Y'HE MGMUW AND msmsmoaa OF
2.. 4amsasammmmoxmcmc ACID IN CANADA THISTLE

Thais fiat H16 0W (4‘ Ph. D.
Mifl‘lfGAN STATE UNEVERSITY
Wes-adimd Hum
1962

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This is to certify that the

thesis entitled

The Mobility and Distribution of 2, 4-Dichlorophenoxy-
acetic Acid in Canada Thistle

presented by

Woodland Hurtt

has been accepted towards fulfillment
of the requirements for

Ph. D. Farm Crops

degree in

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Major professor

DateDecember 29, 1961

 

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LIBRARY

Michigan State
University

 

THE MOBILITY'AND DISTRIBUTION OF
2,4-DICHLOROPHENOXYACETIC ACID
IN CANADA THISTLE

by Woodland Hurtt

Canada thistle plants were found to be very responsive
to the sodium salt of 2.4-dichlorophenoxyacetic acid (2,4—D)
when it was introduced directly into the growing regions.
However, when the herbicide was applied to the foliage of
the plants, the majority of it remained in the leaves and
the plants exhibited negligible responses. By extracting
the leaves with ether and bioassaying the extracts with the
‘gygna straight growth test, it was found that the 2,4-D was
present in the tissue in a biologically active form.

To test the mobility of the 2.4-D in the extracellular
liquid of the leaves, low levels of direct current were
passed through the tissue in an attempt to displace the
herbicide electrophoretically.

In one method the current was applied to opposite
margins of the leaves such that mobility could be studied by
the pattern of induced displacement of the herbicide. The
2,4—D was preferentially displaced from one-half to one
centimeter toward the electropositive contact by the current

levels and time intervals employed.

Woodland Hurtt

In the second electrophoretic technique, which was
used to extract the 2,4—D from the extracellular liquid of
the tissue, the current was applied to the upper and lower
surfaces of the leaves such that the 2,4-D was displaced into
cotton traps. Bioassays of the extracts from the cotton
traps showed that 2,4-D was present in the extracellular
liquid in an active form which was freely mobile under the
influence of the imposed current.

It was concluded that the lack of effectiveness of
2,4-D on the thistle clones used in these experiments was
caused by the lack of adequate transport to the growing
regions of the plants and was not attributable to either a

lack of mobility or activity of the herbicide.

THE MOBILITY AND DISTRIBUTION
OF 2,4-DICHLOROPHENOXYACETIC ACID

IN CANADA THISTLE

BY

Woodland Hurtt

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Farm Crops

1962

ACKNOWLEDGEMENTS

Sincere appreciation is expressed to Dr. C. R. Olien
for his advice throughout the course of this study.
Appreciation is also expressed to Dr. W. F. Meggitt,

Dr. C. M. Harrison, and Professor B. R. Churchill.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 2
MATERIALS AND METHODS . . . . . . . . . . . . . . . 7
Materials 7
Methods 7
RESULTS . . . . . . . . . . . . . . . . . . . . . . 16
The Introduction of 2,4-D and Dye in Canada
Thistle Plants 16
The Electrophoretic Displacement of Dye 19
The Electrophoretic Extraction of Dye 21
The Electrophoretic Displacement of
2.4-D 29
The Extraction of 2,4-D by a Combination
of Methods 36
DISCUSSION . . . . . . . . . . . . . . . . . . . . . 51
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 59
LITERATURE CITED . . . . . . . . . . . . . . . . . . 61

iii

Table

LIST OF TABLES

The amounts of 2,4—D recovered from
cotton pads used as traps in the
electrophoretic extraction of

2,4—D from an excised Canada thistle

The

The

leaf, whose petiole had been immersed
in 2 X 10'3 M 2,4—D for 2.5 hours by an
application of 20 uamperes of current

for seven hours . . . . . . . . . .

amount of 2,4-D recovered from cotton

pads used as traps in the electro-
phoretic extraction of 2,4-D from
excised Canada thistle leaves, whose

petiole had been immersed in 2 X 10‘3 M
2,4—D for 1.5 hours, by application of

20 uamperes of current for two, four,
six and eight hours respectively . .

amounts of 2,4-D recovered by ether

extractions of sections of tissue from
the electropositive and electronegative
sides of an excised Canada thistle leaf
following a two hour immersion of the

petiole in 2 x 10-3 M 2.4—9 with a

parallel application of 10 uamperes of
current followed by an additional four

hours of current . . . . . . . . . .

amounts of 2.4-D recovered from the
extraction apparatus used for
recovering 2,4—D from cotton, with
and without cotton packing. to which

10-2

umoles of 2,4—D had been added

iv

Page

39

41

44

47

Table

5.

Page

The amount of 2,4-D recovered from cotton

The

pads used as traps in the electro-
phoretic extraction of 2,4-D from
excised Canada thistle leaves and

from ether extracts of a portion of
each leaf following a zero, three, six
and twelve hour interval during which
their petioles were immersed in
distilled water. The petioles of all
treated leaves had previously been
immersed in 2 X 10"3 M 2,4-D for two
hours. All leaves were subjected to a
current application of 20 uamperes for
six hours . . . . . . . . . . . . . . . 48

amounts of 2,4—D recovered from cotton
pads used as traps in the electro-
phoretic extraction of 2,4-D from an
excised Canada thistle leaf and from
an ether extract of a portion of the
leaf following a ten hour interval
during which its petiole was immersed
in distilled water. The petiole had
previously been immersed in 2 X 10'3 M
2,4—D for two hours. The leaf was
subjected to a current application of
20 namperes for six hours . . . . . . . 50

Figure

LIST OF FIGURES
Page

Diagram of method of applying an electric
current to an excised Canada thistle
leaf . . . . . . . . . . . . . . . . . . 10

The effect of an excessive application of an
electric current to an excised Canada
thistle leaf 0 O O 0 O O O O O O O O O O 15

Diagram of method of applying an electric
current to an excised Canada thistle
leaf . . . . . . . . . . . . . . . . . . 26

The effect of 1.58 coulombs of current on the
lateral distribution of 2,4—D in a Canada
thistle leaf whose petiole had been
immersed in a 2 X 10'3 M solution of the
herbicide for the first two hours of the
current treatment . . . . . . . . . . . 35

The effect of 1.58 coulombs of current on the
lateral distribution of 2,4-D in a Canada
thistle leaf whose petiole had been
immersed in a 2 X 10-3 M solution of the
herbicide for 2.5 hours prior to the
current application . . . . . . . . . . 37

The response (elongation of treated sections
divided by elongation of control section)
of Avena coleoptile sections to 2,4-D . 69

The frequency distribution of Avena coleoptile
sections grown in a nonsterilized buffer and
in a sterilized buffer without 2,4-D . . 72

The tumbler and bottles used for rotating
Avena coleoptile sections in the constant
temperature cabinet . . . . . . . . . . 74

The response (elongation of treated sections

divided by elongation of control sections)
of Avena coleoptile sections to 2,4-D . 76

vi

LIST OF APPENDICES

Appendix Page
A. THE BIOASSAY PROCEDURE . . . . . . . . . . . 65
B. THE ETHER EXTRACTION PROCEDURE . . . . . . . 79

vii

INTRODUCTION

Although 2,4-dichlorophenoxyacetic acid (2,4-D)
readily controls many perennial weeds, it has not proven
particularly effective in the control of Canada thistle
(Cirsium arvense). 2,4—D has generally been considered to
be freely translocated in plants, but recent evidence
indicates that it is not translocated as freely as once
thought. It was, therefore, the purpose of this study to
obtain information on the mobility and translocation of
2,4-D in Canada thistle in an effort to ascertain why this

plant is resistant to 2,4—D.

REVIEW OF LITERATURE

Probably the most important factor in the resistance
of many perennial plants to herbicides can be attributed to
lack of adequate transport to the meristematic regions in
the roots. Van Overbeek (34) suggested that the effectiveness
of 2,4-D was due to its ability to penetrate into the proto-
plasm of meristems. Swanson (31) studied the histological
responses of kidney beans treated with 2,4—D and reported
that the meristematic tissues and those capable of reverting
to a meristematic condition were the ones most readily
affected by 2,4-D. Tukey gt_§l. (33) treated bindweed and
sow thistle with 2,4—D and found disorganized and greatly
increased cell division in the cambial regions of the rhizomes
of both plants.

According to Crafts (7) much of the available evidence
indicates that the translocation of 2,4-D and similar compounds
occurs in the phloem in the stream of assimilates. Movement
is generally considered to be from regions of food synthesis
to those of food utilization and occurs only if photosynthesis
is going on or if carbohydrate reserves are being hydrolyzed

and moved (9).

3

Crafts and Yamaquchi (lO) reviewed many older papers
on the translocation of 2,4—D and stated that they seemed to
indicate a relationship between the movement of 2,4—D and
the movement of food in the plant. They also reported that
when they applied carboxyl-labeled 2,4—D and several other
herbicides to the leaves of two species of plants their
results suggested that the 2,4-D moved in a common stream
of assimilates from the leaves to various sinks (regions
of utilization).

Vbevodin (35) applied Cl4-labeled 2,4—D to field
plots of Canada thistle and reported that the spraying of
autumn rosettes resulted in increased uptake of 2,4-D in
the roots. When the herbicide was applied to spring rosettes,
the majority of it remained in the above ground parts of the
thistles. These results were attributed to the direction
of movement of the products of photosynthesis.

Corms (6) injected 2,4—D into perennial sow thistle
plants and suggested that its transport was governed by
movement of sap to the young rapidly developing parts of the
plants.

The translocation of 2,4—D in plants has been considered
to occur quite readily (3, 7). Rapid rates of translocation

have also been reported. Day (13), for example, reported

4

an average speed of transport of 2,4-D in kidney bean seedlings
of 50 cm per hour. The results of recent studies, however,
indicate that the transport of 2,4—D does not occur as
readily as previously reported (11). Crafts and Yamaquchi (10)
applied labeled 2,4—D and 2,4,5-T to blocks of potato tuber
tissue and found that they were accumulated and held in the
living cells. On the basis of further studies in which
they applied labeled 2,4—D to the leaves of Zebrina pendula,
they suggested the herbicide was taken up by the living
cells and any that was transported constituted a reSidue
over and above that which was retained by the active cells (10).

