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MECEEGAN STATE UHK‘E'ERSETY

Krishna; Kumar Singh
W68

Erika-db

LIBRARY

Michigan State
University

 

This is to certify that the
thesis entitled

Genetic-physiology of iron-induced manganese-
chlorosis in beans (Phaseolus vulgaris L.)

presented by

Krishna» Kumar Sin gh

has been accepted towards fulfillment
of the requirements for

_l:h’_.D_°_ degree in Manes

Major professor

Date /y\/[ 7/47 5/
/ /

0-169

ABSTRACT

GENETIC-PHYSIOLOGY or IRON-INDUCED MANGANESE CHLOROSIS
IN BEANS (PHASEOLUS VULGARIS L.)

BY

Krishna Kumar Singh

A genetic-physiologic study of iron—induced manganese
chlorosis in beans was initiated. The experiments were con-
ducted in a greenhouse in sand—culture. Soil culture was
used for crossing and seed multiplication only. Approxi—
mately 60 breeding lines of navy beans (Phaseolus vulgaris L.)
were planted under two nutrient conditions; normal (control)
and screening (5.0 ppm iron and zero manganese). Differential
chlorotic symptoms were noted on plants in the screening
solution 5-4 weeks after planting. On the basis of the
appearance, two extremely chlorotic (50 and 80) and two non-
chlorotic lines (40 and 47) were selected for further studies.
The analysis of plant parts indicated that the chlorotic and
nonchlorotic lines differ in the pattern of distribution of
iron and manganese. Chlorotic lines transport relatively
more iron and less manganese to the shoot, whereas the con-
verse is true for nonchlorotic lines.

The F1, F2, and backcross populations were evaluated

for qualitative response to study the mode of inheritance.

Krishna Kumar Singh

The appearance of F1 combinations indicated phenotypic
dominance of the chlorotic type. F2 populations segregated 9
Chlorotic:7 nonchlorotic plants which suggests the involve—
ment of two genes.

Grafts were made in various root/scion combinations to
determine possible sites of control. Based on the appearance
of chlorotic symptoms, the genotype of rootstock line 50 and -
the genotype of scion line 80 are responsible for the develop—
ment of chlorosis. The analysis, however, indicated that
roots of both lines 50 and 80 were efficient in retaining
iron, but they also retained manganese. On the basis of both
appearance and analysis, it is concluded that retention of
iron by roots depends upon the genotype of the scion whereas
manganese holding-capacity depends mainly on the genotype
of the rootstock.

Nonchlorotic lines exuded less sap in the screening
solution than chlorotic lines. Although the concentration
of iron was greater in nonchlorotic lines, the total amount
(conc x g) was less. Both the concentration of manganese
and total manganese were greater in the sap of nonchlorotic
lines but the pattern did not hold when the total amount of
manganese produced under the screening solution was expressed
as a percent of manganese in normal solution. Fean ratio
and pH of the sap were lower for nonchlorotic lines.

In order to relate the genetic and physiological mechan-
isms, it is postulated that the genes function to control the
amount of iron and manganese in the shoot by affecting sap

flow.

GENETIC-PHYSIOLOGY OF IRON-INDUCED MANGANESE CHLOROSIS

IN BEANS (PHASEOLUS VULGARIS L.)

BY

Krishna Kumar Singh

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Crop Science

1968

q-‘x
(\

Q>

t§\ K m

ACKNOWLEDGEMENT

The author expresses his sincere appreciation to
Dr. M. W. Adams for his guidance throughout this study.
Thanks are due also to Drs. F. W. Snyder, L. R. Baker and
B. D. Knezek, who served as guidance committee members

and assisted in preparing the manuscript.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . . . 5
Fe and Mn Interactions. . . . . . . . . . . . . 3
Genetic Differences in Ion Accumulation . . . . 6
A. Qualitative Responses . . . . . . . . . 6
B. Quantitative Responses. . . . . . . . . 7

Possible Mechanisms for the Observed Differ-
ences. . . . . . . . . . . . . . . . . . . 9
MATERIALS AND METHODS. . . . . . . . . . . . . . . . 13

Screening for Genotypes Differing in Their

Responses. . . . . . . . . . . . . . . . . 15
Analytical Procedure. . . . . . . . . . . . . . 16
Genetic Study . . . . . . . . . . . . . . . . . l7
Grafting Experiment . . . . . ... . . . . . . . 18
Exudate Experiment. . . . . . . . . . . . . . . 19
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 20
1. Initial Screening. . . . . . . . . . . . . . 20
2. Genetic Studies. . . . . . . . . . . . . . . 50
5. Grafting Experiment. . . . . . . . . . . . . 59
4. Exudate Experiment . . . . . . . . . . . . . 50
CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . 56
1. Initial Screening. . . . . . . . . . . . . . 56
2. Genetic Studies. . . . . . . . . . . . . . . 56
5. Grafting Experiment. . . . . . . . . . . . . 57
4. Exudate EXperiment . . . . . . . . . . . . . 57
LIST OF REFERENCES . . . . . . . . . . . . . . . . . 58

iii

TABLE

1.

LIST OF TABLES

Proportion and sources of micronutrient elements
in nutrient solutions. . . . . . . . . . . . . .

The concentration of Fe and Mn (ug/g dry wt) in
the roots, stems, and leaves of navy bean lines
grown in normal and screening solutions. . . . .

Fe/Mn ratios in the roots, stems and leaves of
navy bean lines grown in normal and screening
solutions 0 I C O O O C O O O O O O O O O O O O O

Phenotypic appearance of F1 combinations of navy
bean lines grown in screening solution . . . . .

Observed and eXpected numbers of chlorotic and
nonchlorotic plants and Chi-square values for
goodness of fit for the F2 and backcross popula-
tions of navy bean lines grown in screening
solutions. . . . . . . . . . . . . . . . . . .

The genetic constitutions, their proportions,
pattern of Fe and Mn transport, Fe/Mn ratios and
the phenotypes in the F2 and backcross pOpula—
tions according to "heterozygote superiority"
hypothesis . . . . . . . . . . . . . . . . . . .

Observed and eXpected numbers of chlorotic and
nonchlorotic plants and Chi-square values for
goodness of fit for backcross populations

of navy bean lines grown in screening solution,
according to “heterozygote superiority" hypothe-

Sis. O O O I O O O O O C O O O O O O O O O O O O

The appearance of grafted navy bean plants grown
in screening solution. . . . . . . . . . . . . .

The concentrations of Fe and Mn (pg/g dry wt) in

the roots, stems and leaves of grafted navy bean
plants grown in normal and screening solutions .

iv

Page

14

25

27

30

54

37

38

4O

43

LIST OF TABLES - Continued

TABLE

10.

11.

12.

13.

14.

15.

Fe/Mn ratios in the roots, stems and leaves of
grafted navy bean plants grown in normal and
screening solutions. . . . . . . . . . . . . . .

Total wt of exudate in g per bean plant grown in
normal and screening solutions . . . . . . . . .

Fe and Mn concentrations (ppm) in the exudate of
bean plants grown in normal and screening solu-
tions. . . . . . . . . . . . . . . . . . . . . .

Total Fe and Mn (ug) in exudate of bean plants
grown in normal and screening solutions. . . . .

Fe/Mn ratios in the exudate of bean plants grown
in normal and screening solutions. . . . . . . .

pH of the exudate of bean plants grown in normal
and screening solutions. . . . . . . . . . . . .

Page

47

51

52

55

55

54

LIST OF FIGURES

FIGURE Page

1. The appearance of navy bean lines grown in
screening solution. . . . . . . . . . . . . . 21

2. Phenotypic appearance of F1 combinations of
navy bean lines grown in screening solution . 51

5. The appearance of grafted navy bean plants
grown in screening solution . . . . . . . . . 41

vi

INTRODUCTION

A sub—Optimal or supra-optimal concentration of a
nutrient element(s) in the substrate or in plant tissue may
disrupt physiological or biochemical processes which require
the direct or indirect involvement of the element(s). Foliar
manifestations of the disturbances are the deficiency or
the toxic symptoms. The relation between the concentration
of the element(s). morphological manifestations, and the
internal processes is often incomplete and unclear, probably
because of elemental interactions occurring either in the
medium, at the uptake, at the transport, or at the utiliza—
tion sites. Different species and different genotypes
within Species, however, differ qualitatively and quantita—
tively in their responses to these alterations and inter-
actions, owing their capabilities to genetic differences.

