EFFECTS OF MINERAL LEVELS ON

PHYSIOLOGY AND MORPHOLOGY

OF PLANTS

By

JOHANNES BERNARDUS BALTHASAR BROLMANN

A DISSEKTATION PRESENTED TO THE GRADUATE COUNCIL OF

THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1968

AQHU

CULTURAL
UBRARV

UNIVERSITY OF FLORIDA

3 1262 08552 2547

ACKNOWLEDGEMENTS

The author wishes to express his deep appreciation and
sincere gratitude to Dr. Henry C. Harris and Dr. Sherly H.
West for their advice, criticism and encouragement in the
preparation and completion of this manuscript.

The author also wishes to thank Dr. Richard C. Smith
for serving as a member of the supervisory committee and
reviewing the manuscript.

Appreciation is also extended to the Department of
Agronomy for their very generous support which made it
possible to conduct graduate studies at the University of
Florida.

Gratitude is similarly extended to the Department of
Entomology and Nematology for use of their laboratory faci-
lities.

Deepest appreciation must go to his wife Antoinette, and
his mother for their help and moral support during the years
of graduate study.

IX

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

LIST OF TABLES v

LIST OF FIGURES vi

INTRODUCTION 1

REVIEW OF LITERATURE 2

Accumulation of Elements 2

Effect on Uptake and Distribution of Other Ions.. 3

Toxic Effects 5

Cytological Effects 6

Effects on the Germination of Different Seeds.... 7

Tolerance and Adaptation to Heavy Metals 8

Plasmatic Resistance 11

Long-Term Effects 13

Effects on Chromosome Aberrations and Plant

Mutations 14

Interaction with Chelates 19

The Mode of Action on Chromosomal and Cellular

Behavior 21

EXPERIMENTAL AND RESULTS 24

Part One 25

Experiment 1: Effect of Copper and Manganese

Deficiencies on Soybeans 25

Part Two 30

Experiment 2: Effect of Concentrated Solutions
of Copper, Manganese and Zinc Salts on
Seeds of Soybean, Rye, Sorghum and Oats... 30

Experiment 3: Effect of Copper, Manganese and
Zinc Solutions on Germination of Sorghum
Seeds 36

Experiment 4: The Effect of Soaking Oat Seeds
in Concentrated Solutions of Copper,
Zinc and Manganese 38

Experiment 5: Effect of the Anions NO3",
Cl~, S0/^"~ on the Germination of
Portulaca oleracea Seeds 39

Experiment 6: Effect of Various Concentrations
of Copper and Zinc Solutions on Germina-
tion and Further Development of Portulaca
oleracea Seeds 41

111.

TABLE OF CONTENTS (continued)

Page

EXPERIMENTAL AND RESULTS (continued)

Experiment 7: Effect of Lithium Chloride

on Portulaca oleracea 44

Experiment 8: Effect of Various Levels of

Mineral Elements on Plants and Progeny

Seeds of Portulaca oleracea 70

Part Three 78

Experiment 9: Inducing Metal Tolerance in

Portulaca oleracea 78

Experiment 10: Inducing Plasmatic Resistance

in Portulaca oleracea 80

DISCUSSION 82

SUMMARY AND CONCLUSIONS 91

BIBLIOGRAPHY 93

LV

LIST OF TABLES

Page

TABLE

1. YIELD DATA FOR SOYBEAN PLANTS DERIVED FROM

ABNORMAL SEEDS DUE TO COPPER DEFICIENT

TREATMENT AND NORMAL SEEDS 32

2. GERMINATION OF SOYBEANS, RYE, SORGHUM AND OAT

SEEDS AFTER SOAKING IN VARIOUS CONCEN-
TRATIONS OF COPPER, MANGANESE AND ZINC FOR
24 HOURS 34

3. GERMINATION OF SOYBEANS, RYE, SORGHUM AND OAT

SEEDS AFTER SOAKING IN VARIOUS CONCENTRATIONS
OF COPPER, MANGANESE AND ZINC FOR 24 HOURS,
FOLLOWED BY DRYING 35

4. EFFECT OF STRONG SOLUTIONS OF COPPER, ZINC AND

MANGANESE ON GERMINATION AND EARLY DEVELOP-
MENT OF SORGHUM SEEDS 37

5. EFFECT OF DIFFERENT LEVELS OF POTASSIUM NITRATE,

POTASSIUM CHLORIDE AND POTASSIUM SULFATE

SOLUTIONS ON THE GERMINATION OF PORTULACA

OLERACEA SEEDS 40

6. EFFECT OF DIFFERENT CONCENTRATIONS OF COPPER

SULFATE ON GERMINATION, SHOOT LENGTH AND

ROOT LENGTH OF PORTULACA OLERACEA 43

7. EFFECT OF DIFFERENT CONCENTRATIONS OF ZINC

SULFATE ON GERMINATION, SHOOT LENGTH AND

ROOT LENGTH OF PORTULACA OLERACEA 45

8. EFFECT OF TREATMENT WITH DIFFERENT IONS IN

VARIOUS CONCENTRATIONS ON RATE OF SURVIVAL

OF PORTULACA OLERACEA CUTTINGS 71

9. EFFECT OF TREATMENT WITH DIFFERENT IONS IN

VARIOUS CONCENTRATIONS ON RATE OF SURVIVAL

OF PORTULACA OLERACEA SEEDLINGS 73

LIST OF FIGURES

Page

Figure

1. Photomicrograph of longitudinal section through

cotyledon of soybean seed (normal seeds) 27

2. Photomicrograph of longitudinal section through

cotyledon of soybean seed from manganese

deficient plants 28

3. Forked stems in soybean as a result of manganese

deficiency 29

4. Twin plants in soybean as a result of copper

deficiency 31

5. Portulaca oleracea plant derived after seed

treatment with 1 °U lithiimi chloride 47

6. Flowers of wild and mutated types of Portulaca

oleracea 48

7. Flower of mutated type of Portulaca oleracea

produced after seed treatment with 1 7„

lithiiom chloride 49

8. Mutated branch of Portulaca oleracea 50

9. Cutting of mutated branch of Portulaca oleracea,

showing reversion to wild type at base 52

10. Cutting of mutated branch of Portulaca oleracea,

showing young wild shoot at base 53

11. Percent reversion from mutated to wild types in

four generations of clones of Portulaca

oleracea 54

12. Photomicrograph of stomata of lower leaf

epidermis of mutated type of Portulaca

oleracea 60

13. Photomicrograph of stomata of upper leaf

epidermis from wild type of Portulaca

oleracea 61

VI

LIST OF FIGURES (continued)

Figure

Page

14. Photomicrograph of stomata of lower leaf

epidermis from wild type of Portulaca

oleracca 62

15. Photomicrograph of stomata of upper epidermis

from mutated type of Portulaca oleracca 63

16. Photomicrograph of cross section through wild

shoot of Portulaca oleracca 66

17. Photomicrograph of cross section through mutated

shoot of Portulaca oleracca 68

18. Photomicrograph of cross section of half mutated

(left) and half wild (right) stem tissues of
Portulaca oleracea 69

19. Effect of pretreatment of boric acid on percent

germination of Portulaca oleracca seeds 74

20. Effect of pretreatment of copper chelate on

percent germination of Portulaca oleracea

seeds 75

21. Effect of pretreatment of iron chelate on

percent germination of Portulaca oleracea

seeds 76

22. Effect of pretreatment of zinc chelate on percent

germination of Portulaca oleracea seeds 77

via.

INTRODUCTION

The influence of certain mineral deficiencies on the progeny
seed from peanut plants has been described (44) . The results of
those experiments, together with reports in the literature on
mutagenic effects of deficient and excessive mineral levels, prompted
a study of these mineral effects on other plants in more detail.

The effect of mineral deficiencies on seed formation, seed
characteristics and phenotype of subsequent generations in soybeans
was studied here, because earlier reports (43) showed a very strong
response of this plant to mineral deficient treatments.

Furthermore it seemed desirable to conduct experiments in
which many elements were tested to induce mutations, especially
in Portulaca oleracea, a plant especially suitable for such experi-
ments because of its short life cycle.

The pronounced tolerance of certain plants to high levels of
heavy metals (9, 11, 38), suggested the possibility of inducing
metal tolerance in Portulaca oleracea. It seemed desirable therefore
to treat cuttings of Portulaca oleracea with high levels of heavy
metals and to study the adaptation to these high metal levels.

REVIEW OF LITERATURE

Many aspects of the effects of mineral elements on physiology
and morphology of plants have been brought together. Most of the
literature discussed here deals with the effect of high levels of
elements. Accumulation, interaction with other ions and toxic
action are reviewed. Effects on germination and tolerance to heavy
metals are discussed. Most of the literature available on long-
term effects and mutagenic action of elements has been cited. A
short review on the mode of action of various elements on chromosome
and cellular behavior concludes this literature survey.

Accumulation of Elements

Uptake and accxomulation of various elements is strongly
dependent upon the environment and is, furthermore, largely genetic-
ally controlled. Bertrand (4) showed that seeds of monocotyledons
contained less lithium than those from the dicotyledons. The vege-
tative parts were in general four times richer in lithium than the
seeds. Large differences existed also between the various plant
families. Plants in the Papaveracea family, for example, were very
high in lithium while those in the Rubiacea family were very low.

It has been shown by Zimmerman e_t a_l. (Ill) that several
species in the family of Theaceae do accumulate fluoride in large
amounts. In other species fluoride content could only be increased
through additional amounts of fluorides as was shown in celery and

several other legumes (32) . Robinson and Edington (86) reported an
extreme variation in selenium content (from 0.1-15,000 ppm) in plants.
Astralasus species were able to accumulate up to 4,000 ppm Se from
a soil that contained only 2 ppm Se. Seeds of Neptunia from plants
grown on soils rich in selenium contained 123 pg Se per seed (80) .
When these seeds were planted on normal soil the next generation
seeds had only 0.45 mg Se per seed. However, analysis showed that
the total amount of selenium in one plant was + 0.100 mg. This
could have come from entirely mobilized selenium in the seed.

Thomas and Baker (102) provided evidence that the level of
chemical element accumulation in corn is under partial genetic
control. Through plant breeding it is possible to obtain low
accumulating genotypes for some elements. This may be of importance
when varieties are required which accumulate less of a toxic or
radioactive element as with strontium fallout.

Effect on Uptake and Distribution of
Other Ions

A mineral excess or deficiency would be expected to affect the
uptake, distribution and metabolism of other ions. Also the inter-
relationship with other ions probably would differ with the metal
tolerant species. Under copper deficiency young citrus plants

accumulated very small amounts of phosphorus as was shown in auto-

32
radiographic studies with P (79). Excess copper was accompanied

by an almost normal distribution of phosphorus. However, a slight

reduction in uptake of radioactive phosphorus was observed. Excess

boron (22 ppm) also reduced considerably the uptake of phosphorus.

A lead-mine population of Agrosiiis was treated with different

4

levels of calcium and phosphorus (55). The lead-mine population did
not respond to this higher level of fertility, whereas a pasture
population (non-lead tolerant) responded favorably. Cu in concentra-
tion of 5 to 10 ppm had a lethal effect on young citrus roots but
when used in combination with an Fe-EDTA treatment the toxic effect
was reduced (95). Analysis showed that less copper had been taken
up in the latter case.

The manganese and a luminum tolerance of certain alfalfa varieties
was related to the calcium metabolism (77). Results indicated that
toxicity symptoms in both tolerant and non- tolerant plants started
at the same level of toxic concentration in the aerial organs. Ca
ions were found to be particularly high in the roots of the tolerant
species. It was believed that calcium lowered the uptake of manganese
and aluminum or immobilized them within the roots. In strontium-
adapted plants a correlation was found between the amount of
strontium and calcium (21). Relatively low amounts of strontium in
the plant corresponded to high amounts of calcium and vice versa.
Ion substitution did take place within certain plants.

Calcium partially antagonized the toxic effect of lithium
chloride (31). When 0.3 percent calcium nitrate was added to toxic
lithium chloride solutions of 0.1 and 0.3 percent, plants survived
for a longer period of time.

A high heavy metal concentration in a nutrient medium in general
reduced the uptake of iron, resulting in chlorosis (30). This was
shown for oats when Co or Cu was given in excess and for barley when
high amounts of Cu and Ni were applied. With a high cobalt treatment
the uptake of Ca and Mg in oats was higher than in the controls.

Toxic Effects

High levels of many metals and other elements are well known to
be toxic to plants. Otto (76) in 1891 did probably one of the
earliest experiments to prove the toxicity of a CuSO, solution on
corn seedlings. The author worked with a concentration of 5 and
10 ppm Cu. Plant growth was greatly reduced. Analysis showed that
the leaves had taken up only minute amounts of copper. As early as
1896 Haselhoff (46) reported the toxic effect of copper on plants.
Solutions of 5 mg CuO per liter were toxic to Zea mays . Phaseolus
vulgaris grew in a concentration as high as 10 mg CuO per liter
before toxicity symptoms occurred.

The effect of 74 different acids and metal salts on root
growth of Lupinus albus was tested (56). When different salts of
the same element were used, the same concentration of the element
in all cases caused injury. The metal ion action was independent of
the salt used and was only determined by its actual molecular concen-
tration. The same molecular concentration of cobalt, nickel, and
iron gave the same toxic effects. The authors concluded that this
was related to their atomic weights which were the same. Von
Rosen's findings were similar (105). Heald (47) provided further
evidence of the toxic action of nickel, cobalt, and copper on the
seedlings of Pisum sativum and Zea mays . Partial recovery occurred
when the plants were transferred from the toxic solution to distilled
water. It was reported (45) that galvanized iron netting had a toxic
effect on plants. From almost new wire 50 mg Zn per 100 g could
be extracted by immersing the wire for 24 hours in water. Soils
rich in nickel (100 to 400 ppm Ni) caused a considerable reduction

in growth of oats and the plants had a high amount of necrosis and
chlorosis (50). A considerable concentration of nickel occurred in
all parts of the plant, being higher in the young leaves and flowers.
Moderately affected plants contained about 35 ppm Ni in dry matter.
Severely affected plants contained 110 ppm Ni. When J»or_on,was
supplied in supra-optimal quantities to plants, it was transported
directly to the site of injury (74). Boron, in this case, probably
moves mainly with the transpiration stream. Carnations were very
tolerant and lemons very sensitive to high boron supplies. In
general, necrosis may be expected when boron concentration in the
leaves exceeds 1,500 ppm (dry wt.).

