with a mean of4.0 mg g". Thermally processed seed

had on average 8% less seed P than raw seed. This phosphorus either leached into

71

the soak water or the brine, but since these components of the thermally processed

beans were not fractionated, the fate of phosphorus in thermally processing samples

is unknown.

The zinc concentration of raw seed ranged from 24.3ug g" to 46.2pg g'l with a
mean of31.1-ug g". The zinc concentration of the thermally processed seed had
much less variability as compared with the raw seed (Figure A5). The concentrations
ranged from 20.5[rg g'l to 34.5ug g" with a mean of 25.1 pg g". The thermally
processed seed had, on average, 17% less zinc than the raw seed (Table 13). There
was considerable variability in the amount of Zn that leached from the seed during
thermal processing. Fleetwood lost 48% of its seed Zn during processing, while I
Shiny Crow, Black Jack, Laker, and San Fernando lost less than 1% of this mineral
during processing (Table 13). Studies have shown a loss of nutrients in the effluent
from bean processing (Meiners et al., 1976 and Muggio et al., 1985). Rodriguez-
Burger et al. (1998) found limited reduction in seed Zn in black beans following
cooking, but they only investigated one cultivar. The large cultivar differences for
the amount of Zn lost during processing may be related to the location in the seed
that Zn is stored. Based on studies with two black bean cultivars, Moraghan and
Grafton (2002) found 39% to 44% of the total seed zinc was located in the seed coat.
Although no literature could be found on the leaching of zinc from different parts of
the seed, one would expect the zinc in the seed coat to leach more rapidly from the
seed than zinc in the cotyledons and embryo. Despite the reduction in seed zinc
following thermal processing, there existed significant variation for [Zn] among the

cultivars. The genotype P766, a black seeded bean, had 27% more seed [Zn] after

72

after processing than the average of the genotypes. Additional screening of P766 is
necessary to determine its value in a breeding program for improving the zinc status
of dry bean. Breeding for increased seed [Zn] may be a promising way to increase
the bioavailability of zinc for human consumption. According to experiments with
rat models, the % of Zn bioavailability tends to remain constant independent of the
amount of zinc in the seed (Ruel and Bouis, 1998). Therefore, Zn enhanced seed

should have a net increase in bioavailable Zn.

CONCLUSIONS

Screening of the 48 dry bean genotypes for seed Zn, PA-P, and total P, indicated
that substantial variability existed among genotypes for Zn in raw seed but there was
less variability in the thermally processed seed as much of the anas lost during the
processing. The variability for PA-P and total P among the 48 genotypes was
minimal. There was not significant variability in the PA-P of raw or thermally
processed seed, but the level of PA-P was reduced 29% on average by thermal

processing.

73

APPENDIX

74

Table A1. Analysis of variance for phosphorus in the phytic acid form [PA-P],
non phytic acid P [Non PA-P], and total phosphorus concentration in the seed of
Voyager and Albion navy bean cultivars grown under three levels of phosphorus
and five levels of zinc T.

 

Degrees of Mean

 

 

 

 

 

 

 

Trait Source of variation freedom square F statistic Pr > F

PA—P Replication 3 0.07 1.7 0.18
Cultivar 1 0.01 0.22 0.64
P 2 33 742 < .0001
Zn 4 1.80 40 < .0001
P "' Zn 8 0.62 14 < .0001
Cultivar * P 2 0.03 0.64 0.53
Cultivar * Zn 4 0.05 1.04 0.39
Cultivar * P * Zn 8 0.04 0.88 0.53

Degrees of Mean

Trait Source of variation freedom square F statistic Pr > F

Non Replication 3 0.04 1.0 0.38

PA-P Cultivar 1 7.24 209 <.0001
P 2 7.3 210 < .0001
Zn 4 0.83 24 < .0001
P * Zn 8 0.16 4.6 0.0001
Cultivar "‘ P 2 1.08 31 < .0001
Cultivar * Zn 4 0.50 14. <.0001
Cultivar * P * Zn 8 0.10 2.8 0.01

Degrees of Mean

Trait Source of variation freedom square F statistic Pr > F

Total Replication 3 0.06 1.6 0.20

P Cultivar l 6.8 176 <.0001
P 2 71 1848 < .0001
Zn 4 5.1 132 < .0001
P * Zn 8 1.3 33 < .0001
Cultivar * P 2 0.77 20 < .0001
Cultivar "' Zn 4 0.43 11 < .0001
Cultivar * P * Zn 8 0.17 4.4 0.0002

 

T Voyager and Albion are zinc efficient and zinc inefficient cultivars respectively.

75

Table A2. Analysis of variance for content per experimental unit (seed of 3
plants) of phosphorus in the phytic acid form (PA-P), non phytic acid P (non PA-
P), and total phosphorus in Voyager and Albion navy bean cultivars grown
under three levels of phosphorus and five levels of zinc 1'.

 

Degrees of Mean

 

 

 

 

 

 

 

Trait Source of variation freedom square F statistic Pr > F
PA-P Replication 3 173 5.3 0.0021
Content Cultivar 1 4762 146 <.0001
P 2 3074' 945 <.0001
Zn 4 358 1 1 <.0001
P * Zn 8 357 11 <.0001
Cultivar * P 2 1320 41 <.0001
Cultivar * Zn 4 1159 36 <.0001
Cultivar * P * Zn 8 266 8 <.0001
Degrees of Mean
Trait Source of variation freedom square F statistic Pr > F
Non Replication 3 257 l l <.0001
PA-P Cultivar 1 37 1.6 0.22
Content P 2 1000. 419 <.0001
Zn 4 470 20 <.0001
P * Zn 8 242 10 <.0001
Cultivar * P 2 465 19 <.0001
Cultivar * Zn 4 305 13 <.0001
Cultivar * P * Zn 8 153 6.4 <.0001
Degrees of Mean
Trait Source of variation freedom square F statistic Pr > F
Total Replication 3 821 13 <.0001
P Cultivar 1 3959 64 <.0001
Content P 2 7549' 1218 <.0001
Zn 4 1619 26 <.0001
P * Zn 8 1077 17 <.0001
Cultivar * P 2 2341 38 <.0001
Cultivar * Zn 4 1049 42 <.0001
Cultivar * P * Zn 8 6211 13 <.0001

 

T Voyager and Albion are zinc efficient and zinc inefficient cultivars respectively.