Crafts (11) reviewed the previously reported work
of Johnson and Bonner (20) and suggested that the low
mobility of 2,4-D may be explained by the reported active
accumulation of the herbicide by the living cells. These
workers (20) suggested a binding action and reported that
the 2,4-D was accumulated in the tissue to a higher
concentration than that in the external medium.

Leonard (21) discussed the binding factors involved
in the translocation of 2,4—D and postulated that some of
the 2,4-D is weakly bound after entering the leaf or is

precipitated as the acid of 2,4—D.

5

Bach and Fellig (2) studied the uptake and fate of
carboxyl—labeled 2,4-D in bean stem sections and suggest
the 2,4-D was bound in some way to cellular material of the
tissue. They found the peak of the radioactivity was located
only several millimeters from the point of application.

Canny and Katalin (5) used both carboxyl and methylene
labeled 2,4—D to determine the breakdown of the herbicides
in the shoots and roots of tick bean plants. They stated
that the 2,4-D entered the roots of the plants in the trans-
location stream and was probably bound there in complexes
with proteins.

Leonard (21) cited several papers which suggested
that free 2,4—D as such rapidly disappears from the leaf
and becomes bound with proteins.

Pallas (29) studied the effects of temperature and
humidity on the absorption and translocation of labeled 2,4-D
and benzoic acid in red kidney beans. He reported that
movement of 2,4—D, as determined by radioautograms, followed
the route of the assimilate stream out of the leaf and into
the stem, bud, and roots. Further studies involving
extraction and chromatography of the herbicide showed that

it was translocated as free 2,4—D or its dissociable salts.

6
An electrophoretic method was developed by Olien (27,28)

for studying the mobility of electrolytes in the extracellular
liquid of wheat leaves. Anderson gt_al. (1) used this method
to study the transport of Ca45 in bean plants and found a
preferential transport of the calcium towards the positive
electrode. It was theorized that the calcium ion was attached
to a negative carrier which migrated towards the positive

electrode.

MATERIALS AND METHODS

Materials

The monohydrate sodium salt of 2,4—D (Baker Chemical
Company) was used in these experiments. All solutions were
prepared in distilled water and stored at approximately five
degrees centigrade when not in use.

Since there are several known varieties of Canada
thistle (14), the plants used in these experiments were
grown from one source by asexual propagation in order to
eliminate possible varietal differences in response to
2,4—D. The plants were grown from one inch sections of
rhizomes in four inch pots containing a one-third sand and
two—thirds soil mixture. Prior to planting all sections
were soaked for several minutes in a solution of Captan
equivalent to two pounds per hundred gallons of water to
minimize root rot. The resulting plants were watered once
weekly with a complete nutrient solution and supplied with

supplemental lighting when needed.

Methods

Since the lack of effectiveness of the salt form of
2,4-D on Canada thistle plants was thought to be due to its

lack of transport within the plant, an electrophoretic
7

8
method was used to study its mobility.

Prior to using an electrophoretic method for dis-
placing charged materials such as 2,4—D in the extracellular
liquid, it was necessary to determine the voltage and amount
of current that could be passed through leaves without
damaging the tissue.

Contacts. In order to displace a substance having
an electrical charge in the extracellular films of liquid,

a method was needed which would extend these films into an
exterior system suitable for the application of the electric
current. Such a system should introduce the electric
current into the tissue in a uniform manner without causing
concentrated areas of stress and prevent the introduction

of possible toxic substances such as copper from the anode
into the tissue as a result of the current flow.

Excised leaves were used in all of the following
studies because preliminary experiments indicated that the
release of both 2,4-D and the dye from the xylem was similar
to that obtained when 2,4—D and dye were introduced into
intact plants.

Preliminary experiments in which contacts were made
on the tips of two opposite leaves on the stem of each intact

plant showed that electrical displacement was parallel to

 

.4.

-l

the natural path of transport. For this reason the Norite
contacts were made on the margins of the leaves such that
the path of electrical displacement was perpendicular to
the normal path of transport.

Each contact was made by burying 2.5 cm of the leaf
margin about 2 mm deep in a thick charcoal paste consisting
of Norite and water. The moisture content of the paste was
maintained by a wick leading to a water reservoir. A
graphite rod was used to complete the contact between the
wire of the electric circuit and the paste. Two such
contacts were made on opposite edges midway between the
petiole and the tip of an excised leaf whose petiole was
immersed in 3 ml of distilled water, dye, or 2,4-D (Figure l).

Intensity limit. When a direct current of sufficiently
low voltage is passed through normal tissue, the natural
diffusion barriers of the protoplats are not disrupted, and
in such cases the resistance of the tissue is independent of
the voltage (27). At slightly higher voltages a reversible
decrease in the resistance occurs which is indicative of a
weakening of the diffusion barrier, but further increases
in voltage results in an irreversible decrease in resistance
(27). The latter indicates destruction of the diffusion

barrier. For any given section of uniform tissue the voltage

FW+ —

10

Graphite Rod
Charcoal Paste

Water Resereolr
chk

   
   
  

 

   

     
   
 
  

0.0.
Power
Source

uamperemeter

Reservoir for 2.4-0,
Water, or Dye

 

Figure 1. Diagram of method of applying an electric

current to an excised Canada thistle leaf

11

is a function of the length of the current path. Therefore,
the intensity limit is considered to be the maximum voltage
per centimeter that can be applied to the tissue without
affecting the diffusion barrier sufficiently to cause a
decrease in resistance.

In order to determine the voltage maximum for a
Canada thistle leaf, marginal contacts were placed on a
leaf and starting at one volt, succeedingly higher voltages
were applied. At each voltage a reading was made of the
current flow in microamperes and the resistance of the
tissue calculated.

It was found that the resistance of the tissue between
the contacts was essentially independent of the voltage up
to 17 volts per centimeter, at which point it started to
decrease and continued to do so with each increased increment
of voltage. To go beyond this voltage would be unnecessary
because the current started to drift and continued to do so
if this voltage was held for more than a few seconds. The
resistance of the tissue at this point was 1.3 X 106 ohms
per centimeter.

Areas of electrical stress in which the voltage
maximum had been exceeded and the diffusion barriers weakened

or destroyed rapidly became necrotic. Prior to the appearance

12

of visible necrosis the resistance of this area of tissue
decreased and as a consequence the current drifted to higher
levels. As the tissue became necrotic it lost moisture and
became dessicated if it was in a dry atmosphere. This in
turn caused the resistance to increase and the current to
bridge this area with the resulting formation of new areas
of electrical stress on either side of the dessicated tissue.

Necrotic areas of tissue formed as a consequence of
electrical stress were quite different in appearance from
those resulting from 2,4-D treatments. The latter were
always purple or black in color, whereas the former were
light brown to brown in color.

In order to ensure that the maximum voltage drop
of 17 volts per centimeter of tissue was not exceeded in
subsequent experiments, it would have been possible to
measure the voltage drop across the contacts of each leaf.
This, however, would have been more difficult because the
contacts themselves offer resistance to the flow of current.
Since the current flow is constant in all parts of a circuit,
it was more convenient to measure the level of current by
means of a microamperemeter in series with the circuit. By
applying contacts to the same cross-sectional area of each

leaf, the voltage drop per centimeter could be maintained

13
below the maximum level by varying the external resistance
until the desired microampere reading was obtained. This
method compensated for the normal variation in the width of
the leaves which affects the resistance of the tissue
between the contacts.

If inadvertently the maximum voltage value was
reached or exceeded, the experiment was discontinued. The
stress limit was then re-evaluated and subsequent experiments
were carried out within a safe range.

Capacity limit. The application of a sufficiently
high voltage per centimeter of tissue caused irreversible
injury to the tissue. In any given electrophoretic unit
the limit of the electrical treatment at a low electrical
stress is dependent upon the total amount of electricity
passed through the tissue rather than the total energy
(watts) applied (27). The electrical treatments were
therefore measured in coulombs or ampere-seconds.

It was found that approximately 1.0 coulomb was
the upper limit of electrical treatment with the type of
electrophoretic unit employed in these experiments.
Assuming an average leaf thickness of 0.1 cm and a contact
length of 2.5 cm, the maximum limit of 1.0 coulomb was

applied through a cross—sectional area of 0.25 square

14
centimeters. The capacity limit of the tissue was there—
fore considered to be 4.0 coulombs per square centimeter.
The majority of current applications employed a current
level which was 40-50 percent of this value. When the upper
limit of current application had been exceeded and the
tissue collapsed in a dry atmosphere, the current was
diverted and concentrated on the sides of each spot. The
result was the formation of lines or wrinkles of necrotic
tissue which were perpendicular to the current path (Figure 2).
However, when such an excess of current was applied in a
moist atmosphere, the loss of moisture from the injured
tissue was prevented and a general collapse of the entire

area of tissue between the contacts occurred.

 

Figure 2.

15

 

The effect of an excessive application of an
electric current to an excised Canada thistle
leaf

RESULTS

The Introduction of 2,4—D and Dye in Canada Thistle Plants

Preliminary experiments were carried out to obtain
observational information on the uptake and translocation
of 2,4-D and dye in Canada thistle plants. Several methods
were used for applying the herbicide to the plants. One
consisted of pipetting 10, 50, or 100 1 drops on the surface
of one or several leaves. In some cases the 2,4-D was
confined within a small area by means of a ring of stopcock
grease and in others it was spread over the entire surface
of the leaf. A second method consisted of immersing a
leaf in a small vial containing the herbicide. In some
instances the leaf tip was excised prior to immersion. An
injection technique was also used in which a hypodermic
needle was inserted in the stem and the 2,4-D was added
through the hollow shank of the needle. A red dye, Amaranth,
was used in conjunction with the 2,4-D in the experiments
concerned with translocation. The dye was introduced into
the plants in the same manner as was the 2,4-D.

The majority of these experiments were carried out
under relatively low light intensities in the greenhouse

with supplemental incandescent light or in the laboratory

16

 

 

rn

’TY

17

under fluorescent lighting. A few experiments were conducted
in the greenhouse in late spring or early summer under high
light intensities. The plants used in these experiments
were 12-16 cm tall and approximately two months old.

The application of 2,4-D to the surface of the leaves
either resulted in no response or in very slight epinasty
in the upper leaves. The only exception were treatments in
which some of the herbicide ran back into the axils of the
leaves but even then, death of the plants never occurred.