Zinc in concentrations of 1 to 10 ppm in polluted river water
is a potential danger to vegetation on the side of rivers (71).
Zinc from mine dumps was carried into the rivers as zinc sulphate.
Terrestrial vegetation was severely damaged by smelter ftmies which
contained high amounts of sulfur (36) . In some regions one million
tons of sulfur per year were released into the air. Atmospheric
fluoride in the form of cryolite particles produced no visible
injury to plants (65). Physiologically, cryolite is a rather in-
effective form of fluoride. Gaseous forms of fluoride are much more
toxic (67) .

Cytological Effects

A few studies have dealt with the effect of a mineral stress
on fine structure of particular cell components. It was shown (103)
that the absence of zinc blocked the full development of the grana
fret\</ork in the chloroplast. Redistribution of zinc did not seem to

take place, as the older leaves appeared to be normally developed.
Chloroplasts of iron deficient leaves of Xanthium (7) showed a
lamellar structure considerably different from normal ones. The
lamellae were reduced from a group of ten per granum to a group of
two or three.

A cytological effect of calcium deficiency in the shoot apex
of barley has been studied by Marinos (62). A frequent occurence of
an unevenness in the contour of the nuclear envelope, which became
vesiculated with large gaps, was observed. Other cell membranes be-
came disorganized under calcium stress. The evidence is fairly
convincing that calcium is essential for the maintenance of structural
integrity of different membrane systems.

Effects on the Germination of Different Seeds

In earlier studies the remarkable resistance of seeds to metal-
lic poisons has been shown (19) . Cress seeds were able to germinate
in a 1/75 N zinc-sulfate solution. Copper proved far more toxic
than zinc. The after-effect of strong copper solutions (1/75-1/600 N)
was detrimental to the seedlings. On transfer to normal solutions
the germinated seeds progressed no further. Ernst (26) found that
seeds from zinc-tolerant plants of Silene and Minuartia species
would germinate in very high concentrations of zinc. Sixty percent
of Silene cucubalus seeds were still able to germinate in a 10,000
ppm Zn-solution. In contrast, the control plant Epilobium augusti-
folium was unable to germinate at 500 ppm Zn. Seeds of metal-toler-
ant plants also germinated faster and their total rate of survival
was higher than in any other metal-sensitive plant.

An instant toxic effect has been reported for thallium where
very low levels destroyed the vitality of the seeds (19). It has
been suggested (38, 82, 84, 104) that the reaction of protoplasma
with heavy metal causes the protein to precipitate in the outer
layers of cells (shock-reaction). Further entry of the toxic metal
is thus prevented. Seeds of Sabal minor were able to germinate
after being immersed in seawater for four weeks (12).

Imbibition of seeds will be strongest in the lower salt concen-
trations where osmotic pressure of the solution is low. In very high
concentrations imbibition is arrested which may result in a complete
exclusion of the toxic ions.

Kisser and Lettmay r (59) tested the uptake of various metal
ions on numerous agricultural seeds. They found that the differences
in uptake are closely associated with the morphology of the seeds.
The ion-absorption was strongly dependent on the rate of water uptake
by the seeds. The faster this uptake, the more ions were absorbed.

Tolerance and Adaptation to Heav^'- Metals

Adaptation of plants to high metal concentrations has been
studied by many investigators (11, 13, 27, 37). Most of these studies
were concerned with so-called mine-plant populations. The diversity
of the mining areas around the world has contributed very much to the
knowledge and understanding of plant behavior under mineral excess.
Increasing air and water pollution have become significant factors
in providing excessive amounts of elements to plants (36, 65, 71).

Bradshaw e_t a_l. (10) discussed the evolutionary significance
of heavy metals and the role they may have played in the evolution.

They pointed out the importance of the industrial environment where
accumulation of certain waste products may result in certain selection
pressures. The development of metal tolerance in Asrostis tenuis
is one of adaption as a result of evolutionary changes. Tolerant
and non- tolerant populations have sharp bounderies even if they grow
close to each other. Wind pollination facilitates in such cases
an ample gene exchange. The heavy metals of this environment play
only a selective role and cannot in this case be considered as a
mutagenic agent. There are several species of Agrostis , which are
tolerant to high concentrations of Cu, Ni, Pb, and Zn (9, 10). The
plants which were tolerant to these heavy metals originated from
mine-plant populations. The Ni and Zn tolerance were correlated
in one direction only. Zinc-tolerant plants were always tolerant
to nickel but nickel-tolerance could be developed without tolerance
to zinc.

Iron content was determined for several plants which grew in
a soil very rich in iron (45 to 50 percent Fe) close to an old iron
mine (8) . Some plants here were particularly high in iron.
Taraxacum officinale had four times as much iron as when grown in
normal soil. Other plants such as Cirsium arvense had normal iron
contents. The differences in iron uptake in both cases illustrate
that the mechanism of tolerance must also be different.

The zinc-tolerant types of Silene, Campanula, Plantago and other
plants have smaller cells than the normal types (92) . Since these
plants also grew on a very poor and dry soil, selection pressure in
the direction of phenotypical smaller cells is very likely. The
zinc-tolerant types were not in the least tolerant to lead, indicating

10

a very specific metal tolerance. Schwanitz and Hahn (91) found a
difference in size of epidermis cells and of nucleus in various zinc-
tolerant and normal forros of the same species of Silene . When both
forms were grown under normal conditions the differences in size
of cells and nuclei were maintained. A cop_per-resistant form of
Silcnc inf lata did not appear to be resistant to zinc. Broker (11)
demonstrated that there was no correlation between the morphology
of Silene inf lata and the tolerance to high amounts of zinc. When
grown under normal conditions, Silene inf lata did not lose this
tolerance. However, the plants were altogether bigger, with long
stems and broad leaves in contrast with the more dwarf type grovm
on zinc soils. The tolerance to zinc is genetic in this particular
variety, and is carried over dominantly.

Tillers of lead-tolerant Agrostis and tillers of pasture plants
were transplanted to soil collected from an abandoned lead mine
with 1.0 percent lead and 0.03 percent zinc (9). The pasture plants
did not grow at all and 50 percent died. The lead-tolerant Agrostis
grew normally. It was shown (27) that heavy-metal tolerance is
particularly frequent in the Silene, Minuartia and Armeria species.
Minuartia verna growing on a soil containing 4,626 ppm Cu accxomulated
1,033 ppm Cu in the seeds. Silene cucubalis seeds accumulated only
374 ppm Cu from the same soil.

It has been found also (2) that the excess amount of zinc which
is taken up by the .zinc-tolerant_species of Silene inflata Sm. is
correlated to increased photosynthesis. CO2 assimilation was not
affected when non-tolerant plants were treated with higher levels of
zinc. Prat (81) described a uopper-resistant Agrostis and Melandrium
variety, growing on a sandy soil very rich in copper. Melandrium

11

silvcstre seeds were able to germinate in a mixture of 25 percent
CuCOo and 75 percent soil. Seeds from the same variety grown in a
low copper locality did not germinate at all. According to Prat,
these copper resistant varieties can only be obtained through natural
selection. In the F generation the plants have kept this high
copper tolerance. Extensive studies (25) have been made of the
copper vegetation in Katanga. Total copper in this copper-rich
region is present in amount from 500-100,000 ppm. Although the
copper concentration is very high in the rhizosphere, the absorption
is always limited. Differences however exist. Copper-resistant
plants are able to exclude the copper. The so-called cuprophytes
are able to accumulate 20 to 50 times the normal amount.

Very high amounts of strontium were reported in plants which
had grown on strontium sulfate mines (21) . The effect of different
levels of zinc on the germination of seeds from various zinc-tolerant
plants was determined (26) , Germination was little affected when
the nutrient mediiom contained less than 100 ppm Zn, but between
10,000 and 50,000 ppm Zn there was a rapid reduction in percent
germination.

Plasmatic Resistance

It has been found by several authors (38, 84, 87) that plants
which are tolerant to high metal concentrations also have a high
plasmatic resistanceor tolerance for the same metal. This means
that cells of tolerant plants are able to perform normal plasmolysis
after treatment with high levels of metal ions. Cells of metal"
sensitive plants are instantly killed by such a treatment and con-
sequently plasmolysis is no longer possible.

12

Gries (38) tested the resistance of protoplasm of different
tolerant and non- tolerant species against high concentrations of
zinc salt. Through plasmolysis experiments the author was able to
show the great differences in zinc- tolerant and zinc-sensitive
plants. The cytoplasm of zinc-tolerant forms withstood 100 to 1,000
times stronger concentrations of zinc than that of normal plants.
The criterion for survival was the ability of the epidermal cells to
perform plasmolysis in 1.0 M glucose solution.

The resistance of cell plasma against specific concentrations
of vanadivrai varies from plant to plant (6) . For some plants (Trades-
cantia zebrina) 0.0001 percent vanadiiim sulfate was harmful to the
epidermis cells. Others (Rhoeo discolor) were able to support
up to one percent vanadivim sulfate. Various plants known to be Cu-
tolerant showed high plasmatic resistance against toxic copper
concentrations (84) . Tije effect of heavy metals on plasmolysis has
also been studied in several Bryophyta and higher plants (82). It
was noticed that in these otherwise non- tolerant plants notable
differences exist in plant resistance.

Otto (76), in experiments on copper toxicity, came to the
conclusion that the living protoplasm formed__a barn' pt for excessive
amounts of copper. Only very small amounts of copper were found in
the shoot and root of corn seedlings when treated with high levels
of copper sulfate solution.

When the concentration of metal, e.g., copper, is very high^
an impermeable layer is formed on the outside of protoplasm pre-
venting further entrance of the toxic copper (104). There are
suggestions (84) that plasmatic resistance can be built up, though no

13

experiments of this nature have ever been conducted, Vitis vinifcra
showed a high degree of Cu tolerance (84) . Although the grape is
propagated vegetatively it could well be that the many repeated copper
sprays so commonly used in this culture have resulted in this high
plasmatic resistance.

Growth retardant chemicals increased the tolerance of soybean
plants against toxic levels of salt (64). The retarded plants,
which could withstand an excessive amount of fertilizer, produced
viable seed. Internal plasmatic changes may be responsible for this
reaction. Similar results were obtained on kidney bean plants (75).
Plants treated with the growth retardant B 995 tolerated twice as
much salt as the untreated ones.

Long-Term Effects

Most of the experiments dealing with the prolonged effect of

elements on plants have been limited to the effect of N P K treat-

32
ments or radioactive element treatments. When applications of P

were made to young barley plants, fertility was reduced in the

treated plants and in offspring (63). In subsequent generations

the plants appeared to be normal and no long-term effect could be

observed. Durrant and Tyson (24) reported an after-effect of

N P K treatments on wheat in the first generation. The effect in

the generation after treatment on yield depression was found for

potassium deficiency, particularly. The effect was not correlated

to differences in seed weight. In the second generations no effects

of the original treatment could be detected. With different N P K

treatments small and large forms of Linum plants, were obtained (23).

14

These variations remained stable for six generations. The heritable
change is not directly associated with gene-mutations but is explained
as possibly due to alterations in nuclear substances closely associa-
ted with chromosomes. Some comparison may be drawn here between this
response and the induction of drought resistance in plants. Accord-
ing to some investigators drought hardening persisted through
several generations (33) . Differences in nitrogen base composition
of nucleic acids and in composition of amino-acids were found (109) .

Effects on Chromosome Aberrations and
Plant Mutations

Chromosome aberrations and mutations have been obtained by
many workers when plants were grown under mineral excess or stress
conditions. Stockberger (101) in 1910 made a detailed study of the
effect of CuSO^ on mitosis in the root tips of Vicia faba. Dilute
as well as strong CuSO^ solutions, from 1/20,000 N - 1/12 N, were
used. The weaker solution gave the same result as the stronger
but it took a much longer time. Microscopic examination revealed
an inhibited mitosis, a disorganization or interrupted formation of
the ^£itidie__f4,bers , anon-continuous cell plate, and often an unusually
- large vacuole.

The mutagenic effect of a combination of copper and ethyl
methane sulphonate (EMS) has been shown (1). When copper alone was
used in concentrations of 10 M and 10 M no significant aberrations
could be detected in the root tip cells of wheat, but when combined
with EMS there was a very strong synergistic action resulting in many
chromosome mutations. It seems here that the stimulating effect of
Cu on plant growth in these low concentrations promotes the mutagenic

15

activity of EMS. Similar results were obtained with Vicia fab a (69).

In contrast, when barley seeds were treated with EMS in the
presence of Cu or Zn ions the total chromosome aberrations did not
increase (68). However, the relative proportions of chromosome
aberrations were changed. Effects of EMS alone, and in combination
with Cu and Zn ions, were also studied on chlorophyll mutation rate
in Arabidopsis (5) . It was found that neither Zn or Cu in a
concentration of 10" M induced genetic changes, but in combination
with EMS they enhanced the mutagenic action. The maximum synergistic
effect depended, furthermore, on the pH of the solution, and was
found for the zinc solution at pH 7 and for the copper solution at
pH 9. Cytological observations of onion root tip cells treated
with sub-lethal concentrations of boric acid (1/100 M - 1/25 M)
showed an arrested mitosis (22). Effects of some other ions (Ba,
Ca, Mg) on the development of pollen mother cells of Tradescantia
reflexa were reported (110) . The reactions were described as a
reversible coagulation of the chromosomes. The otherwise invisible
chromosomes became visible in a light microscope without staining.
Several chromosome aberrations could be induced in Allium cepa
when roots were treated with a lO" solution of sodijim_fluoride (66).
The presence of many bridges as a probable result of dicentric
chromosomes indicates that fluoride may act on the chromosome during
interphase. In another study the same authors (67) showed that
permanent chromosomal aberrations could be induced when tomato plants
were treated with gaseous fluoride. In the F^ generation abnormal
phenotypes were found. Hydrogen fluoride can, for this reason, be
considered as a mutagenic agent.

16

Hyde and Paliwal (51) showed the effect of ethylene diamine tetra
acetic acid (EDTA) and various levels of Ca and Mg on chromosome
behavior. An excess of calcium, as well as a deficiency of calcium
and magnesium, increased the chiasma frequency in Plantago ovata.

Deficiencies of N, P, K, Ca, Mg and S caused abnormalities in
the chromosomes of Vicia faba (29). A shortage of K, P and Ca
resulted in hyperchromatic chromosomes. The chromosomes, in this
case, became shorter than usual with a general reduction in size of
the nucleus and nucleoli. Hyperchromatic chromosomes were the
result of sulfate stress. Three times as many mutations in the F2
generation of Antirrhinum ma jus were obtained when the plants were
grown under nitrogen, phosphorus and magnesium stress (20).

Abnormal cytological effects in Tradescantia were reported
when plants were grovm at deficient levels of sulfate (4 and 8 ppm)
(97). The effect was primarily observed in the meiosis where
tripolar spindles were formed. The presence of micronuclei in
mitosis indicated a breakage of chromosomes.