Iron (Fe) and manganese (Mn), two of the essential
micronutrients, are antagonistic and when present in an im-
prOper balance, toxic concentrations of the one induces
symptoms resembling deficiency of the other and vice versa.
Researches have revealed that the two elements are function-
ally related and interfere with each other during uptake

and transport. Modes of genetic control and the relation

between the genetic and physiological mechanism(s) which
induces the symptoms are not understood.

This investigation was initiated to study and to relate
the genetic and physiological mechanism(s) responsible for

the iron-induced manganese chlorosis in beans (Phaseolus

 

vulgaris L.).

LITERATURE REVIEW

Fe and Mn Interactions

Twyman (48), in his review, cites that plants were
grown by altering the balance between Fe and Mn as far back
as 1848 by Salm-Horstmar, who noted the symptoms of the
so-called “grey-speck" disease in oats, when the nutrient
solution consisted of a high amount of Fe and little or no
Mn. The description given by him is regarded as the first
recorded symptoms of Mn deficiency. Twyman (48) also
referred to Pugliese, who raised cereals in water-culture
using Knop's solution with varying amounts of ferrous sul-
phate and manganese salts. Pugliese's results indicated
that plants could tolerate larger doses of Mn in the presence
of Fe.

Tottingham and Beck (47), using KnOp's solution minus
ferric phOSphate, observed that high concentrations of Mn
depressed the stimulating effect of Fe and vice versa, while
at low levels, Mn, in the presence of Fe, caused chlorosis
in wheat. They also suggested that Mn interfered with Fe
in the formation of chlorophyll.

Somers and Shive (45) considered that symptoms due to

excessive Fe were identical with those when Mn was deficient

and vice versa. They found that high concentrations of
soluble Mn in the tissue invariably were associated with
low concentrations of soluble Fe and vice versa, thus they
concluded that Fe and Mn were functionally related. They
further suggested that the oxidation of ferrous to ferric
ions by active Mn resulted in an inactivation and precipi-
tation of Fe in the form of a ferric-organic complex.
Weinstein and Robbins (55), using green and albino scions
grafted to green rootstocks in solution culture, similarly
noted Fe deficiency symptoms on the plants supplied with a
high level of Mn, as well as with a low level of Fe. On the
basis of biochemical analysis, they concluded that a high
level of Mn in the presence of low Fe induced a true Fe
deficiency. Mn deficiency was noted, however, only when

Mn in the substrate was low but it was not noted on plants
supplied with high Fe.

In contrast, Berger and Gerloff (4) observed Mn toxicity
(stem-streak necrosis) in potatoes, when Mn was present in
toxic concentrations. However, the Fe in the tissue gradual-
ly decreased as the Mn in the tissue increased. Morris and
Pierre (28) reported that lespedeza exhibited Mn toxicity if
grown in a nutrient solution containing low Fe. An increase
of Fe in the culture solution, up to a certain level, markedly
reduced the toxicity of a given concentration of Mn. The
beneficial effect of Fe in reducing Mn toxicity resulted from

a decrease in Mn absorbed by the plant rather than an increase

in Fe absorption. Similarly, Agrawal et al.(1) noted
symptoms of Mn toxicity in barley grown in sand culture when
an excess supply of Mn was accompanied by a low level of Fe.
These toxicity symptoms were distinct from Fe—deficiency
chlorosis. Excess Fe did not produce any characteristic
effect.

Interference in uptake and transport have been reported
by some investigators (12,41,49). According to Chapman (12),
Bertrand proposed that by converting Fe to an insoluble
ferric form, Mn prevented its transport in or from the
leaves. Sideris (41) observed the deposition of Fe in the
exodermal root tissue of Ananas comosus, when a high concen-
tration of Mn was supplied. The oxidation of Fe by Mn was
suggested as a possible explanation. Vlamis and Williams
(49) reported a reduction in Mn content of roots consistent
with increased Fe supply in barley. Mn at low levels stimu—
lated the uptake and transport of Fe while at the higher
concentrations it exerted an inhibitory effect in soybeans
(24), and in tomatoes (39). The inhibition, however was not
very effective as compared to zinc (24).

Antagonism between Fe and Mn has been further emphasized
by Gerloff et al. (15) in an interaction study between
Fe-Mn-Mo. They observed that Mo accentuates Fe chlorosis in
tomatoes induced by excess Mn in the nutrient medium. Fe
content of the tissue decreased with increasing concentra-

tions of either Mn or Mo in the external medium. In a similar

study, Kirsch et al. (22) reported that at a low Fe level,
Mn decreased Mo uptake while at a higher Fe level, Mn in-
creased the uptake of Mo, indicating the antagonism between

Fe and Mn.

Genetic Differences In:I9n Accumulation

A. Qualitative ReSponses

 

Differential visual symptoms in response to differing
levels of nutrient elements have often been noted and used
as criteria to study the mode of inheritance. Weiss (54)
noted that two soybean varieties differed in their iron re-
quirement and reported that the efficiency to utilize Fe was
governed by a single dominant gene. Bell et al. (2,3) re-
ported a homozygous yellow stripe mutant (ysl/ysl) of corn
incapable of utilizing iron efficiently. The inefficiency of
the mutant was due to its inability to utilize ferric iron at
the root tips.

Brown (7) grew iron-deficient (yellow stripe-1, ysl/ysl)
and iron-efficient (Pa 4) corn on calcareous soil and found
that Fe deficiency was accentuated by phosphate (P04). The
two corn genotypes differentially absorbed and translocated
P04 from organic phOSphate, a factor that could be associated
with Fe-utilization.

Wall and Andrus (51), comparing the uptake and transport
of boron in two varieties of tomatoes, found no differences

in uptake; but translocation was inefficient in T 3258 as

compared to Rutgers, and this inefficiency was caused by a
single recessive gene.

Pope and Munger (55,56) observed that two celery varie-
ties were differentially susceptible to low boron (0.01 ppm)
and to low magnesium (2.5 ppm). Efficiency was dominant in
both cases and was shown to be conditioned by a single
dominant gene.

Bernard and Howell (5) studied the inheritance of dif-
ferential responses of certain soybean varieties to high P
in nutrient solution. Segregation in the F2 and F3 genera-
tions indicated the existence of a major gene pair, Mp and
pp, responsible for the differences.

Polson (34) reported that two genes are involved in the
differential response of two dry bean varieties to a low
level of zinc. Differences in the response were also observed
at a high level of zinc (5.0 ppm), but the number of genes
could not be established for this level because of a limited

pOpulation sizes.

B. Quantitative Responses
Differences in concentration of elements or the total
accumulation within plants has been noted. An approximate
mode of inheritance has been reported for a number of cases,
establishing that such processes are under genetic control.
Gorsline et al. (16) found differential ear-leaf
accumulations of Ca, Mg and K in various single crosses and

inbreds of maize. For Ca and Mg, the differences were highly

heritable on an additive basis, but for K, nonadditive ele-
ments also were included. Additivity accounted for different
levels of calcium and strontium accumulation between geno-
types of corn (17). The inheritance of the concentration of
a number of elements in the corn leaves and grain by diallel
crosses was studied (18). Nonadditive gene action was indi-
cated for ear-leaf concentration of P, Mg, Cu and B and for
the grain concentration of K. Probable number of loci and
the effect of each gene pair have been established (19) for
the ear-leaf concentration of Sr—Ca, Mg, K, P, Zn, Cu, B,
Al-Fe, and Mn.