When Amaranth was injected into the stems of plants
a characteristic pattern of staining resulted. Generally,
the first and thereafter every third leaf was stained.
A similar pattern of necrosis occurred when plants were
treated with 2,4—D. The movement of the dye and 2,4—D was
found to be upward and outward into the leaves. Neither
lateral nor downward translocation of 2,4-D and dye occurred
since leaves on the opposite side of the stem or below the
point of injection were never stained or necrotic.

The lack of downward transport was further substantiated

by repeated applications of 2 X 10-3 M 2,4-D through the

shank of a needle inserted and left in the stem for 13 days.
At the conclusion of this interval only the leaves above the

point of injection were injured or dead. Other plants on the

18
same rhizome exhibited no symptoms of 2,4-D injury.

Immersing intact leaves and those with the tip
excised in 2,4-D generally had no lethal effect. Such
treatments usually caused epinasty of the upper portion of
the plant. Neither necrosis nor epinasty was observed in
leaves below the ones immersed in the 2,4-D. Immersion of
leaves in the dye, with or without the tip excised, usually
failed to produce any visual evidence of translocation out
of the treated leaf.

The leaf immersing procedure revealed that the
occurrence of epinastic responses in the plant was dependent
on the development of necrosis up to the petiole of the
leaf which was immersed in the 2,4—D solution. This was
favored either by high concentrations of 2,4—D or the use of
small plants (3-5 inches tall). The observation of
crystalline particles of 2,4-D on such necrotic leaves
seemed to indicate that the mechanism of uptake and transport
was not one involving the vascular system but merely one
resulting from the absorption and free diffusion of 2,4—D
in the damaged tissue.

The above observations were substantiated by artificially
injuring a leaf with a small flame. The tip of the leaf was

then placed in a solution of 4 X 10-3 M 2,4-D for 34 hours.

19

After 24 hours the top two leaves of the plant exhibited
typical epinastic effects. However, at 34 hours when the
leaf was removed from the solution of 2,4-D it was approxi-
mately 80 percent necrotic and the upper stem of the plant
was exhibiting severe epinasty.

Sectioning of plants in which dye had been introduced
revealed that it was transported in the xylem. Because of
similar behavior of 2,4-D to the dye, it was assumed to move

in the xylem also.

The Electrophoretic Displacement of Dye

The uptake of dye by excised leaves. This series
of experiments was conducted to characterize the uptake and
movement of dye in excised Canada thistle leaves.

The dye was introduced by placing the leaf petioles
in 3 ml of an aqueous solution of Amaranth (3.5 9/1).

In one-half to one hour the dye was visible in most
of the trichomes of the leaves. This was the first visual
indication of uptake of dye by the leaves and occurred
consistently in all experiments. After one, or at most two
hours, the dye was observed in the xylem vessels by placing
the leaf over a light source and by observations under the

microscope of cross sections of the leaves. The time interval

20
required for staining of the interveinal areas varied between
four and six hours.

Examination of sections of the leaves under the
microscope at intervals showed that the interveinal areas
were stained by the gradual diffusion of the dye from the
smaller network of vessels.

Displacement. The following experiments were made to
determine if a low voltage direct current applied transversely
through an excised leaf by marginal contacts could effect a
displacement of the negatively charged dye.

The petioles of several leaves were placed in the
dye solution and marginal contacts were applied. The current
was then adjusted to 10 uamperes in each circuit.

After several hours the results of the imposed current
were very evident in the visible gradient of dye distribution
through the leaf. A definite band of concentrated dye
paralleled the positive contact. The dye between this band
and the midrib of the leaf appeared to be much less
concentrated. On the electronegative side of the midrib the
dye was fairly concentrated in the tissuetg>to within
several millimeters of the negative electrode. Paralleling
the negative electrode was a narrow strip of tissue which

was free of any visible dye. This strip was comparable

21

in size to the area paralleling the positive contact in
which the dye was concentrated.

This experiment was repeated except that a current
level of 30 uamperes was used. After one hour the dye was
visible in all of the larger vessels but there was no
differential distribution between the electropositive and
electronegative sides of the leaves. After two hours dye
accumulated at the end of a vessel next to the positive
contact. At approximately three hours the accumulation of
dye on the positive side of the leaf was quite evident.

A current level of 10 pamperes for approximately
three hours was ample to cause a lateral distribution of the
dye between the electropositive and electronegative sides

of excised leaves.

The Electrophoretic Extraction of Dye

Vertical displacement. The results of the previous
experiments established that it was possible to displace
the dye within the plant tissue. Further experiments were
designed to extend this procedure to ascertain if an electric
current could displace the dye in a vertical path through
living tissue without injury.

A pad of Norite was constructed on a glass plate

22

with the negative electrode and a wick buried below the
surface. Ten layers of 1 cm square filter paper were placed
on top of this pad. The top five layers were cut and
separated in such a manner that the midrib of an excised leaf
fitted in the resulting groove. Approximately ten layers of
1 cm square filter paper were placed on top of the leaf. A
Norite pad containing the positive electrode and wick was
constructed on top of this layer of paper. The petiole of
the leaf was placed in a container of water and the wicks
were placed in small vials containing distilled water. The
water level in each vial was adjusted such that a siphoning
action resulted and kept the Norite contacts from drying
out. The current was adjusted to 60 uamperes and passed
through the tissue for eight hours after which one small
spot of dye was found on the top layer of paper.

This experiment and others of a similar nature
established that the dye could be transported vertically
and accumulated in filter paper on the electropositive side.
However, the current levels were high and generally
resulted in injury to the leaves. The dye exhibited a
tendency to accumulate in these injured areas. It was
thought that the upper cuticle of the leaf might be a
barrier to the movement of the dye from the upper side of

the leaf.

23

For this reason attempts were made to remove the
cuticle from excised leaves without injuring the underlying
tissue. One square inch of the upper surface of five leaves
were scrubbed with a pad of cotton soaked in one of the
following materials: soap, Tween-20, a one percent solution
of Tween-20, ether, and a half and half water and ether mixture.
One drop of concentrated dye was placed on each treated area.
The petioles of the leaves were placed in water and moist
cotton pads complete with the previously described system
of wicks and vials of water were put in place.

After 11 hours the pads were removed and the leaves
were examined. It was found that both ether treatments were
effective in removing the cuticle and allowing the dye to
penetrate into the upper tissue of the leaf. However, both
of these treatments caused fairly large necrotic areas.

The soap and the concentrated Tween-20 treatment allowed
penetration of the dye and did not injure the leaf. One
percent Tween-20 was ineffective. Although ether was the
most effective in removing the cuticle, Tween-20 was selected
as the most desirable treatment because it did not cause
visible injury.

The initial experiments concerned with the vertical

displacement of dye through tissue were repeated with the

24

following innovations. Rectangular cotton pads replaced
the filter paper and a 2 X 3 cm area of the leaf surface
was treated to remove the cuticle prior to the application of
the pads. One leaf was treated with soap and another with
Tween-20. The dye was added to the lower pad and a widk and
carbon electrode were placed in each pad. The pad containing
the positive electrode was placed over the treated area on
the upper surface of each leaf. The petioles and wicks were
placed in their respective containers of water and the
current adjusted to 40 uamperes.

After two hours it was found that the cotton pad
on the electropositive side of the leaf treated with
Tween—20 was appreciably stained with the dye. Examination
under the microscope of sections from this leaf showed that
the dye appeared to be in the extracellular fluid around the
palisade and epidermal cell walls. Since there was no dye
on the upper pad on the leaf treated with soap, this
experiment was continued for an eight hour period and still
exhibited no sign of dye on its upper surface or in the pad.

A current level of 40 uamperes was, therefore,
sufficient to displace the dye vertically through the leaf,
providing the cuticle was removed under the contacts. The

observations suggested that the dye had moved in the

25
extracellular fluid in the tissue in its passage through
the leaf.

Extraction. Having established that it was possible
to transport dye in a vertical path through living tissue with
an electric current, attempts were made to extract the dye
from leaves which had previously been treated by immersing
the petioles in 3 ml of the dye solution. A Norite pad was
constructed as in previous experiments. The leaf was removed
from the dye solution and treated as previously with Tween-20
to remove the cuticle. The margins of the leaf opposite this
area were coated with a thin film of silicone stopcock grease
to prevent moisture films from creeping around the leaf. The
upper surface of the leaf was divided into two equal parts by
a 3 cm long ridge of stopcock grease placed over the midrib.
The leaf was then centered on a Norite pad and its petiole
placed in water. Two cotton pads approximately 2 cm square
were prepared containing the widks and the positive and
negative electrodes respectively. Each pad was then placed
on the surface of the leaf such that they were separated by
the silicone ridge. The wicks were placed in their respective
vials and the current was adjusted to the desired level. The
arrangement of wicks and electrodes is shown in Figure 3.

The result of the above arrangement was the establish-

ment of three regions of charge separation. The cotton

26

Carbon
Electrode

 
  
    

 

 

 

  

Wick Thistle Leaf

 

 

 

 

Water Reservoir Charcoal Paste
0. C.
Power
Source

 

 

Figure 3. Diagram of method of applying an electric
current to an excised Canada thistle leaf

27
pad containing the negative electrode was electronegative
to the Norite underneath this area of the leaf. This portion
of Norite was electronegative to the portion of Norite below
the opposite side of the leaf which in turn was electro-
negative to the cotton pad containing the positive electrode
directly above. It can thus be seen that a diffusible
substance with a negative charge in the tissue under the
negative electrode would tend to be drawn into the Norite
below, whereas a similar substance in the leaf under the
cotton pad containing the positive electrode would tend to
be drawn up into the cotton pad.

The design of this electrophoretic unit was based
principally upon observations that the diffusion of water
from the cotton pads into the tissue had a slight tendency
to wash the dye out of the tissue under the cotton pads
if no current was applied.

Previous observations indicated the necessity of
cuticle removal. The upper epidermis seemed more resistant
to the injurious effects of cuticle removal than the lower,
and the upper cell structure of the leaves was more dense
and uniform than that of the lower.

For these reasons the electrophoretic unit was

designed such that the contacts were applied to comparable

28
regions of tissue of the upper leaf surface. Thus, there
was no greater tendency, other than that caused by the
electric current, for a charged substance to diffuse into
or out of one cotton pad than the other.

