Title: Effects of mineral levels on physiology and morphology of plants
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00097788/00001
 Material Information
Title: Effects of mineral levels on physiology and morphology of plants
Physical Description: 103 leaves : ill. 28 cm. ;
Language: English
Creator: Brolmann, Johannes Bernardus Balthasar, 1920-
Copyright Date: 1968
 Subjects
Subject: Plant physiology   ( lcsh )
Plants, Effect of minerals on   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Johannes Bernardus Balthasar Brolmann.
Thesis: Thesis (Ph. D.)--University of Florida, 1968.
Bibliography: Includes bibliographical references (leaves 93-101).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00097788
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000430835
oclc - 37704156
notis - ACJ0222

Downloads

This item has the following downloads:

effectsofmineral00brolrich ( PDF )


Full Text












EFFECTS OF MINERAL LEVELS ON
PHYSIOLOGY AND MORPHOLOGY
OF PLANTS











By

JOHANNES BERNARDUS BALTHASAR BROLMANN


A DISSERTATION 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
















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.












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,
CI~, S04- 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







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













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 % lithium 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 %
lithium 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







LIST OF FIGURES (continued)


Page

Figure

14. Photomicrograph of stomata of lower leaf
epidermis from wild type of Portulaca
oleracea...................................... 62

15. Photomicrograph of stomata of upper epidermis
from mutated type of Portulaca oleracea....... 63

16. Photomicrograph of cross section through wild
shoot of Portulaca oleracca................... 66

17. Photomicrograph of cross section through mutated
shoot of Portulaca oleracea................... 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 oleracea 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














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 accumulation 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 et al. (111) 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.

Astralagus 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 ig 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-

radiographic studies with 32P (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 Agrostis was treated with different







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 nijanese and aluminum 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.




5

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 CuSO4 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 boron 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 fumes 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

fretwork 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 Lettmayr (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 Heavy 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 et al. (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 Agrostis tenuis

is one of adaption as a result of evolutionary changes. Tolerant

and non-tolerant populations have sharp boundaries 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







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 forms 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 cooner-resistant form of

Silence inflata did not appear to be resistant to zinc. Broker (11)

demonstrated that there was no correlation between the morphology

of Silene inflata and the tolerance to high amounts of zinc. When

grown under normal conditions, Silene inflata did not lose this

tolerance. However, the plants were altogether bigger, with long

stems and broad leaves in contrast with the more dwarf type grown

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 accumulated

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 copper-resistant Agrostis and Melandrium

variety, growing on a sandy soil very rich in copper. Melandrium







silvestre seeds were able to germinate in a mixture of 25 percent

CuCO3 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 F1 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 medium 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 resistance or 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.







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 vanadium varies from plant to plant (6). For some plants (Trades-

cantia zebrina) 0.0001 percent vanadium sulfate was harmful to the

epidermis cells. Others (Rhoeo discolor) were able to support

up to one percent vanadium sulfate. Various plants known to be Cu-

tolerant showed high plasmatic resistance against toxic copper

concentrations (84). The 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 barrier 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







experiments of this nature have ever been conducted. Vitis vinifera

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-

ments or radioactive element treatments. When applications of 32P

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).







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 CuSO4 on mitosis in the root tips of Vicia faba. Dilute

as well as strong CuS04 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 spidlefbers, 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 10-6 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







activity of EMS. Similar results were obtained with Vicia faba (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 10-6 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
-2
when roots were treated with a 10-2 solution of sodium 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 F1 generation abnormal

phenotypes were found. Hydrogen fluoride can, for this reason, be

considered as a mutagenic agent.







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 majus were obtained when the plants were

grown under nitrogen, phosphorus and magnesium stress (20).

Abnormal cytological effects in Tradescantia were reported

when plants were grown 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. Anthers fomf_ 1LAts gown _in vr, w

calcium concentrations had only-abhr-tjve 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







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 reflexa 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 volume. 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 (NO3)2 and AgNO3 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

concentrations of the metals used ranged from 10-5 M to 10-2 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







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(NO3)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 AlCl3 and

Al(NO3)3 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

-3
103 M AC 3.

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 Pisum 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







recognizable mutations in summer wheat were obtained after treatment

with A1C1 (90). Although aluminum chloride was very toxic, it had

a high mutagenous effect. Most frequent mutations were square head

types ears with short stem. A1C13 was introduced by the so-called

"notching method" of Oehlkers (73), whereby part of the stem was

split and immersed in AlC13 solution. Haarring (39) obtained

several mutations in a hexaploid summer wheat variety using AlCI3

in concentrations of 10- 10-2 M. Zschege (112) used the same

wheat variety for cytogenetical investigations and found several

chromosome aberrations. Antirrhinum majus when treated with 10-2

M AlCI3 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

faba. 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






result here in binding elements such as calcium with a direct

effect on cell division and chromosome behavior.