Massey and Loeffel (26) determined zinc content (58.4-
15.5 ppm) of grain from inbred lines of corn and prOposed
that genes control the ability of the plant to absorb zinc.
or to translocate it to the kernels. They (27) related the
high concentrations in the kernels to the net transfer of
zinc from stalk and leaves to the grains, the effectiveness
of which depended on the general zinc content of the plant.

Kleese (25) established that 898r and 45Ca accumulation
in soybean seeds was largely controlled by the genotype of
the stem. In the terminal 12-15 Cm, however, 89Sr and 45Ca
accumulation depended on both the genotype of the root, and
the stem, with the stem being relatively more important.

Rasmusson et al. (58) found large and significant geno—
typic differences for strontium accumulation in wheat and

barley. The pattern of inheritance was not reported.

By including two pairs of isogenic lines in their studies,
they established that the genes or gene responsible for 89Sr
accumulation was not associated with the gene for the condi-
tion of starch (normal starch and waxed starch), but the

gene controlling row number or genes closely linked to it
played an important role. Substantial genetic control of

the process of 89Sr accumulation in the grains of barley was
indicated by a heritability study involving F1, F2, and back-
cross populations (57). With few exceptions, however, con—
centration in the grain was accounted for by additive gene

effects.
Possible Mechanisms for the Observed Differences

In a number of cases, the observed differential responses
have been accounted for by differences in anatomical or
physiological mechanisms. It is possible, therefore, that
the genes exert their effects on ion—accumulation via affect-
ing the physiological mechanisms involved.

Differential reSponses of inbred lines and crosses of
maize to variations in P and N (42), and to P (25) were
attributed to variations in root-morphology. Plants with
greater proportions of secondary to primary roots were more
efficient.

Vose (50) in his review, emphasized that the factors of
nutrient absorption, translocation, and metabolism could

account for the varietal differences in mineral nutrition.

10

Recently, strains of snapbeans have been shown to differ
in K utilization (40). Since differences were not caused by
variations in seed size or root absorption capacity, they
may be associated with transport or utilization at the cellu-
lar level.

Numerous investigators have utilized root/scion grafts
to differentiate betWeen the root and the shoot as possible
sites of control for the observed differences (25,8,14,54).

Kleese (25) established that the genotype of the soybean
stem was more effective in controlling strontium accumulation
in the seed and in the apical 12-15 Cm region than the geno-
type of the root. Brown et al. (8) associated the genotype
of the rootstock with efficiency of Fe absorption in soybeans,
where Hawkeye rootstocks were more efficient than PI-54619—5—1.
They (9) also reported that the efficient genotype was more
capable of reducing Fe+++ to Fe++ at the root surface and
therefore the reductive capacity of the roots, their capacity
to absorb Fe, and the susceptibility of these plants to Fe
chlorosis were all related. Foote and Howell (14) observed
the differential response of soybean varieties Chief (tolerant)
and Lincoln (sensitive) to high P. By grafting tolerant shoot
on sensitive root, the tolerant shoot was found to develop
P-toxicity. Conversely, sensitive shoots did not develOp
symptoms when grafted on the tolerant root. Polson (54)
noted that the genotype of the scion was responsible for the

differential tolerance of two navy bean varieties to high

11

zinc (5.0 ppm). He also found that the variety, Saginaw,
tolerant to low zinc, transferred a greater volume of plant
sap in a given time than Sanilac. The concentration of zinc
was approximately three times greater in the exudate of
tolerant than in the sensitive variety.

Brown et al. (11) used the split-root technique to
demonstrate the internal inactivation of Fe in PI-54619m5—1
soybeans, principally from the combined effects of P and Ca.
Ca stimulated root growth, increased absorption and trans-
location of P and Ca to the above ground parts, but decreased
the absorption and translocation of Fe in the presence of P.
Hawkeye remained green under conditions which induced
chlorosis in PI-54619-5-1.

Wallace et al. (52) reported that the PI soybean variety
inhibited 59Fe uptake by Hawkeye when both were grown in the
same nutrient solution but Hawkeye had no effect on
PI. This may be considered as an indication that PI exudes
an inhibitory factor into the medium which decreases the
absorption of Fe by Hawkeye.

The carrier concept of ion uptake, originally proposed
by Epstein and Hagen (15), has gained further support by the
work of Pardee (52) and Pardee et al. (55), who isolated a

sulphate binding protein from Salmonella. Differential

 

affinity of carrier sites for different elements and the
competition between elements for the available sites accounted
for the different proportions of various elements entering

the plant root.

12

Hiatt (20) proposed that organic and amino acids play
an important role in ion accumulation by providing nondifr
fusible charges which may bind or retain inorganic ions
within the cell, also the nonadditive component of ion uptake
which becomes important at salt concentrations higher than
1 mM is a result of diffusion of neutral salts according to
Donnan phenomena. Ion uptake by this mechanism would not
necessarily involve the action of carriers.

Fe has been shown to exist as Fe-malate, Fe-malonate
and Fe-citrate in stem exudate (44,45,46). On this basis,
these natural chelating agents have been postulated to act
as transport carriers. The quality and the quantity of the
chelating agents, the involvement of similar carriers for
various elements, or the competition between ions for the

same carrier also might affect differential transport.

MATERIALS AND METHODS

The genetic and physiological studies were conducted
in sand—culture in the greenhouse. Soil-culture was used
for crossing and seed multiplication only. The general
procedure of raising plants in sand involved the following.

Polyethylene bags were placed inside clay pots to avoid
contamination from the pot, and a hole was made at the bottom
to facilitate drainage. Acid washed sand was put in the pots.
Seeds were then planted at a depth of one-half to one inch.
Sand was moistened with deionized water and deionized water
was supplied till germination, if necessary to prevent
desiccation. Following germination, a routine of nutrient
application was initiated which consisted of applying the
nutrient solution every second day and deionized water on
alternate days. All the culture pots were flushed with
deionized water once a week.

Two types of nutrient solutions were prepared, i.e.,
normal (control), and high iron minus manganese (screening).
Slightly modified Hoagland's (21) macronutrient solution was
used as the basal solution. Chemicals and their quantities

are listed below:

15

14

 

Chemical Quantity (g/liter)

Ca(N03)2-4H20 1.401

KH2P04 0.0795

KN03 0.5284

MgSO4-7H20 0.4525

NH4H2PO4 0.670

KCl 0.0454

Composition of the micronutrient solution also was based

on Hoaglan
in accorda
of various

(Table 1).

Table 1.

d's (21) formula but slight variation was adapted
nce with the objective. The proportion and sources

elements are presented for both the solutions

PrOportion and sources of micronutrient elements in
nutrient solutions.

 

 

 

 

Element Source Concentration (ppm)
Normal Screening

Boron H3B03 0.5 0.5
Manganese MnClg-4H20 0.5 None
Zinc ZnSO4-7H20 0.05 0.05
COpper CuSO4-5H20 0.02 0.02
Molybdenum H2MOO4'H20 0.01 0.01
Iron Fe-citrate 0.5 5.0

 

15

Reagent grade chemicals were used to make the nutrient
solutions. The pH of the final solution varied between 5.5
and 5.7. Temperature and light conditions were those of

the greenhouse environment.
Screening for Genotypes Differing in Their Responses

Approximately 60 breeding lines of navy beans (Phaseolus
vulgaris L.) were grown in sand with either normal or screen-
ing solution. The entire experiment was replicated twice.
Three to four weeks after planting, differential chlorotic
symptoms developed on plants where screening solution was pro-
vided. In this study, no attempt was made to determine
whether the chlorosis was due to Fe toxicity or due to Mn
deficiency. The term chlorosis in this thesis refers to the
appearance of chlorotic lesions on the foliage of certain
genotypes when supplied with screening nutrient solution.

On the basis of appearance, two extremely chlorotic lines 50
and 80 and two nonchlorotic lines 40 and 47 were selected
for further studies.