In the first experiment the petioles of the leaves
'were soaked in the dye for four hours prior to making the
contacts. After five hours of current application at
40 uamperes a noticeable red color was evident on the
positive pad. The current was adjusted to 20 uamperes and
continued for 3.5 hours longer. At this time the distribution
of red dye in the positive pad was somewhat greater and more
intense in color. Examination of the leaf surface with a
lens revealed that the dye appeared to be more concentrated
in the area under the positive pad than in surrounding parts
of the leaf or under the negative pad.

Petioles of several other leaves were soaked in the
dye solution for six hours. One leaf was selected for
electrophoretic extraction at 20 uamperes. After two hours
a faint red color was present only on the positive pad.
Examination of the leaf under the microscope revealed that
the dye was concentrated in the vessels and seemingly in
regions bordering the upper and lower epidermis. Sections

from tissue which had been directly under the positive pad

29
showed that the dye appeared to be more concentrated near
the upper epidermis, but the dye in a similar section taken
from the tissue under the negative pad appeared equally
distributed. The upper epidermis of the leaves was considered
to be more of a barrier to the movement of the dye than was
the lower even when the cuticle was removed from both.

Methylene blue, a positively charged dye, was selected
for similar experiments to those performed with Amaranth
as a check for electroosmotic displacement. A concentration
of 6.5 g/l was found adequate to produce reasonably stained
leaves after five hours of soaking.

The results obtained with methylene blue were the
opposite of those obtained with Amaranth. The negative pad,
rather than the positive, was found to be stained with the
dye. Thus, electroosmosis was not considered to be a
factor in respect to electrophoretic displacement and

extraction.

The Electrophoretic Displacement of 2,4—D

The uptake of 2,4—D by excised leaves. The methods
evolved and the results obtained were sufficient to suggest
that a negatively charged diffusable substance such as

2,4-D could be displaced in the extracellular liquid of a

30

leaf by a noninjurious low level of current. The following
experiments were designed to establish the appropriate levels
of current and 2,4—D.

The method used to introduce the 2,4—D into the
excised leaves was identical to that used for the dye.
Leaves similar in size and shape were excised and their
petioles were placed in 3 ml of solution containing the
desired concentration of herbicide.

In order to determine the most desirable concentration
of the herbicide, an exponential series of 2,4—D solutions

were prepared ranging in concentration from 8 X 10-3 M to

8 x 10‘6

M (2088 ppm - 2 ppm). A water control was also
included. Five leaves were excised and placed in the solutions.
Observations were made at 12 and 24 hours.

In 12 hours the 8 X 10-4 M concentration of 2,4—D
was the most dilute solution that caused necrosis. The
leaf in the 8 X 10-3 M solution was entirely necrotic and
varied in color from purple to black. After 24 hours, the
control leaf in water and the leaves in the two most dilute
concentrations of the herbicide were normal in appearance.
The leaf in the 8 X 10-4 M concentration of 2,4—D was

severely wilted and had large black necrotic areas scattered

over its surface.

31

Additional work was done to determine optimum periods
of immersion in 2,4-D for an intermediate injurious effect.
The concentration of 2,4—D selected for use was 2 X 10-3 M
(522 ppm). Four to six hours were required before the first
symptoms of 2,4-D injury appeared in the form of several
small necrotic spots, one of which invariably was located on
the periphery of the leaf. With increasing time more
necrotic spots appeared, and the necrotic areas at the leaf
tip and those on the margin coalesced to form a complete
or almost complete rim of purple to black tissue surrounding
the now mottled inner leaf area.

The effects observed when the leaves were removed
from their respective solutions were as follows. The leaf
which had been in the 2,4—D for the shortest time period
(eight hours) had four small necrotic spots on its surface.
The first necrotic spot, which was marginal in location,
developed at five hours. The leaf which had been in the
2,4—D nine hours at the time of removal had approximately
20 necrotic spots on its surface plus almost complete marginal
and leaf tip necrosis. The leaves subjected to the longer
periods of treatment were almost completely necrotic.

Twenty-four hours after the experiment was begun,

or nine hours after all the leaves had been placed in

32
distilled water, all of the leaves were shriveled and
completely necrotic except the one which had been in the
2,4-D solution for the shortest interval (eight hours).
The only difference between this leaf and the others was
that the necrotic areas had not yet coalesced. Thus, the
shortest time interval used for immersing the petioles of
excised leaves in 2 X 10-3 M 2,4-D was found to be too long.

Subsequent work showed that two hours of petiole
immersion in 2 X 10-3 M 2,4-D produced intermediate symptoms
of 2,4-D injury which developed more slowly and did not cause
such a rapid collapse of the tissue.

In order to ascertain if the development of necrotic
symptoms on excised leaves was a sufficiently valid criterion
for symptoms of 2,4—D injury, a solution containing 2 X 10-3 M
acetic acid was prepared. Two leaves were excised, the
petiole of one was placed in 2 ml of the acetic acid solution,
and the other in a comparable molar concentration of 2,4-D.

After ten hours the leaf whose petiole had been in
the acetic acid solution was perfectly normal in external
appearances, and the other leaf was completely necrotic.

The results of this series of experiments established

the molar concentration of 2,4—D used in all subsequent work

and the boundaries used for later variations in the intervals

33
of immersing in both the herbicide and water. Observations
of the development of necrosis in excised leaves over hourly
intervals very closely paralleled the movement of dye
obtained in previous work in which a similar peripheral
movement was repeatedly observed.

Displacement. These experiments were based on the
assumption that the herbicide taken up by the leaf was
carrying a negative charge such as the dye previously used.
Evidence of electrophoretic displacement was based upon the
differential development of necrosis in the leaf between the
positive and negative sides.

Two types of experiments were carried out. In one
the petioles of the leaves were placed in 2 X 10—3 M 2,4-D
at the same time the current application was made. In the
second the petioles of the leaves were immersed in 2,4—D
before the current was applied. The previous procedure was
modified to conduct both experiments in a dessicator over
water to maintain a high relative humidity.

In the first experiment the petiole of a leaf was
placed in the 2,4-D solution at the same time 10 uamperes
of current was applied. After two hours the petiole was

removed from the 2,4—D solution and placed in distilled water

with a continued application of current.

34

After six hours of current application no symptoms
of 2,4—D injury were observed. At eight hours faint marginal
necrosis was evident. At 19 hours, marginal necrosis was
evident in addition to three small necrotic spots next to
the anode. At 31 hours these spots had enlarged somewhat,
but at 44 hours, after a total of 1.58 coulombs of current
had been applied, these spots had enlarged to such an extent
that they were 4 X 10, 3 X 10, and 6 X 7 mm in size
respectively (Figure 4). There was no indication of current
injury to the leaf, nor were there any necrotic areas next
to the electronegative contact.

In the previous experiment the 2,4-D was presumably
swept along in the path of the current. This would have
allowed little time for the herbicide to react with the
tissue or to be altered in some manner in the extracellular
liquid. In order to allow the herbicide to diffuse out of
the vascular elements the following modification was made.

The petiole of an excised leaf was placed in
2 x 10-3 M 2,4—D for 2.5 hours and then removed from the
2,4—D solution and washed. The leaf was then set up with
marginal contacts and a 10 uampere current was applied.

After 20 hours of current had been applied the only

necrosis present was on the electropositive side of the leaf.

35

 

 

 

Figure 4. The effect of 1.58 coulombs of current on the
lateral distribution of 2,4—D in a Canada
thistle leaf whose petiole had been immersed in
a 2 X 10-3 M solution of the herbicide for the
first two hours of the current treatment

36
The purple to black color of the necrotic area was typical
of that caused by 2,4—D rather than that of an excessive
current application. At 37 hours a faint trace of marginal
necrosis was evident and a necrotic area approximately 3 cm
wide had developed on the positive side of the midrib. The
results after 44 hours at 10 uamperes may be seen in Figure 5.
The necrosis at the leaf tip was slightly more severe and
the necrotic area on the positive side of the leaf was
very prominent.

The differential development of necrosis on the
electropositive side of the leaves indicated that 2,4-D was
laterally displaced in the leaves toward the positive contact.
The displacement in this and other leaves varied between

0.5 and 1.0 cm.

The Extraction of 2,4-D byra Combination of Methods

In the following experiments water extractions were
made of the cotton contacts following the predetermined
interval of current used for electrophoretic displacement.
Each pad was separately shredded and packed in the stem of
a funnel. The funnel was placed in a suction flask such
that the liquid drawn through it was caught in a small vial
graduated to contain 5 ml. After a vacuum.was applied,

distilled water was slowly dripped through the cotton until

37

 

Figure 5. The effect of 1.58 coulombs of current on the
lateral distribution of 2,4—D in a Canada thistle
leaf whose petiole had been immersed in a 2 X 10‘3
M solution of the herbicide for 2.5 hours prior
to the current application

38

a total of 5 ml was obtained which was refrigerated and
later bioassayed with the Avena straight growth test which
is described in Appendix A. Prior to assaying the solution
was diluted one to one with a phosphate-citrate buffer.
The control consisted of an identical extraction procedure
with leaves whose petioles had been immersed in distilled
water instead of 2,4—D. By comparison with a standard
curve, the amount of 2,4—D extracted from.the cotton was
determined.

Extraction with one interval of current. The petiole
of an excised leaf was immersed in 3 ml of 2 X 10-3 M
2,4—D for 2.25 hours, removed from the solution, thoroughly
washed in water, and placed in distilled water. A current
application of 20 uamperes was applied for seven hours
and water extractions were made of the positive and negative
cotton pads.

The results (Table 1) show that nine times as much
2,4-D was extracted from the positive pad as from the
negative pad. It was observed that the extractions from
the positive and negative pads of the control leaf did not

differ from each other nor did they differ from the buffer-

water control.

39

TABLE 1. The amounts of 2,4-D recovered from cotton pads
used as traps in the electrophoretic extraction
of 2,4-D from an excised Canada thistle leaf,
whose petiole had been immersed in 2 X 10' M
2,4-D for 2.25 hours, by an application of
20 uamperes of current for seven hours

 

 

Treatments Coleoptile* E ** 2,4-D recovered

elongation t/E (umoles x 103)

C

 

Control leaf

Positive pad 17 1.00
Negative pad 17 1.00
Treated leaf
Positive pad 39 2.29 1.8
Negative pad 26 1.53 0.2
Buffer-Water control 17

 

*Mean elongation expressed in grid units (64 g.u. =
1 cm), coefficient of variation for all treatments = 6.5%.

