 

HESTOLOGICAL CHANGES AND NUTRIENT *
LOSSES FROM 9mm mums UNDER _ .
MIST PROPAGATION» _ ‘ “ V

11min for the Degree cf mo.
MICHIGAN STATE UNIVERSITY .
Dale V. Sweet ' '
1956~ -

THEas

This is to certify that the
thesis entitled

HISTOLOGICAL CHANGES AND NUTRIENT LOSSES
FROM PLANT FOLIAGE UNDER MIST
PROPAGATION

presented by

Dale Vernon Weet

has been accepted towards fulfillment ‘
of the requirements for

ii..— clegree in _ HorticultUr e

Major professor

Date March 30, 1956

0-169

 

 

 

HIST OLOGICAL CHANGES AND NUTRIENT LOSSES FROM

PLANI‘ F OLIAGE UNDER MIST PROPAGATION

By

DALE v.' SWEET

A THESIS
Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science

in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

l 956

HIST OLOGICAL CHANGES AND NUTRIENT LOSSES FROM

PLANT FOLIAGE UNDER MIST PROPAGATION

BY

DALE V. SWEET

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1956

Approved M / )éif/W,

DALE V. SWEET ABSTRACT

The effect of a mist treatment on the leaching of root and non—root

32 86

absorbed radioactive phosphorus , potassium”, and rubidium from plant
foliage was investigated to determine the nutrient losses from cuttings of
certain horticultural plants which were placed under the mist method of
propagation. Studies of misted and non—misted cross sections of leaves and

stems of Phaseolus vulgaris (variety Cranberry), Malus Silvestris (Malling IX),

 

 

and Prunus mahaleb (clone 3-16) were included to observe any histological

 

changes which may have occurred during the water-mist treatment.

Rosa odorata (variety Better Times), and Phaseolus vulgaris

 

 

(variety Cranberry) absorbed radioactive phosphorus32 for forty—eight hours
through the cut surfaces of their stems prior to being placed under a water—
mist spray for a similar period of time. The leachate was collected and
analyzed for radioactive phosphorus32 which was expressed in per cent of
total uptake of the isotope in the misted cuttings.

Phaseolus vulgaris (variety Cranberry), and Ipomea batatas
32

 

 

(variety Gold Coast) which had absorbed phosphorus through their root
systems were =.;Ot leached of the tracer under the water-mist treatment.

Root and non-root absorbed radioactive potassium42 was recovered

in greater quantities from the leachate of Phaseolus vulgaris (variety Cran—

 

berry), Euphorbia pulcherima (variety Albert Ecke), and Ipomea batatas

 

 

my

DALE V. SWEET - 2 ABSTRACT

(variety Gold Coast) when absorption had occurred in the dark prior to the
water-mist treatment. The greatest amount of leaching of radioactive
potassiumé‘2 occurred when the isotope was absorbed in the dark through
the cut stem surfaces of plant materials.

Comparative percentages of the leaching of root-absorbed

rubidium86 from softwood cuttings of Phaseolus vulgaris (variety Cranberry)

 

which had received different environmental conditions prior to a forty-eight
hour mist treatment indicated that the greatest loss of the isotope occurred

86 at full nutrient levels in the dark as

after absorption of the rubidium
compared to low nutrient cultures. Those plants which had absorbed
rubidium86 in the light under either high or low nutrient levels were not

I significantly different in per cent of leaching of the tracer during the water-
mist treatment.

Histological changes were observed between misted and non-
misted cross sections of leaves and stems of plant materials. An absence
of reserve foods in the palisade layers of the leaf sections was accompanied
with early maturation and accumulation of carbohydrate products in the stem

tissues of misted plants as compared to non-misted plants of the same

physiological age.

TABLE OF CONTENTS

 

 

 

 

 

 

Page

INTRODUCTION . . . ........... . . . . . . l
REVIEWOFLITERATURE............... 3
MATERIALANDMETHODS............... 13
A. PreliminaryTrials . . . . . . . . . . . . . . . l3
B.GeneralProcedure 16

C. Leaching Studies with Radioactive Phosphorus32 . . 20

l. Rosa odorata (stem absorption). . . . . . . . 20

2. Phaseolus vulgaris (root absorption) . . . . . 21

3. Phaseolus vulgaris (stem absorption). . . . . 23

4. Ipomea batatas (root absorption). . . . . . . 24

D. Leaching Studies with Radioactive Potassium42 . . . 25

l. Phaseolus vulgaris (stem absorption in darkness) 25

2. Phaseolus vulgaris (stem absorption in daylight) 26

3. Phaseolus vulgaris (root absorption in darkness) 27

 

4. Euphorbia pulcherima (root absorption in darkness) 27

 

5. Ipomea batatas (root absorption in daylight). . 28

E. Leaching Studies with Radioactive Rubidium86. . . 29

 

l. Phaseolus vulgaris (different environmental condi-

 

tions)................ 30

F. Histological Studies. . . . . . . . . . . . . . . 32

ACKNOWLEDGMENT

The author wishes to express his sincere appreciation to
Dr. Charles L. Hamner for his kind guidance and enthusiastic en-
couragement during the course of this investigation.

Thanks are due to Dr. H. B. Tukey for his valuable
advice and suggestions.

The author also expresses appreciation for the generous
help and advice of Dr. L. W. Mericle, Dr. William G. Long, and

Dr. R. F. Carlson in the development of the thesis.

1.

INTRODUCTION

Fluctuations of nutrient levels within the plant has been under
careful investigation since the time of Leibig. The causes of'nutrient im-
balance have occupied a large part of the scientiﬁc literature.

During the last one hundred years, suspicion of nutrient losses
from plants due to rain, fog, or dew has been suggested from time to
time. These investigators have attempted to gather the leachates from
trees, or separated leaves exposed to excessive moisture conditions, to
determine the extent of this loss.

Evidence of foliar absorption of nutrients practiced in recent
times may further support these theories. The fact that plants can absorb
minerals through their leaf surfaces, suggests the possibility of certain
mechanisms, which would allow the leaves to lose nutrients under periods
of precipitation.

Gradual adjustment of plants to these environmental conditions
is shown by observations of leaf morphology of rain forest foliage compared
to the structural arrangement of cell types in the leaves of plants native to
regions having normal amounts of rainfall.

Recently, new attention to the loss of nutrients from plant foliage

by leaching has developed with the use of continuous or intermittent sprays

'r.

.4 . I. .I
.
. . L
'i\ '.-. L J. .

 

in the vegetative propagation of plants. This new method for the root-
ing of cuttings accelerates root growth and increases the number of roots
in certain species to a greater extent than that which is possible with
similar cuttings rooted under high humidity conditions.

However, nutrient deﬁciency symptoms on mature leaves growing
under mist treatment and chlorosis appearing in the young leaves of newly
rooted plants are commonly observed when cuttings are rooted under
mist.

The purpose of this investigation was to determine, by the use
of radioactive tracers, the leaching of certain nutrients from plant foliage
during periods of mist. Histological studies of leaf and stem tissues of
certain plants were used to observe any morphological changes which
took place in the tissues, and which function to increase or decrease the

leaching of nutrients during periods of mist.

. .

.\

'."_C

 

REVIEW OF LITERATURE

During the latter part of the nineteenth century, the ash con-
tent of plants was actively investigated in an effort to determine the exact
fertilizer needs of important field crops.

German chemists noted apparent differences in total ash con-
tent of plants in various stages of growth. Immature plants increased in
mineral content until the onset of ﬂowering. Following the vegetative con-
dition in which highest activity of nutrient absorption decreased was a
period of loss in ash content representing up to fifty per cent of the total
amount of some elements at the time of harvest.

Investigators attempted to explain this phenomenon in various
ways. Outstanding among these concepts, as reviewed by Mes (17), was
the theory of Wilfarth, Roemer and Wimmer (26), who suggested that as
the plant matured, the surplus minerals not needed in metabolism includ-
ing certain nitrogenous materials used by the plant during active growth,
were translocated through the stems and roots and "excreted" into the soil.
Other workers considered that the loss of leaves due to diseases, insects
or defoliation were responsible for a decreased mineral content at maturity.