Induction of chromosome breakage at meiosis was obtained by a
magnesium (96) and calcium deficiency (98). Tests were performed
on clonal material of Tradescantia (96). Chromosome fragments were
nine times more frequent in the Mg deficient material. The high
percentage of micronuclei was directly correlated to the high fre-
quency of aborted pollen. Airthexs_-l£om_2j^jn^ts__gjwr^
calcium concentrations had pnly-ahflr-tujye cells (98). Microspore
division was halted. In somewhat higher levels (10 ppm Ca) the
pollen grain contained micronuclei 18 times more frequently than in
normal cells. Since micronuclei are mostly formed from the acentric

17
chromosome fragments, their frequency is a good index for abnormal
chromosome behavior. In another study (99) it was shown that calcium
is able to affect the radiosensitivity in Tradescantia. In calcium
deficient treatment both interchange and interstitial deletions were
40 percent higher than in the controls. Chromosomes are subject
to breakage at low levels of calcium when irradiated.

In the experiments of Sigenaga (94) the young leaf epidermis
and petal cells of Tradescantia ref lexa were used. Both neutral
salts and heavy metals were applied to plant material. The neutral
salts in a concentration of 0.1 M - 0.3 M caused a decrease in the
spindle voliome. The chromosomes increased in refractivity and failed
to migrate to the poles. Heavy metals, such as Pb , Zn, Fe, Sn, Ag
and Cu, caused several abnormalities when used in relatively low
concentration. Pb (N03)2 and AgN02 yielded trinucleate cells and
anaphase and telophase bridges. High concentration resulted in
death of the cells.

Considerable research has been done on the action of several

metals on the mitosis of root cells of Vicia faba (34, 35). The

-5 9
concentrations of the metals used ranged from 10 M to 10 M.

In the lowest concentration, an increase in the mitotic activity
was obtained, comparable to a minor element activation. The
reactions described were very specific for each metal. Cadmium
gave the highest percentage of pyknosis, whereby a complete des-
truction of the nucleic acids took place in the chromosomes. Cobalt
gave the most achromatic chromosomes, copper increased considerably
the stickyness, chromium treatment had the' highest number of trans-
locations, manganese seemed to interfere strongly with the coiling

18
of the chromosomes, and nickel affected the spindle formation
resulting in tri- and multipolar cells. In general Mn and Zn gave
the highest percentage of fragmentation. Here, fragmentation and
translocation are the only chromosome aberrations considered as
prerequisites for mutation. With the metal treatments the centromere
of the SAT- chromosomes also became visible.

It has been reported (48) that a concentration of 0.1 - 0.2
percent Co(N03)2 had a cytostatic effect on mitosis. A thicken-
ing and despiralization of the chromosomes in the root tip cells
of Vicia faba was obtained. Scheibe (89) has shown that AICI3 and
A1(N0_). could produce mutations in Melilotus albus. In an ingenious
way the solutions were given directly to the plant by means of a
split stem. One part of the stem sustained the plant as usual, the
other split part was brought into the solution. Toxic solution
could reach the cells in this way before meiosis took place. Most
of the mutations were found in the second generation after treat-
ment, especially the chlorophyll mutations where a single recessive
factor is involved. Other types of mutations produced were low
coumarin-containing plants especially found after treatment with
10"-^ M AlCl .

In the experiments conducted by Von Rosen (106, 107), the
cytological effect of 55 elements of the periodic system on the
mitosis in the rootlets of Pi sum abyssinicum have been studied.
Plants from treated seeds showed in the second generation chlorophyll
aberrations and mutations such as a waxless type and a so-called
Gigas-plant, with enlargement of all plant parts. Similar types of
mutations could also be produced by radiation. Several phenotypically

19

recognizable mutations in summer wheat were obtained after treatment
with AlCl (90). Although aluminum chloride was very toxic, it had
a high mutagenous effect. Most frequent mutations were square head
types ears with short stem. A1C1„ was introduced by the so-called
"notching method" of Oehlkers (73), whereby part of the stem was
split and immersed in AlCl solution. Haarring (39) obtained
several mutations in a hexaploid summer wheat variety using AlClo
in concentrations of 10 - 10 M. Zschege (112) used the same
wheat variety for cytogenetical investigations and found several
chromosome aberrations. Antirrhinum ma jus when treated with 10
M AlClo also showed a considerable increase in mutation rate (73).

Interaction with Chelates

Introduction of chelates in plant studies has thrown new light
on the role of metals in chromosomes. Cohn (15) studied the effect
of chelation on the production of chromatid aberrations in Vicia
f aba. In these experiments, 2,2, bipyridine which complexes easily
with iron was used. The result of the treatment was chromosome
breakage in the heterochromatic regions of the chromosome. Found,
also, were a significant number of achromatic lesions and a marked
clumping of the chromosomes. If ionic linkages exist between the
protein component of the chromosome and the DNA then iron may be
one of them. These linkages could then be disturbed by a chelate
such as bipyridine. It has been found that versene (a tetrasodium
salt of ethylene diamine tetracetic acid) induced chromosome sticki-
ness in neuroblast cells of the grasshopper (88) . The rate of
mitotic divisions was reduced. The chelating action of versene may

20

result here in binding elements such as calcium with a direct
effect on cell division and chromosome behavior.

Rieger e_t a_l. (85) studied the interaction of ethylene diamine
tetra-acetic acid (EDTA) and triethyl melamine (TEM) in root tips
of Vicia faba . Simultaneous action of these two components increased
the chromosome aberrations by 20 percent above the expected value.
EDTA alone has only a weak mutagenic effect. Introduction of metal

ions before or after treatment inhibited in some cases the actions

•I
of EDTA (complex forming). The mode of action of the chelate could

be a direct one in the sense of a metal inactivation or metal removal

leading to chromosome aberrations, or an indirect action by means

of an inactivation of the enzyme system or by changing the ionic

balance.

The specificity of certain chelates in the cells has enabled
Phallusia mamillata to concentrate vanadium a million times more
than the vanadium level in sea water (3) . Bayer (3) synthesized a
highly selective chelate for gold by polycondensation of di- and
triaminothiophenols with glyoxal. In fact, gold could be removed
from sea water without interference of other metals. A similar
mechanism may be operative in plant cells.

The bivalent ions are also able to form chelates with protein
(105). They belong to the group of metals with atomic weight of
less than 65. Metals of higher atomic weight, such as mercury, are
highly poisonous to the cells.

The introduction of heavy metal leads to alteration of the
total balance of chelates with a direct effect on nucleic-acid and
nucleotide metabolism (106). On the other hand, it was found (54)

21

that the resistance of plants to heavy metals was probably due to the
reaction of certain chelates with these metals.

The Mode of Action on Chromosomal and_
Cellular Behavior

The effect of Cu is probably related to a direct reaction with
the SH-groups of the spindle fibers (101), In prophase of mitosis,
spindle fibers contain only SH-groups which later in anaphase form
solid S-S-bonds and these maintain fiber integrity. Also, the absence
of sulphur probably affects the fiber chemistry of the spindle,
resulting in its abnormal behavior (97), Histochemical tests (51)
specific for sulfhydryl groups, gave a deeper staining of the nuclei
in EDTA treated plants of Plantago ovata than in those not treated
with EDTA, This leads to the inference that the structure of the
sulphur containing proteins was modified, loosening the bonding
between chromosomal RNA and associated proteins.

Selenium, since it is able to substitute for sulphur, may
affect the structural chromosomal proteins (108), This could explain
why a reduction in the rate of crossing over in barley was found
when plants were treated with 0.5 ppm selenium.

The inhibitory effect of boron on starch phosphorylase was
shown (93), Boron complexes either at the active site of this
enzyme or with glucose-1-phosphate, the substrate.

It was suggested (22) that the formation of a boron ribonucleic
acid complex in the interphase cell is responsible for blockage
of mitosis. No chromosome aberrations were observed. The inhibiting
effect of fluoride in concentrations of 2 x 10" and 10 M on
■ growth of corn seedlings was demonstrated (14), This effect could

22
be related to inhibition of DNA-synthesis since less DNA was found
in the fluoride treatment. Higher protein content in the roots of
orange seedlings counteracted the toxic action of copper (95). Thus,
more iron could be taken up. It was suggested that insoluble
nitrogenous material could act as an absorbing complex for Cu.

Peterson and Butler (80) studied the incorporation of radio-
active selenium into Neptunia, a selenium accumulator, and into
non-accumulating species of wheat, rye grass, and clover. Although
the amounts of selenium taken up from a culture solution was
found to be the same for all plants the incorporation into various
compounds was different. In plants of wheat, rye grass, and clover
there was an extensive incorporation of selenium into the protein,
as seleno-amino-acids. In Neptunia, however, the proportion of

Se bound to protein was much lower. Only a small proportion
of the total radioactivity could be extracted by proteolytic
enzymes (trypsine, chymotrypsine) . A high percent was soluble
in bromine water^ indicating a high proportion of an insoluble
inorganic selenium compound.

Radioactive calcium was incorporated into Lilium longif lorum
(100), showing that on germination of the radioactive pollen the

Ca was firmly bound to the nuclei, indicating a possible binding
with the chromosomes. It was suggested that the binding site for
these and other metals is probably on the phosphate groups of the
DNA.

Mutations, as they occur after certain mineral deficiencies,
were explained as a result of stress in the so-called "Zuwachsphase"
(20) . The duplication of chromosomal material in this phase is

23

short of the necessary elements and chromosome aberrations may
occur. The direct effect of the mineral stress on the nucleic
acid metabolism was correlated with the chromosome anomalities
(29).

EXPERIMENTAL AND RESULTS

The experimental data presented here are divided as follows:

The first part deals especially with the effect of copper and
manganese deficiencies on soybean plants. Abnormalities in progeny
seed and plants are described.

The second part consists of a number of experiments dealing
with the direct and prolonged effect of high mineral levels on
plants and seeds from treated plants. Two approaches were made
here.

First, seeds of sorghum, rye, oats and soybean and seeds of
Portulaca oleracea were treated with various concentrations of
mineral salts. Germination and further development of seedlings
were investigated. Germination of Portulaca oleracea was also
tested at various levels of potassium sulfate, potassium nitrate
and potassiiim chloride to explore the levels of toxicity of the
anions. Several generations in Portulaca were studied to obtain more
information on the mutagenic effect of the elements. The rooting
experiments with cuttings and leaves provided further information
on the characteristics of the mutation. Grafting experiments were
performed to investigate the possibility of a bacterium or viral
infection. Morphological and histological differences between a
mutated type of Portulaca (mutation induced by Li CI) and the wild
type were examined. Chromosome studies and leaf epidermis studies
were conducted to obtain more information on the type of mutation produced,

24

25

The second approach was an attempt to induce mutations by
direct treatment of seedlings and cuttings of Portulaca oleracea
with higher levels of mineral elements. Seeds collected from
treated plants were tested for germination and further development.
Several generations were grown to find out if mutations had occurred.

The third part relates to the effect of various heavy metal
ions on the induction of plasmatic resistance and metal-ion tolerance
in Portulaca oleracea . The first experiment deals with the build up
of this tolerance within the plant cell and the plasmolysis technique
to test the viability of the treated cells. The second experiment
relates to the tolerance of the whole plant as affected by a pre-
treatment with heavy metals.

Since the methods varied for the different experiments, the
details of the methods will be explained under the respective
experiments. Throughout these experiments any mention of nutrient
solution refers to the no. 2 solution of Hoagland and Arnon (49).

The results will be given following each experiment.

Part One

Experiment _1 : Effect of Copper and Manganese Deficiencies on Soybeans
General methods

Soybeans, var. Hardee, were germinated on paper towels moistened
with deionized water. The young seedlings were planted in big glass
jars and grown in the open. Nutrient solution which was added to
the jars Xv^as made up of all the usual major and minor elements, minus
one element for a particular treatment. Minor element deficiencies
were tested for Cu, Mn, Zn, Mo and Ca. A special study was made of the
copper and manganese deficiencies.

26

Experimental pi'occdure and results with manr^ancsc do Eicicncy

Many seeds from plants which had grown under minus manganese
conditions showed a very specific brown spot at the side of the
cotyledons. Location of this particular spot was the same for each
affected seed. To investigate the nature of this necrotic spot,
microscopic examinations of the seeds were made. Seeds were soaked
for 18 hours in water, fixed in Randolph's Modified Navashin fluid
(52) and dehydrated with ethyl- and butyl-alcohol. Thereafter, seed
was embedded in paraffin and cut into serial sections ten microns in
thickness. A quadruple stain combination of Johansen (52) was used.
Microscopic studies indicated that the area which was affected
in the manganese deficient seeds, consisted of a specific group of
cells directly connected to the vascular bundles through thin
parenchymatous cells (Figures 1 and 2) . The size of these cell
groups varied. On the average they were 20 to 30 cells long, 10 to
15 cells wide and 4 to 6 cells deep. Cell walls of these cells
were thin compared to those of surrounding cells. The direct linkage
of these cells with the vascular system suggested that they were
involved in some way in water transport during development of the seed
from the ovule. Deterioration of these cells with a manganese
deficiency is not well understood. Plants grown under Cu, Zn, Mo,
or Ca deficiency did not produce any seed of this kind. In a few
cases the minus manganese treatment changed the morphology of the
cotyledons. Tri-cotyledons were produced. In the check treatments
hundreds of seeds were tested and no abnormalities were found.
Upon further development most of the tri-cotyledon seeds grew into
plants with twisted or forked stems (Figure 3) . These deviations
were never observed in the check plants.

27

Figure 1. -- Photomicrograph of longitudinal section through

cotyledon of soybean seed (normal seeds). Thin paren-
chymatous cells are shown in A. This cell group is
connected through cells of B with vascular system of the
cotyledon (darker tissues) . (95x)

28

U»^

Figure 2. -- Photomicrograph of longitudinal section through cotyledon
of soybean seed from manganese deficient plants. White area
indicates large destruction of cells. (95x)

29

Figure 3. -- Forked stems in soybean as a result of manganese

deficiency. Picture at bottom shows a twisting of the
forked stem.

30

Experimental procedures and results v;ith copper deficiency

Seeds of soybean plants which had been grown in a nutrient
solution without copper did not show any necrosis. However, many
cotyledons were malformed, and plants grown from these seeds showed
various abnormalities. There were many plants with a typical
forked stem. In other cases two plants had developed from one
seed. Sometimes the stems of these plants were united at the base
or in the middle, forming twins. \^iere these twins were not united
the roots were simply twisted around each other (Figure 4).

For further studies, six abnormal plants were retained and
transplanted to containers filled with Leon fine sand. Nutrient
solution at one half strength was added at the beginning. Tap
water was used thereafter. From each plant, seeds were collected and
pods and beans weighed. The results are shown in Table 1.