Plants were harvested and divided into roots, stems
and.leaves. Samples were rinsed in 0.5N HCl and deionized
Ivater and left for drying. Dried plant samples were ground
ill a 20-mesh Wiley mill and then analyzed for Fe and Mn
(Kantents. Both of the replications were analyzed separately.
Selected lines were replanted, harvested and analyzed again.
II'hus the results reported in the initial screening section

are the average of three analyses.

16

Analytical Procedure

 

Known amounts of the ground material were placed in
porcelain crucibles and ashed at 600 C for about 10 hours.
Temperature was raised gradually to prevent ignition of the
samples. Ash was moistened with deionized water and 5 ml
of 2N HCl was added. The solution was filtered, using
Whatman number 2 filter paper. Deionized water was poured
through the filter paper in aliquots, the latter of which
rinsed the crucible, and adjusted to a given volume. In
some samples, sand remained on the filter paper. These were
preserved and weighed. The weight of sand was subtracted
from the previously recorded weight to determine the actual
weight of the plant material.

The solutions were analyzed for Fe and Mn content in a
Perkin-Elmer model 290 atomic absorption Spectrophotometer.
Standards of known concentrations were used and since read-
ings (percent absorption) were linear, it was possible to
calculate a constant (K) by dividing the reading by ppm
concentration. The percents of absorption were converted to

09/9 dry wt, according to the following formula:

K x Percent absorption x Volume of solution

ug/g dry wt. = Weight of the sample

Part of the investigation was completed using the Perkin-
Ifllmer model 505. The procedure was the same, except that the

:readings (percent absorption) were converted to absorbance

17

first, according to the table published by Perkin-Elmer
Corporation, Connecticut, U. S. A. Conversion to ug/g

dry wt was accomplished by the same formula.

Genetic Study

Selected lines were planted in soil in the greenhouse.
Crosses were made in all possible combinations. Seeds of
reciprocals were not kept separately but pooled together.
Resulting F1 seeds were planted in sand in two replications
for each nutrient solutions, i.e., normal and screening.

Two seeds of each parent and their F; were planted in the
same pot. At the same time, some F1 seeds were planted in
soil to produce F2 seeds and to make backcrosses. As the
chlorosis appeared in parents, phenotype of the F1 was
recorded and photographs were taken.

The F2 seeds were collected from 40 x 50 and 47 x 80
combinations only, thus four backcrosses involving the F1
and their respective parents, i.e., (40 x 50) x 40,

(40 x 50) x 50, (47 x 80) x 47, (47 x 80) x 80, were made.
Seeds of F2 and backcrosses were planted in sand in screen-
ing nutrient solution only along with the parents. After

5-4 weeks, when the parents exhibited differences, individuals
of segregating populations were evaluated and categorized

as chlorotic or nonchlorotic, based on phenotypic appearance.
I\ Chi-square test was used to evaluate the differences

statistically.

18

Grafting Experiment

Grafts in different scion/root combinations were made
in an attempt to localize possible sites of control. Seeds
of the chlorotic and nonchlorotic lines were planted side
by side in sand in the same pot. After germination and
before the primary leaves had fully eXpanded, grafts were
made in the following scion/root combinations: 40/40, 40/50,
50/40, 50/50, 47/47, 47/80, 80/47 and 80/80.

The stems were cut diagonally between the cotyledonary
node and the sand surface. A small piece of toothpick was
inserted into the pith which supported the scion of the de-
sired combination. After putting the stock and scion to-
gether, a small piece of latex bandage was placed around the
graft to facilitate reunion and to prevent desiccation.
Deionized water was supplied and the pots were covered with
plastic bags. Subsequently all the grafts were transferred
to a moist chamber, where they remained for about 5 days.
During this period, they received deionized water as needed.
After 5 days, the bags were removed and grafts of each combi-
nation were divided into two groups for nutrient application.
After about 4-5 weeks, chlorotic symptoms appeared; photo-
graphs were then taken, and the plants harvested immediately
for analysis. Plant parts from all the grafts of one combi-

nation were ground together and two analyses were made.

19

Exudate Experiment

Seeds of chlorotic and nonchlorotic lines were planted
in the same pot in sand in two replications. Following
emergence, the number was reduced to 5 plants of each line
in one pot. Partial shading was provided during germination
and the week following to elongate the hypocotyl. Nutrient
conditions and routine of application were as previously
described. A week before collecting the exudate, however,
the set which had been receiving screening solution was
supplied with the normal concentrations of Mn. Four weeks
after planting, leaves were removed, cut surfaces were
covered with vaseline and stems were cut at a height of
approximately nine inches from the sand surface. Cut stems
were bent over and placed into small plastic vials. Exudate
was collected for a period of 24 hours. The weight of
exudate, and the Fe and Mn contents were used to determine
differences in rate of transport. The pH of each sample

also was recorded.

RESULTS AND DISCUS SION
1. Initial Screening

Genotypes of beans consistently differed in their
visual symptoms in response to screening solution; lines 50
and 80 developed chlorosis, while 40 and 47 did not (Figure
1). The onset of chlorosis occurred about 5 weeks after
planting, although distinct differences were evident until
the end of the fourth week; therefore, this was considered a
critical period in the present investigation.

The primary purpose of analyzing plant parts for Fe and
Mn contents was to establish a relation between the visual
symptoms and concentration of the elements. Thus the mode
of inheritance could be studied by basing the evaluation of
progeny performance both on the appearance and the analysis
of plant parts. Since the F2 and backcross pOpulations were
grown in screening solution only, the initial problem centered
around identifying genetic differences in the screening solu—
tion alone without involving the comparison with normal.

Analytical data are summarized in Tables 2 and 5.

20

Figure 1. The appearance of navy bean lines grown in
screening solution.

 

 

A. Line 40

 

 

B. Line 47

Figure 1 (cont'd)

 

 

 

C. Line 50

 

 

 

D. Line 80

25

.Amfi mmmm mmmv momwamcm woman mo mmmno>¢a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N.mm o.m¢ >.maa o.>¢ ¢.¢m H.M> m.am m.¢m N.mm om
>.>m m.¢m d.¢ma o.mm ¢.¢H m.am m.mm o.mm N.N> om
m.mm m.>m N.®MH m.m> $.Nm m.m¢ >.>w w.om m.mm he
N.ma m.mm m.ama o.mm H.am >.mw m.mw o.wm a.mm ow c2
m.mmm o.dfim w.ama a.>ma ¢.m¢ m.mm m.mmm N.ooe m.m¢a om
o.wmd o.mma >.ama >.mma 0.05 >.N¢ N.m>a ¢.H>H m.>m om
m.m¢a m.oam m.>¢fi m.NHH m.0d m.wm o.m¢m m.0fim m.om he
m.amfi >.mmfi m.¢md H.m¢fi H.Hm o.aw w.m¢m m.mfim N.mm os om
Hmeuoc one Hmfiuoz Adamo: mca HmEHoz HmEHoc mca Hmauoz
mo R Icmouom mo R Icmmuom mo R Icmmnum
mm mca mm mca mm mad
Icoouum Icmmuom Icmmuom
mmoq Emum poom mmcaq ucmEmHm
.mcoausaom mcacmmuum can HMEHo: ca :3oum mmcaa coon m>mc
mo mo>mmH pcm .mEopm .muoou may ca dau3 wan m\miv :2 pam om mo coauwuucmocoo one .N manna

24

Table 2 shows the Fe concentration in roots, stems, and
leaves. It is clear that the genotypes growing in screening
solution had more Fe in various plant parts than the same
genotypes cultured in normal solution. Although the concen-
tration of Fe was 10 times greater in the screening solution
(5.0 ppm) than in the normal (0.5 ppm), concentration in
the plant parts did not vary in same prOportion. This may be
the result of preferential exclusion of ions at the root
surfaces. In normal solution, leaves usually contain a
greater concentration of Fe than roots with the exception of
line 80 which has more in the root and the least amounts are
in the stems. In the screening solution, roots always have
greater concentrations followed by leaves and stems. This
may indicate that uptake increases with supply to a certain
extent, but transport does not keep pace. Uptake and trans-
port are two separate components of the accumulation process.
Part of uptake may be accounted for by diffusion, whereas
transport is largely active. DisprOportionate uptake and
transport of Fe may then result during metabolic disturbances,
which, in this case was induced by altering the composition
of the nutrient solution. The comparison between chlorotic
and nonchlorotic lines does not reveal a pattern for Fe
neither in the screening solution nor in comparison with
normal. It is possible that the Fe concentration pg£_§§_is

not directly associated with the deveIOpment of chlorosis.