Kent and Patrick (1 l) grew corn from the green mature to the

mature stage, analyzing samples at four different times. Ash, fat, fiber

and compounds containing nitrogen decreased in the leaves, husks, and
stalks. The kernels increased in all these constituents. Although no
rain fell during the entire period, considerable dew was evident. There
was a certain amount of mechanical loss due to defoliation in harvesting
the crop.

LeClerc and Breazeale (14) were the first to conclude that
nutrients were washed from plants by the action of rain or dew. In a
series of controlled greenhouse experiments with potted plants of wheat,
barley, rice, oats and potatoes, they attempted to show through elaborate
sampling methods that the nutrients moved upward, not downward, or
were translocated to reproductive structures or growing points during the
growth processes. The analyses seemed to demonstrate that as the part

of an organ dies, the nitrogen and phosphoric acid tend to move from the

dying organ into the living tissues and toward seed producing areas, where- ‘

as potassium moved only into the living stems and leaves. The upper
nodes were found to be richer in ash constituents than the lower nodes. It
was believed that if a downward movement of nutrients had occurred in
the plant, the lower portion should increase in mineral content. There
was no evidence of increased nutrients in the root area from ﬂowering

to harvest.

Convinced that the loss of ash constituents was not caused by

 

t

.

 

 

the downward translocation of materials, that is, a physiological process,
LeClerc and Breazeale carried out experiments on cereals and potatoes
to observe the effect of natural and artiﬁcial rains, and short time soak-
ings on plant parts. Results of these experiments indicated losses of
plant constituents, which usually increased with increasing maturity, from
full bloom to the time of maturity. The exposure of plants to natural and
artiﬁcial rains had no appreciable effect upon the loss of nitrogen during
the early stages of plant growth, but as the plants approached maturity or
at the dead ripe stage, as much as seven per cent of nitrogen was lost.
When plants were in the ﬂowering stage, phosphoric acid was not always
leached; however, when the plants were in the mature stage, losses up to
thirty- six per cent of phosphoric acid occurred following leaching. Approx-
imately ﬁfty per cent of the potassium, soda, magnesia, and chlorine
which was present in plants at the time of heading, was washed from the
plants by the action of rain. In the case of continued rains, administered
intermittently, most of the leaching usually occurred during the ﬁrst rain-
fall.

The investigators ﬁnally concluded that a portion of the nutrients
used by the plants are returned to the soil, but no complicated physiological
process is involved. Nutrient losses occurred by the simple “dissolving"

action of rain water on the salts which have been "exuded" upon the surface

 

 

of leaves. They calculated that the analysis of mineral constituents of
harvested plants are not a true estimate of the amount of nutrients taken

up by the plants from the soil, because some leaching may have taken place
at earlier periods or in larger amounts than others.

This evidence appeared to end the active research on leaching
for nearly two decades. Mann and Wallace (16) reported on some deficiency
symptoms appearing on the leaves of Coxes Orange Pippin apple trees.
Purple bloches eventually became necrotic and defoliation followed in severe
cases. These indications of leaching were presumed to have been caused by
an extremely wet summer of 1924. They were able to duplicate the results
artificially the following summer by immersing the apple leaves in cold
water for short periods of time. Analyses of the leaves indicated losses in
dry matter of 30 per cent, ash content of 56 to 60 per cent, and 80 to 90
per cent of the potassium. By limiting the amount of soaking, the leaves
would return to a clear green color.

Lees (15) observed that the previous season's rainfall may have
affected apple yields. The leaching of nutrients from the leaves may have
had some inﬂuence upon the subsequent formation and development of ﬂoral
primordia and this affecting fruiting.

Arens (1), working with the apple, found that the leaves which

were soaked for twenty-four hour periods would lose the equivalent of fifty

 

 

 

 

per cent of their total ash content, depending upon the environmental
factors and the species of the plant. The action was decreased by the
use of wax coatings over the leaves. He concluded that excretion was
controlled by the epidermal cells, explaining that the phenomenon was
an ec010gical- geographical function.

Lausberg (13), by arranging daily soakings of living material
over a period of eighteen days, found that the total excretion in the case
of potassium and calcium amounted to two and three times that quantity
left in the leaves at the conclusion of the experiment. Soft rains re-
moved more than hard rains. The adaxial surface excreted larger
amounts than the abaxial epidermis. Mature plants lost more nutrients
than those in active growth.

The composition of rainfall collected underneath the crowns
of forest trees was investigated by Tamm (22). He reported that large
amounts of organic matter, calcium, potassium and lesser quantities
of phosphorus and nitrogen were found in the leachate as compared to
rainfall collected in an open field.

Mes (17) reported extensive experiments on potted plants of
tobacco, tomato, potato, bean, pea, and maize plants which were sub—
jected to natural and artificial rain and soaking of leaves for very short

periods. The plants were grown under several conditions of controlled

   

 

light and temperature. Radioactive phosphorus32

was absorbed through
the root systems of certain plants.

She was unable to show any connection between the proportion
of ash contained in plants and their size or weight. Those plants protected
from rain contained more ash than exposed plants. However, natural
rainfall, supplemented by artiﬁcial rains did not further increase the
amoxmts of nutrients lost from plants. Leaves of plants growing in water
culture which were soaked for ten minutes, released about ten per cent
of the phosphorus32 which had previously been absorbed by the plant. No
correlation existed between the amount of the isotope taken up by the
plants and the quantities released by rain or by the soakings. The amount
of "recretion" showed a closer relationship to the time of year than to the
total uptake.

Mes used the term "recretion" as deﬁned by Frey-Wyssling (7),
stating that it refers to the elimination of substances which have not been
assimilated or changed in metabolism of plants, namely mineral nutrients
that are excreted by tuttation and glands, insoluble calcium compounds in
cells, silica and other deposits capable of being washed out of cell walls.
On the other hand, “secretion“ should be only organic substances and enzymes

which are direct products of assimilation, and "excretion" is named for

dissimilation products of metabolism.

 

The theory developed by Mes of recreted substances was based
on the work of Strugger (21), who found that ﬂuorescence dyes such as the
sodium salt of oxypyrenetrisulphonic acid and berberine sulphate were not
transported from cell to cell, but were observed to be rapidly transported
upward through the cell walls while they moved into the cells at a slower
rate. The velocity of transport depended upon the intensity of the trans-
piration. The dye accumulated in the epidermal cell walls or hairs at
the surface. As the water moved out of the plant leaf by evaporation, the
dye particle was left behind on the surface.

These spots could be located under ultra violet light with the
use of a ﬂuorescence microscope. She claimed that there was no reason
to doubt why mineral salts, traveling upward in the intermicellular spaces,
could not move in a similar fashion to the dye particle, eventually accum-
ulating in the epidermis and cuticle. Thus, the minerals may be recreted
by the action of rain or dew. No mention was made of the method by which
the organic substances reported in the literature to be leached from plants
were lost.

Will (27) studied the loss of mineral nutrients from tree crowns
by rain. Considerable amounts of sodium, potassium and magnesium with
lesser quantities of phosphorus and calcium were found to be present in

collected water.

10.

Dalbro (5) recently has calculated the loss of nutrients from
mature apple trees during one season of rainfall to be approximately 800
to 900 pounds to the acre. Approximately ten pounds is calcium, ten
pounds is sodium, twenty to thirty pounds is potassium, and the balance
of the loss is organic matter.

As far as can be determined, few reports have appeared in the
literature to date, speciﬁcally outlining the loss of nutrients due to leach-
ing of plants under the mist method of rooting.

The fact that mist propagation has been slow to develop may
account for the absence of information on the leaching of nutrients from
cuttings. Although Raines (l9) mentioned the unsuccessful efforts of
Spencer to root cacao cuttings under mist in 1936, it was not until O'Rourke
(l8) and Watkins (24) introduced suitable apparatus for the use of mist that
this method of rooting was brought to the attention of propagators.

The present method for the rooting of cuttings under mist does
not differ widely from the humidiﬁcation system which has been in use for
many years. Both methods require similar equipment. The mist treatment
involves the use of an atmosphere of excessive moisture beyond saturation,
and provides a constant film of water to the leaf surface. The mist prevents
excessive transpiration from the cuttings, and also induces a cooling effect

upon the foliage. This mist treatment may permit greater exposure to light

 
 

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11.

than would be possible by humidiﬁcation which requires a closed system.