Rieger ct al. (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

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)







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-l-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 ih concentrations of 2 x 10- and 10-3 M on

growth of corn seedlings was demonstrated (14). This effect could







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

75Se 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 longiflorum

(100), showing that on germination of the radioactive pollen the

45Ca 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 potassium 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 Cl) and the wild

type were examined. Chromosome studies and leaf epidermis studies

were conducted to obtain more information on the type of mutation produced.







The second approach was an attempt to induce mutations by

direct treatment of seedlings and cuttings of Portulaca oleracca

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 was 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.






Experimental procedure and results with manganese deficiency

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.



















































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)



































"- a 4 -. .., .. t ,.- s,'-

- ..'. '" t" a """.--" " w
.-- lip! -ft
oz-)














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





















































Figure 3. -- Forked stems in soybean as a result of manganese
deficiency. Picture at bottom shows a twisting of the
forked stem.







Experimental procedures and results with 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. Where 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 twin plants with twisted 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 Two

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, sorghum 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























































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


































u-l \0
LC)







co

CM4


ir) CN
-1 -1


0











a-4
HZ


SH









0E-

MC











H


Cj



c P

OU
0 4J
ow(


4.


a,
C 0
'2 P


1r- CM 0 oo i-
-IT ~1) 00 00 Co







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 80 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.











































0


ZZ


C-.




< 0w

Hl C14
cl)OZc/






(N r -:
.'I4 H C* 'J

H n


00 N
0 co3
cON

H~Z







-4 H
0 H
p~~


U)
co




Q)
4-j
(a




r.

0
a,


E:
.r-4
4i
co


C.
Ur


4J

0










00







Q
(I3













Q





















0

0
(I


Q

E
Q


0
4
C.)


CO




CO


U


D C 0 0 00 0 0







o0 0 Co






00L L) 0 z %0 Ul 0




























C- C)
0 0000000 C:
















SU N
C-. E E
E E E a



%0 C00 CO -1 00 Uf
0) Cl) (N 00 r. -4 -1











































O

0
HmO

oQ
0



0 M








0-4 0










E-40
S OHQ<

m OP









S-4
H


e 0


4'



m
ro

4-4


03


44
I


*T-


a
41


0




r.9


0
u


0 O 0 0 0 0 0
o o oo o Co oo o












0o o 0 o 0 0 0
00 0 0 0 Lt) 0 00
r- r- U-I











0 0 0 0 0 0 0




















O c
?0 N




aO CO C00 -~ 00 l
o cn CM oo C~- <
5 4 5-ML
OmlO5






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

Material and methods

Since the sorghum seeds sccmu 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.












































C) .i" CM


















NL CM O Nt
CL

N



OI
CM








0 l

o
*H CM


4-J



Co
4-W

a
O

u


CM





0

0

0 0
a0 0

CC
U)







Experiment 4: The Effect of Soaking Oat Secc(' in Concentrated
Solutions of Copper, Zinc and Manganese

Materials and methods

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.






Experiment 5: Effect of the Anions NO3 C1 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

concentratiorshere 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 millilitersof 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.








TABLE 5

EFFECT OF DIFFERENT LEVELS OF POTASSIUM NITRATE, POTASSIUM
CHLORIDE AND POTASSIUM SULFATE SOLUTIONS ON THE
GERMINATION OF PORTULACA OLERACEA SEEDS



Germination % after
Treatment 6 days 9 days


Control (Water) 75 90


NO3 20,000 ppm 0 0

10,000 ppm 43 62

2,500 ppm 76 88

625 ppm 77 91

300 ppm 68 96


Cl 20,000 ppm 0 0

10,000 ppm 19 43

2,500 ppm 55 76

625 ppm 52 72

300 ppm 69 98


SO4 20,000 ppm 0 33

10,000 ppm 20 27

2,500 ppm 40 84

625 ppm 50 62

300 ppm 48 63







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 life 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 250 C..







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 two

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































I-4 1-41 -4


UL tt


/-*
0
0

CO
S

0












4-1
a


















C
< -1















4-J 4








M
( 41


o o o o C -4 o o o








0- 000000 0-






















0 0 coJ -tn 00 e













S- co 4 o
N ri







(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 7: 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,




























0 0


00 .I .I 0 0


\ 00 00 0o0 0 00 0


C0 -It r- -t o
-I


Z
M





c.


H0
NO



PN
O,





Z O
OM





ZZ






rH







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 where 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.



















































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


















































Figure 6. -- Flowers of wild and mutated types of Portulaca oleracea.
Left: Flowers of wild type. Leaves are here obovate and
petals lobed. Right: Two smaller flowers of the mutated
type. (Approximately natural size.)





















































Figure 7. -- Flower of mutated type of Portulaca oleracea produced
after seed treatment with 1% lithium chloride. Leaves are
eliptical and margin of the petals is incised. (5x)










C"R


C"RI


C"R'


C'R


C'R'r


i th.e tex .


C' II \l'*-



CL' iZ







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







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 number 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 two directions.

The only case in which it is possible to obtain seeds from the mutated











































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


),/ 1
1004"q




















































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.)