25

Table 2 also contains the Mn concentrations in roots,
stems, and leaves. Mn is higher in plants grown in normal
solutions than those grown in screening solution. In spite
of the fact that cultures grown in screening solutions were
not provided with Mn, Mn was always detected in plant tissue.
This may be the residual Mn from seeds and/or contamination
from various chemicals used in preparing the nutrient solu-
tions. Although chlorotic lines had more Mn in roots, the
concentration in line 50 (58.0 ppm) was slightly greater than
the nonchlorotic lines (50.4 and 54.0 ppm). Stems and leaves
do not exhibit significant differences. If the Mn content
of plants grown in screening solution is expressed as percent
of normal, nonchlorotic lines tend to have lower values in
the roots and leaves and higher values in the stems as com-
pared to chlorotic lines. Apparently, nonchlorotic lines
have the capacity of transporting Mn to the foliar parts
efficiently especially when exogenous Mn is very low, whereas
the chlorotic lines lack this capability or are not equally
efficient. If the distribution of Mn is associated with
chlorosis, as has been assumed, leaves, being the utilization
site, should have a higher value in nonchlorotic lines, but
actually they had less. This discrepancy may result from the
mobile nature of Mn. In such a situation the Mn of the stem
should not be considered separate from the Mn of the leaf.

It may be reserve and/or transitional, capable of being moved

to the organ where needed. It is not possible, however, to

26

draw a firm conclusion on the basis of this study since total
uptake is not available, yet this may be considered as an
indication that the distribution of Mn is different in the
two sets of lines.

If the distribution is different for Fe and Mn in chloro—
tic and nonchlorotic lines, it should be eXpressed in the
Fe/Mn ratios. This would emphasize the importance of the
relative balance of the two elements and at the same time
could be used as a reliable guide for evaluating inter-
genotypic differences.

In the screening solution, the comparison of Fe/Mn ratios
between the chlorotic and nonchlorotic lines does not reveal
a pattern (Table 5). When the ratios from screening solution
are expressed as percent of ratios in normal solution, roots
of nonchlorotic lines show higher values and stems the lower
than the chlorotic lines, whereas the leaves do not show a
consistent pattern. It seems in agreement with the previous
assumption and findings, i.e., that higher Fe/Mn ratios in
the roots in screening solution indicate that the nonchlorotic
lines retain relatively more Fe and less Mn in the roots than
the chlorotic lines.

The develoPment of chlorosis is an end result of a series
of internal disorders occurring in response to the screening
nutrient solution. An ionic imbalance will affect various
processes requiring direct or indirect involvement of Fe

and/or Mn. This may also affect other processes via affecting

.ADB mun m\miv :2\Au3 mun m\m1v mmd

 

27

 

 

 

 

 

 

 

 

m.wmw 0.5 o.a o.o¢N N.d m.o m.mmm m.> m.m om
N.o>¢ o.m o.H o.mmm o.m m.o >.¢mm m.¢ m.a om
N.mmm m.m o.a o.omH N.H m.o m.wa> H.0d ¢.H we
m.mmm m.m m.o m.mmd o.d m.o o.om¢ m.m m.H ow
HmEHoc one HmEHoz HmEHoc mca Hmauoz HmEHoc mcfl Hmeuoz
mo R mm Icmmuum mo R on Icmmnom mo R mm Icmmnum
mcflcmmuum mcflcmmuum mcflamouom
moon Emum uoom mmcflq

 

 

 

 

.mGOHuSHom mcflcmmuom can Adamo:
ca czoum mocHH coon >>mc mo mo>moa cam .mEmum .muoou on» CH awoaumn c2\mm .m manna

28

either the availability or the utilization of other elements
due to interactions with Fe and/or Mn. Since it is possible
to make only limited qualitative observations, a reliable
quantitative guide should be established. Tissue concentra-
tions of Fe and Mn were chosen for this purpose.

On the basis of the composition of nutrient solution,
both Fe toxicity and Mn deficiency may be eXpected to develop.
Although attempts were not made to identify real cause, simi-
lar symptoms were noted throughout the investigation with
little variation in the intensity of the chlorosis that
developed. Visually observed chlorosis may be correlated
with distribution of the two elements within the plant.
Studies involving single elements have revealed that the
capacity of certain genotypes to tolerate toxicity under high
levels is a result of their inability to transport the ele-
ment to the shoot (51), while the capability to escape the
deficiency at low supply is due to efficient transport (54).
The concentrations of Fe and Mn and Fe/Mn ratios may indicate
that the differences observed in this study are of a combined
nature; an efficient Fe transport and an inefficient Mn trans-
port in the chlorotic lines and the converse in nonchlorotic
lines. In conclusion, therefore, the appearance of chlorotic
and nonchlorotic conditions may be related to the differential
distribution of Fe and Mn within the plant; relatively high
Fe and low Mn in the foliar parts is conducive to chloroSis

and the converse for nonchlorosis. Differences in Fe

29

transport were noted by Brown (6) in two soybean varieties
and the involvement of oxidative phosphorylation was postu-
lated as a likely cause. The involvement of natural organic
acid chelates (10,44,45) often has been associated with Fe
stress. The differences in the quality or the quantity of
these chelates have been postulated to account for the dif-
ferences in Fe tranSport. Mn has been shown by Munns et al.
(29,50) to exist in various forms in the roots. The size and
the turn-over rate of labile forms has been shown to account
for the differences in shoot-Mn among oat varieties. Similar
systems may be Operative in bean plant for Fe and Mn, but it
is not possible to elucidate the nature of the mechanism(s)
involved. An inverse relation between Fe and Mn may be a
result of direct antagonism occurring during transport, as
has been reported (12,41). This may also arise due to a
greater uptake of interfering ions, e.g., phosphorus, which
might render Fe inactive in the root system (11) and promote
Mn transport. It is also possible that Fe and Mn are trans-
ported independent of each other, in a manner depending on
the genetic constitution. Then it should be possible to
identify recombinant segregates with transport combinations
different from the parents in segregating pOpulations, and
also, certain genetic combinations should tolerate the
antagonistic effects of Fe and Mn.

It is apparent from the results presented that the

chlorotic and nonchlorotic lines exhibit quantitative

50

differences. In most instances these differences are not
expressed in data from the screening solution alone, but
require a comparison with data from normal solutions. Since
it was not possible to establish an index to reveal genotypic
differences in screening solution alone, genetic studies were

based on visual symptoms.

2. Genetic Studies

 

Plants in F1, F2, and backcross populations were evalu-
ated for qualitative responses in order to determine an
approximate mode of inheritance. All the F1 combinations
were planted in screening, as well as normal, solution.
Phenotypic appearance of various hybrids in the screening

solution is presented in Table 4 and Figure 2.

Table 4. Phenotypic appearance of F1 combinations of navy
bean lines grown in screening solution.

 

 

 

Cross Phenotype
40 x 47 (nonchlorotic x nonchlorotic) nonchlorotic
40 x 50 (nonchlorotic x chlorotic) chlorotic
40 x 80 (nonchlorotic x chlorotic) chlorotic
47 x 50 (nonchlorotic x chlorotic) chlorotic
47 x 80 (nonchlorotic x chlorotic) chlorotic
50 x 80 (chlorotic x chlorotic) chlorotic

 

51

Figure 2. Phenotypic appearance of F1 combinations of navy
bean lines grown in screening solution.