No definite conclusion can be drawn from these data since only
single plants were used. Seeds from tv.'in plants with t;^7isted roots
yielded the lowest average weight for beans plus pods. From each
plant, forty seeds were planted in rows in the field. In all cases
normal plants developed. It appears that the effect of the copper
deficient treatment was only associated with the first generation
plants.

Part Tx^'o

Experiment 2: Effect of Concentrated Solutions of Copper, Manganese
and Zinc Salts on Seeds of Soybean, Rye. Sorghum and Oats

Material and methods

Mature seeds of soybean, rye, sorghvim and oats were soaked for

24 hours in deionized water (control) and in strong solutions of copper,

manganese, and zinc salts. The treatments were not aerated. Copper

31

Figure 4. -- Twin plants in soybean as a result of copper deficiency.
All plants developed from one seed. Left: Roots tvv^isted.
Middle: Stems united in middle. Right: Base of stems grown
together.

32

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and zinc were applied as sulfate, manganese was added as a chloride.
Concentrations used were: for copper 36 and 288 ppm, for manganese
888 and 7104 ppm, and for zinc 188 and 1500 ppm. Half of the
seeds from each treatment were transferred to Petri dishes on wet
filter paper (series A) and germinated, while the other portion
(series B) was dried without a preliminary washing and germinated
at a later date. Germination was evaluated after 7 days. Criterion
for germination in all cases was a distinct development of the radicle
and appearance of the shoot.
Results

Percent germination after 7 days for each treatment is indicated
in Tables 2 and 3. After 7 days no further increase in germination
took place. The results indicate clearly that the sorghum seeds are
very tolerant to high metal concentrations, especially in the series A,
An SO to 100 percent germination was found in these strong metal
solutions. The oats in these series for all the treatments, gave
a zero percent germination.

Comparison of the two series showed that drying the seeds after
soaking with the toxic solutions had a favorable effect on the
germination, especially in the case of oats and rye (compare Tables
2 and 3) . Since drying took place on filter paper it is possible
that a part of the toxic solution was drawn out. This could explain
the differences between the two series. Highest percentage of germin-
ation for oats was found in the series B for the manganese treatment.
It is possible that manganese is removed more easily from the oats than
the copper and zinc. This confirms earlier reports (60) where absorbed
manganese could be completely removed from peas and wheat seeds by wash-
ing. Absorbed zinc and copper ions could only be removed to a certain
extent.

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Experiment: 3: Effect of Copper, Mr , anesc and Zinc Solutions on
Germination o L" Sort'.hum Seeds ~

Material and methods

Since the sorghum seeds seemca more tolerant to the heavy
metal solutions than the other seed:, used (see Experiment 1),
further experiments were done with sorghum seeds. Seeds of sor-
ghum var. Lindsey 77 F, were soaked in concentrations of 288 ppm
copper, 7,104 ppm manganese and 1,500 ppm zinc salt. Copper and
zinc were used as sulfate, manganese as chloride. A total of
287 seeds were used for the copper treatment, 214 seeds for the
manganese treatment and 454 seeds for the zinc treatment. After
six days the seeds were taken out of the solutions and contrary
with the first experiment thoroughly washed with distilled water and
planted in glass trays which contained gravel saturated with
nutrient solution. Germination was evaluated seven days after
planting. Part of the sorghum plants derived from seeds that
survived the treatments were later set out in crocks filled with
vermiculite. Nutrient solution at one half strength was added.
Results

Total germination after seven days amounted to about 75 per-
cent in the copper and zinc treatment and only 25 percent for the
manganese treatment (see Table 4). The last results are not in
accord with the first data (Tables 2 and 3). However, in this
experiment the seeds were kept in the toxic solutions much longer.
This could account for the differences found in the manganese
treatments, especially since the manganese concentration was
extremely high. Upon further development of the sorghum plants
no abnormalities were found.

37

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Experiment 4: Tl^c Effect of Soakinp; Oat Sect's in Concentrated
Solutions of Copper, Zinc and Manganese

Materials and med^ods

Oats were soaked for four weeks in concentrations of 375
ppm Zn, 1,776 ppm Mn and 72 ppm Cu. Zn and Cu were applied as
sulfate, Mn was added as chloride. The strong concentrations of
the solutions prevented germination of oats. After four weeks
seeds were washed once with tap water and germinated on moist
paper towels in glass trays filled with gravel. A part of the
seeds would not germinate. The part that did germinate produced
viable seedlings. These were transplanted in crocks filled with
Leon fine sand. Crocks were placed outside and protected by wire
netting to prevent bird damage. There were 14 plants per crock, and
2 crocks per treatment.
Results

These experiments showed a direct influence of the previous
heavy metal treatment on the time of flowering in the plants.
Blooming occurred 10 days earlier in the plants grown from seeds
treated with 375 ppm Zn than those of the check treatment. The
copper treatment did not appear to affect further plant develop-
ment. The manganese treatment in contrast retarded the blooming
by about ten days as compared with the check.

The reason for these differences must be directly or indirectly
related to the accumulation of heavy metals in the seeds. No dif-
ferences in plant height were observed between the treatments. Leaves
of plants from copper treated oat seeds were somewhat darker than
leaves from the other treatments.

39

Experiment 5^: Effect of the Anions NO3 , ^ , SO/ on the Germi-
nation of Portulaca oleracea Seeds

Material and Methods

To explore the level of toxicity of the anions, seeds of
Portulaca oleracea were treated with different levels of potas-
sium nitrate, potassium sulfate and potassium chloride. The
concentratiorehere used for each salt in ppm were 20,000, 10,000,
2,500, 625 and 300. Approximately 200 seeds were placed in Petri
dishes. Five milliliters of one of the different levels of potas-
sium salts were added to each dish. - Germination was determined after
6 and 9 days. After 9 days the seeds were thoroughly washed with
tap water and left in beaker glasses for further observation.
Results

In the nitrate and chloride treatment (Table 5) there was a
complete inhibition of germination with the 20,000 ppm treat-
ment. At this level 30 percent of the seeds germinated after 9
days in the sulfate series. However seedlings were small. In
the nitrate and chloride series germination starts at a level
of 10,000 ppm. It can be seen that the germination of Portulaca
oleracea seeds was better in the nitrate series than in the others.
It was found that after washing, the seeds did not germinate any
further.

Anions can in general be tolerated in much higher concentra-
tions by plants than the metal ions. It is obvious that when very
high concentrations of metal salts are used, the anion part
becomes also toxic. Any mutagenic or toxic effect at lower
levels of metal salts solutions can be attributed solely to the
action of metals.

40

TABLE 5

EFFECT OF DIFFERENT LEXTilLS OF POTASSIUM NITRATE, POTASSIUM

CHLORIDE AND POTASSIUM SULFATE SOLUTIONS ON TliE

GERMINATION OF PORTULACA OLERACEA SEEDS

Germination % after

Treatment 6 days 9 days

Control (Water) 7 5 90

NO3 20,000 ppm 0

CI 20,000 ppm 0

SO^ 20,000 ppm 0

0

10,000 ppm 43 62

2,500 ppm 76 88

625 ppm 77 91

300 ppm 68 96

0

10,000 ppm 19 43

2,500 ppm 55 76

625 ppm 52 72

300 ppm 69 98

33

10,000 ppm 20 27

2,500 ppm 40 84

625 ppm 50 62

300 ppm 48 63

<hl

Experiment 6: Effect of Various Concentrations of Copper and Zinc
Solutions on Germination and Further Development of Portulaca
oleracea Seeds

In these experiments Portulaca oleracea was used. This plant
has a short lifa cycle of six to seven weeks from seed to production
of seed, with many advantages as well. Portulaca is a good seed
producer. There are approximately fifty seeds in one seed head.
Relatively small plants can easily produce a thousand seeds.
Furthermore, the plant can be propagated very easily by cuttings.
Clones of uniform material can be obtained in short time. Ten to
twelve days after flowering the seeds are ripe. There is no
dormancy and a close to 100 percent germination occurs in most
cases. The plant requires little care and is able to survive
through long periods of drought. Portulaca has a pronounced Circad-
ian rhythm (57, 61) and is excellent for studying photoperiodicity
(58, 61).
The effect of the copper solutions

Material and methods. -- One hundred seeds of Portulaca oleracea
were placed in each Petri dish on Whatman no. 2 filter paper, using
three papers per dish. Five milliliters of one of the copper sulfate
concentrations were added. The following concentrations of copper were
used in ppm: 4.5, 9, 18, 36, 72, 144, 288 and 576. To reduce the
amount of evaporation, Petri dishes were placed on a moist sheet of
filter paper and completely enclosed in a large plastic bag. It
was found that the solution evaporated very fast when these pre-
cautions were not taken. Twelve hours of light and twelve hours
darkness were provided for germination since Portulaca seeds germin-
ated better under these conditions. Temperature was constant through-
out the experiment at 25° C..

42

Germination was evaluated after 10 days and average shoot length
and root length determined. Thereafter all the seeds, germinated and
non-germinated, were transferred to water and washed. Further
germination and development of seedlings was checked. Seeds were
considered as germinated when either radicle or shoot or both had
pierced through the seed coats. Portulaca seeds which had been
killed in a 1 percent mercuric chloride solution, were not able
to perform any similar breakthrough.

Results . -- The development of the seedlings in the first t\>70
treatments (4.5 and 9 ppm Cu) was not different from those of the
check (see Table 6) . However at 18 ppm Cu considerable reduction
in root growth was observed. Increasing the amount of copper further
reduced the root growth and at 36 ppm the shoot growth was affected.
The germination percentage remained high until a concentration of
576 ppm Cu was reached. Higher concentrations inhibited strongly.

After washing, all seedlings developed in the 72 ppm Cu treat-
ment died within a few days. This was also true for the seedlings
grown in the 144 ppm Cu and 288 ppm Cu treatments. After washing
a further germination took place of the seeds not germinated before.
In the 576 ppm Cu treatment 70 percent of the seeds germinated and
produced viable seedlings. These data showed that concentrated
solutions of copper were able to inhibit strongly the germination
of Portulaca oleracea seeds. No apparent damage was done to the
seeds. Inhibition was removed as soon as the seeds were thoroughly
washed in water. The seedlings treated with lower concentrations of
copper (4.5, 9, 18, and 36 ppm Cu) all survived after being washed
with water. Most damage was done in the middle concentrations

43

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44

(72, 144 and 228 ppm Cu) where copper concentrations were relatively
high.

The effect of the zinc solution s

Material and methods. -- Similar experiments were carried out
as in the preceding one, this time however with various concentra-
tions of zinc sulfate solutions (see Table 7). Portulaca oleracea
seeds were planted and treated in the same way as in the former
experiment. Germination was determined after 10 days and root
length and shoot length measured. All the seeds and seedlings were
washed with tap water after 10 days and further development was
checked.

Results. Data indicated (Table 7) that the percentage of
germination was not greatly affected by the concentration of the
treatment, but root and shoot growth were reduced. The first
toxic effects were visible in the 94 ppm Zn solution. Tips of
roots and cotyledons turned brown. From the 188 ppm Zn and
stronger treatments all the roots and shoots became black, with
■a further reduction in size. When seedlings were later washed
with water, all died which had been treated with higher than 47
ppm Zn. In the highest treatment of 1,500 ppm Zn, no further
germination took place of the seeds that were inhibited before.
Experiment 1_: Effect of Lithium Chloride on Portulaca oleracea
Material and methods

Portulaca oleracea seeds were treated with different concen-
trations of lithium chloride (1 - 2%). Two hundred seeds were
soaked for 20 days in these solutions . Only part of the seeds
germinated. After washing the seeds for two days with tap water,

45

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46

germinated and non-germinated seeds were planted in small pots

filled with sterilized soil.

Results

The non-germinated seeds started to germinate after four
days. About 50.0 percent of the plants that developed from the
seeds treated with 1 percent lithium chloride, bore branches with
modified leaves and flowers (Figure 5) . Leaves were more pointed
than oval shaped. Differences were also found in the margins of
the leaves. Margin cells of modified leaves were considerably
reduced in size, giving a smooth appearance to the edge of the leaf.
The petals of flowers on modified branches were incised, instead of
having only one notch in the middle, as is the case with wild
flowers (Figures 6 and 7) .
Further experimental procedures and results

Rooting of cuttings. -- In order to check the constancy of the
mutated branches, cuttings were made. A record was kept for each
plant from where cuttings were taken and drawings made (Figure 8).
On each drawing was indicated from v;here the cuttings originated.
Main top shoots were indicated by A, B, C, etc. Going towards base
of plant, leaf axil indicated as A' , A", etc. Main top shoot of
left branch from A' is A'L. The different leaf axils on this branch
indicated were as A'L, A'L", etc. Left and right branches from A'L'
are then A'L'r and A'L'l branches. Following this system it is easy
to label the whole plant. In this way all the clones have a known
origin, and they carry the same letters as the branches from where
they were derived.

It was found that most cuttings kept their morphological features.

47

Figure 5. -- Portulaca oleracea plant derived after seed treatment
with 1 7o lithium chloride. Note the t\^7o mutated branches
at the left and right. In the middle the original wild
type is shown.

48

Figure 6. -- Flowers of v^7ild and mutated types of Portulaca olcracca,
Left: Flowers of wild type. Leaves are here obovatc and
petals lobed. Right: Two smaller flowers of the mutated
type. (Approximately natural size.)

49

Figure 7. -- Flower of mutated type of Portulaca oleracea produced
after seed treatment with VL lithium chloride. Leaves are
eliptical and margin of the petals is incised. (5x)

50

C'L'V

i

C'RI

C"R

Z

k

/^v-S^

CWr "^(b

<^'

^^5*

^iL\ C'R*.

<::?<I^

\r

C"R'

/vCi'

\

O

C'll

^^cr

Figure 8. -- Mutated branch of Portulaca oleracea. Cuttings were
taken at the dotted lines. Numbering system is explained
in the text.

51

In approximately 30 percent of the cuttings, however, branches of
the original wild type reappeared (see Figures 9 and 10). In four
generations of cuttings tested it was never possible to obtain a
pure clone of the modified Portulaca type. The percentage of
reversion for four generations is shown in Figure 11 for four
different clones. The high percentage of reversion in the begin-
ning is probably due to the low niamber of observations since only
a few cuttings were available. With the next generation the number
of cuttings increased. Consequently, more observations could be
made, resulting in more reliable percentages.