Consequently, the physical conditions involved in mist spray
may be similar to the soakings, immersions, and soft rain applications
which have been reported previously in the literature to be the causes
of nutrients lost from the foliage of plants.

Evans (6) reported that cacao cuttings under mist developed a
yellowish green appearance. He associated it with a slow breakdown of
chlorophyll. Analysis of the leaf tissues showed that nitrates and phos-
phates were leached during the first two weeks, while potassium was
lost constantly from the foliage.

Snyder (20) mentioned a yellowish- green appearance on the new
gowth of Taxus cuttings rooted under mist; that less leaching occurred
when mist was applied intermittently.

Wells (25) noted that excess water used with constant mist did
not appear harmful to cuttings. It caused accelerated rooting and improved
the root system, provided that good drainage was secured to remove the
excess water. However, he points out that various plants respond differently
to the mist treatment. Varieties differ in their ability to remain alive under
extreme moisture conditions.

Recently, many modifications in the application of mist to cuttings

have been developed, such as intermittent systems (3), timing devices (8),

 

 

 

12.

and the electronic leaf (9) for improving the rooting of cuttings. Little
effort has been made to analyze the plant parts histologically to deter-
mine the effects that excess applications of water in the form of mist

may produce in the cuttings during the rooting period.

 

 

 

MATERIALS AND METHODS

A. Preliminary Trials

 

During the summer of 1953, an open-air mist propagation
system was installed at the Michigan State University horticultural farm
at East Lansing, Michigan. Cuttings of East Malling 11, VII and IX apple

rootstocks and Prunus mahaleb cherry clones were placed in beds to ob-

 

serve rooting conditions under continuous mist.

Probelms such as air circulation, exposure of the cuttings to
full sunlight under mist, drainage, and the leaching of nutrients were
studied. Side screens were erected to control wind currents which caused
drifting of the mist away from the cuttings, while exposing them to the full
sunlight. Under these conditions, leaf burning and defoliation of the cut-
tings resulted. Drainage of excess water from the mist beds was accom-
plished by installing propagation boxes containing quarter-inch hardware
cloth on the under side, allowing the water to drain from the medium.

As a result of the mist treatment, nutrient deﬁciency symptoms
were observed on the leaves of cuttings during the period of rooting. Brownish
spots appeared on apple foliage, which in severe cases developed into black
necrotic areas covering one—third of the entire leaf blade (Figure l). ‘Defol-
iation resulted when the cuttings were left under mist conditions for extended

periods. Removal of the new plants from the mist to pots of soil immediately

13.

 

 

 

Figure l. The toxic effect of a prolonged mist treatment on

the leaves of Mallng 11, VII, and IX rootstocks.

 

 

after rooting controlled this condition. If the transplanted cuttings were
not protected from full sunlight for a period of three days after potting,
the leaves wither and turn brown. Defoliation did not always occur in
these cases; the dried leaves sometimes remained on the stems. When
the transplants were shaded from the full sunlight for three days, they
initiated new growth and the old leaves remained green and intact upon
the stems in high light intensities.

Other trials of £118 serrulata (variety Kwanzan) and %
odorata (variety Better Times), which were rooted under mist in green—
house benches in the summer required shading from full sunlight for a
three day period after transplanting. When direct sun was allowed to
shine on the leaves for a few hours, extreme leaf burn appeared during
the ﬁrst three days. Subsequent sunlight of any intensity failed to cause
burning of leaves or necrotic areas. Where the shading technique was
employed, the old leaves remained green and intact on the young growing

plants.

15.

 

 

 

16.

B. General Procedure

 

It was suggested that work be conducted with radioactive mater-
ials in an effort to determine if certain essential nutrients were leached
from the leaves of plant materials under the mist method of propagation.

Certain advantages could be gained by the use of isotopes in
the investigation. Comar (4) states that when a tracer is introduced into
a plant, it will distribute itself in the proportion to the stable form present
in the organs of the plant. Simple measurements of its activity give more
information in a comparatively short time than could be obtained in a more
difﬁcult and often impracticable chemical analysis. Within this particular
study, the fact that the period of entry of the isotope into a plant can be
controlled, allows the investigator to determine the leachability of a nutrient
in relation to the time and place of absorption. In addition to this, minute

quantities of the isotOpe may be measured with considerable accuracy.

32 42

Accordingly, radioisotopes phosphorus , potassium , and
rubidium86 were selected as tracers for the leaching experiments. Addi-
tional studies on the histological sections of leaf and stem tissues of the
treated and non-treated plant materials were undertaken.

Plant materials used for the investigations were either secured

from existing stock in the plant science research greenhouses or were grown

from seed and moved into the experimental greenhouses when they were

 

 

 

l7.

needed. Softwood cuttings of Rosa odorata (variety Better Times) were

 

taken from ﬂowering wood of greenhouse stock plants. Garden bean seeds

Phaseolus vulgaris (variety Cranberry) were germinated and grown in

 

four-inch clay pots ﬁlled with washed white quartz sand”. These were
fed Hoagland's complete nutrient solution (10) twice each week until
ready to use, unless special nutrient solutions were required. When
growing plants for speciﬁc nutrient cultures, the necessary concentra—

tions were fed to the different groups. Sweet potato, Ipomea batatas

 

(variety Gold Coast) were grown from cuttings. These were planted in
five-inch clay pots containing white quartz sand and supplied with nutrient

solution once each week. Poinsettia plants, Euphorbia pulcherima (variety

 

Albert Ecke) were rooted from greenhouse stock plants. They were
planted in four-inch pots containing quartz sand, and kept in active growth
with nutrient solution. The materials used for the histological studies

were taken from leaf and stem sections of cherry, Prunus mahaleb (clone

 

3-16), and apple, Malus sylvestris (East Malling IX) obtained from propa—

 

gating stock on the University farm.
The misting apparatus for the radioactive work was constructed
of chemically-treated wood—frame, three feet wide by four feet long and
four feet high. It was covered inside and outside with 0. OOS—inch polyethylene
ﬁlm. A hinged door, twelve inches high, covered the entire front of the

—~—¢--‘-——-_.———~———o

*American Graded Sand Company, Chicago, Illinois

a

vr

 

 

18.

frame and ﬁtted securely to prevent any mist from escaping. Inside the
gable of the mist chamber, a series of three atomizer nozzles were attached
to a three-eighth inch galvanized iron pipe line, which was connected to the
compressed air supply. The nozzles were directed downward to stainless
steel tanks, eight inches wide by twelve inches long and sixteen inches

high for housing the treated plant materials. The tanks were open at the

top for receiving the mist spray. Pyrex tubing was connected to the drain-

 

age nipple on each tank. The tubing passed through the mist chamber to
the collecting carboys below. Any mist which was not conﬁned to the steel
tanks within the mist chamber was collected in another carboy for in-
spection.
The leachate was passed through ion exchangers to separate the
isotope from the solution. Amberlite IR-120 and Dowex 2X-10, 50 to 100
mesh were used in 10 x 300 mm pyrex chromatographic tubes. The ﬁnes
were removed by the decanting method and the resin prepared according to
company directions. To increase the ﬂow of liquid through the cation and
anion tubes, a water suction pump was used to facilitate the movement of
the leachate through the columns. A similar apparatus was used for regen-
eration with six per cent hydrochloric acid and ﬁve per cent sodium hydroxide
or ammonium hydroxide, depending upon the tracer elution from the columns.
Dry weight of oven—dried leached and unleached plant materials

was determined to the nearest tenth of a milligram.

 

 

Wet ashing of the plant materials followed the method described
by Toth gt a}: (23) with certain modiﬁcations. Ten milliliters of concentrated
nitric acid were added to each beaker containing dried substance. The
beakers were covered with watch glasses and were heated on the steam
bath under a hood until digestion was complete. Two and one-half milli-
liters of seventy per cent perchloric acid were added to each sample for
further clearing. They were heated gently until the liquid was evaporated
to one to three milliliters in volume. The solutions were transferred to
numbered twenty-five milliliter volumetric ﬂasks using three per cent
hydrochloric acid for washing and bringing the liquid to volume.

Triplicate one milliliter aliquots were lifted from each ﬂask
with pipettes into one-inch porcelain planchets. The aliquots were dried
on a simmer hot plate under the hood.