 

 

A. Cross 40 x 47

 

 

 

B. Cross 40 x 50

52

Figure 2 (cont'd)

 

C. Cross 47 x 80

 

D. Cross 50 x 80

 

55

The results (Table 4) indicated phenotypic dominance of
the chlorotic condition, since crosses involving at least
one chlorotic parent exhibited chlorosis and a nonchlorotic
hybrid arose only if both the parents were nonchlorotic
(40 x 47). The two chlorotic lines behaved identically as
did the two nonchlorotic lines, judged by the appearance of
different F1 combinations. This may indicate that the genetic
constitutions of both chlorotic lines, 50 and 80, are identi-
cal, and similarly for the two nonchlorotic lines, 40 and 47.

Individual plants in each of the two F2 and four back-
cross pOpulations were examined visually and categorized as
chlorotic or nonchlorotic. Chi-square values for goodness of
fit to theoretical ratios are presented in Table 5. Both of
the F2 populations fit a 9 chlorotic:7 nonchlorotic segrega-
tion pattern, indicating the involvement of two genes. If
the genotypes of chlorotic and nonchlorotic parents are
assumed to be AA_§§ and §§_§b respectively, the genetic con-
stitution of the F1 plants will be A§_§b, giving a chlorotic
phenotype. The backcross of a F1 plant to a nonchlorotic
parent (Aa Bb x §§_bb) will produce four genotypes (§§_§Q.
A§_§b, §3_§b and gg_2§),giving an eXpected phenotypic ratio
of 1 chlorotic:5 nonchlorotic. The chi-square values and
probabilities were compatible with the theoretical eXpecta—
tions. With this genetic hypothesis, the backcross involving
the F1 and the chlorotic parent (Aa_§b_x AA_B§) should produce

all chlorotic plants with the following genotypes: AA BB,

 

 

 

 

 

 

 

 

 

 

 

 

 

UHuOHoHso
II II HH4 0 an m mm cm x am Aom x hev
oa.olom.o mm.m mud mm m mm we me x am Row M bwv
Om.oumm.o Nwoo.oo Sum 0.0m ¢.mm Hm mm mm Aom x may
UHuOHoHQU
II II Ham 0 en 0d em om x Hm “om x owv
4.
5 oa.onom.o mm.m mua em . m om NH Os x am Aom x owv
om.onom.o 05.0 bum a.¢m m.om «m «m «m Aom x owv
oauonoHno vapouoHSO UHuOHoHSU UHuOHoHSO
Icoz Icoz
MuHHHQ osam> oaumu oouummxm oo>ummno
Imnoum mumsvmlanu ooEDmm¢ mucmHm mo HoQEsz :oHumasmom
.coausaom mcacmmuom ca GBOHm moCHH
coon >>mc mo mGOHumHsmom mmonoxomn paw mm map How paw mo mmmcooom How mosHMP
mumsvmlflso pom mucmHm UADOHOHQUCOG ocm oauouoano mo mquEsc pouoomxo pom oo>uomno .m manna

55

AA Bb, Aa BB and Aa Bb. Chi-square values could not be calcu-
lated because of the unexpected appearance of nonchlorotic
plants.

Since the nonchlorotic plants were noted in both the
backcross populations involving F1 and their respective
chlorotic parents in considerable numbers, the assumed genetic
constitution(s) or the mechanism(s) may be an oversimplifica-
tion of the more complex mechanism really involved. An
alternate hypothesis is being proposed.

The analysis of plant parts in the initial screening
section indicated that the distribution of Fe and Mn differs
in the two sets of lines and also, the relative balance of
the two elements is more important than the absolute concen-
tration of either one. Although, total amount of Fe and Mn
is not known, nevertheless, the ratio of total Fe/total Mn
in the foliar parts may be a reliable index for the inter-
genotypic differences. Thus, the characteristics of the
chlorotic and nonchlorotic lines may be as follows:

Chlorotic High Fe and Low Mn transport High Fe/Mn
ratio

Nonchlorotic Low Fe and High Mn transport Low Fe/Mn
ratio

Genotypes were initially assumed to be AA_§§ and aa bb
for chlorotic and nonchlorotic parents respectively. This
assumption is not supported by the backcross pOpulations
derived from the F1 and the chlorotic parents. It is then

postulated that the genotype of a chlorotic parent is AA bb

56

(AA- High Fe transport, bb- Low Mn transport), and of a non-
chlorotic parent is aa BB (aa- Low Fe transport, BB- High

Mn transport). Heterozygosity at the A locus (Aa) results

in the transport of more Fe than AA, and BB is completely
dominant over bb. An additional assumption is being made,
i.e., Aa with BB or Bb and aa bb produce nonchlorotic plants
since the Fe/Mn ratio would not be too high. The implications
of this postulation are discussed.

An F; with genetic constitution Aa Bb would be chlorotic.
The Fe/Mn ratio would be higher than the chlorotic parent
because Fe transport is accentuated in the hybrid more so
than Mn transport. The range of ratios in the F2 population
should exceed the lower and higher limits imposed by the
parental genotypes because of different transport recombinants.
The proposed genetic constitutions, their prOportions,
pattern of Fe and Mn transport, Fe/Mn ratios and the pheno-
types in the F2 and backcross pOpulations are given in
Table 6.

Chi-square values were calculated for backcross popula-
tions according to the expected ratios (Table 7). The values
are non-significant indicating that the assumed ratios and
the genetic constitutions can be accepted.

On this assumption, genotypes of F2 could be arranged
on the basis of the Fe/Mn ratio in shoots in ascending order

(classes 1-6) as follows:

57

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oauouoHao swam sou nonmam a name
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oauouoHno ream 30g swam a peas
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oHuouoHno nmflm Bog Harman m name
capoungo swam sou swam a pass
oauouoaso swam 30g “mamas a 9mm«
oauouoaso swam 304 umnmflm N mmmé
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mocmumommfi oaumu cz\mm mmocmmcmz couH coauuomoum nonhuocmw coaumHsmom

omuommxm cumuumm HHOQWGMHB

 

 

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58

 

 

 

 

 

 

 

 

 

 

 

 

 

om.ouom.o Sm.o Hum m.m m.mm m mm om x am
mo.onoa.o m>.m and ma ma mm we we x am
8m x Z;
om.ouo~.o em.o dam m.m m.mm oa «N om x am
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Aom x oev.
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Icoz Icoz
muHHan mwsHm> oaumu omuuoaxm om>nmmno
Innoum mumsvmlano omEDmm< mucmHmlmo Hmnfisz coaumasmom
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aa BB aa bb AA BB AA bb (Aa BB Aa bb
aa Bb AA Bb Aa Bb
1 2 5 4 5 6

Classes 1, 2, and 5 are eXpected to be nonchlorotic
(7/16) and classes 4, 5, and 6 chlorotic (9/16). Furthermore,
8/16 of the F2 population (classes 5 and 6) should have a
higher Fe/Mn ratio than the chlorotic parent and 2/16 (class
6) higher than the F1. The backcross to the chlorotic parent
should produce plants in a 1 : 1 : 1 : 1 prOportion in
classes 5, 4, 5, and 6, respectively. Backcross with non-
chlorotic parents should have one-half of the pOpulation
similar to the nonchlorotic parent and the other half like
the F1 (chlorotic).

The alternate hypothesis proposed here seems plausible
and explains the appearance of nonchlorotic plants in back-

cross pOpulations with chlorotic parents.
5. Grafting Experiment

Grafts in different root/scion combinations were made
to localize possible sites of control. Established grafts
were divided into two groups for nutrient application, i.e.,
normal and screening solutions. The appearance of grafted
plants after five weeks was recorded and the observations
are presented in Table 8 and Figure 5.

It is clear (Table 8) that the genotype of the root-
stock is responsible for the appearance of chlorosis in line

50, since on this root even the scion of nonchlorotic line 40

40

Table 8. The appearance of grafted navy bean plants grown
in screening solution.