**Elongation of treated sections divided by elongation
of control sections.

Extraction with varying intervals of current. The
purpose of this experiment was to determine the effect of
four intervals of current on the amount of 2,4—D recovered
from excised leaves.

The petioles of four leaves were immersed in 3 ml
of 2 x 10-3 M 2.4—]: for 1.5 hours. The control leaf
consisted of a leaf whose petiole was immersed in distilled

water for 1.75 hours. A current application of 20 uamperes

was applied to the leaves for two, four, six, and eight

40
hours respectively. An intermediate value of four hours of
current was used for the control leaf. Extracts from these
pads were stored under refrigeration and later bioassayed.
The results are shown in Table 2. The low value

obtained for elongation of the coleoptile sections with the
extract from the positive pad of the control leaf was dis-
regarded because of a known contamination in the extract.
Since the previous elongations for the positive and negative
extracts of the control leaf were equal (Table 1), the value

for the negative extract in this experiment was used for

E
calculating the t/

Ec ratios (elongation of treated sections
divided by elongation of control sections) for the electro—
phoretic treatments.

Since the quantities of 2,4-D extracted from these
leaves were rather small (Table 2). a two hour period was
used for immersion of the petioles in future experiments.
When the electrophoretic extraction was extended up to the
longest interval of eight hours it did not result in the
extraction of any material having physiological activity
from the negative pad.

The recovery obtained at two hours indicated that

as much 2,4-D could be recovered from the leaves with this

interval as could be obtained with an eight hour interval.

41

TABLE 2. The amounts of 2,4-D recovered from cotton pads
used as traps in the electrophoretic extraction
of 2,4—D from excised Canada thistle leaves,
whose petioles had been immersed in 2 X 10‘3 M
2,4-D for 1.5 hours, by applications of 20 uamperes
of current for two, four, six, and eight hours

 

 

respectively
Treatments Hrs. of Coleoptile* E ** 2,4-D recovered
current elongation EC (umoles x 103)

 

Control leaf

Positive pad 4 13***
Negative pad 4 16 1.00
Treated leaves
Positive pad 2 28 1.75 0.4
Negative pad 2 17 1.06
Positive pad 4 23 1.44 0.2
Negative pad 4 17 1.06
Positive pad 6 21 1.31 0.2
Negative pad 6 17 1.06
Positive pad 8 27 1.69 0.4
Negative pad 8 16 1.00
Buffer-Water control 15

 

*Mean elongation expressed in grid units (64 g.u. =
1 cm), coefficient of variation for all treatments = 7.0%.

**Elongation of treated sections divided by elongation
of control sections. E
t/

***This value not used in calculating EC because
of a known contaminant.

42
It was thought, however, that at the eight hour interval an
equilibrium had been reached in which the amount of 2,4-D
extracted was balanced by the amount which was being inactivated
at the electropositive electrode. Since a two hour interval
might not be long enough to allow for possible changes in
the mobility of the herbicide, a six hour interval was
selected for use in subsequent experiments.

Electrophoretic displacement and extraction bypether.
Experiments with charged dyes and 2,4-D indicated that these
materials were displaced horizontally in the leaves toward
the oppositely charged electrodes. This experiment was
designed to obtain information of a more quantitative nature
on the displacement of 2,4-D. The amount or relative amounts
of material displaced toward the respective electrodes
should give an indication of whether the mobility or the
physiological activity of the herbicide was altered in the
extracellular liquid.

For the purpose of determining the amount of 2,4—D,
selected areas of a leaf were excised following the electro-
phoretic treatment. This tissue was then ground in a Waring
blendor, boiled, acidified, and extracted with ether in
accordance with the technique described in Appendix B.

Following the usual procedures the petiole of an

43
excised leaf was placed in 2 X 10-3 M 2,4—D with current
of 10 uamperes applied through marginal Norite contacts.
After a two hour interval the leaf was removed from the
2,4-D solution and placed in distilled water. Following a
six hour interval of current, the contacts were removed and
1.5 X 3 cm sections of tissue were excised from the leaf
adjacent to the positive and negative contacts respectively.
These sections of tissue were subjected to the ether
extraction procedure.

The results indicated that considerable response
was obtained in the bioassay of the ether extracts from
both sections of tissue (Table 3). However, the extraction
of the tissue on the electropositive side of the leaf
resulted in the recovery of approximately twice as much
2,4-D than that obtained from the electronegative side of
the leaf.

Electrophoretic and ether extractions. varying time
intervals were used in this experiment between the conclusion
of the 2,4-D treatments and the beginning of the extraction
procedures in order to obtain information on the inactivation
or altering of the 2,4-D in the extracellular liquid with
increasing incriments of time. Since it is conceivable that

the 2,4—D could either lose its electrical charge or acquire

a positive charge as a result of a chemical reaction or

44

TABLE 3. The amounts of 2,4-D recovered by ether extractions
of sections of tissue from the electropositive
and electronegative sides of an excised Canada
thistle leaf following a two hour immersion of
the petiole in 2 X 10"3 M 2,4—D with a parallel
application of 10 uamperes of current followed
by an additional four hours of current.

 

 

 

Treatments Coleoptile* E ** 2,4—D recovered

elongation /Ec (umoles X 103)
Positive side of leaf 35 2.06 5.0
Negative side of leaf 31 1.82 2.6
Control 17 1.00

 

*Mean elongation expressed in grid unit (64 g.u. =
1 cm), coefficient of variation for all treatments = 7.2%.

**Elongation of treated sections divided by elongation
of control sections.
complexing with constituents in the extracellular liquid,
the results obtained from electrophoretic extractions could
be expected to differ at each increment of time or perhaps
no response would be obtained after a sufficient interval
had elapsed. A comparison of the amount of physiologically
active substance extracted by the electrical and ether extraction
techniques should yield additional information on the fate
of the herbicide. If inactivation of the herbicide were
occurring, no response could be expected in the bioassay
of the ether extractions of the tissue. This would be

anticipated at the longer time intervals.

45

The petioles of excised leaves were uniformly
immersed in 3 ml of 2 x 10'3 M 2.4—1) for two hours, removed,
thoroughly washed, and placed in distilled water. Four
time intervals were employed between the conclusion of the
2,4-D treatments and the beginning of the electrophoretic
extraction. These intervals were as follows: zero, three,
six, and twelve hours. Appropriate controls were included.
At the conclusion of each electrophoretic treatment
extractions were made of the cotton pads and the terminal 3 cm
portion of the leaf by water and ether respectively.

Treatments were included to obtain an approximate
evaluation of the recovery of 2,4—D from the extraction
apparatus, with and without cotton packed in the stems of
the funnels, when known amount of 2,4-D were added. The
procedure was as follows. A funnel was placed over one of
the 5 ml graduated vials which was used for collecting the
water extract from.the pads. In this case, however, a pad
was not shredded and packed in the stem of the funnel.
Twenty-five microliters of 3.9 X 10-4 M 2,4—D was pipetted
into the funnel and distilled water was added drop by drop
in the same manner as when the stem of the funnel was
packed with cotton. The resulting 5 m1 of solution was

refrigerated and then bioassayed with the other extracts.

46
This procedure was repeated and a second 5 m1 of solution
was obtained. The entire process was repeated with cotton
in the stems of the funnels. In this case, however, 25 A
of distilled water was added to two funnels and 25 1 of the
2,4—D solution was added to two other funnels.

It may be seen in Table 4 that the 2,4—D solution
was approximately one half as active as it theoretically
should have been. Since this solution of 2,4-D was quite
old, the low response was not considered as an experimental
error. However, the adsorption of such a small volume of
herbicide (25 A) to the surface of the glassware was also
considered as a contributing factor. Assuming that some-
what less than 5 X 10-3 umoles of 2,4-D was obtained from
the extraction apparatus for bioassaying (Table 4), the
recovery of 7 X 10-4 umoles is approximately equal to a
15 percent recovery. Thus, it must be assumed that the
responses obtained from the water extraction of the cotton
pads used as contacts in the electrophoretic extractions of
treated leaves is only indicative of approximately 15 percent
of the actual materials present.

The results of the series of extractions employing
the four time intervals are presented in Table 5. From a

standpoint of comparison of the amounts of 2,4—D recovered

47

TABLE 4. The amounts of 2,4—D recovered from the extraction
apparatus used for recovering 2,4—D from cotton,
with and without the cotton packing, to which
10"2 umole of 2,4-D had been added

 

 

Treatments Material Coleoptile* E ** 2,4—D recovered
added elongation /Ec (umoles X 103)

 

Extraction Apparatus

Without cotton 2,4—D 42 2.62 5.1

Without cotton 2,4-D 41 2.56 4.5
Buffer-Water Control 16 1.00
Extraction Apparatus

With cotton 2,4-D 20 1.54 0.7

With cotton water 13 1.00

With cotton 2,4-D 22 2.00 0.8

With cotton water 11 1.00

 

*Mean elongation expressed in grid units (64 g.u. =
1 cm), coefficient of variation for all treatment = 7.3%.

**Elongation of treated sections divided by elongation
of control sections.
by the ether extractions of the tissue with the amounts
electrophoretically extracted, it can be seen that considerably
more recovery was obtained by the ether extractions. It
should be noted, however, that the area of tissue under the
electrical contacts was considerably smaller than the
area of tissue subjected to the ether extractions.

Because the slightly lower response obtained from

the water extract of the positive pad at the 12 hour interval

(Table 5) suggested the possible formation of a neutral

48

 

 

 

TABLE 5. The amounts of 2,4—D recovered from cotton pads
used as traps in the electrophoretic extraction
of 2,4—D from excised Canada thistle leaves and
from ether extracts of a portion of each leaf
following a zero, three, six and twelve hour
interval during which their petioles were immersed
in distilled water. The petioles of all treated
leaves had previously been immersed in 2 X 10‘3 M
2,4-D for two hours. All leaves were subjected
to a current application of 20 pamperes for six
hours.