All counts were made with a Model SC 1 B Autoscaler (T racerlab)

2 mica end-window infinite life Geiger-Mueller

equipped with a l. 4 mg/ cm
tube (Nuclear) in a Mark 3 Model 15 (RCL) iron sample chamber. A model
P-lO (Tracerlab) preamplifier was used in the imput circuit to the G. M.

tube. The counts were taken in triplicate and averaged. The standard

deviation was less than ten per cent.

19.

 

   

C. Leaching Studies With Radioactive Phosphorus32

 

Experiments using radioactive phosphorus32 were conducted

separately on Rosa odorata, Phaseolus vulgaris, and Ipomea batatas

 

plant materials to determine the amount of the isotope which could be re—
moved from the foliage under a water-mist treatment, similar to the
mist method used in the rooting of cuttings.

l. Ro_sa (M (stem absorption)

Twenty softwood 3E odorata cuttings (variety Better Times)
were taken from ﬂowering wood of stock plants in the (greenhouse. They
were selected for uniformity and were trimmed to four lateral leaﬂets
on each of two nodes.

Phosphorus32 isotope, amounting to 0.01 microcurrie, was
added to 100 milliliters of distilled water solution containing 681 milli-
grams of KH2P04. The solution was placed in a SOC-milliliter beaker
which was kept inside another container for safe handling. An "airstone"
aerator was inserted into the solution. It was attached to the compressed
air line by a pyrex tubing for bubbling air through the solution.

The basal portions of the cuttings were immersed the length
of two centimeters into the solution contained in the beakers.

The cuttings absorbed the liquid for a period of forty-eight

hours, after which they were trimmed. The two lower leaﬂets were

20.

 

 

 

removed from the lower petiole and three centimeters of the basal stem
were cut off to eliminate contaminated parts. The cut surfaces were
dried on the open bench for two hours to prevent exudation into the leachate.

The prepared cuttings were divided into two groups; ten were
placed in the mist chamber for treatment and the remaining cuttings were
secured into paper bags as non-misted control plants.

The mist-treated cuttings were removed after forty-eight hours
from the chamber. Each plant was placed in a paper bag. All plant mater-
ials were oven—dried for weighing.

The total leachate collected in carboys during the misting period
was passed through cation and anion resin tubes to separate the phos—
phorus32 from the collected water. The elution was removed from the
resin column with six per cent hydrochloric acid until the monitor failed
to record any tracer in the tube. The solution was brought to standard
volume in a ZOO-milliliter ﬂask and amount of radioactive phosphorus32
determined.

2. Phaseolus vulgaris (root absorption)

 

An attempt was made to determine if phosphorus32 could be
leached from the foliage of bean plants when the active solution was ab-
sorbed through the root systems before the cuttings were placed under

mist.

21.

 

 

 

 

22.

Six pots of Phaseolus vulgaris (variety Cranberry) containing

 

two plants in each pot, were selected from plants receiving bi-weekly

applications of one-half concentration of nutrient solution. The plants

were selected for uniformity of growth with the first trifoliate leaf ex-
tending two inches beyond the primary leaf nodes.

The six pots of plants were placed in individual enameled
containers on the experimental greenhouse bench. Ninety milliliters of
the active solution, containing 0. 01 M KH2P04, and 0. 19 microcurries
of radioactive phosphorussz, was applied to the surface of the sand in
each bean pot. The plants had been previously watered, leaving them
at approximate ﬁeld capacity with the addition of the active liquid.

During the absorption period, the sand was moistened with
distilled water sufficiently to maintain turgidity in the plants.

Forty- eight hours later, one cutting from each plant was
taken from the pots above the cotyledonary nodes. The basal stems were
dipped in warmed paraffin to seal the cut surfaces. One cutting from each
pot was placed under mist in the chamber for a period of forty—eight hours.
An effort was made to secure an even ﬂow of mist upon the plant material
at all times.

After the period of misting was completed, the treated plants

were removed, bagged, and oven-dried with the non-misted cuttings taken

 

 

23.

from the second plant in each pot.
Later, individual plants were weighed, wet—ashed, and tri-
plicate aliquots prepared and counted.
The leachate was passed through resin columns. The elution
was checked as before for radioactivity.
3. Phaseolus vulgaris (stem absorption)

It was thought possible that phosphorus32

which had previously
been absorbed through the cut stem surfaces of cuttings, could later be
leached-from the foliage of garden beans when placed under mist condi—
tions.

Ten Phaseolus MES (variety Cranberry) seedlings were
selected for uniformity in weight and habit of growth. Cuttings were
taken one-fourth inch above the cotyledonary nodes and placed immediately
in the active solution which contained 0. 01 M KH2P04 with 0. 2 micro-
curries of radioactive phosphorus32.

Forty-eight hours later the cuttings were removed. The basal
portions were trimmed to prevent contamination of the leachate. The
stems were dipped in warmed parafﬁn to limit exudation of plant juices
into the leachate from cut surfaces.

Five cuttings were placed under mist for forty—eight hours.

The leachate was collected in glass carboys.

 

 

24.

After the mist period, all plants were secured in paper bags
and oven-dried.

The leachate was passed through resin columns and the eluate
brought to standard in a volumetric ﬂask.

The plant materials were weighed, wet ashed, and assayed for
radioactive phosphorus32 in the usual manner.

4. Ipomea batatas (root absorption)

 

 

An active solution of 100 milliliters containing 0. 2 microcurries

of radioactive phosphorus32 in 0. 01 M KHZPO solution had been applied

4

to the sand surfaces of two pots of Ipomea batatas (variety Gold Coast) for

 

a period of fifteen days. The G. M. monitor recorded high activity in
all leaves and growing points of the plants.

After the absorption period, four mature leaves with petioles
attached and five-inch growing point were selected from each plant. The
basal portions of the petioles and stems were dipped in warmed paraffin.

The leaves and growing point from one plant were placed under
mist for a period of twelve hours. The other plant materials were reserved

as controls.

 

D. Leaching Studies with Radioactive Potassium42

 

In reviewing the literature on potassium lost from the foliage
of plants by the action of rain or dew, it was decided to investigate the
loss of potassium from cuttings which were placed under mist with the
use of potassium‘}2 as the tracer. Because of the short half-life of
this isotope (12. 4 hoursT 1/2), it was suggested that the experiments
be limited in'duration, and that the analysis be confined to necessary
detail. Consequently, the plant materials were treated in groups and
the data gathered using shortest possible procedures.

When the active material arrived, the solutions werepre-
pared simultaneously. The three liters of O. 01 M KH2P04 solution con-
tained 42. 5 millicurries of potassium42. The reference solution was

checked on the Autoscaler to be 325, 91? counts per minute of the tracer.

l. Phaseolus vulgaris (stem absorption in darkness)

 

Twenty cuttings of ten-day Phaseolus vulgaris plants, variety

 

Cranberry, were selected for uniformity in weight and habit of growth.
They were placed in a beaker containing 250 milliliters of potassium
solution as described. The cuttings were allowed to absorb the solution
in the dark until the leaves were found to contain high activity’l‘. The

basal stems were trimmed and sealed with warmed paraffin. The cuttings

——————————————_——~_———.-

*10, 000 cpm by G. M. monitor reading.

25.

 

 

 

were divided into two equal weight groups, one-half of them were placed
under mist for three hours, and the other half reserved as controls.
After the mist treatment, the plant materials were taken to
the laboratory for analysis. Each group was placed in 100 milliliter
beakers and digested with twenty milliliters of nitric acid. One milli-
liter aliquots were placed in planchets, dried and counted when cool.
Five liters of the leachate was collected from the chamber.

It was passed through ion exchangers, aliquots taken and counted.

2. Phaseolus vulgaris (stem absorption in daylight)

 

An additional twenty Phaseolus vulgaris (variety Cranberry)

 

cuttings were taken early in the morning, placed in an absorption beaker,
and allowed to take up the active solution of potassium42 during daylight
hours. In the evening, ten cuttings were trimmed and sealed with par-
affin in the usual manner. They were placed under mist for a period
of three hours.

The treated and the control cuttings were taken to the labora-
tory for analysis. Plant materials were digested with nitric acid, ali-
quots prepared and counted. Seven liters of leachate were passed

through ion exchangers, and the elution counted in the usual manner.

26.