Plants grown from seeds collected from the mutated branches
did not show abnormalities but resembled the original wild type.
Also, no difference was found in the second and third generation
plants. It seems likely that we have here a chimera phenomenon.
Part of the somatic tissue is probably mutated. If all cells were
mutated in a deviated branch then we should at least find some
plants of the mutated type in the next generation. Most gene
mutations go from dominant to recessive (89), so that in most cases
we cannot, in the first generation, recognize the modified types.
In the next generation, however, double recessive types are readily
recognizable as the mutated ones. In the case of a chimera, especi-
ally the periclinal type, it will never be possible to obtain seeds
reproducing the same modified tissue.

Microspores and megaspores are initially formed from the sub-
epidermal cell layers in the corpus. The tunica cells only divide
in one direction. The cells of the corpus divide in b>7o directions.
The only case in which it is possible to obtain seeds from the mutated

52

Figure 9. -- Cutting of mutated branch of Portulaca oleracea,
showing reversion to wild type at base. Left branch
with obovate leaves. (Approximately natural size.)

53

Figure 10, -- Cutting of mutated branch of Portulaca oleracea,
showing young wild shoot at base. The young shoot
developed in the axil of the third pair of leaves is
from the mutated type. (Approximately natural size.)

54

7o REVERSION
50

2326

2 3

GENERATION

02210

..0^328

iiO

Figure 11. -- Percent reversion from mutated to wild types in four
generations of clones of Portulaca oleracea. Code number
for each clone is indicated at left of curve.

55

tissue will be when tunica cells are given off to the corpus cells.
Usually this does not occur.

Rooting of leaves . -- Leaves can also be rooted easily by
sticking them in coarse gravel moistened with nutrient solution in
a tray covered with plastic. If the petiole is left on the leaf it
will root much better. Cutting off the petiole and the lower part
of the leaf makes the chimera type leaves more difficult to root
than the wild ones. However, an addition of 15 ppm indole acetic
acid (lAA) to the nutrient solution will considerably promote the
rooting of both types. It is not necessary to dissolve the lAA in
alcohol as is usually done. When added directly to the concentrated
nutrient stock solution (ten times Hoagland's solution) it is
easily dissolved by stirring with a magnetic rod.

Results indicated that roots were formed at the base of the
leaf from the vascular bundles. In three weeks these leaves grew
much larger and increased their fresh and dry weight five times
or more. Sometimes new sprouts developed at the base of the leaf.
These sprouts usually had the same leaf type as the mother leaf.
Seeds were collected from such branches and the progeny studied.

It was found that seeds obtained from the mutated leaf sprouts
reproduced only the wild types. This indicates that two different
cell tissues, wild and mutated, must be present in these leaves.
The periclinal chimera type continued in the structure of the leaves.

Virus studies and grafting. -- When making clones from the
chimera and wild form an occasional infection showed up in the new
growth. This was probably caused by a virus or bacterium. The new
leaves were considerably reduced in size and typically elongated.

56

Infection could have taken place by a contaminated razor blade or
directly from outside into the cut surface.

In order to check the possibility of a viral or bacterial in-
fection being responsible for the formation of the pointed leaf type
in Portulaca, several plants of the wild type were mechanically
infected with a leaf sap extract. The diseased leaves were
ground in a porcelain mortar together with a few milliliters of
0.05 M 1<H2P0^. The juice was applied to the cut surface of a stem
directly above a branch or to the cut surface of a leaf base where
a young bud was just beginning to develop.

Tobacco plant leaves were also treated with the extract. The
leaves were first dusted with a light cover of carborundum (600
mesh) and the infected sap applied by a slight pressure with a
piece of cheesecloth. The same procedure was repeated using
chimera type leaves.

After several weeks of growing no signs of virus infections
were observed. The treated plants developed in a normal way
and no leaf deformity could be detected.

In addition to these experiments several graftings were made.
If a virus had been responsible for this modified leaf type the
chances of transmission through the graft v;ould have increased.
The easiest way to perform the grafting was to insert the stem
portion into a pre-made hole in the stock. The opening of the hole
should be slightly smaller than the diameter of the scion. Graft-
ings can be made onto any part of the receiving plant. It is not
necessary to bind the grafted sections together with string or to
use waxed tape.

57

The results indicated a normal development of the grafts.
The wild form was not affected and continued to develop normal
wild branches. The grafted pointed leaf type kept the typical
morphological features in the same way.

Chromosome studies. -- It was to be expected that chromo-
some differences exist between the two types of Portulaca oleracea.
The extreme smallness and high number (2n = 54) of chromosomes in
Portulaca oleracea made it difficult to detect variations, unless
the mutation occurred in the form of a polyploid. The adventi-
tious root tips of Portulaca formed an excellent material to study
the chromosomes. These roots in general were thicker and easier
to handle than the finer roots of the seedlings. Rooting of the
cuttings started in about 5 days. Roots developed fast at the
beginning. The best stage for taking material was when the roots
were about a half inch long.

When dealing with a chimera, in which a periclinal mutation
existed, it was difficult to obtain root tips from both mutated
and wild tissues. Adventitious roots in Portulaca oleracea
originated in the pericycle and interfascicular cambium.
Unless some mutated tissue was present in these regions no dif-
ference would be found in chromosomal composition in the root
tips of both types. Nevertheless an attempt was made to analyze
root tip material of Portulaca on chromosome abnormalities.
Root tips were fixed in a mixture of five parts ethyl alcohol
(957o) , one part chloroform, one part glacial acetic acid, and one
part formalin (40% formaldehyde) for five minutes. After fixation
root tips were washed and hydrolyzsd in 1 N KCl solution for ten
minutes at a temperature of 60° C. To avoid fluctuation in

58

temperature the small vials containing the root tips were placed
in a pyrex tray of sand and kept in an oven at a constant tempera-
ture of 60 C. After hydrolysis, root tips were transferred to
Schiff's reagent (52). A deep red color developed after 10 to
15 minutes after which the root tips were ready for examination
of chromosomes. Root tips were then transferred to a slide, a
drop of aceto-carmine added, and the tips squashed between slide
and cover slip.

Several methods for preparation of Schiff's reagent were
tested. The best combination, with longest preserving time and
fastest reaction was prepared as follows: Dissolve 0.5 gram
basic fuchsin in 50 ml boiling water, filter at 60 to 80 C and
add one gram sodium metabisulf ite together with 10 ml IN
hydrochloric acid. Let stand overnight to decolorize and filter
after shaking for one minute with 300 mg powered charcoal.

The results indicated that the wild type and the mutative
type had the same number of chromosomes, namely 27. Since chromo-
somes are very small it was not possible to determine any abnormal-
ities.

Studies on leaf epidermal cells . -- It is known that stoma ta
of polyploid forms are larger than the ones from the same diploid
types (13). If the mutant in Portulaca was the result of poly-
ploidy then it should be possible to recognize this by the larger
stoma ta in the leaf blades. For this reason comparisons were made
on the microscopic structure of the leaf epidermal cells of both
types of Portulaca.

Epidermal cells were removed very easily by a small scalpel.

59
Staining was performed at the same time by dipping the scalpel in
a 1.0 percent Erythrosine solution. After the stain had dried, the
scalpel was worked under the epidermis which was cut off with a
sharp razor blade on both sides, A perfect, live stained section
was thus obtained. Transfers and damages were avoided this way.
Sections were transferred to a drop of water on a slide. Water to
which a detergent had been added was used. This released surface
tension and avoided air bubbles. For better contrast and observation,
sections were dried several hours under cover glass. This intensi-
fied the colors and sharpened the contrast between the cell walls
and the rest of the cells.

Examination of the epidermis showed that the stomata were more
abundant in the upper epidermis than in the lower epidermis ( 1 1/2-
2 as many). All epidermal cells, including guard cells and accessory
cells, were slightly smaller in the upper epidermis. Per unit of
surface there was approximately the same number of stomata in both
Portulaca types. No difference in size or shape of comparable
cells could be detected (see Figures 12, 13, 14 and 15). Since
the corpus cells contribute to the inner structure of the leaves and
tunica cells determine the structure of epidermis, it is evident that
two kinds of tissues in a leaf of chimera must be present.

Furthermore, it can be seen that when rooting such a leaf, it
must have been possible to obtain both types of shoots, depending
on the organization of the two different tissues. Rooting experi-
ments with Portulaca oleracea leaves have indeed shown that both
types of shoots could be produced.

60

Figure 12. -- Photomicrograph of stomata of lower leaf epidermis
of mutated type of Portulaca oleracea. (540x)

61

Figure 13. -- Photomicrograph of stomata of upper leaf epidermis
from wild type of Fortulaca oleracea. (540x)

62

Figure 14. -- Photomicrograph of stomata of lower leaf epidermis
from wild type of Portulaca olcracea. (540x)

63

Figure 15. -- Photomicrograph of stomata of upper epidermis from
mutated type of Portulaca oleracea. (540x)

64

Histological differences in the chimera and wild type. --In
order to have a better understanding of the chimera phenomenon, it
was necessary to study the initial development of the shoot. The
apical meristcm is located at the top of the shoot. The peripheral
portion of the apical meristem is formed by repeated cell division
of the apical meristem. Foliar primordia and cortex originate
from this peripheral portion of the apical meristcm.

Leaf primodia are formed from the surface layers. If the
tunica is several cell layers deep the entire leaf may be derived
from this zone. Leaf primordia may also originate in the corpus.
In the case of a chimera this offers various possibilities. Leaves
of different origin may be found on the same branch opposite each
other. In case of the chimera type Portulaca oleracea this typical
situation has repeatedly been found by the author in young shoots.
After giving rise to a pair of pointed leaves the shoots of Portulaca
subsequently produced only the wild type.

The anatomy of the vascular connections beti^een leaf and stem
can be best studied in the region between the internodes. The
vascular bundles (leaf traces) may travel as independent bundles
through several nodes. Somewhere they are linked to another part
of the vascular system. However, the branch traces are located in
the nodal region with vascular bundles connecting the main stem with
the branch.

In Portulaca branch traces are formed slightly below the in-
sertion of a leaf. Cross sections through the nodes reveal the
various aspects of branching.

Axillary buds are formed in the axils of the leaves. In general,

65

they originate in the superficial layers of the young stem. Peri-

clinal and anticlinal divisions precede the protrusion of the buds.
Axillary buds may develop later into lateral shoots having the same
general structure on the main stem. Since different cell layers
can participate in the formation of lateral shoots it is evident
that, in case of periclinal chimeras, wild types as well as
mutated types may originate from these sites.

Many comparisons at microscopic level have been made between
the t^^ro types of Portulaca. However, when young tissues were examined
no differences could be established. Axillary buds in early stages
of development could not be recognized externally as wild or mutated.
Consequently, microscopic comparisons of those structures were
useless.

The older stems of both types did not show any anatomical
differences. Also, the flower buds have essentially the same cell
structure with no particular deviation being detected. However,
when young mutated and wild branches originating from the same
mutated stem were compared definite anatomical differences could
be established. Most of these differences were found in the zone
where the shoot joins with the main stem. Thin wall parenchymatous
cells fill the gap between the two vascular bundles of main shoot and
lateral shoot.

Orientation and structure of the first four to five cell layers
of the cortex were not the same for both types of shoots. In wild
shoots these cortical cells are flattened and form a distinctive
ring around the vascular bundles (see Figure 16) . In many places
these cells are broken. This leaves the impression that the cell

66

Figure 16. -- Photomicrograph of cross section through wild shoot

of Portulaca oleracea . Note typical ring of flattened

cells around the pcricycle. In a few places (A) cells
are disrupted. (150x)

67

walls are thinner and arc more easily damaged by the cutting
process. Outside this ring the cortical cells appear to be normal.
Measured across, toward the epidermis, this zone has about five
to six cells.

In the mutated shoot we do not find this ring structure of
flattened cells (see Figure 17) . The cell arrangement is more
open without further differentiation of the cortical cells.

In further development this structural difference is lost.
Sections from ^^7ild lateral shoots above the axil cannot be recog-
nized from those of mutated lateral shoots.

Cross sections were also taken from stems which carried the
two different kinds of leaves at the same level opposite each
other. It was found that one-half of the section had disrupted
cells primarily derived of epidermis and cortical cells (see
Figure 18). These differences must be linked to the two different
cell tissues present in this section. One part carried the mutated
leaf, the other part carried the wild leaf type. Disruption of the
cells could be the result of thin wall cells in one part of the
section. In the process of cutting or dehydration these cells
are probably damaged. Thus, there must be a correlation between
the disrupted cells and the appearance of wild leaves or shoots
in altered branches.

Microscopically, cell differences can be detected only in
the young developing wild tissues within a chimera. In the older
tissues, however, structural differences between the two Portulaca
types cannot be established.

68

Figure 17. -- Photomicrograph of cross section through mutated shoot
of For tu lac a oleracoa . Cortical cells show a normal
development. Note the many starch grains that fill cells
of the first cortical layers. (150x)

69

Figure 18. -- Photomicrograph of cross section of half mutated

(left) and half wild (right) stem tissues of Portulaca
oleracea . Note the disruption of epidermis and cortical
cells on the right. (150 x)

70

Experiment 8 : Effect o^ Various Levels of. Mineral Elements on
Plnnts nnd Progeny Seeds of Portulaca oleracea

Effect on cuttings and seeds thereof

Material and methods. -- Cuttings of Portulaca oleracea were
grown for 4 weeks in various concentrations of salts of different
elements. Cu was supplied as sulfate, Li and Zn as chlorides, B
as boric acid and Se as seleniumoxychloride. All dilutions were
made in one-half strength nutrient solution. Five cuttings were
placed in each jar with 135 ml of solution through a 2 mm opening
in the lid. Concentrations and elements used are listed in Table
8. A plus sign indicates that all plants did survive and were
able to form seeds. A minus sign indicates that all plants died.
Where flowers were formed and seed setting took place, the seeds
were harvested. Further germination of seeds from the above plants
and development was tested. Young plants were set out in a green-
house in small plastic pots filled with sterilized soil.

Results. -- In the course of the experiment it was found that
extremely high concentrations could be tolerated by the cuttings.
Copper was relatively toxic and zinc to a lesser degree. Seeds
that were harvested from the treated plants showed in most cases
a normal development.

Selenium in seleniumoxychloride proved to be very toxic. In
the higher concentrations plants died within a few hours. Many
seedlings died after the two cotyledons appeared. Cotyledons were
very dark red in color in contrast to a normal dark green. High
amounts of selenium were probably incorporated in the seeds.
This could explain its devastating action on germination.

71

TABLE 8

EFFECT OF TREATMENT WITtl DIFFERENT IONS IN VARIOUS

CONCENTRATIONS ON RATE OF SURVIVAL OF

PORTULACA OLERACEA CUTTINGS

Treatment

Cu ppm

B ppm

Zn ppm

Rate of
Survival

4.5

+

9

+

18

+

36

+

72

-

144

09.9.