76

Table A3. Analysis of variance for seed yield, seed zinc concentration [Zn], seed
zinc per experimental unit, and percent phosphorus in the phytic acid form for
Voyager and Albion navy bean cultivars grown under three levels of phosphorus
and five levels of zinc. T

 

Degrees of Mean

 

 

 

 

 

 

 

Trait Source of variation freedom square F statistic Pr > F
Seed Replication 3 52 14 <.0001
Yield Cultivar 1 477 130 <.0001
P 2 961 262 <.0001
Zn 4 207 56 <.0001
P * Zn 8 89 24 <.0001
Cultivar * P 2 68 18 <.0001
Cultivar * Zn 4 96 26 <.0001
Cultivar * P * Zn 8 21 5.8 <.0001
Degrees of Mean
Trait Source of variation freedom square F statistic Pr > F
Seed Replication 3 6.4 3.2 0.03
[Zn] Cultivar l 476 238 <.0001
P 2 2704 1353 <.0001
Zn 4 405 202 <.0001
P * Zn 8 30 15 <.0001
Cultivar * P 2 45 23 <.0001
Cultivar * Zn 4 25 12 <.0001
Cultivar * P * Zn 8 16 7.9 <.0001
Degrees of Mean
Trait Source of variation freedom square F statistic Pr > F
Seed Replication 3 4829' 14 <.0001
Zinc Cultivar 1 7491 218 <.0001
Content P 2 81 12' 24 <.0001
Zn 4 3891' 113 <.0001
P * Zn 8 2526 7.4 <.0001
Cultivar * P 2 1416 0.41 0.66
Cultivar * Zn 4 7263 2.1 0.09
Cultivar * P * Zn 8 7356 2.1 0.04

 

77

 

Degrees of Mean
Trait Source of variation freedom square Fstatistic Pr>F

 

% Replication 3 25 1.2 0.30

P as PA Cultivar 1 974 48 <.0001
P 2 995 49 < .0001
Zn 4 3.5 0.17 0.95
P * Zn 8 31 1.5 0.16
Cultivar * P 2 162 8.0 0.0006
Cultivar * Zn 4 64 3.2 0.02
Cultivar * P * Zn 8 9.7 0.48 0.86

 

1' Voyager and Albion are zinc efficient and zinc inefficient cultivars respectively.

78

4.0 ..

r = -0.80**

3.5 -

3.0 -

PA-P (mg g ")

 

 

 

Zn (us g“)

Figure A1. Correlations between seed zinc concentration [Zn] and the
concentration of phosphorus in the phytic acid form [PA-P] of Albion navy beans
grown under three levels of phosphorus and five levels of zinc.

** significant at P £0.05

79

 

lL5-

 

 

4.0 - ' .
- I r = '0087**
3.5 - I I
. ' II
3.0 - .
'7; i - I \ '
I
w 2.5 — n . I \ u
E I I
v i . I
e,.20—
E ' II '
I
1.5 - I
‘ ' I I ‘- I
I I
1.0 - "
q I
05-
‘ I ' I ' I ' I ' I r I ' I 1
10 15 20 25 30 35 40 45
-1
Zn (us 8 )

Figure A2. Correlations between seed zinc concentration [Zn] and the
concentration of phosphorus in the phytic acid form [PA-P] of Voyager navy
beans grown under three levels of phosphorus and five levels of zinc.

"‘* significant at P $0.05

80

 

 

 

 

 

 

2-5 a I Raw Seed
0 Thermally Processed Seed
. I I

2.0 - . I. I
9.. I
:9. . I ' . I '
u i I I
a A I O. I . . - -
.g'ko I of" I l..:) qllf
E? 1'57 o 0 ""lo- 5

I0 O ' '9' <5 I
m o 0 00 o
O O O
o o O O I O OO O 00
1.0 't O O O
o 0 O
o O o O o
O C)
I
48 Dry Bean Lines

Figure A3. Phytic acid phosphorus concentration [PA-P] of raw and thermally
processed seed of 48 diverse genetic stocks of dry bean grown in a nursery in

1999.

81

 

 

 

 

 

 

59- _ I Rawseed
O Thermally processed seed
5.5-
I
8 5.0-
3 I I I
.:.-"A .. I I .
8 w 45‘ I ' I
e 5’ ' "' '0 " ° ° ' ' a '
v j .63 O O 0 “I00 I
g l O o I . o .08 'I o I
U) 4.0- O OO ' - 06'l .0 DO.
0 O 000 o 0 0000 00 OO
4 O O 6
354 O O O
O
3.0
48 Dry Bean Lines

Figure A4. Phosphorus concentration [P] of raw and thermally processed seed of
48 diverse genetic stocks of dry bean grown in a nursery in 1999.

82

 

I Raw seed
50 _ o Thermally processed seed

 

 

 

Seed Zn
(us 9")
w
01
l
D
I
I
I

 

 

48 Dry Bean Genotypes

Figure A5. Zinc concentration [Zn] of raw and thermally processed seed of 48
diverse genetic stocks of dry bean grown in a nursery in 1999.

83

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