 

 

 

 

 

3. Phaseolus vulgaris (root absorption in darkness)

 

Ten pots of Phaseolus vulgaris (variety Cranberry), each one

 

containing two plants, were placed in a dark room in porcelain containers.

42 solution were applied to

Fifty milliliters of the radioactive potassium
the sand surfaces of each pot. After sixteen hours, cuttings from the
plants were taken above the cotyledonary nodes. The basal stems of ten
cuttings were sealed with liquid parafﬁn and placed in the mist house

for three hours. Eight liters of the leachate were collected and taken

to the laboratory with the plant materials for analysis. The two groups
of cuttings were digested with nitric acid, aliquots prepared and counted.

The cations were collected in the resin and the elutate brought to volume.

One milliliter aliquots were taken and counted in the usual manner.

4. Euphorbia pulcherima (root absorption in darkness)

 

Eight Euphorbia pulcherima plants were selected for uniform

 

size and shape. They were placed in porcelain containers in a dark room.

Fifty milliliters of the active potassium‘j:2 solution were applied to the
sand surfaces of each of the pots. The plant roots remained in the tracer
for eighteen hours.

Four cuttings from the treated plants were taken of equal size,
their basal stems sealed in warmed parafﬁn and divided by weight into

two groups. Two cuttings were placed under mist for three hours.

 

 

 

 

 

 

 

 

 

28.

All cuttings were prepared for analysis. One milliliter aliquots were
placed in planchets, dried and counted. The eight liters of leachate col-
lected were passed on cation resin, and the elution was brought to volume.

Aliquots were taken, dried and counted.

5. Ipomea batatas (root absorption in daylight)

 

Ipomea batatas plants (variety Gold Coast) growing in ﬁve-inch

 

pots were used for the studies. Two pots were placed in porcelain con-

. . . . 42 . .
tainers to prevent the radioactive potaSSium from contaminating the

 

greenhouse bench. One hundred milliliters of potassium42 solution were
applied to the sand surface of each pot.

When 10, 000 cpm activity was monitored in the leaves of the grow-
ing plants, leaves with petioles attached and growing points were selected
for their physiological age and uniformity and cut from the vines. All cut
surfaces were sealed with warm paraffin.

Cuttings were divided into two equal groups, based on their green
weight, and selected for the experiment. One group was placed under mist

for three hours, and the other remained as controls. Eight liters of leachate
were collected in carboys.

All materials were removed to the laboratory for analysis. The cut-
tings were digested with nitric acid, aliquots taken and counts recorded. The
leachate was passed through a cation column. The cations were removed and

brought to volume. One—milliliter aliquots were placed in porcelain planchets,

dried and counts recorded.

 

E. Leaching Studies with Radioactive Rubidium86

 

In reviewing the results of the previous experiments, it was
considered probable that the amount of nutrients lost from plant parts
subjected to mist treatment may be associated with environmental
conditions, such as the absorption of the isotope by the plant while
growing under normal daylight conditions, or placed in continuous
darkness, and also, may be dependent upon the concentration of min—
erals taken up in the soil solution.

Accordingly, an experiment was planned in which plants
could absorb two levels of nutrient solution both in normal daylight
and in continuous shade for forty-eight hours prior to placing the cut-
tings under mist.

Because of the difficulty of working with radioactive potas-
sium‘,‘2 over an extended period, radioactive rubidium86 (l9. 5 daysT 1/2)
was selected for the tracer. It was assumed that the metabolic reactions
involving either of the two univalent cations were of a similar nature

within the plant.

29.

 

 

 

 

l. Phaseolus vulgaris (different environmental conditions)

 

Phaseolus vulgaris (variety Cranberry) were germinated and

 

grown without nutrients added in clay pots filled with white quartz sand
until the primary leaves were fully expanded. The plants were divided
into two groups on the greenhouse bench. One group was fed a complete
Hoagland's nutrient solution. The other pots received one part Hoagland's
solution to nine parts distilled water.

When the plants were fourteen days old, the first trifoliate
leaf began to expand rapidly. Their roots were washed free of sand and
the plants were placed in water-culture containers which held seven
liters each of their corresponding levels of nutrient solution. Sixteen
crocks containing four plants each were placed in two rows of eight crocks
on the experimental bench. All cultures were equipped with aerators.
The plants were fitted in the top plate with styrofoam fasteners, allowing
the root systems to be completely covered by their solutions.

The crocks were randomized in each row which contained four
solutions of high level nutrients and four of low level water cultures. A
black shade cloth was drawn on wires over one row to produce continuous
darkness. The other row of bean plants received the normal daylight.

All plants were allowed to adjust to their solutions for twenty-

four hours. After the period, 100 milliliters aliquots of radioactive

30.

 

 

   

 

rubidium86, containing Rb2C03 as a carrier, were added to each solution.
The absorption period continued for forty-eight hours.

Eight cuttings were taken from each of the four treatments.
The cut surfaces were dipped in warmed paraffin to prevent exudation
into the leachate. Each group received four hours of mist spray and the
separate leachates were collected in glass carboys.

Similar groups of cuttings were taken from the remainder of
the plants at the same time as controls.

The leachates were passed through resin columns and the
cations were removed to volumetric ﬂasks. Three aliquots of each sam-
ple were placed in porcelain planchets for counting.

All groups of plant materials were oven-dried, weighed, and
wet-ashed with nitric acid. Aliquots were taken in triplicate for count- .

ing.

31.

 

 

32.

F. Histologcal Studies

 

Samples were taken for histological preparations of treated and
control plants which were identical in physiological age. Two cuttings

of garden bean, Phaseolus vulgaris (variety Cranberry) were placed

 

under constant mist. Two cuttings of the same group of seedlings, sel—

ected for like habit of growth, were placed in a beaker of distilled water

 

under the same temperature and light environment. After ﬁve days, two
square centimeter samples of treated and non-treated leaves were placed
in formalin, acetic acid, and alcohol (F. A. A. 1:1:18) killing and ﬁxing
solution. Similarly, samples of misted and non—misted apple leaves,

Malus sylvestris (Malling IX), and cherry leaves, Prunus mahaleb (clone

 

 

3 - 16) were placed in F. A. A. solution after a mist period of twenty-
ﬁve days.

All solutions were immediately taken to the laboratory and
aspirated to remove air from the tissues. Dehydration was accomplished
through the ethyl-tertiary—butyl alcohol series and the tissues were in-
ﬁltrated and imbedded in Fisher's tissuemat.

The paraffin blocks were sliced at sixteen and twenty microns
thickness on a hand-operated rotary microtome. Sections of misted and
non-misted tissues of each plant species were mounted on the same glass

slides. The inﬁltrated agent was removed. The slides were passed

 

 

33.

through Conant’s quadruple staining procedures. Permanent mountings
were accomplished with clarite under cover glass.

Microphotographs were made from Adox KB - 17 film using
Ethol ultra ﬁne grain developer. Contrast of tissues was improved

with the aid of an orange gelatin ﬁlter.

 

 

 

 

RESULTS

I. Radioactive Phosphorus32

a. Rosa odorata (stem absorption)

 

32 was leached from

It was evident that radioactive phosphorus
rose cuttings which were placed under a mist of distilled water for a
period of forty-eight hours. Upon examination, the leachate was found
to contain the isotope which was originally supplied to the cuttings be-
fore treatment (Table I).

A wide variation existed in the amounts of the tracer actually
absorbed by individual cuttings regardless of their individual weights

(Table 1). However, when the amount of phosphorus32

recovered from
the leachate was compared with the total amount of tracer absorbed by
the plants for forty-eight hours before the mist treatment, the result

indicated a considerable loss of the nutrient. The radioactive material
recovered in the leachate amounted to 12. 8 per cent of the total isotope
absorbed by the treated plants. This was obtained by adding the tracer

recovered in the leachate to the amount remaining in the plant materials

(Table II).

34.

 

 

 

35.

TABLE I

32
The Leaching of Non-root* Absorbed Phosphorus from Softwood Cuttings
of Rosa odorata (Variety Better Times) During a Forty-eight Hour Water-
Mist Treatment.