~

7

+

15

+

31

+

62

+

125

+

250

+

500

+

24

+

47

+

94

+

188

+

375

-

750

-

1,500

Treatment

Li ppm

Be ppm

Rate of
Survival

26

+

52

+

104

+

208

+

416

+

833

+

1,666

+

4.5

+

9

. +

18

+

36

+

72

-

144

-

288

-

72

Effect on seed] inp;s and seeds thereof

Material and metlunls . -- In a similar experiment young seed-
lings of Portulaca oleracea were used. Roots were cut to a length
of one-half inch. Concentrations and elements used are indicated
in Table 9. All the dilutions were made up with one-quarter strength
nutrient solution. Copper, zinc, and iron were given as chelates,
lithium as chloride, aluminum as sulfate, boron as boric acid, and
selenium as seleniiomoxychloride. Observations were made at regular
intervals of three days.

Results . -- After 14 days a definite separation could be made
into surviving and dying plants (see Table 9) . A plus sign indicates
that no noticeable effect from die solution was observed. A minus
sign indicates that all plants died. A plus and minus sign means
that plants were slightly affected. After 20 days most of the
plants which survived started to bloom and seeds were harvested
20 days later.

Further experimental procedure. -- From each of the above
treatments that produced seed 25 seeds were taken and planted in
small pots filled with sterilized soil. A square piece of carton,
perforated with 25 small holes, served as a guide for planting.
Seeds are extremely small and they need to be guided in order to
obtain a regular planting. Percentage of germination was determined
after 7, 14, and 21 days.

Results . -- Results are given in Figures 19, 20, 21 and 22,
Pretreatment of iron and boron had a direct effect on the germination
at the beginning. It took the seeds from these treatments much
longer to germinate than those from the plants which were treated

73

TABLE 9

EFFECT OF TREATMENT WITH DIFFERENT IONS IN VARIOUS

CONCENTRATIONS ON RATE OF SURVIVAL OF

PORTULACA OLERACEA SEEDLINGS

Al ppm

8

+

27

+

68

-

135

-

270

-

B ppm

25

+

62

+

125

+

250

+

500

-

Zn ppm

4

+

8

+

24

+

82

+

410

-

Rate of

Rate of

Treatment

Survival

Treatment

Surv: val

Cu ppm

Li ppm

4

+

42

+

8

+

208

-

24

+

416

-

80

-

833

-

400

1,666

4

+

8

+

21

+

70

-

350

-

Se ppm

12

46

115

231

1,156

+

74

% GERMINATION
lOOh

90
80

70
60

50

40

30

?-0

10

0

PRETREATMENT
BORON

25ppm
/62ppm

7 14

DAYS

ai

Figure 19. -- Effect of pretreatment of boric acid on percent
germination of Portulaca oleracea seeds.

75

% GERMINAT ON
100"

90

PRETREATMENT

80

COPPER

y 4ppm
^^- 8 PpRi

70

80

^* *^'' /

50

/

40

/

30

/

20

/

10

-

0

■ 1

I

7 14

2i

DAYS

Figure 20. -- Effect of pretreatment of copper chelate on percent
germination of Portulaca oleracea seeds.

76

% GERMINATION
100-

90
80
70
60
50
40

30

20

JO

0

PRETREATMENT
I RON

. 4 ppm

I
I

/ / 8 ppm
/ /•'2I ppm

7 I'

DAYS

21

Figure 21. -- Effect of pretreatment of iron chelate on percent
germination of Portulaca oleracea seeds.

77

% GERMINATION

100

90
80
70

60
50
40

30
20

10

0

4 pprn

''24ppm
/ ,'- 8ppm

/ PRETREATMENT
/ Zl NC

7 14

DAYS

21

Figure 22. -- Effect of pretreatment of zinc chelate on percent
germination of Portulaca oleracea seeds.

78

with an excess of copper or zinc. The data showed, furthermore,
that the total percentage of germination, which remained constant
after 21 days, was very much affected by the concentrations of the
solutions and of the elements used. Final germination percentage
for the boron pretreatment was rather low (647o) . Pretreatment
with zinc did not seem to affect the total germination. Upon
further development of the plants none showed abnormalities.

Part Three

Experiment _9: Inducing Metal Tolerance in Portulaca oleracea

The possibility was investigated whether the resistance of
cells to high concentrations of metal salts solutions could be
artificially induced. For this reason several concentrations of
copper, zinc, and iron chelate solutions were prepared. The
idea was to allow Portulaca oleracea cuttings to develop in high
concentrations of metal ions for several weeks. Thereafter,
cuttings of these plants were to be transplanted to the same or
even higher salt concentrations. If some cellular resistance to
high salt concentrations had been developed, the cuttings of the
treated plants would then probably do better in concentrated
solutions of metal ions than non-treated plants.
Material and methods

Chelated copper was used for this experiment in concentrations
of 8, 24, and 80 ppm, chelated zinc in concentrations of 24, 82 and
204 ppm, and chelated iron in concentrations of 21, 35 and 70 ppm.
Preliminary experiments, in which a wide concentration range of
metal chelates were used, indicated that these concentrations
were most suitable for this experiment. The various

79

concentrations were made up in one-quarter strength nutrient
solution. Cuttings of clonal material of Portulaca oleracca
were taken and planted four per jar in 135 ml of the solution.
Plants were kept in this solution for two months. These experiments
were carried out in duplicate.
Results

In the iron series root development took place on all the
cuttings. However, there was a gradual retardation of plant
growth in the higher concentrations. In the copper series only
the 80 ppm solution showed a severe inhibition on growth. In
spite of this inhibited growth, roots were formed even in this high
concentration of Cu. Plants showed severe retardation in growth
in the concentration of 204 ppm zinc solution. Roots were formed
but six of the eight plants died.
Further experimental procedures and results

Cuttings of plants which had grown in the 8 ppm Cu solution

were placed in a Cu concentration of 80 ppm and 160 ppm. A similar

scheme was worked out for the rest of the treatments. In summary

they were:

Cuttings from 8 ppm Cu solution placed in 80
' and 160 ppm Cu

Cuttings from 24 ppm Cu solutions placed in 80
and 160 ppm Cu

Cuttings from 35 ppm Fe solution placed in 70
and 140 ppm Fe

Cuttings from 70 ppm Fe solution placed in 70
and 140 ppm Fe

Cuttings from 24 ppm Zn solution placed in 82
and 164 ppm Zn

Cuttings from 82 ppm Zn solution placed in 82

and 164 ppm Zn

80

For comparison, cuttings of control plants were included and
received the same treatment as the cuttings derived from the metal
chelate solutions.

After two weeks, observations were made on root development and
general appearance. The results indicated that the control plants
developed slightly better in the 82 ppm Zn and 140 ppm Fe solutions.
In the 70 ppm Fe and the 160 ppm Cu solutions, cuttings from plants
grown in high Cu and Fe solutions grew better.

Definite conclusions cannot be drawn from this experiment.
It seems that the control plants grew better in some cases. This
might be because they were stronger at the start of the test. The
plants which had grown in the high concentrations of metal ions
had suffered definite growth retardation. On the other hand, it
was shown that cuttings from plants having grown in 24 ppm Cu
were able to form complete roots in a solution of 160 ppm Cu.
Control plants did not develop roots in this high concentration, but
produced a stunted growth. The leaves also came off these plants.
This indicates that a certain tolerance against high concentrations
of copper, at least at the beginning, may have been developed.
Experiment 10: Inducing Plasmatic Resistance in Portulaca oleracea
Material and methods

Cuttings of Portulaca oleracea were grown for two months in
a concentrated solution of 82 ppm Zn made up in a 1/4 strength
nutrient solution. After this the cuttings were transferred from
the greenhouse and set outside, in the sun to develop enough antho-
cyanin. The epidermis cells which turn red through pigmentation
of the vacuolar contents were then easier to examine for further

81

plasmolysis. Cell membrane may be seen pulling away from the cell

wall.

Small pieces of stem from control plants, and from plants having

received 82 ppm Zn, were excised. They were put into zinc sulfate

1-2 "^
solutions of 1, 10' , 10 and lO""^ M strength. After 24 hours

pieces were taken out. Epidermis cells were removed and treated
with 5.0 percent KNOo solution to test the viability of the cells.
Deplasmolysis was performed with a 3.0 percent sugar solution;
In order to obtain a good section of the epidermis for examination
the piece of stem was held steady with a needle in the center and
small incisions were made with a razor blade about 1 mm apart.
The epidermis layer was then removed with a small scalpel between
the points of incision.
Results

It was found that epidermis cells from plants treated with 82
ppm ZnSO, still plasmolized in a 5.0 percent KNO3 solution after
a 24 hour treatment with 10 M zinc sulfate. Epidermis cells
from control plants could not always be plasmolyzed and if they
could the plasmolysis reaction time was slowed down. After plasmoly-
sis all cells deplasmolyzed with a 3.0 percent sugar solution.

' -1 -2

In the 1 M, 10 M, and 10 M zinc sulfate solution most

of the cells died. Anthocyanin had leaked from the cells. A

few single cells derived from the 82 ppm zinc treatment under-

_2
went plasmolysis after treatment with 10 molar zinc sulfate

solution.

Results of this experiment indicated that a certain cell tol-
erance for zinc could be built up by growing the plants in concen-
trated zinc solutions.

DISCUSSION

Whenever elements are found in excess or deficient quantities,
it is to be expected that they affect plant growth; the nutrient
balance is disturbed and result in abnormalities in growth. This
direct effect has been studied by many authors (44, 50, 76).

The prolonged effect on seed and following generations is
more difficult to interpret. We have to distinguish between cell
disturbances in seeds as a result of a particular excess or
deficiency in the parent plant and between the genetic effects of
such a treatment. Seeds are probably affected directly by a
mineral excess or deficient treatment. Several reports have dealt
with the effect of a mineral deficient treatment on seed quality
and germination (42, 44). The "hollow heart" defect of boron
deficient peanuts may in severe stages destroy the tips of the
young plumule (44). Peanut plants grown under calcium deficient
conditions produced seeds which showed severe vascular deteriora-
tion.

It is evident that in most cases the germination of such seeds
is also affected. Harrington (42) found that seeds from carrots
and lettuce grown on a low calcium level did not germinate as well
as the ones from a complete nutrient solution. Deficiency or
excess of certain ions may result in bad or retarded germination.
It may affect the formation of the young embryo, resulting in
deformed cotyledons (41) , or in some cases it may affect the apical

82

83
system. Shoot formation becomes abnormal, unusual branching and
twisting of stems occurs.

In the experiments conducted in part one on soybeans, many of
these disturbances in seeds have been found with a manganese or
copper deficient treatment. The occurrence of twin embryos in
soybean, as a result of a copper deficient treatment, has never
been reported before. The natural occurrence of twin plants in
a particular line of soybean was described by Owen (78) . In many
other soybean varieties tested no twin plants were found. The
twins that were grown to maturity showed an independent segregation
of genetic characteristics. This means that the twin plants were
not identical and probably two eggs must have been fertilized
independently .

It was known that treatments such as 2-4 D could produce
twinning in plants (40). However, where such plants have a common
root, we may assume that the apical meristem is damaged, resulting
in a take-over by the lateral shoots. The young embryo is here
affected in the later stages of development. This is also the
case where very young developed seeds of Eranthis hiemalis were
treated with excess lithium carbonate (41) .

Polyembryony as a result of a treatment with growth- promo ting
substances has been reported by Johri (53). A certain combination
of casein hydrolysate, yeast extract and indolacetic acid induced
polyembryony in young excised pollinated embryos of Anethum graveolens.

Where soybeans were grown under copper stress, the young embryo
is probably affected in a very early stage, before any differentiation
of cotyledons took place. A longitudinal cleavage of the multi-cellular

84
embryo gave rise to these twins. They may be completely separated
or may remain attached with a few cells in the middle (incomplete
cleavage). A deficiency of copper at the very early stages of embryo
development was probably responsible for this cleavage of cells.
Since copper is a component of ascorbic acid oxydase, it might well
be that the function of this enzyme was inhibited by a copper
deficiency. Ascorbic acid oxydase along with numerous other enzymes
was found in the primary cell wall of the plant (70) . Although the
proper function of ascorbic acid oxydase in cell walls is not known,
it might be involved in synthesis of wall components. This could
possibly explain why under a copper stress a cleavage of cells may
occur. On the other hand, since polyembryony in soybean may result
from double fertilization (78) the possibility cannot be excluded
that a deficiency of copper promotes this anomality.

The symptoms of manganese deficient soybeans can be related
to the destruction of certain cell tissues in the early phases of
seed development. The impression is that tissues of the apical
meristem and certain areas of the cotyledons are particularly
affected. The typical forked stem, which sometimes remains twisted,
is a direct proof that the apical meristem has been damaged. We
must here clearly distinguish between the later branching where a
main shoot is present and between the forked stem where no main shoot
is found.

In contrast to the copper deficiency in soybean the deficiency
of manganese is here manifest at a much later stage of embryo develop-
ment. Particular cell groups, probably fully developed, are disturbed
in the cotyledons.

85

In the experiments of part two some of the direct effects of

metal excess on seed germination have been investigated. It was
found that germination of different seeds in various metal salt
solutions was not the same for all the seeds. Some seeds like
sorghum were able to germinate in much higher concentrations of
metal salts than rye, oats or soybean. This does not necessarily
indicate that the sorghum plant is more tolerant to high concentra-
tions of metals than the other plants tested. However, a certain
relationship may exist between the ability to germinate in a con-
centrated metal salt solution and the tolerance to this specific
metal.

In the experiments conducted here it was found that very high
concentrations of certain mineral elements prevented the germination
of Portulaca oleracea seeds. The vitality of these seeds however
remained normal. It is a common fact that unfavorable conditions,
as they often occur in nature, inhibit the germination of seeds.
Seeds of Sabal minor, the stemless palmetto, survived a four weeks'
soaking in seawater (12).

Genetic effects of a mineral excess or deficient treatment have
been studied by many authors (89, 90, 106, 107). Several mutations
have been obtained. The mutations could also be produced by means
of irradiation or other agents (107) . This means that the mutation
caused by a treatment of heavy metals is not necessarily tolerant
to this metal.

Results of the experiment conducted here indicated that the
Portulaca mutants obtained after treatment with l.Opercent lithium
chloride was less tolerant to high concentrations of lithium chloride
than the wild type.

86

We must sharply distinguish between the mutagenic action of an
element and the selection pressure executed by the same element.

It was already known that lithium produced abnormalities in
the cotyledons of plants (41) . Malformations resulting from
excess of lithium were also found in young animal embryos (83) . It
was reasonable to believe therefore that similar results might be
expected when young seeds of Portulaca oleracea were treated with
high amounts of lithium chloride. It could also be seen that since
the seed contains a completely developed embryo it would be
difficult genetically to affect the mainshoot and cotyledons. All
abnormalities observed' in these regions are probably due to the
direct toxic action of lithium chloride.