Treatments Hrs. Coleoptile* E ** 2,4-D recovered

in elongation /E (umoles X 103)
water c
Control leaf
Positive pad 6 16 1.00
Negative pad 6 17 1.00
Tissue 6 13 1.00

Treated leaves***

Positive pad 0 31 1.94 1.0
Tissue 0 27 2.08 8.0
Positive pad 3 24 1.50 0.5
Tissue 3 30 2.31 13.0
Positive pad 6 28 1.75 0.7
Tissue 6 26 2.00 6.5
Positive pad 12 19 1.19 0.3
Tissue 12 31 2.38 15.0
Buffer-Water Control
For tissue 16
For pads 15

 

*Mean elongation expressed in grid units (64 g.u = 1

cm), coefficient of variation for all treatments = 7.3%.

**Elongation of treated sections divided by elongation
of control sections.

***Values for negative pads omitted because responses
were not greater than control.

49

complex or the loss of electrical charges as a consequence
of an alteration of the molecule, the previous experiment
was repeated. An interval of ten hours was selected as an
intermediate period between the end of the 2,4—D treatment
and the beginning of the electrophoretic extraction. All
procedures, solutions, and conditions were the same as in
the previous experiment.

The results, as given in Table 6, show'that
considerably more 2,4—D was recovered from the positive pad
than was recovered from the positive pads in the previous
experiment (Table 5). This value, however, is not indicative
of any change in the mobility of the herbicide.

The recovery obtained by the ether extraction compared

favorably with those shown in Table 5.

50

 

 

 

TABLE 6. The amounts of 2,4—D recovered from cotton pads
used as traps in the electrophoretic extraction
of 2,4-D from an excised Canada thistle leaf and
from an ether extract of a portion of the leaf
following a ten hour interval during which its
petiole was immersed in distilled water. The
petiole had previously been immersed in 2 X 10"3 M
2,4-D for two hours. The leaf was subjected to
a current application of 20 uamperes for six
hours.
Treatments Coleoptile* Et ** 2,4-D recovered
elongation /Ec (umoles X 103)
Control leaf
Positive pad 15 1.00
Negative pad 17 1.00
Tissue 12 1.00
Treated leaf
Positive pad 31 2.07 1.8
Negative pad 18 1.06
Tissue 25 2.08 9.2

Buffer-Water control 16

 

*Mean elongation expressed in grid units (64 g.u. =

1 cm), coefficient of variation for all treatments = 7.0%m

**Elongation of treated sections divided by elongation
of control sections.

DISCUSSION

The electrophoretic method used in this study was
found to be effective in extracting 2,4-D from the extra-
cellular liquid of Canada thistle leaves. Several limitations
of the method, however, had to be considered. One of these
was the possibility of electrically induced flow of the extra—
cellular liquid. The results of experiments with oppositely
charged dyes indicated that electroosmosis was not a major
factor in electrical displacement. The low recoveries obtained
from cotton pads to which known amounts of 2,4—D had been
added was a more serious limitation of the method. However,
the relative differences between the recoveries obtained from
the positive and negative pads furnished the desired information
concerning the possible changes in the mobility of the herbicide.
The method could probably be modified to make it more quanti-
tative. One such method of achieving this end might be the
utilization of a continuous flow system of liquid contacts
in which the liquid was retained in a suitable trap for later
assaying or chemical analysis. Such a method would eliminate
the retention of the herbicide by the cotton pads.

Although the leaves used in these experiments were
selected on the basis of uniform size and position on the

plants, there was considerable variation in the amounts of

51

52
2,4—D and dye taken up by them. In some cases this variability
was as high as sixfold, but in most instances the range of
variability was between two and threefold. The petiole
immersion method of introducing the 2,4-D into the excised
leaves was employed in spite of this variation because it
provided a means of introducing the herbicide directly into
the extracellular liquid. Improved environmental control
might have resulted in more uniform uptake of the dye and
herbicide.

The results of the experiments utilizing the electro-
phoretic extraction technique were in agreement with those of
observational experiments in which the 2,4-D and dye were
found to be transported in the extracellular water. The 2,4-D
was repeatedly recovered from the positive contacts. In only
one instance was evidence obtained which suggested a change
in the electrical charge of the growth regulator (Table 1).

In this instance approximately nine timesas much active
material was recovered from the positive pad as was obtained
from the negative pad. All subsequent data from extractions
of cotton pads indicated that the herbicide was not altered
by an interaction with substances in the extracellular liquid.
The preferential displacement of an applied electrolyte to an
electrode of the same charge has recently been reported by

Anderson et a1. (1). An oppositely charged carrier was

53

suggested as the explanation for this response. However, the
conflicting value in Table l was probably the result of dif-
fusion of the 2,4-D outside of the electrical field as a
result of a poor contact.

When ether extractions were made of portions of a
leaf from the positive and negative sides following an electro-
phoretic displacement, it was found that a response was obtained
from the tissue on the negative side (Table 3). This response
is readily explained when it is recalled that a similar dis-
placement of dye indicated that the midrib was a barrier to
transverse movement. Since the 1.5 X 3.0 cm portion of excised
tissue from each side of the leaf included areas near the
midrib, it was probable that part of this tissue on the negative
side of the leaf included an area in which the herbicide was
concentrated as a result of the barrier imposed by the midrib.

The responses obtained from ether extractions of the
terminal 3 cm portion of leaves over varying intervals of time
(Table 5) suggested that inactivation of the herbicide over
periods up to 12 hours did not occur. When this experiment
was repeated with a ten hour interval the recovery obtained
(Table 6) was in good agreement with the data in Table 5. The
data from ether extractions in Table 5 and 6 indicate gradual
increases in the amounts of extractable material over in-

creasing time intervals. This substantiated previous

54

observations (obtained from parallel experiments with dye and
2,4-D) that the 2,4—D was transported toward peripheral areas.

It should be noted that in these experiments it was
considered essential to work only with tissue which was normal
in appearance. Any tissue which had become necrotic was dis—
carded because of the possible release and subsequent extraction
of inhibitory substances-

The recoveries obtained from the positive cotton pads
up to and including the six hour interval were essentially
constant (Table 5). This was interpreted as evidence that the
herbicide was not readily taken up by the living cells and was
not involved in the formation of an immobile complex in the
extracellular liquid. HOwever, the somewhat lower value
obtained for the 12 hour interval did not negate the latter
possibility. This lower response could not be validly inter-
preted as inactivation since the response from the tissue
extract for this interval was quite high. When the experiment
was repeated with an intermediate interval of ten hours (Table 6),
the recovery obtained was considerably higher than those given
in Table 5 but was in exact agreement with that given in Table l
for the positive pad. On the basis of these results, it was
considered unlikely that the 2,4—D formed a neutral complex.
The higher recoveries obtained in the last experiment were

probably caused by a greater uptake of the herbicide by the leaf._

55

The possibility of the 2,4-D molecule forming a complex
in the extracellular liquid such that it had an effective
positive charge was disproven since no response was obtained
in any of the extracts from the negative pads at any of the
time intervals.

The transport of 2,4—D which occurred in these experi-
ments was principally, if not entirely, confined to the xylem
vessels. That the vessels were capable of functioning in
transport was evidenced by the observations of dye transport
in the vessels and by the observation of lack of protoplasm
and the presence of secondary walls in the form of annular and
spiral rings.

The peripheral movement of the herbicide to the margins
of the leaves to which it had been applied was clearly indicative
of xylem transport. A similar peripheral movement of a herbicide
from the point of application on Canada thistle leaves has
previously been reported (19). The upward and outward movement
of the herbicide which occurred regardless of the method of
introduction was quite typical of transport of a material in
the transpirational stream. The parallel movement of the dye
and its presence in the xylem, as found by examination of
sections under the microscope, were considered as additional

evidence of xylem transport. Corms (6) studied translocation

of 2,4-D in perennial sow thistle and suggested a similar

56
xylem pattern of movement within the plant but also reported
extensive downward movement.

The same pattern of response was obtained when leaves
whose tips had been excised were immersed in 2,4—D. Movement
in the former was attributed to a reversal of the transpiration
stream. Since essentially the same responses were obtained by
both methods, the same avenue of transport was considered to be
operative in both cases. A similar movement of 2,4-D in the
transpiration stream as a result of leaf tip excising has been
reported (37).

The enhanced transport of 2,4-D that was obtained when
the leaf and petiole were injured or when a portion of the
applied 2,4—D ran back down the petiole of the leaf into the
axil was considered to be evidence of placement of the herbicide
nearer the vigorous upward transpirational stream and nearer to
larger vascular bundles. There is evidence in the literature
that the position of placement of a herbicide on a plant leaf
has considerable bearing on the effectiveness of such an
application (7, 21, 36). More extensive growth inhibition was
obtained when 2,4-D was applied at the base of the leaves than
when it was applied nearer the apex. These results, however,
may be interpreted as either enhanced xylem or phloem transport.

The explanation for the lack of phloem transport in

these plants could be attributed to a number of causes. One

57
of which concerns the position of the treated leaf on the stem.
Crafts (11, 12) reported that applications of 2,4-D to the
lower leaves of plants resulted in the downward translocation
of 2,4-D whereas applications to upper leaves resulted in
transport to the upper regions of the plants. It was further
reported that applications of 2,4—D to the middle leaves of
morning glory plants resulted in either downward or both upward
and downward transport (8). Since the applications of 2,4—D
to the Canada thistle plants used in these experiments were
always made on leaves intermediate in their position on the
stems and since the roots of these plants were actively growing,
some transport into the roots was anticipated. Such a downward
path of transport, however, was never observed.

Further explanations for the lack of adequate transport
of 2,4-D could involve a binding mechanism or active accumu-
lation with the tissue (2, 11, 20, 21). Since the evidence
obtained with the electrophoretic technique used in these
experiments indicated that the 2,4-D was freely mobile in
the extracellular liquid, it can only be assumed that if
such a mechanism occurred it must have resulted in the
formation of strong bonds between a small portion of the
2,4—D and the cellular materials or else such a mechanism

occurred within the protoplasm out of the path of the electric

58
current.

The most probable explanation for the lack of transport
and hence lack of effectiveness of 2,4—D on these Canada
thistle plants is that it was not taken up by the functioning
sieve elements. This lack of uptake is attributed either to
the polar nature of the sodium salt and the apolar nature
of the lipoidal properties of the protoplasm or to the lack
of the proper activated metabolic processes necessary for the
accumulation of 2,4—D in the phloem and its transport once
in the phloem, or possibly to both. Evidence for the
necessity of metabolic energy in the transport of 2,4—D
has been presented in several publications (16, 18, 23).