 

 

 

 

 

 

Misted Non-Misted
No. Dry weight cpm cpm No. Dry weight cpm cpm
grams cuttings g. wt. grams cuttings g. wt.
1 0. 751 5530 7364 l 0. 906 6166 6806
2 0. 733 720 983 2 0. 672 4494 6688
3 l. 099 6116 5565 3 0. 794 8919 11233
4 0. 730 2954 4047 4 l. 038 7065 6806
5 0. 917 4860 5300 5 1. 055 7692 7291
6 l. 343 4751 3538 6 0. 981 5136 5235
7 l. 301 7611 5850 7 1. 049 5622 5359
8 0. 822 4304 5236 8 0. 676 3113 4605
9 0. 759 8872 11689 9 0. 768 2491 3244
10 l. 785 10817 6060 10 l. 567 11506 7343

Total 10.240 56535 55632” Total 9.506 62204 64610**

I

1.

*All plant cuttings absorbed radioactive phosphorus32 through the cut
surfaces of their stems for forty-eight hours prior to being placed
under mist for a similar period of time.

**t value equals 0. 8134.

 

 

 

 

36.

TABLE II

The Percentage of Non-root Absorbed Phosphorus32 Leached from Softwood
Cuttings of Rosa odorata (Variety Better Times) during a Forty-eight Hour
Water-Mist Treatment.

f
——:r

 

 

Total cpm Total cpm Per cent isotope

 

 

cp c uttings leachate leachate
Control cuttings 62, 226 l, 555, 650
Misted cuttings 56, 568 l, 414, 200
Leachate (44 l.) l, 039 207, 892
Initial absorption 12. 8

 

 

 

37.

b. Phaseolus vulgaris (root absorption)

 

Analysis of seventy—two liters of leachate collected from plant
cuttings which were held under mist for forty-eight hours indicated that
the loss of phosphorus32 was negligible, when the isotope was absorbed
by the root systems of garden bean plants previous to mist treatment
(Table III).

The average of one milliliter aliquots of a 100 milliliter solu-
tion of the elution from the anion resin column amounted to approximately
two counts per minute over background (Table V). At the same time,
one milliliter aliquots of the reference solution contained an average of
12, 467 counts per minute over background.

Variations occurred in the amounts of absorbed phosphorus
in relation to their gram weights of both treated and non—treated cuttings

(Table III).

c. Phaseolus vulgaris (stem absorption)

 

The analysis of the eluate of forty liters of leachate collected
from the mist spray falling on five bean cuttings over a period of forty-eight

32 was leached from the foliage, when

hours indicated that radiophosphorus
the cuttings had absorbed the isotope through their cut stem surfaces

previous to treatment.

 

 

38.

TABLE III

The Leaching of Root-Absorbed Phosphorus32 from Herbaceous Cuttings of
Phaseolus vulgaris (Variety Cranberry) During a Forty-eight Hour Water-
Mist Treatment.

 

 

 

 

 

 

 

Misted Non-Misted

No. Dry weight cpm cpm No. Dry weight cpm cpm
grams cuttings g. wt. grams cuttings g. wt.

1 . 3090 1714 5547 1 . 2105 1345 6390

2 . 2532 1563 6173 2 . 2085 1651 7918

3 . 2108 1709 8107 3 . 2435 1946 7992

4 . 3358 1860 5539 4 . 2048 1632 7969

5 . 3635 1878 5166 5 . 2443 1630 6672

6 . 1985 1234 6217 6 . 2860 1752 6126
Total 9958 36749“ Total 9956 43067“

 

*t value equals 1. 5320

 

 

 

The actual amount of isotope collected in the leachate was 1. 54
per cent of the total absorption of the tracer in the misted plant materials

(Tables IV and V).

d. Ipomea batatas (root absorption)

 

 

When sweet potato, Ipomea batatas (variety Gold Coast) cuttings,
which had previously absorbed radioactive phosphorus32 through the root
system of the mother vine, were placed under mist, none of the isotope
could be found in the leachate.

One milliliter aliquots, taken from the eluted solution of the
leachate, were monitored by a count rate meter (Nuclear model no. 1615).

No significant activity above background was noted.

39.

 

 

The Leaching of Non—Root Absorbed Phosphorus32
of Phaseolus vulgris (Variety Cranberry) During a Forty-eight Hour Water-

Mist Treatment.

TABLE IV

from Herbaceous Cuttings

 

 

 

Misted Non—Misted

No. Dry weight cpm cpm No. Dry weight cpm cpm
grams cuttings g. wt. grams cuttings g. wt.
1 0. 399 7099 17, 792 l 0. 361 9605 26, 607
2 0. 413 6584 15, 942 2 0. 363 6525 17, 975
3 0. 315 5654 17, 949 3 0. 302 6651 22, 023
4 0. 333 5570 16, 727 4 0. 336 10055 29, 906
5 0. 417 7468 17, 909 5 0. 369 11345 30, 745
Total 1. 877 32375 86, 319 Total 1. 769 44181 127, 276

 

 

 

 

 

 

 

 

41.

TABLE V

Comparative Leaching of Root Absorbed and Non-Root Absorbed Phosphorus32
from Herbaceous Cuttings of Phaseolus vulgaris (Variety Cranberry) During a
Forty-eight Hour Water-Mist Treatment (Percentages).

 

 

 

cpm Total cpm Total cpm Per cent isotope
cuttings leachate leachate

 

Root Absorbed

 

Control cuttings 43, 067 l, 076, 675

 

Misted cuttings 36, 749 918, 725
Leachate (72. l) 0 0
Initial abosrption 0. 0*

Non—Root Absorbed

 

Control cuttings 44, 181 1, 104, 525

Misted cuttings 32, 375 8 09, 375
Leachate (40. l) 505 12, 625
Initial absorption 1. 54

 

 

a:
0. 0 value is not necessarily zero, but is not signiﬁcant above back—
ground count rate.

 

 

 

42
II. Radioactive Potassium

a. Phaseolus vulgaris (stem absorption in darkness)

 

When radioactive potassium42 solution was absorbed through
the cut stems of ten day garden bean seedlings for five and one-half hours
in the dark, ﬁfty-ﬁve per cent of the absorbed tracer was leached from

the foliage within a three hour period of mist spray treatment (Table VI).

b. Phaseolus vulgaris (stem absorption in daylight)

 

Ten day garden bean seedlings which had absorbed potassium‘j‘2

solution through the cut stems under normal daylight conditions for a
twelve hour period before the mist was applied to the foliage, leached
thirty-five and one-half per cent of the tracer from the cuttings during

three hours of mist (Table VII).

c. Phaseolus vulgaris (root absorption in darkness)

 

Phaseolus vulgaris (variety Cranberry) which had absorbed

 

. . 42 . . .
radioactive potassmm through their root systems for Sixteen hours in
the dark, lost nine and two-tenths per cent of the total tracer in the cut-

tings during three hours of mist treatment (Table VIII).

(1. Euphorbia pulcherima (root absorption in darkness)

 

Poinsettia plants in active growth, which had absorbed radio-

active potassium42 solution through their root systems for eighteen hours

42.

 

   

 

TABLE VI

The Leaching of Potassiumlll2 Which was Non- Root Absorbed in the Dark from
Herbaceous Cuttings of Phaseolus vulgris (Variety Cranberry) During a Three
Hour Water— Mist Treatment.

 

 

Total cpm Total cpm Per cent isotope
cuttings leachate leachate
Non-misted cuttings 1580
Misted cuttings 840
Leachate (5 l.) 1050

Initial absorption 55. 5

 

43.

   

44.

TABLE VII

The Leaching of Potassium” which was Non-Root Absorbed in the Light
from Herbaceous Cuttings of Phaseolus vulgaris (Variety Cranberry) Dur-
ing a Three Hour Water-Mist Treatment.

 

 

 

Total cpm Total cpm Per cent isotope
cuttings leachate leachate

 

Non-misted cuttings 48, 475
Misted cuttings 34, 560
Leachate (7 1.) 19, 062

Initial absorption 35. 5

 

 

 

 

 

 

 

 

 

 

45.

TABLE VIII

The Leaching of Potassium42 which was Root Absorbed from Herbaceous
Cuttings of Phaseolus vulgaris (Variety Cranberry) During a Three Hour
Water-Mist Treatment.