In the initial stage of branching, buds are formed from cells
in the superficial layers of the tunica and deeper layers of the
corpus. It is probably at this stage, where only a few cells are
involved, that lithium chloride can act. The first four branches
in Portulaca oleracea are in general much more developed than the
later ones.

It was among these four branches only that the mutations were
found, after Portulaca oleracea seeds had been treated with a 1
percent lithium chloride solution. The number of mutated branches
in each plant varied from one to three.

Reversion to wild type in the mutated branches indicated that
a typical chimera type was produced. Therefore mutation took
place only in the superficial cell layers of the tunica. Chlorophyll
mufe<«tl'"iiiri In j'ny-.uL-.yr. c;,l.ofao<,? c)l>':'«i.iii-!i,; flvv:!r ivciatmont with i por-
cciit lir.hiim chliride, indi-catcd a t.ii.iil.jr mutagenic action on cells
in the tunica.

87

Dermen (17) in studies on periclinal cytochimeras in cranberry
could clearly distinguish between the different polyploid types
which were obtained from treatment with colchicine. Histological
studies on the apical dome and tissues of the stem showed the
various differences in structural make-up of the cell layers of the
chimeras.

In the experiments with Portulaca a similar attempt was made
to identify the various tissues in young growing buds. Comparisons
were made between the wild and mutated types. If the Portulaca
chimera was the result of polyploidy then distinct histogenic layers
must be found. The polyploid layers have in general larger cells
than the diploid cell layers (18). The same is true for stomata
of the leaf epidermis (17). If this cell layer was derived from
polyploid cell tissue then the stomata should have been larger than
normal. The results of microscopic examinations of different
tissues and epidermis cells in Portulaca showed no difference in
cell size, when similar cells were compared in wild and mutated
types.

This leads to the conclusion that the Portulaca chimera type is
not the result of polyploidy.

The action of lithium chloride was rather specific. That is,
besides some chlorophyll mutations, only one type of somatic mutation
namely an eliptical leaf type, was produced. Since polyploidy is
probably not involved here, the mutagenic action must have been on
a specific gene.

There are a few instances in which spontaneous mutations have
been found in the Portulacea. Somatic mutations in Portulaca

88
Rrandi flora have been described by Faberge and Beale (28) . That
mutation was caused by an unstable gene which was responsible
for production of colored spots on stems and petals. The mutation
rate was considerably reduced with rising temperatures.

Spontaneous mutations in epidermis cells of adult leaves of
Portulaca grandiflora have also been described by Czeika (16).
Mutations produced were of polyploid nature and occurred during
mitosis of adult epidermis cells.

The effect of an excess mineral element treatment can sometimes
be related to a deficiency of certain elements. It was found that
chlorosis often occurs as a result of toxic action of a high metal
concentration (30). Heavy metals interfered with the uptake of

iron.

It was found in the experiments of part two (the second ap-
proach) that percentage germination of Portulaca seeds grown from
plants treated with high concentrations of copper, zinc, iron and
boron was considerably reduced. The effect was specific for each
ion. The time required for germination seemed much more prolonged
than in control treatment. It took about seven days for normal
treated Portulaca seeds to reach a germination percentage of ninety.
After 14 days a 30 - 40 percent germination was found for seeds
from plants treated with different iron chelate solutions. There
was a retardation which could also be observed in the other treat-
ments. It is not known how much of the different elements had been
taken up by the seeds. But it seems likely that some accumulation
took place.

Seeds of Ncptunia grown on soils rich in selenium contained

89

123 itig selenium per seed (80). The normal amount is only 0.45
ug per seed.

It was found that Portulaca seeds derived from the plants
treated with high levels of selenium produced a high percentage
of empty seeds. Many seeds which did germinate died in an early
stage of growth. It seems that a heavy accumulation of selenium
by the young seed must be responsible for the early death of the
seedling. Selenivim is extremely mobile in plants. The effect of
its uptake is very pronounced. In only a few hours after intro-
duction of selenium into plants, the leaves are discolored and pro-
duce the typical odor of a selenium solution. It must be emphasi-
zed that under very toxic conditions fertilization could be
arrested. Incompletely developed embryos may also be produced.

In the experiments conducted in part three a particular
metal tolerance could be built up by the plant cell if plants were
grown in high levels of mineral solutions. It should be emphasized
that this induction is not an alteration of the genetic consti-
tution of the cell. The greater resistance of the cell to certain
metals is probably due to a protoplasmic reorganization. This
build up of tolerance to a certain metal under excess metal conditions
can be compared with the effect of other environmental factors
resulting in a structural reorganization of the cell. Since high
tolerance to metals corresponds to high metal content in the
plant (87) , we may assume that the mechanism for tolerance probably
involves a precipitation or adsorption of the toxic ions. Where
adsorption takes place, special organic chelates are probably
involved. It was also found that the presence of anthocyanin

90
increased the metal tolerance in plants (87). Anthocyanin may
act as a metal binding agent. High osmotic pressure in ccllsap
could also be partly responsible for the increased metal tolerance.

SUMMARY AND CONCLUSIONS

The effect of various mineral levels on physiology and morpho-
logy of different plants was studied.

In a copper and manganese deficient nutrient meditmi, soybean
plants produced seeds which were typically affected. Syncotyledons
and tricotyledons were produced. A' high percentage of twin plants
developed from these abnormalities in the copper deficient treatment.
In the manganese deficient treatment many so called "forked stems"
developed from these seeds. Effect of the manganese deficient
treatment could be related to the destruction of certain cell
tissues in the young embryo. Effect of the copper deficient treat-
ment may inhibit cell wall formation resulting in an early cleavage
of the zygote and formation of twin plants.

The effect of various salt solutions on germination of seeds,
particularly on seeds of Portulaca oleracea, was studied. It was
found that high concentrations of salt solutions inhibited the
germination without destroying the vitality of the seed. Sorghum
seeds were able to germinate in much higher concentrations of metal
salts than rye, oats or soybean seeds.

Soaking oat seeds in concentrated solutions of zinc and mangan-
ese retarded the blooming in the zinc treatment and advanced the
blooming in the manganese treatment.

When Portulaca oleracea seeds were treated with a 1 percent
lithium chloride solution, mutations were produced with eliptical

91

92

leaves and incised petals. Reversion to the wild type in the cuttings
indicated a chimera characteristic. The mutation was shown not to
be a result of polyploidy. At certain stages of development marked
his tological differences could be shown in the cortical cells of
both the mutated and wild types. Grafting experiments performed
between the two types of Portulaca, also showed that the modifica-
tion found in the leaves and flowers of Portulaca, was not a result
of virus or bacterium infection.

Plants which had grown in high concentrations of metal ions
produced seeds that showed a characteristic reduction and retard-
ation in germination. No mutation effects were observed.

It was found that a tolerance for zinc can be built up in the
cells of Portulaca oleracea, when growing these plants in concen-
trated zinc solutions. Plasmo lysis technique was used to measure
the viability of the cells. Pretreatment of Portulaca oleracea
cuttings with concentrated solutions of heavy metals increased
the resistance of the cuttings to high metal concentrations in
some cases. Cuttings from plants having grown in 24 ppm Cu were
able to form complete roots in a solution of 150 ppm Cu. Control
plants did not develop any roots in this concentration.

In conclusion, when plants are grown under excessive or
deficient levels of mineral elements, abnormalities in the progeny
seed may result. As another alternative, mutations may occur,
especially after treatment of seeds with high levels of mineral
elements. The induction of tolerance to high levels of heavy
metals is probably due to protoplasmic reorganization.

BIBLIOGRAPHY

1. Bari, Ghulam. 1963. The mutagenic effect of ethyl methane

sulphonate alone and in combination with copper on wheat.
Caryologia 16:619-624.

2. Baumeister, W. 1955. Uber den Einfluss des Zinks bei Silene

inflata Smith. Ber. Deut. Bot. Ges. 67:205-213.

3. Bayer, E. 1964. Structure and specificity of organic che-

lating agents. Angew. Chem. Int. (Eng. ed.) 3:325-332.

4. Bertrand, Didier. 1959. Nouvelles recherches sur le lithium

des graines. Compt. Rend. Acad. Paris 249:331-332.

5. Bhatia, C. R. , and K. R. Narayanan. 1965. Genetic effects of

ethyl methane sulfonate in combination with copper and
zinc ions on Arabidopsis thaliana. Genetics 52:577-581.

6. Biebl, R. 1949. Uberdie Resistenz pflanzlicher Plasmen gegen

Vanadium. Protoplasma 39:251-259.

7. Bogorad, L. , G. Pires, H. Swift, and W. J. Mcllrath. 1958.

The structure of chloroplast in leaf tissue of iron
deficient Xanthium. Brookhaven Symp. Biol. 11:132-137.

8. Bollmann, A., and F. Schwanitz. 1957. Uber den Eisengehalt

in Pflanzen einer eisenreichen Standortes. Z.f. Bot.
45:39-42.

9. Bradshaw, A. D. 1952. Population of Agrostis tenuis resistent

to lead and zinc poisoning. Nature 169:1098.

10. Bradshaw, A. D., T. S. McNeilly, and R. P. G. Gregory. 1965.

Industrialization, evolution and the development of heavy
metal tolerance in plants. In Ecology and the Industrial
Society. 5th Brit. Ecol. Soc. Symp.: 327-343. Blackwell
Scientific Publications.

11. Broker, Werner. 1963. Genetisch-psysiologische Untersuchungen

liber die Zinkvertraglichkeit von Silene inflata Sm. Flora
153:122-156.

12. Burk, Carl John. 1966. Dynamics of plant distribution. 1.

Sabal minor. In Advancing Frontiers of Plant Sciences
14:1-4.

93

94

13. Cannon, Helen L. 1960. Botanical prospecting for ore deposits.

Science 132:591-598.

14. Chang, Chong W. , and C. Ray Thompson. 1966. Effect of fluoride

on nucleic acids and growth in germinating corn seedling roots,
Physiol. Plant. 19:911-918.

15. Cohn, N. S. 1961. The effect of chelation on the production of

chromatid aberrations in Vicia faba. Exp, Cell Res. 24:
596-599.

16. Czeika, G. 1958. Uber das spontane Auftreten von Mitosen und

inaqualen cytokinesen in der endopolyploiden Epidermis von
Portulaca grandif lora. Planta 51:566-574.

17. Dermen, H. 1947. Periclinal cytochimeras and histogenesis in

cranberry. Amer. J. Bot. 34:32-43.

18. Dermen, H. , and H. F. Bain. 1944. A general cytological study

of colchicine polyploidy in cranberry. Amer. J. Bot.
31:451-463.

19. Billing, W. J. 1926. Influence of lead and the metallic ions

of copper, zinc, thorium, beryllium and thallium on the
germination of seeds. Ann. Apl. Biol. 13:160-167.

20. Dorung, H. 1937. Uber den Einfluss der Ernahrung auf die

Mutationshauf igkeit bei Antirrhinum ma jus . Ber. Deut.
Bot. Ges. 55: (167)-(182).

21. Drescher-Kaden, F. K. , and F. Schwanitz. 1956. Uber den

Strontium und Calcium-gehalt in Pflanzen eines Strontium-
haltigen Standortes. Z.f. Bot. 44:501-504,

22. Du Bois, Anne M. , Gilbert Turian, and Alain Gonet. 1957.

Recherches sur 1' action mitostatique de I'acide borique
dans le meristeme radiculaire d' Allium cepa L. Caryologia
10:102-110.

23. Durrant, A. 1962. The environmental induction of heritable

change in Linum. Heredity 17:27-62.

24. Durrant, A., and H. Tyson. 1964. The effects of fertilizers

on two subsequent generations of winterwheat, Der ZUchter
34:41-45.

25. Duvigneaud, P., and S. Denayer-de Smet. 1963. Cuivre et

ve'getation au Katanga. Bull. Soc. Roy. Bot. Belg. 96:
93-231.

26. Ernst, Wilfried. 1965. liber den Einfluss des Zinks auf die

Keimung von Schwermetallpf lanzen und auf die Entwicklung
der Schwermetall-pf lanzengellschaf t. Ber. Deut. Bot. Ges.
78:205-212.

95

27. Ernst, Wilfricd, 1966. Okologische-soziologischc Untcrsuchun-

gen an Schwermetallpf lanzengesellschaf ten siidfrankreichs
und des bstlichen Harzvorlandes . Flora (B.) 156:301-318.

28. Faberge, A. C, and G. H. Beale. 1942. An unstable gene in

Portulaca: mutation rate at different temperatures. J.
of Genet. 43:173-187.

29. Fink, Hanneliese. 1950. Experimentelle Untersuchungen {iber die

Wirkung des Nahrsalzmangels auf die Mitose der Wurzelspitzen
von Vicia faba. Chromosoma 3:510-565.

30. Forster, W. A. 1954. Toxic effects of heavy metals on crop

plants grown in soil culture. Ann. Appl. Biol. 41:637-651.

31. Frerking, H. 1915. iJber die Giftwirkung der Lithiumsalze auf

Pflanzen. Flora 108:449-453.

32. Garber, K. 1962. Pflanze und Fluor. Qual. Plant, et Mater.

Veg. 9:33-44.

33. Genkel, D. A., R. S. Morozova, and N. D. Pronina. 1962,

Synthetic ability of drought hardened tomato plants.
Fiziol. Rastenii 9:57-61.

34. Glass, E. 1955. Untersuchungen iiber die Einwirkung von

Schwermetallsalzen auf die Wurzelspitzenmitose von
Vicia faba. Z.f. Bot. 43:359-403.

35. Glass, E. 1956. Die verteilung von Fragmentationen und

achromatischen Stellen auf den Chromosomen von Vicia
faba nach Behandlung mit Schwermetallsalzen. Chromo-
soma 8:260-284.

36. Gorham, E., and A. G. Gordon. 1960. The influence of smelter

fumes upon the chemical composition of lake waters near
Sudbury, Ontario, and upon the surrounding vegetation.
Can. J. of Bot. 38:477-487.

37. Gregory, R.P.G., and A. D. Bradshaw. 1965. Heavy metal

tolerance in populations of Agrostis tenuis Sibth.
New Phytologist 64:131-143.

38. Gries, Brunhild. 1966. Zellphysiologische Untersuchungen

liber die Zinkresistenz bei Galmeiokotypen und Normalformen
von Silene cucubalus Wib. Flora 156 (B) : 271-290 .