The results obtained in these experiments also
indicated that there was no appreciable metabolism or
inactivation of the 2,4-D up to the 12 hour interval used.

If the 2,4-D was metabolized to any extent, it must be
assumed that it evoked the formation of material of similar

physiological activity and mobility.

SUMMARY AND CONCLUSIONS

An electrophoretic method was used to study the

mobility of the sodium salt of 2,4—D in the extracellular

liquid of Canada thistle leaves since the ineffectiveness

of the herbicide might be attributed to the formation of an

inactive or immobile complex. Extraction of the 2,4-D was

accomplished electrophoretically and by an ether extraction

procedure. The concentration of the herbicide in the extract

was assayed by the Avena straight growth test.

These studies provided the following conclusions:
The transport of 2,4-D was found to occur principally
in the extracellular liquid of the tissue. Because
of the identical peripheral movement of the 2,4-D
and a water soluble dye (Amaranth) and the visual
presence of the dye in the xylem vessels, it was
concluded that the transport of the 2,4—D was
confined to the xylem.

Transport in the phloem was considered to be
negligible.

On the basis of electrophoretic studies there was
no evidence of a change of or a lack of mobility of

the 2,4—D in the extracellular liquid. The herbicide

59

60
was consistently extractable at the electropositive
contact.
There was no loss of biological activity of the
herbicide up to and including a 12 hour interval
as a result of metabolism or other detoxification
processes.
The limited uptake of the herbicide by the living
protoplasm was thought to be the principal factor

in the lack of transport.

10.

LITERATURE CITED

Anderson, J. R., Wittwer, S. H., and Bukovac, M. J.
Effect of electrical currents on the transport of
radiocalcium (Ca45) in Phaseolus vulgaris.
Physiologia Plant. 14:548-553. 1961.

Bach, M. K., and Fellig, J. The uptake and fate of
2,4-dichlorophenoxyacetic acid in bean stem
sections. In: Plant Growth Regulation (Klein,
R. M., editor) Iowa State Univ. Press, Ames.
pp. 273-87. 1961.

Blackmann, G. E., Bruce, R. S., and Holly, K. Studies
on the principles of phytotoxicity. V. Interrelation-
ships between specific differences in spray retention
and selective toxicity. Jour. Exptl. Bot. 9:175-205.
1958.

Bonner, J. The action of the plant growth hormone.
Jour. Gen. Physiol. 17:63-76. 1933.

Canny, M. J., and Katalin, M. The breakdown of 2,4-
dichlorophenoxyacetic acid in shoots and roots.
Australian Jour. Biol. Sci. 13:486-500. 1960.

Corns, W. G. Translocation of 2,4-D in injected flowering
perennial sow thistle. Canadian Jour. of Res. Sect.
C. Bot. Sci. 26:239-48. 1948.

Crafts, A. S. Translocation of herbicides. I. The
mechanism of translocation: Methods of study with

C14—1abe1ed 2,4-D. Hilgardia 26:287-334. 1956.

. Translocation of herbicides. II. Absorption

 

and translocations of 2,4—D by wild morning glory.
Hilgardia 26:335—65. 1956.

. The chemistry and mode of action of herbicides.

 

Advances in Pest Control Research 1:39-79. 1957.

, and Yamaquchi, S. Comparative test on the

 

uptake and distribution of labeled herbicides by
Zebrina pendula and Tradescantia fluminesis.
Hilgardia 27:421-54. 1958.

61

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

62

. Further studies on comparative mobility of
labeled herbicides. Plant Physiol. 34:613-20. 1959.

 

. New research on the translocation of

 

herbicides. Proc. NEWCC 13:14-17. 1959.

Day, B. E. The absorption and translocation of 2,4-
dichlorophenoxyacetic acid by bean plants. Plant
Physiol. 27:143-54. 1952.

Fernald, M. L. Gray's Manual of Botany. 8th ed.
American Book Co., New York.

Hancock, C. R., and Barlow, H. W. B. The assay of
growth substances by a modified straight growth
method. Rep. E. Malling Res. Sta. 40:88-94. 1953.

Hay, J. R. Experiments on the mechanism for the
translocation of 2,4-D. Plant Physiol. 30 (Sup.):
V, 1955.

, and Thimann, K. V. The fate of 2,4-
dichlorophenoxyacetic acid in bean seedlings. I.
Recovery of 2,4-dichlorophenoxyacetic acid and its
breakdown in the plant. Plant Physiol. 31:382-87.
1956.

 

, and . The fate of 2,4-dichloro-

 

phenoxyacetic acid in bean seedlings. II. Trans-
location. Plant Physiol. 31:446-51. 1956.

Herrett, R. A. Studies on the absorption, translocation,
and metabolism of 3-amino-1, 2, 4-triozole in
perennial plants. Ph.D. thesis, University of
Minnesota, St. Paul, 1959.

Johnson, M. P., and Bonner, J. The uptake of auxin
by plant tissue. Physiol. Plant. 9:102-18. 1956.

Leonard, O. A. Studies on the absorption and trans-
location of 2,4-D in bean plants. Hilgardia 28:115-60.
1958.

Leopold, A. C. Auxins and Plant Growth. University
of California Press, Berkeley and Los Angeles.

pp. 13-59. 1955.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

63

Loomis, W. E. Resistance of plants to herbicides. In:
Origin of Resistance to Toxic Agents (Sevag, M. G.,
Reid, R. D., and Reynolds, 0. E., editors). Academic
Press, New YOrk. pp. 99-121. 1955.

Luckwill, L. C. Growth-inhibiting and growth-promoting
substances in relation to the dormancy and after-
ripening of apple seeds. Jour. Hort. Sci. 27:53-67.
1952.

Nitsch, J. P. Free auxins and free tryptophane in the
strawberry. Plant Physiol. 30:33-39. 1955.

, and Nitsch, Collette. Studies on the

 

growth of coleoptile and first internode sections.
A new sensitive, straight-growth test for auxins.
Plant Physiol. 31:94-111. 1956.

Olien, C. R. Studies on necrosis in the infection of
Khapli emmer by stem rust fungus, Puccinia gpaminis

var. tritici. Diss. Abs. 17:1171. 1957.

. A method of studying stresses occurring

 

in plant tissue during freezing. Crop Science
1:26-28. 1961.

Pallas, J. E., Jr. Effects of temperature and humidity
on foliar absorption and translocation of 2,4—
dichlorophenoxyacetic acid and benzoic acid.

Plant Physiol. 35:575-80. 1960.

Showacre, J. L., and du Buy, H. G. The relation of
water availability and auxin in the growth of Avena
coleoptiles and its meaning for a theory of tropisms.
Am. Jour. Bot. 34:175-182. 1947.

Swanson, C. P. Histological responses of the kidney
bean to aqueous sprays of 2,4-dichlorophenoxyacetic
acid. 107:522-31. 1946.

Thimann, K. V., and Bonner, W. D., Jr. Experiments on
the growth and inhibition of isolated plant parts.
I. The action of iodo acetate and organic acids
on the Avena coleoptile. Am. Jour. Bot. 35:271-81.
1948.

33.

34.

35.

36.

37.

64

Tukey, H. B., Hamner, C. L., and Imhoff, B. Histological
changes in bindweed and sow thistle following
applications of 2,4-dichlorophenoxyacetic acid in
herbicidal concentrations. Bot. Gaz. 107:62-73. 1945.

Van Overbeek, J. Use of synthetic hormones as weed
killers in tropical agriculture. Economic Bot.
1:446-59. 1947.

Voevodin, A. V. The decomposition of 2,4-D in the
tissues of Canada thistle. Fiziol. Rastenii
[Translated] 7:259-62. 1960.

Weaver, R. J., and DeRose, H. R. Absorption and
translocation of 2,4-dichlorophenoxyacetic acid.
Bot. Gaz. 107:509—21. 1946.

Weintraub, R. L., and Brown, J. W. Translocation of
exogenous growth-regulators in the bean seedling.
Plant Physiol. 25:140-49. 1950.

APPENDIX A

THE BIOASSAY PROCEDURE

The Avena straight growth test was used to determine
the amounts of 2,4—D present in extracts obtained from
treated Canada thistle leaves by ether extractions of the
tissue (Appendix B) and by water extractions of cotton pads
which had been used as traps in the electrophoretic extraction
procedure.

The oats, Avena sativa var. Brighton (Bio—Lab
Equipment Company) were grown in a constant temperature
cabinet at 25 degrees centigrade and approximately 90 percent
relative humidity. The cabinet was equipped with a small
fan to provide a uniform circulation ofair and a source
of red light 30 cm above the plant bed. The light source
consisted of two General Electric Ruby red bulbs. During
the interval in which the lights were on the temperature
rose approximately three degress and remained at this level
until the light period ended. The normal range in temperature
variation that occurred between the heating and cooling
cycles was approximately one-half of a degree centigrade.

Brighton oats were selected for use rather than the

more commonly used Victory variety because it had been

65

66
established that they were a suitable substitute and
eliminated the dehulling procedure (25). The method, as
first employed, was essentially the same as that outlined
by Leopold (22). Literature citations are given to major
deviations from the outlined method.

The seed were placed in a suction flask, soaked in
water under a vacuum for two hours, removed and placed on
glass strips covered with paper towels such that the grooved
side was down and the embryo end projected over the side.
The strips were placed in a glass tray with sufficient
distilled water to cover the bottom to a depth of 5 mm and
placed in the dark constant temperature cabinet.

Twenty-four hours later the seed were exposed to
two hours of red light and 72 hours later, or when the
coleoptiles were 2.0 to 3.0 cm long, they were removed from
the cabinet in the dark. All subsequent manipulations were
performed under a low intensity Ruby red light. One 5 mm
section was cut from each coleoptile 3 mm from the apical
end and placed in distilled water. The primary leaf was
pushed out and the hollow sections were threaded on sealed
glass capillaries and placed in small glass boats after the
method of Luckwill (24). The boats contained 1.0 m1 of

the solution to be tested and 1.0 ml of a pH 5.0 buffer to

67
avoid variations in growth due to pH effects. The composition
of the buffer as given by Nitsch and Nitsch (26) was as

follows: citric acid monohydrate, 2.038 g/l; K2 4

sucrose, 40 g/l. The boats, containing two capillaries with
five sections each, were placed in Petri dishes in the
constant temperature cabinet.