 

 

 

Total cpm Total cpm Per cent isotope

 

 

cuttings leachate leachate
Non-misted cuttings 528, 240
Misted cuttings 412, 920
Leachate (8 l.) 41, 750

Initial absorption 9. 2

 

 

 

 

in the dark, lost thirty-one and four-tenths per cent of the tracer from

the foliage when placed under mist for three hours (Table IX).

e. Ipomea batatas (root absorption in daylight)

 

When leaves and growing points of sweet potato vines were
subjected to mist spray for three hours, it was found that they lost two
and eight -tenths per centof the radioactive potassium‘l‘2 which had
been taken up by the roots during the absorption period a few hours

before leaching (Table X).

46.

 

 

 

 

47.

TABLE IX

The Leaching of Potassium42 which was Root Absorbed in the Dark from
Softwood Cuttings of Euphorbia pulcherima (Variety Albert Ecke) During
a Three Hour Water-Mist Treatment.

 

 

Total cpm Total cpm Per cent isotope

 

 

cuttings leachate leachate
Non-misted cuttings 3520
Misted cuttings 2875
Leachate (7 l.) 1316

Initial absorption 31. 4

 

 

 

 

 

 

 

 

 

 

TABLE X

48.

The Leaching of Potassium42 which was Root Absorbed in the Light from
Herbaceous Cuttings of Ipomea batatas (Variety Gold Coast) During a

 

Three Hour Water-Mist Treatment.

 

 

 

Total cpm Total cpm Per cent isotope
cuttings leachate leachate
Non-misted cuttings 47, 100
Misted cuttings 46, 320
Leachate (8 1.) l, 300
Initial absorption 2. 8

 

 

 

 

 

 

 

49. t

86
III. Radioactive Rubidium

a. Phaseolus vulgaris (different environmental conditions)

 

The greatest loss of rubidium86 when mist was applied for

four hours to Phaseolus vulgaris (variety Cranberry) occurred from those

 

plants which were grown in high nutrient levels in darkness for forty-

eight hours prior to the mist. The loss amounted to fourteen and four

 

tenth per cent of the total tracer absorbed. The plants growing under
low nutrient conditions in darkness for forty-eight hours prior to the
mist treatment lost only six and six tenths per cent of the isotope

(Tables XI and XII).

The plants grown under normal daylight during the experi-
mental period had synthesized more dry matter and absorbed more iso-
tope than those grown in darkness. However, they lost only five per
cent of the active element when their cuttings were placed under a four
hour mist. There was no apparent difference in the amount of leaching
between plants grown in high or low levels of nutrients under normal
light conditions (Tables XI and X11).

There were visual differences between the plants growing
Imder the various environmental conditions. The plants growing under

low nutrient levels in normal daylight grew rapidly in size, although

 

 

 

TABLE XI

The Leaching of Root-Absorbed Rubidium86 from Softwood Cuttings
of Phaseolus vulgaris (Variety Cranberry) which had Received Differ-
ent Environmental Conditions Prior to Forty-eight Hours of Water—
Mist Treatment.

 

 

 

 

 

Misted Non-Misted
Dry weight cpm (8) cpm Dry weight cpm (8) cpm
grams cuttings g. wt. grams cuttings g. wt.

 

1. Complete Hoagland's Solution under Continuous Darkness

 

l. 207 75, 000 62, 138 l. 313 112, 600 85, 758

2. One-tenth Hoagland's Solution under Continuous Darkness

 

l. 065 124, 800 117, 174 l. 190 147, 600 124, 034

3. Complete Hoagland's Solution under Normal Daylight

 

1. 850 164, 800 89, 081 1. 814 189, 300 104, 355

4. One-tenth Hoagland's Solution under Normal Daylight

 

2. 026 1, 321, 200 652, 122 l. 832 l, 257, 500 686, 408

 

 

50.

 

 

 

 

TABLE XII

Comparative Percentages of Leaching of Root-Absorbed Rubidium86 from
Softwood Cuttings of Phaseolus vulgaris (Variety Cranberry) which had
Received Different Environmental Conditions Prior to the Forty-Eight
Hour Water-Mist Treatment.

 

 

 

Total cpm Total cpm Per cent isotope
cuttings leachate leachate

 

1. Complete Hoagland's Solution under Continuous Darkness

 

Non-misted cuttings 112, 600
Misted cuttings 75, 000 10, 475 14. 4

2. One-tenth‘Hoagland's Solution under Continuous Darkness

 

Non-misted cuttings 147, 600
Misted cuttings 124, 800 8, 275 6. 6

3. Complete Hoagland's Solution under Normal Daylight

 

Non-misted cuttings 189, 300
Misted cuttings ' 164, 800 4, 575 4. 9

4. One-tenth Hoagland's Solution under Normal Daylight

 

Non-misted cuttings l, 257, 500

Misted cuttings l, 321, 200 33, 275 4. 9

 

 

51.

 

 

 

52.

the trifoliate leaﬂets were light green in color. The plants growing
under high nutrient levels and normal daylight were darker green,
and exhibited less elongation than the low nutrient plants. The plants
growing under continuous darkness grew less rapidly than those under
normal daylight.

Some curling of the primary leaves occurred, perhaps

indicating a loss of carbohydrates.

 

 

53.

IV. Observations from Histological Studies

Cross-sections of bean leaves of plants placed under a continu-
ous mist were compared with those from non-misted plants. The sections
used for the investigation were selected from tissues of the same physiolo-
gical age (Figure 2). Observations were made with special reference to

the relationship of size and number of cell types within a given radius,

 

special arrangement of respective tissues, and general thickness of leaf
blades.

The general size of the various types of cells in the mist-treated
samples were found to be considerably smaller than the cells in the non-
misted control samples. This was especially noticeable in the epidermal
tissue. However, the leaf blade thickness of mist—treated leaves was ap-
proximately thirty per cent greater than the non-misted leaves. Upon
close examination, this apparent difference may be explained by the greater
number of intercellular spaces in the interior of the leaves of the misted
plant. This effect of mist-treated leaves resembled the effect which is
produced when a seed swells from the imbibition of water.

The average number of cells of the respective tissues were
compared, and it was found that within a given area the control section

contained twenty per cent more cells than the treated leaves.

 

 

 

Figure 2. Microphotographs of cross-sections of Phaseolus

 

vulgaris (variety Cranberry) leaves of the same physiolo—

gical age. Top: non—misted leaf. Bottom: leaf given ﬁve

days of water—mist treatment.

 

   

 

54.

 

 

 

The mist-treated sections did not stain as deeply as the control
sections. This was not alone due to the general density of the controls.
When viewed under high power lens, the individual cells were highly
stained in the untreated tissues, particularly in the palisade layers. The
misted tissues were more translucent with the nuclei clearly outlined.

When slides of apple and cherry leaf sections, which were

misted for a period of twenty-five days, were examined under the micro-

 

scope, a similar depth of color was noted inthe control sectiOns as
compared to the treated tissues. The palisade layers of apple sections
of non-misted leaves were dense in color and were regular in form.
Similar layers of mist-treated sections were lightly stained, nuclei
prominent, and the form irregular. The cells appeared swollen and
crowded with few intercellular spaces in the misted sections.

In the non-treated material, the mesophyll cells were well-
spaced, orderly, and distinctly visible. Similar tissue which was sub-
jected to mist appeared swollen, indistinct, and occupied all of the
intercellular spaces.

In general, the thickness of the mist-treated leaves were
equal to the controls. However, with the increase in cell size, the
misted tissuesapparently filled the intercellular spaces causing an ir-

regular pattern in the leaf section (Figure 3).

 

 

 

 

 

Figure 3. Microphotographs of cross-sections of Malus
sylvestris (Malling IX) leaves of the same physiol—
ogical age. Top: non-misted leaf.

Bottom: leaf given twenty-ﬁve days of water—mist

treatment .

 

56.

 

 

 

 

 

57.

This ﬁlling of the intercellular spaces in the mesophyll by
swollen cells was especially noticeable in the sections of mist -treated
leaves of cherry. Palisade tissues in the control sections were deeply
stained as compared with the lightly stained misted tissues. It will be
recalled that both treated and controlled sections were passed through
the staining procedures on the same slide. Also, the phloem of the veins

of the mist-treated materials were generally stained deeply. This was

 

not evident in the control tissues.