39. Haarring, R. 1962. Mutationauslosung durch Chemikalien bei

Weizen. 3. Unterschiedliche Folgen einer mutagenen Behand-
lung von verschiedenartigen Zellen. Z.f. Pflanzenz. 48:
117-142.

40. Haccius, B. 1955. Experimentally induced twinning in plants.

Nature 176:355-356.

96

41. Haccius, B. 1956. Uber die Beeinf lussung der Morphogcnese

pflanzlicher Embryonen durch Lithium-ioncn. Bcr. Dcut.
Bot. Ges. 69:87-93.

42. Harrington, James F. 1960 Germination of seeds from carrot,

lettuce and pepper plants grown under severe nutrient
deficiencies. Hilgardia 30:219-235.

43. Harris, Henry C., 1965. The interrelated effects of mineral

nutrient deficiencies, environment, and heredity on the
nitrogen metabolism of plants. Ann. Rep. 1965, Fla. Ag.
Exp. Sta. 59.

44. Harris, Henry C, and John B. Brolmann. 1966. Comparison of

calcium and boron deficiencies of the peanut. 2. Seed
quality in relation to histology and viability. Agron. J.
58:578-582.

45. Harris, T. M. 1946. Zinc poisoning of wild plants from wire

netting. New Phytologist 45:50-55.

46. Haselhoff, E. 1892. Uber die schadigende Wirkung von Kupfer-

sulfat- und kupfer-nitrathaltigen Wasser auf Boden und
Pflanzen. Landwirt. Jahrb. 21:261-271.

47. Heald, F. D. 1896. On the toxic effect of dilute solutions

of acids and salts upon plants. Bot. Gaz. 22:125-153.

48. Herich, Rudolf. 1965. The effect of cobalt on the structure

of chromosomes and on the mitosis. Chromosoma 17:194-198.

49. Hoagland, D. R. , and D. I. Arnon. 1950. The water-culture

method for growing plants without soil. California Agr.
Exp. Sta. Circ. 347 (Rev.).

50. Hunter, J. G. , and 0. Vergnano. 1952. Nickel toxicity in

plants. Ann. Appl. Biol. 39:279-284.

51. Hyde, B. B. , and R. L. Paliwal. 1958. Studies on the role of

cations in the structure and behavior of plant chromo-
somes. Amer. J. Bot. 45:433-438.

52. Johansen, Donald A. 1940. Plant microtechnique. Mc-Graw-

Hill Book Company, Inc., New York.

53. Johri, B. M. , and C. B. Sehgal. 1963. Chemical induction of

polyembryony in Anethum graveolens L. Naturwissenschaf ten .
50:47-48.

54. Jowett, D.,1958. Populations of Agrostis spp. tolerant of heavy

metals. Nature 182:816-817.

55. Jowett, D. 1959. Adaptation of a lead tolerant population of

Agrostis tenuis to low soil fertility. Nature 184:43.

97

56. Kahlenbcrg, Louis, and Rodney H. Ture. 1896. On the toxic

action of dissolved salts and their electrolytic dis-
sociation. Bot. Gaz. 22:81-124.

57. Karve' A. D. , and S. G. Jigajinni. 1965. Circadian rhythm in

Portulaca. Z. Pf lanzenphysiol. 53: 169-172.

58. Karve', A. D., and S. G. Jigajinni. 1966. Leaf movements of

Portulaca under different photoperiods . Z. Pf lanzenphysiol.
54:270-274.

59. Kisser, J., and K. Lettmayr. 1933. Untersuchungen uber die

Absorption von Salzen durch Samen. Z. f. Pf lanzenernahrung,
Dungung und Bodenkunde. 29A: 195-210.

60. Kisser, J., and K. Lettmayr. 1934. Untersuchungen uber die

Auswaschbarkeit der von Samen absorbierten Salze und
ihre Bedeutung fifr die Samenstimulation. Z.f. Pf lanz-
enernahrung, Dungung und Bodenkunde. 34A: 172-181.

61. Lebrun, J. 1965. Le mouvement d'ouverture et de fermeture

des fleurs chez diverses Por tulacace'es. Bull. Soc. Roy.
Bot. Belg. 90:19-35.

62. Marines, N. G. 1962. Studies on submicroscopic aspects of

mineral deficiencies 1. Calcium deficiency in the shoot-
apex of barley. Amer. J. Bot. 49:834-841.

63. Marschner, H. 1960. Uber die Nachwirkung von P"^^ in der

2 Generation von Sommerger ste . Die Kulturpf lanze
8:90-92.

64. Marth, P. C., and J. R. Frank. 1961. Increasing tolerance of

soybean plants to some soluble salts through application
of plant growth retardant chemicals. J. Agr. Food Chem.
9:359-361.

65. McCune, D. C., A. E. Hitchcock, Jay S. Jacobson, and Leonard

H. Weinstein. 1965. Fluoride accumulation and growth of
plants exposed to particulate cryolite in the atmosphere.
Contrib. Boyce Thompson Inst. 23:1-11.

66. Mohamed, Aly H. , Howard G. Applegate, and James D. Smith.

1966. Cytological reactions induced by sodium fluoride in
Allium cepa root tip chromosomes. Can. J. Genet. Cyt.
8:241-244.

67. Mohamed, Aly H. , James D. Smith, and Howard G. Applegate.

1966. Cytological effects of hydrogen fluoride on tomato
chromosomes. Can. J. Genet. Cyt. 8:575-583.

68. Moutschen, J., A. Moes, and J. Gilot. 1964. Some meiotic

consequences of ethyl-methanesulfonate and the inter-
action of copper and zinc. Experientia (Basel) 20:494-495.

98

69. Moutschen-Dahmen, J., and M. Moutschcn-Dahmen. 1963. Influence

of Cu++ and Zn """ ions on the effect of ethylmcthancsulf-
onate (EMS) on chromosomes. Experientia (Basel) 19:144-145.

70. Newcomb, E. H. 1963. Cytoplasm-cell wall relationships. Ann,

Rev. Plant Physiol. 14:43-64.

71. Newton, Lily. 1944. Pollution of the rivers of West Wales by

lead and zinc mine effluent. Ann. Appl. Biol. 31:1-11.

72. Niethammer, A. 1930. Studien liber die Beeinf lussung der

Pflanzen durch Schwermetallverbindungen. Protoplasma
8:50-57.

73. Oehlkers, Friedrich, 1956. Die Auslosung von Mutationen durch

Chemikalicn bei Antirrhinum ma jus. Z. Vererbungslehre
87:584-589.

74. Oertli, J. J., and H. C. Kohl. 1961. Some considerations

about the tolerance of various plant species to excessive
supplies of boron. Soil Sci. 92:243-247.

75. Ota, T, 1964. Increasing tolerance of kidney bean plants to

toxic levels of sodium chloride and ammonium sulfate
through application of B 995. Plant and Cell Physiol.
5:255-258.

76. Otto, R. 1893. Untersuchungen iiber das Verhalten der Pflanz-

enwurzeln gegen Kupfersalzlosungen. Z. f. Pf lanzenkrankh.
3:322-334.

77. Ouellette, G. J., and L. Dessureaux. 1958. Chemical composition

of Alfalfa as related to degree of tolerance of manganese
and aluminium. Canad. J. Plant Sci. 38:206-214.

78. Owen, F. V. 1928. Soybean seeds with two embryos. J. Heredity

19:373-374.

79. Parkash, Ved, and B. V. Subbish, 1965. Radioisotopes in micro-

nutrient studies. 1. Distribution pattern of P in
citrus seedlings under deficient or toxic conditions of
different micronutrients. Soil Sci. and Plant Nutrition
11:1-8.

80. Peterson, P. J., and G. W. Butler. 1962. The uptake and

assimilation of selenite by higher plants. Aust. J. Biol.
Sci. 15:126-146.

81. Pra't, S. 1934. Die Erblichkeit der Resistenz gegen Kupfer.

Ber. Deut. Bot. Ges. 52:65-67.

82. Pringsheim, E. G. 1924. Uber Plasmolyse durch Schwerraetall-

salze. Beih. Bot. Zentralbl. 41:1-14.

99

83. Raven, Chr. P. 1952. Lithium as a tool in the analysis of

morphogenesis in Limnaea stagnalis . Experientia (Basel)
8:252-257.

84. Repp, G. 1963. Die Kupferresistenz des Protoplasmas hoherer

Pflanzen auf Kupferbo'den. Protoplasma 57:641-659.

85. Rieger, Rigomor, Hristo Nicoloff and Arnd Michaelis. 1963.

Chelatbilding als Sensibilisierungfactor bei der chemischen
Auslosung von Chromosomenaberrationen. Biol. Zentralbl.
82:393-412.

86. Robinson, W. 0., and G. Edington. 1945. Minor elements in

plants and some accumulator plants. Soil Sci. 60:15-28.

87. Ruther, F. 1967. Vergleichende physiologische Untersuchungcn

uber die Resistenz von Schwermetallplf lanzen. Protoplasma
64:400-425.

88. Sarkar, Ira. 1957. Effects of versene-induced calcium defici-

ency in living neuroblasts of the grasshopper embryo.
Cytologia 22:370-379.

89. Scheibe, A., and G. Hiilsmann. 1958. Mutations- auslosung durch

Chemikalien beim Steinklee (Melilotus albus) . Z.f. Pflanzenz.
39:299-324.

90. Scheibe, A., and H. Meyer. 1962. Mutationsauslosung durch Chem-

ikalien bei Weizen. 1. Die Anschnittmethode und erste
Ergebnisse. Z. f. Pflanzenz. 47:181-192.

91. Schwanitz, Fritz, and H. Hahn. 1954, Genetisch-entwicklung-

physiologische Untersuchungen an Galmeipf lanzen. 1.
Pflanzengrosse und Resistenz gegen Zinksulfat bei Viola
lutea Hudson, Alsine verne L. und Silene inf lata Sm.
Z. f. Bot. 42:179-190.

92. Schwanitz, Fritz, and H. Hahn. 1954. Genetisch-entwicklung-

sphysiologische Untersuchungen an Galmeipf lanzen. 2.
liber Galmeibiotypen bei Linimi cartharticiom L. Campanula
rotundifolia L. , Plantago lanceolata L. , und Rim:iex acetosa
L. Z.f. Bot. 42:459-471.

93. Scott, E. G. 1960. Effect of supra-optimal boron levels on

respiration and carbohydrate metabolism of Helianthus
annus. Plant Physiol. 35:653-661.

94. Sigenaga, M. 1944. Experimental studies of abnormal nuclear

and cell division. 1, Observation with living cells of the
effects of neutral salts and heavy metal salts. Cytologia
13:380-404.

95. Smith, P. F. 1953. Heavy metal accumulation by citrus roots.

Bot. Gaz. 114:426-436.

100

97,

98,

99,

96. Steffenson, D. 1953. Induction of chromosome breakage at meiosis
by a magnesium deficiency in Tradescantia. Proc. Nat. Ac
Sci. 39:613-620. ~

Steffenson, D. 1954. Irregularities of chromosome division in
Tradcscantia grown on low sulfate. Exp. Cell Res. 6-
554-556.

Steffenson, D. 1955. Breakage of chromosomes in Tradcscantia
with a calcium deficiency. Proc. Nat. Ac, Sci. 41:155-160.

Steffenson, D. 1958. Chromosome aberrations in calcium deficient
Tradcscantia produced by irradiation. Nature 182:1750-1751.

100. Steffenson, D. 1959, Divalent metals in the structure of

chromosomes. In The cell nucleus: 216-221. Proc, Faraday
Society, Butterworths & Co., London, publisher, Ed. J. S.
Mitchell.

101. Stockberger, W, W, 1910, The effect of some toxic solutions

on mitosis. Bot. Gaz. 49:401-429.

102. Thomas, W. I., and D, E, Baker. 1966. Application of bio-

chemical genetics in quality improvement and plant nutrition.
2. Studies in the inheritance of chemical element accumu-
lation in corn (Zea mays L.) , Qual. Plants Mater. Veg.
13:98-104.

103. Thompson, William W., and T. E. Weier. 1962. The fine structure

of chloroplasts from mineral deficient leaves of Phaseolus
vulgaris. Amer. J. Bot. 49:1047-1055.

104. Url, W. 1956. Uber Schwermetall- zumal Kupferresistenz einiger

Moose. Protoplasma 46:768-793.

105. Von Rosen, G. 1954. Breaking chromosomes by the action of

elements of the periodical system and by some other
principles. Hereditas 40:258-263.

106. Von Rosen, G. 1957. Mutations induced by the action of metal

ions in Pisum. Hereditas 43:644-664.

107. Von Rosen, G, 1964, Mutations induced by the action of metal

ions in Pisum, 2, Further investigations on the mutagenic
action of metal ions and comparison with the activity of
ionizing radiation, Hereditas 51:89-134,

108. Walker, G. W. R., and fch-Ping Ting. 1967. Effect of selenium

on recombination in barley. Can, J, Gen. Cyt, 9:314-320.

109. West, S. H. 1962, Protein, nucleotide and ribonucleic acid

metabolism in corn during germination under water stress.
Plant Physiol. 37:565-571.

101

110. Yamaha, G., and T. Ishii. 1932. ilber die lonenwirkung auf die

Chromosomen der Pollenmutterzellen von Tradescantia reflexa.
Cytologia 3:333-336,

111. Zimmerman, P. W., A. E. Hitchcock, and J. Gwirtsman. 1957.

Fluorine in food with special reference to tea. Contrib.
Boyce Thompson Inst. 19:49-53.

112. Zschege, Christa. 1963. Mutationsauslbsung durch Chemikalien

bei Weizen 4. Cytogenetiscjhe Untersuchungen an Mutanten.
Z. f. Pflanzenz. 49:122-160.

BIOGRAPHICAL SKETCH

Johannes Bernardus Balthasar Brolmann was born November 20,
1920, in Hilversum, The Netherlands. He was graduated from
High School in Hilversxim in 1939, and received States Diploma
for Physics and Mathematics in 1941 at the Hague, the Netherlands,
entering the University of Wageningen, the Netherlands, in 1941.
His studies were interrupted during World War II and he reentered
the University of Wageningen in 1945, receiving the Master of
Science degree in Tropical Agriculture with a major in plant
breeding and soil chemistry in 1952. From 1952 until 1960 he
worked as plant breeder and chemist at the Agricultural Experiment-
al Station near Aries, Southern France. He entered the University
of Florida in 1962, while being employed as Senior Laboratory
Assistant at the Agricultural Experiment Station and has been
employed in this capacity to the present time.

Mr. Brolmann was married to the former Antoinette Van Rhyn
in 1952, and is the father of four sons.

This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to
the Dean of the College of Agriculture and to the Graduate Council,
and was approved as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

August 1968

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Dean, Graduate School

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Chairman

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