 

 

 

Graft combination (scion/rootstock) Appearance
40/40 (nonchlorotic/nonchlorotic) nonchlorotic
40/50 (nonchlorotic/chlorotic) chlorotic
50/40 (chlorotic/nonchlorotic) nonchlorotic
50/50 (chlorotic/chlorotic) chlorotic
47/47 (nonchlorotic/nonchlorotic) nonchlorotic
47/80 (nonchlorotic/chlorotic) nonchlorotic
80/47 (chlorotic/nonchlorotic) chlorotic
80/80 (chlorotic/chlorotic) chlorotic

 

develops chlorosis while the scion of line 50 grafted on the
rootstock of line 40 does not. Conversely, susceptibility
in line 80 seems to be determined by the genotype of the
scion because it always develOped chlorosis whether grafted
on a chlorotic or a nonchlorotic rootstock. In addition,
the rootstock of line 80 does not seem to be inefficient
since chlorosis does not develOp when a scion of line 47 is
grafted onto a rootstock of line 80.

The concentrations of Fe and Mn in roots, stems and
leaves are presented in Table 9.

Table 9 contains the concentrations of Fe and Mn (pg/g)
in roots, stems and leaves. The values for plants grown in

screening solution are also eXpressed as percent of normal.

41

Figure 5. The appearance of grafted navy bean plants grown
in screening solution.

   

‘ I so
4
H.FE,0

 
   

5'6
-MN H.FE>"M~

 

A. 40/40 and 50/50

 

50

 

B. 40/50 and 50/40

42

Figure 5 (cont'd)

 

C. 47/80

-

 

D. 80/47

45

 

 

 

 

 

 

 

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m.mm m.mm m.mmfi m.wm o.oom o.mam mood
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mo & mm Icmmuom mo R up Icmwuom
mcacwmnum maficmmnom puma
ommcmmcmz COHH ucmHm ummnw

 

 

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44

.Ama some some mommamcm 035 no mmmum>ad

 

 

 

 

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5.m¢ ¢.mm m.mm m.dmd 5.HOH w.m0 Emum
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m.mfi m.5d $.5NH 0.60 ¢.fi5d 0.0dm mmmq
¢.mm 0.0H 0.00 m.d5 H.05 d.mOH Emum
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45

The concentration of Fe in the roots in screening solu-
tion does not reveal a pattern for root/scion combination.

If the concentration of Fe in roots in the screening solution
is expressed as percent of Fe in roots in the normal solution,
lines 40 and 50 seem to behave oppositely than what was noted
in the screening experiment. Yet, however, the capacity of
both the chlorotic lines 50 and 80 to retain Fe in the root
is enhanced when grafted in combination with the nonchlorotic
scions, 40 and 47. This suggests that probably the capacity
of roots to retain Fe or to transport it to the shoot is
governed by the genotype of the scion, and the nonchlorotic
scions, 40 and 47 are more efficient for Fe retention by the
roots than the chlorotic scions, 50 and 80.

Considering Fe concentration in the leaves, all the com-
binations having nonchlorotic scions, e.g., 40/40, 40/50.
47/47 and 47/80, have less Fe than when the chlorotic scion
is involved. The pattern is not clear, however, if the con-
centration in screening solution is expressed as percent of
normal. This again tends to show that if a nonchlorotic
scion is involved in the graft, the concentration of Fe is
lower in the leaves than when the scion is of chlorotic line.
.Also, the concentration of Fe p§£_§e_does not seem to be the
sole factor in the development of chlorosis.

Mn in roots does not reveal a pattern. The combination
40/50 has the greatest concentration in the root. Rootstock

of line 80 always has high concentration of Mn, showing the

46

efficiency of line 80 rootstock in retaining Mn. However,
the pattern is not clear for the chlorotic symptom.

In the leaves, Mn concentration does not reveal a
consistent pattern. In the grafts of line 40 and 50 combi-
nations, both the nonchlorotic grafts have more Mn than the
chlorotic combinations. In the graft combinations of line
47 and 80, when the rootstock 80 is involved, leaves have
more Mn than when the rootstock is line 47. If the values
of plants grown in screening solution are eXpressed as per-
cent of normal, the nonchlorotic combinations tend to have
higher values than the chlorotic combinations with the
exception of combination 47/47. It seems that the concen-
tration of Mn is an important factor in the development of
chlorosis, though it may not be the sole factor.

Fe/Mn ratios are presented in Table 10. If the scion
is line 40, the ratio is lower in the roots than if the
scion is of line 50 in the graft combinations of line 40 and
50. In line 47 and 80 graft combinations, if the rootstock
is line 47, the ratio is higher than if the rootstock is
line 80, but the pattern fails to hold if the ratios of
screening solutions are expressed as percent of ratios in
the normal solution. In comparison with normal, however,
both the nonchlorotic combinations, 47/47 and 47/80 have
higher values than the chlorotic combinations.

In the leaves, all the nonchlorotic graft combinations

have relatively lower Fe/Mn ratios than the chlorotic

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48

combination, though the pattern is not clear if compared
with normal. This agrees with the previous assumption that
the relative balance between Fe and Mn is more important in
the deveIOpment of chlorosis than the absolute concentra—
tion of either one. High ratios are always associated with
the chlorotic condition. However, it is not possible to
state a critical ratio which separates the chlorotic and
nonchlorotic condition, according to this study.

The results of initial screening indicated that the
nonchlorotic lines (40 and 47) are efficient in transporting
Mn to the shoot and inefficient in transporting Fe. The
converse is true for chlorotic lines which upsets the rela—
tive balance between Fe and Mn. Results of the grafting
experiment show little deviation from what was noted earlier
in the screening experiment. It should be re—emphasized,
however, that due to grafting the appearance of chlorosis
was delayed about a week and plants were harvested only after
the symptoms were noted. According to the observations in
the screening experiment, this was beyond the critical period.
It is possible that the visual appearance of chlorosis is
delayed because of the effect of grafting. Extreme devia-
tions are shown by lines 40 and 50 and they seem to behave
oppositely in some respects. It may be that the degree of
resistance in line 40 is relatively low or the differences

between line 40 and 50 are not of large magnitude.

49

Results indicate that rootstock line 50 is capable of
holding Fe but it is particularly efficient in retaining Mn.
The graft of 40/50 develops chlorosis because line 50 root-
stock not only retains Fe but at the same time it also
retains Mn, and therefore, the balance in the foliar parts
is upset. The reciprocal graft of 50/40 does not develop
chlorosis because of the inefficiency of line 40 rootstock
in retaining Mn and its efficiency in holding Fe. Similarly,
rootstock of chlorotic line 80 seems to have the capacity to
retain Fe but it also holds Mn, behaving in this reSpect
like line 50.

It seems probable that the Fe holding capacity of both
lines 50 and 80 rootstocks is not inherently inefficient
but that Fe retention is governed by the genotype of the
scion. Both lines 40 and 47 scions are efficient in retain-
ing Fe in the roots of any genotype to which they serve as
scions. They probably are deficient in some process involved
in the transport of Fe. It is difficult, however, to describe
the nature of the mechanism. The results are not very clear
for the lines 40 and 50, yet the efficiency of line 50 root-
stock in retaining Fe is greatly enhanced if the scion is of
line 40. Also, if a line 47 scion is placed on a line 80
rootstock, this root can retain Fe equally well. Conversely,
a line 47 rootstock does not hold Fe if the scion is line 80.
At the same time, Mn holding capacity is largely dependent

Upon the genotype of the rootstock; both lines 50 and 80 are

50

efficient. The develOpment of chlorosis then seems to de-
pend upon the genotypes of both the rootstock and the scion.
If the rootstock holds Mn, chlorosis develops depending upon
the Fe status of the shoot, and this is governed by the geno-
type of the scion.

0n the basis of chlorotic symptoms on plants of various
graft combinations, lines 50 and 80 are different. The ana-
lytical data, however, suggests that the physiological
mechanism(s) involved is similar in both the chlorotic lines,
i.e., both of them retain Mn and are efficient in retaining
Fe. This supports the conclusions of the initial screening

experiment.