Twenty-four hours later the sections were removed
and the elongation measured with a binocular microscope
equipped with an eyepiece micrometer (4). One centimeter
was equal to 64 grid divisions.

The results of the initial experiments indicated
that insufficient red light had been used to suppress the
elongation of the first internode. As a result of subsequent
experiments it was established that four hours of red light
from two 25 watt bulbs provided the optimum intensity and
duration of light needed.

Growth in the Avena straight growth test, as originally
suggested by Bonner (4), is approximately proportional to the
log of the concentration of the applied auxin. In order to
determine a range of concentrations of 2,4-D that would give
a linear response a serial dilution series of 2,4—D was
prepared ranging in concentration from 2 X 10.-3 umoles/l to

2.0 X 102 umoles/l and a bioassay was run on these solutions.

68

The results as shown in Figure 6 in which the response
of the sections is expressed by dividing the elongation of
the treated sections by the elongation of the control
sections. The control sections in this and all subsequent
experiments were grown in equal proportions of distilled
water and the phosphate-citrate buffer. On the basis of
these responses other bioassays were performed using the
concentrations of 2,4—D which corresponded to the linear
range shown in Figure 6. As a result of these experiments
it was established that growth was approximately proportional
to the log of concentration between 5.0 X 10-2 umoles/l
and 1.95 umoles/l.

The standard deviations of the various treatments in
these bioassays were quite large. In fact, in many cases
the coefficient of variation obtained for some treatments
approached values as high as 15 percent. It was theorized
that microbial contamination of the seed might be a factor
and that the procedure of soaking them under a vacuum might
be a contributing factor in infecting the seed with what-
ever contamination that might have been on the surface. To
test this hypothesis the following three treatments were
employed; one group of seed was subjected to the usual

soaking under vacuum, a second group was washed continuously

69
21%r

24+»

23H»

2.20

2+»

2'Oi

V

r9<

V

P8~

V

l l A l n
V ' U T

-002 -02 '2 2 20 200
Concentration of 2,4—D (umoles/l)

 

Figure 6. The response (elongation of treated sections
divided by elongation of control sections) of
Avena coleoptile sections to 2,4—D

70
in running tap water for two hours, and the third group
was surface sterilized for one minute in one percent
NaOCl. Each group of seed was then plated on a potato
dextrose agar.

After 24 hours it was found that the seed treated
with NaOCl were free of microbial growth, but the seed from
the other treatments were covered with microbial growth.

Of the latter two, the vacuum treatment was decidedly the
most undesirable. As a result of these findings the vacuum
treatment was abandoned and in all subsequent bioassays the
seed were washed in running water and surface sterilized
with one percent NaOCl.

In previous bioassays the buffer solution was
autoclaved after using, and the unused portion was refrigerated
and used in later bioassays. Since the possibility of
microbial contamination in the buffer as well as on the
glassware could affect the growth of the sections, the following
modifications were made in the procedure. The seed were
prepared for planting as described using the NaOCl. A
new buffer solution was prepared and autoclaved along with
a container of water and all of the glassware to be used.
During the preparation of the sections all equipment which

came in contact with the sections was rinsed in sterile water

71
following frequent surface sterilizations with NaOCl. After
the cutting operation, the sections were floated on sterile
water until this operation was complete. One-half of the
sections were grown in a one to one dilution of sterile water
and sterile buffer, and the other half were grown in a
similar dilution of nonsterilized water and buffer. After
24 hours the sections were measured.

The results showed that there was little difference
between the mean lengths of the two groups of sections, but
the standard deviation of the sections grown in the non-
sterilized buffer was 50 percent larger than that of the
sterilized buffer treatment. A histogram showing the
distribution of the individual section lengths around the
mean is shown in Figure 7.

Freshly prepared and autoclaved buffer solutions,
as well as sterilized glassware, were used in all of the
following bioassays.

Although these modifications aided in reducing the
variability of the growth of the Aygga sections, there were
inherent problems associated with growing the sections on
capillary rods in glass boats which often caused erratic
responses. The major problems were the curvature of the

sections, the injury of the sections which occurred when the

72

 

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73
primary leaf was pushed out, and the imperfect contact between
the sections and the solution as a result of the capillaries
becoming closely orientated or crossed when the boats were
placed in the constant temperature cabinet.

In order to avoid these difficulties certain modifi-
cations were made. To prevent injury to the sections and to
simplify the procedure the primary leaf was left inside the
coleoptile (15). A tumbler was used to rotate the sections
to prevent their orientation in the liquid surface and subsequent
curvature. Since it had been reported that aeration of the
sections was necessary (30, 32), the bottles in which the
sections were grown were equipped with glass tubes which
allowed a free exchange of air. The ends of the tubes were
covered with a thin layer of silicone stopcock grease to
prevent them from becoming filled with liquid. The tumbler
with the bottles in the constant temperature cabinet is shown
in Figure 8.

The bioassays which were performed using these
modifications were greatly improved over the previous ones.
Curvature of the sections was essentially eliminated and the
standard deviations were reduced. However, occasionally
erratic responses were obtained which resulted in nonlinearity.

The procedure was then altered such that the sections were

74

 

 

Figure 8. The tumbler and bottles used for rotating
Avena coleoptile sections in the constant
temperature cabinet

75

floated on glass distilled water containing 1.5 ppm MnSo4.H20
for two hours prior to using (26).

As a result of the last modification, the response
of the sections to 2,4—D was increased and much of the
variability in growth was reduced. The coefficients of
variation of the treatments were greatly reduced and seldom
exceeded five percent. As a result of the increased response,
a new dilution series of 2,4-D was used in which the most
concentrated solution was 1.0 umoles/l rather than 1.95
umoles/l as previously used.

The results from the first bioassay using all of the
above modifications are shown in Figure 9. The response was
essentially linear over the entire range of concentrations of
2,4-D used. The coefficient of variation of the most con-
centrated solution of 2,4—D was 4.5 percent.

As a result of skill acquired in performing the
intricate manipulations of the procedure and the establishment
of a strict time schedule, the sensitivity and reliability of
the procedure were further increased. Because of the greater
sensitivity an additional dilution was added to the series of
2,4-D solutions. The response of the sections was approximately

proportional to the log of the concentration of the 2,4-D

solutions between 2.6 X 10-2 umoles/l and 1.0 umoles/l.

76

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77

E
In order to allow an adequate margin of error, t/E

c
values lower than 1.3 were usually not considered when
bioassays were performed on extracts of unknown concentration.

A value of 1.3 normally corresponded to a concentration of

3.0 X 10-2 umoles/l (8 ug/l). When an extract having a volume
of 5 ml was diluted one to one with the buffer and assayed,

a response of 1.3 was equivalent to 3.0 X 10"4 umoles (0.1 ug)
of 2,4-D.

Briefly, the procedure in its final form was as follows:
the oats were screened to obtain a uniform size fraction, washed
in running water for one hour, surface sterilized in one
percent NaOCl for one minute, and placed on paper covered
glass strips with the grooved side down and the embryo end
projecting over the side. The glass strips were placed in a
large glass tray with added distilled water and placed in the
dark constant temperature cabinet at 25 degrees centigrade and
90 percent relative humidity. Twenty-four hours later the
seed were exposed to four hours of light from two 25 watt
Ruby red bulbs which were 30 cm above the germinating tray.

After 80 hours, or when the coleoptiles were 2.0-2.5
cm long, the coleoptiles were removed under a low intensity

red light. All subsequent operations were performed with

sterilized equipment. One 5 mm section was cut from each

78

coleoptile 3 mm from the apical end and placed in distilled
water. When all sections were cut they were transferred to
a dish containing glass distilled water and 1.5 ppm of
MnSO4-4H20. The sections were presoaked in the solution for
one hour in total darkness and then for an additional hour
in a fresh solution under red light.

At the end of this period the sections were placed
in 30 m1 bottles containing 5 ml of the solution to be
bioassayed and 5 m1 of the pH 5.0 phosphate-citrate buffer
containing four percent sucrose. The bottles were fitted
with ground glass vent tubes and placed on the tumbler which
rotated then around the horizontal axis at 20 revolutions
per minute. The tumbler was placed in the constant temperature
cabinet at the same temperature and humidity as used for
growing the coleoptiles.

After 24 hours of rotation, the sections were measured

under a binocular microscope equipped with an eyepiece

micrometer in which one centimeter equaled 64 grid divisions.

APPENDIX B

THE ETHER EXTRACTION PROCEDURE

The method used for extraction of 2,4-D from Canada
thistle leaves was derived from the method given by Hay
and Thimann (17). All attempts to use the method exactly
as outlined were unsatisfactory because of the low recoveries
obtained when known amounts of 2,4—D were added to leaf
tissue and extracted. The major difficulty was thought to
be adsorption of the 2,4—D on the filter paper and residue
in the filter paper.

The method evolved for extraction is as follows:
The tissue sample (0.5-1.0 g) was ground for two minutes in
a Waring blendor with 15 m1 of distilled water, washed into
a small beaker with a minimum of water, boiled for one
minute, allowed to cool for ten minutes, acidified to pH 1.0
with concentrated sulfuric acid, and transferred to a small
centrifuge tube fitted with a rubber stopper covered with
aluminum foil and a stopcock to release the pressure when it
became excessive. The liquid was extracted with two 10 m1
portions of diethyl ether and centrifuged at a low speed
for ten minutes.

The ether phase was then pipetted off, leaving the

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80
aqueous portion and tissue residue behind, and transferred
to a 60 ml separatory funnel and extracted with two 10 m1

aliquots of 0.2 M NaHCo The aqueous portion of this

3.
separation was acidified to pH 1.0 and re-extracted with two
10 m1 aliquots of ether. The ether fraction was evaporated
in a hood. Twenty millileters of distilled water were added
to the residue and the solution adjusted to pH 5.0 by adding
0.1 M NaOH. The solution was diluted to 25 ml and stored

at 5 degrees centigrade until it was bioassayed. When

necessary, serial solutions were made of the extract.

R0034 USE (Di-ELY