% REVERSION


50




40




30-




20-

O


10-



0.


*2328
0/


02210


3110


S2 3 4
GENERATION

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.







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 (IAA) to the nutrient solution will considerably promote the

rooting of both types. It is not necessary to dissolve the IAA 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.







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 KH2PO4. 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 would 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.







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

(95%), one part chloroform, one part glacial acetic acid, and one

part formalin (40% formaldehyde) for five minutes. After fixation

root tips were washed and hydrolyzed in 1 N HC1 solution for ten

minutes at a temperature of 600 C. To avoid fluctuation in






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 600 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 800 C and

add one gram sodium metabisulfite together with 10 ml 1 N

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 stomata

of polyploid forms are larger than the ones from the same diploid

types (18). If the mutant in Portulaca was the result of poly-

ploidy then it should be possible to recognize this by the larger

stomata 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.







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.



















































Figure 32. -- Photomicrograph of stomata of lower leaf epidermis
of mutated type of Portulaca oleracea. (540x)























































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


Aoq ;


















































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




















































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







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 meristem 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 meristem.

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 between 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,






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 two 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




















































Figure 16. -- Photomicrograph of cross section through wild shoot
of Portulaca oleracea. Note typical ring of flattened
cells around the pericycle. In a few places (A) cells
are disrupted. (150x)







walls are thinner and are 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 wild 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.




















































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




































4 .4 $
-rr
I

al


I ~ r
L ~~i*i '*


t


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)


'p
-


. r






Experiment 8: Effect of Various Levels of Mineral Elements on
Plants and Progeny Seeds of Portulaca oleracea

Effect on cuttings and seeds thereof

Material and methods. -- Cuttings of Portulaca oleracca 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 WITH DIFFERENT IONS IN VARIOUS
CONCENTRATIONS ON RATE OF SURVIVAL OF
PORTULACA OLERACEA CUTTINGS




Rate of Rate of
Treatment Survival Treatment Survival


Cu ppm Li ppm

4.5 + 26 +
9 + 52 +
18 + 104 +
36 + 208 +
72 416 +
144 833 +
288 1,666 +


B ppm Se ppm

7 + 4.5 +
15 + 9 +
31 + 18 +
62 + 36 +
125 + 72
250 + 144
500 + 288


Zn ppm

24 +
47 +
94 +
188 +
375
750
1,500






Effect on seedlings and seeds thereof

Material and methods. -- 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 seleniumoxychloride. 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 fromthe 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






TABLE 9

EFFECT OF TREATMENT WITH DIFFERENT IONS IN VARIOUS
CONCENTRATIONS ON RATE OF SURVIVAL OF
PORTULACA OLERACEA SEEDLINGS


Rate of Rate of
Treatment Survival Treatment Survival


Cu ppm


4
8
24
80
400


Al ppm


8
27
68
135
270


B ppm


Zn ppm


4
8
24
82
410


Li ppm

42
208
416
833
1,666


Fe ppm


4
8
21
70
350


Se ppm

12
46
115
231
1,156

















GERMINATION



PRETREATME NT

BORON


%
100


90-


80


70


60


50-


40


30


I I I


DAYS


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


25ppm
,/' 62 ppm
/
I
I
I
I
I
I
I
/
/
I
/
I
I
I
/
/
ol


20


10-













GERMI NATION

PRETREATMENT


COPPER



- ---


4ppm

,-- 8 ppm


7 14 21
DAYS


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


100

90

80-

70-

60-

50-

40-

30-

20-


IO0
















PRETREATMENT


IRON


S4 ppm
/

/ 8ppm
,2i ppm


DAYS


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


% GERMINATION


80

70

60

50

40

30

20












GERMINATION


I%
I0
1001.


4 ppm


- 24 ppm


8 ppm


PRETREAT ME NT


7 14
DAYS


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


90

80


60

50

40


20







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 (64%). 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 oleracca

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







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
and 160 ppm Cu

Cuttings from 35 ppm Fe solution placed in 7(
and 140 ppm Fe

Cuttings from 70 ppm Fe solution placed in 7(
and 140 ppm Fe

Cuttings from 24 ppm Zn solution placed in 8:
and 164 ppm Zn

Cuttings from 82 ppm Zn solution placed in 8:
and 164 ppm Zn


0


0


2


2


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







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 -3
solutions of 1, 10-1, 10 and 10- M strength. After 24 hours

pieces were taken out. Epidermis cells were removed and treated

with 5.0 percent KNO3 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 ZnSO4 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 102 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







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-promoting

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







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.






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.0percent lithium

chloride was less tolerant to high concentrations of lithium chloride

than the wild type.






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



cetiL liJ.iL.m chl(ridc, indicated a ,.im;ilnjr mutagenic action on cells

in the tunica.







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







grandiflora 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 Neptunia grown on soils rich in selenium contained






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

pg 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. Selenium 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 cellsap

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 medium, 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







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. Plasmolysis 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 160 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.




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - Version 2.9.7 - mvs