4. Exudate Experiment

 

Results in the previous sections suggested that the
chlorotic and nonchlorotic lines differed in transporting
Fe and Mn to the shoot. In order to determine the differences
in the rate of transport, exudate was collected and analyzed.
The data are presented in the following Tables 11—15.

Table 11 contains data on the total weight of exudate.
The values indicate that in normal solution excised plants
of nonchlorotic lines deliver more sap in a given time, whereas
in screening solution they deliver less. If the values in
screening solution are eXpressed as percent of normal, plants
of chlorotic lines have higher values than the nonchlorotic

lines. In nonchlorotic plants, a more efficient root system,

51

Table 11. Total wt of exudate in g per bean plantl grown
in normal and screening solutions.

 

 

 

Lines Normal Screening Screening as %
solution solution of normal
40 1.8 0.6 33.3
47 1.7 0.8 47.0
50 0.8 1.0 125.0
80 1.5 1.1 73.3

 

1Average of 4 plants.

greater capacity of xylem system and differential osmotic
pressures in the steler tissue, than in chlorotic plants which
have been postulated (54), may account for the different rates
of sap flow of plants grown in normal solutions. Reduced sap
flow by plants of nonchlorotic lines in screening solution
may be a physiological mechanism for adapting to the altered
composition of the nutrient solution. It is possible that
the over all salt accumulation in these lines is low, and
this, by affecting water uptake and transport, also affects
the volume of sap exuded.

Fe and Mn content (ppm) are given in Table 12.

The Fe concentration of plants grown in screening solu-
tion does not reveal any pattern. When the values are

expressed as percent of normal, however, nonchlorotic lines

52

Table 12. Fe and Mn concentrations (ppm) in the exudate of
bean plants grown in normal and screening solutions.

 

 

 

 

 

Lines Fe Mn
Screening Screening
Screen- as % of Screen- as % of
Normal ing normal Normal ing normal

40 0.45 0.72 166.60 0.60 0.16 26.60
47 0.86 1.45 166.60 0.60 0.20 55.50
50 0.57 0.86 150.00 0.90 0.08 8.88
80 0.86 1.14 155.50 1.10 0.06 5.45

 

 

 

 

 

 

 

show higher values than chlorotic lines. The Mn concentration
is higher in plants of nonchlorotic lines grown in screening
solution as well as in comparison with plants grown in normal
solution than chlorotic lines. High Fe concentration in non—
chlorotic lines is in disagreement with the earlier results.
Total Fe and Mn were then calculated by multiplying ppm con-
centration by the weight of exudate. These values are given
in Table 15.

Total Fe does not show distinct differences in screening
solution alone, but if expressed as percent of normal,
chlorotic lines have higher values than nonchlorotic lines.
Mn is high in nonchlorotic lines grown in screening solution.
If the values are expressed as percent of normal, however,

differences are not clear.

55

 

 

 

 

 

Table 15. Total Fe and Mn (ug) in exudate of been plants
grown in normal and screening solutions.
Lines Fe Mn
Screening Screening
Screen- as % of Screen- as % of
Normal ing normal Normal ing normal
40 0.78 0.48 64.10 1.10 0.11 10.00
47 1.51 1.17 77.48 1.06 1.16 15.44
50 0.45 0.89 197.77 0.71 0.08 11.70
80 1.55 1.27 94.07 1.74 0.07 5.85

 

 

 

 

 

 

 

given in Table 14.

The Fe/Mn ratios in the exudate

were calculated and are

 

 

 

Table 14. Fe/Mn ratios in the exudate of bean plants grown
in normal and screening solutions.
Lines Normal Screening Screening as %
of normal
40 0.71 4.40 615.50
47 1.40 7.15 510.71
50 0.65 10.70 1698.00
80 0.78 19.00 2455.00

 

Chlorotic lines have high ratios in screening solution.

They also show high values if the ratios of screening solution

54

are expressed as percent of normal. This supports the find-
ings reported previously.
The pH of the exudate was measured. Table 15 contains

these values.

Table 15. pH of the exudate of bean plants grown in normal
and screening solutions.

 

 

Lines Normal Screening Screening as %
of normal

 

40 5.50 6.00 109.10
47 6.50 6.00 95.25
50 5.85 6.15 105.15
80 5.90 6.55 111.01

 

In screening solution the chlorotic lines have a higher
pH, but there is no pattern if compared with plants cultured
in normal solution. Both the nonchlorotic lines tend to have
a pH around 6.00, and this may be considered a best pH for
physiological adaptation to the altered composition of
nutrient solution.

The findings from the exudate experiment are consistent
with the view that the reduction in the amount of sap being
delivered by nonchlorotic lines is a physiological adaptation
to ionic imbalance. The Fe content (ppm) of nonchlorotic

lines is higher than chlorotic lines, but when total Fe

55

(conc x g of exudate) is calculated, chlorotic lines have
more Fe, i.e., more Fe is transported in a given time. Mn is
higher in nonchlorotic lines than chlorotic lines, but the
differences are not clear when total Mn in screening solution
is expressed as percent of Mn in normal. This discrepancy
may be because a normal amount of Mn was provided one week
prior to exudation and during exudation and it is possible
that the lines differ in their abilities to recuperate after
stress. The Fe/Mn ratio was highest for the chlorotic lines
and this agrees with the previous findings. It is difficult
to relate pH with the differences in Fe and Mn transport due
to limited data. It seems then that the nonchlorotic lines
adjust to the ionic imbalance by reducing total Fe transport
via reducing the sap delivery. At the same time sap is
relatively more concentrated for Mn and therefore the balance
between Fe and Mn is not upset.

With respect to the relation between the genetic and
physiological mechanisms, it is suggested that the genes do
not control the concentration p§£.§g_but the accumulation or
the total amount of an element via affecting the rate of sap
flow. The differences are not always clear for Mn, but the
amount of Fe shows a distinct pattern. Higher Fe transport
is associated with a greater amount of exudate, which results

in a higher Fe/Mn ratio which eventually induces chlorosis.

CONCLUSIONS

The findings may be summarized as follows:

1. Initial Screening

 

Lines of beans responded differentially to the screening
solution; lines 40 and 47 were nonchlorotic whereas lines 50
and 80 develOped chlorosis. The analysis of plant parts
indicated that the chlorotic and nonchlorotic lines differed
in the pattern of distribution of Fe and Mn in root and shoot--
relatively low Fe and high Mn transport to the shoot in the
nonchlorotic lines and the converse in chlorotic lines. The
quantitative differences could not be used for genetic study,
since they involved comparisons with plants grown in a normal
solution, and such comparisons were not possible on individual

plants of segregating populations.

2. Genetic Studies

The appearance of the F1 combinations indicated pheno-
typic dominance of the chlorotic type. Both the chlorotic
lines behaved identically as did the two nonchlorotic lines.
Both F2 pOpulations segregated 9 chlorotic:7 nonchlorotic

plants indicating the involvement of two genes.

56

57
5. Graftipg Experiment

Visual appearance of the graft combinations indicated
that the genotype of the rootstock line 50 and the genotype
of the scion line 80 were responsible for the develOpment
of chlorosis. Tissue analysis, however, suggested that the
roots of both chlorotic lines were efficient in retaining Fe
but at the same time they also retain Mn. On the basis of
the appearance and the analysis, it was postulated that the
capacity of roots to retain Fe or to transport it to the
shoot is dependent on the genotype of the scion, whereas the
capacity to retain Mn is due mainly to the genotype of the

rootstock.
4. Exudate Experiment

The nonchlorotic lines had lower rate of sap flow when
grown in screening solution than chlorotic lines. Although
the concentration of Fe was greater in the exudate of non-
chlorotic lines, yet the total amount delivered was less.
The Mn concentration of nonchlorotic lines was greater than
that of chlorotic lines, but the pattern was not distinct
for the amount. The Fe/Mn ratios and pHS were greatest in
the exudate of chlorotic lines.

Genes were postulated to control the amount of Fe and

Mn via affecting the rate of sap delivery.

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