Cross-sections of apple stems of identical physiological age
of misted and non-misted plant materials are illustrated in (Figure 4).
A higher degree of maturation was found in the misted stem sections
than in the non-treated stems. This was apparent by the thickening of
ﬁber cells and outer cortical parenchyma tissue. The phloem cells in
the vascular bundles and the vascular ray cells were deeply stained in the
mist-treated material suggesting additional storage of carbohydrate
products in the stems.

Cross-sections of misted cherry stems showed maturation
and heavy carbohydrate accumulation in the vascular tissues and the sur-
rounding cells as compared to non-misted sections which remained in a

general meristematic condition.

 

 

Figure 4. Microphotographs of cross—sections of Malus
sylvestris (Malling IX) stems of the same physiol-
ogical age. Top: non-misted stem.

Bottom: stem given twenty-five days of water-mist

treatment.

 

 

58.

 

 

 

Figure 5. Microphotograph of cross-sections of Prunus
mahaleb (clone 3-16) leaves of the same physiological

age. Top: non—misted leaf. Bottom: leaf given twenty-

five days of water-mist treatment.

 

   

     
 

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Figure 6. Microphotographs of cross-sections of Prunus
mahaleb (clone 3—16) stems of the same physiological

age. Top: non—misted stem. Bottom: stem given

twenty—five days of water-mist treatment.

 

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Figure 7. Microphotographs of cross-sections of Prunus
mahaleb (clone 3-16) stems of the same physiological

age. Top: non—misted stem. Bottom: stem given twenty-

ﬁve days of water-mist treatment.

 

 

 

        

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62.

DISCUSSION

Much of the evidence reported on the leaching of nutrients
from plants by the action of water on plant foliage has been based on
the differences on a per gram dry weight basis of an ash constituent
between the treated and non-treated materials. One exception to this

32 reported by Mes

method was the work with radioactive phosphorus
(17). Her values were based upon the per cent of tracer leached from
the plant with that originally absorbed by the plant. She was unable
to show any appreciable loss of phosphorus32 by this method regardless
of the length of time of the action on the plant. However, in other ex-
periments, using differences based on per gram dry weight between
leached and non-leached materials, considerable loss was evident.
Other workers have concluded similar losses of many nutrients based
on the differences in the plant materials in dry weight measurements
after exposures (Table XIII).

The differences in per cent between the amounts of the ele-
ment found present in the leached and non-leached materials on the per
gram dry weight basis is demonstrated to be a poor criterion for meas-

urement probably due to the differences in absorption of the tracers by

individual plants. It has been mentioned previously that LeClerc and

 

63.

 

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64.

Breazeale had concluded that the ash constituents of plant materials
were not an exact estimate of the minerals used by the plant.

The amount of rubidium86 absorbed by the plants depends
on the concentration of the nutrient solution applied to the root systems
(Table XI).

The environmental factors which surround the plant during
growth, the stage of development, the time of year, and the amount of

nutrients available to the plant have each been pointed out in the liter-

 

ature as playing a role in determining the amount the loss of nutrients
sustained by a particular plant during periods of rain or dew, or mist.
Living plants, which have absorbed their isotopes through
the cut stem surfaces, show a greater per cent of loss due to the
mist treatment than those plants which have absorbed the tracer only
through their root systems. These data may support the theories of
Mes and Strugger, that nutrients traveling through the intermicellular
spaces of a plant may be simply washed from the surfaces of plants
without any physiological process involved. In this particular case,
travel through the plant may have been accomplished by diffusion or
inhibition into the cut surfaces.
In the histological observations reported, the more deeply

stained tissues of the control plants were taken as evidence of storage

 

 

   

65.

materials metabolized from carbohydrates manufactured in the leaves
and of minerals accumulated in the surrounding tissues.

Evidence of nutrient losses from cuttings under mist has
been established by radioactive tracers. However, the rooting of cut-
tings has been accelerated and improved under the mist method as shown
in the literature.

Histological observations of leaf and stem sections of cuttings

rooted under mist have shown accumulation of storage products in the

 

stem tissues and a lack of nutrients in the palisade layers. The vas-
cular system was deeply stained indicating that perhaps the carbohydrates
are moving out of the leaf into the stem. Leaf and stem sections rooted
under non—misted methods show carbohydrate compounds in the temporary
storage layers of the leaf with lesser amounts collecting in stem tissue.
Other evidence of advanced maturation of stem tissues and
axillary ﬂower bud initiation is shown in previous work with cherry cut—
tings rooted under mist. When the cuttings were held under mist for
extended periods after the necessary rooting time, ﬂower initiation was
evident. The initiation of ﬂower primordia was not necessarily due to
maturity as rooted cuttings from the same stock later in the season de-

veloped good vegetative growth when transplanted to the soil.

 

 

   

66.

Lees noted that the previous season's rainfall may affect
ﬂower bud initiation in the apple. Other investigators have found that
certain mist-rooted cuttings when transplanted have a tendency to de-
velop shoots from the transitionary zones of the plant; never again
do they initiate growth from the old stem.

Kobel (12) has pointed out that ﬂower bud initiation in fruit
trees is associated with the presence of increased amounts of carbo—

hydrates in the immediate vicinity of the axillary buds.

 

The increased transport of storage materials from the
leaves into the stem tissues of cuttings placed under mist may have a
direct bearing on the early initiation of roots and also the large number
of roots per stem. It is generally accepted that auxin travels with
carbohydrates. These may in turn be carried with the sugars into the
stem areas where root initials may originate.

The fact that plant parts with mature leaves root well under
the mist system would seem to preclude that the amount of nutrients
lost by leaching have not impaired the ability of the cuttings to form
roots. Biddulph (2) shows accumulation of minerals within the leaf in
excess of the quantities used in the plant's metabolism. The possibi—
lity of surplus nutrients and organic matter not being vitally connected

with the plant processes which motivate root formation and future ,

 

 

 

67.

growth of the new plant, and which may reside in the intermicellular
spaces, as Mes and others point out, may be leached from the leaves
of plants without vital injury to them, provided certain precautions are
taken.

The observations of the behavior of newly transplanted cut-
tings rooted under mist in the preliminary trials required a definite
shading for a period before they were exposed to the direct rays of the
sun in order to prevent leaf burning. These conditions suggested the
necessity for renewal of the food supply in the leaf which was depleted
of its carbohydrates and minerals to low levels by the action of mist.

Mist—treated transplants growing under reduced light intensi-
ties may synthesize a small reserve of food sufﬁcient to withstand the
added demands made upon it with increased respiration due to high
temperature within the leaf under direct sunlight.

In addition to this, the accumulation in the leaf of certain
essential elements such as potassium and phosphorus may be required
for fundamental growth processes. The loss of these elements has
occurred as demonstrated in the experiments with the leaching of radio-

active isotopes under the mist method of propagation.

 

 

 

 

68.

SUMMARY

1. The effect of a mist treatment on the leaching of root and

32
stem absorbed radioactive phosphorus , potassium”. and rubidium86

from plant foliage was investigated.
2. Different quantities of radioactive phosphorussz, radioactive

42

potass1um , and radioactive rubidium 6 were independently obtained from

collected water that was allowed to fall in the form of mist on cuttings

 

which had absorbed the isotope through their cut surfaces, or which were
taken from plants that had absorbed the tracer through their root systems.

42 were leached from cut-

3. Greater quantities of potassium
tings under mist when the isotope was absorbed through their cut surfaces
than through the root systems of the parent plant.

4. Phosphorus32 was not recovered from the mist which fell on
bean and sweet potato cuttings when the tracer had been absorbed pre-
viously by the root systems of the parent plants.

5. Phosphorus32 was found in the leachate from rose and bean
cuttings which had previously absorbed the isotope through the cut sur-
faces of their stems.

6. Garden bean cuttings leached greater quantities of potassiuméf2

86 , . . .
and rubidium under mist when the respective isotopes were absorbed in

the dark at full nutrient levels.

 

69.

7. The per cent of a nutrient leached from plants under mist
based on the difference per gram dry weight between the leached and the
non-leached materials did not correspond with the per cent of the tracer
actually lost from the cuttings due to the difference in the relative ab-
sorption rates of the individual plants.

8. Prepared slides of thin cross-sections of young bean leaves
which remained under mist for five days were "twenty per cent larger in
size and the cells appeared stretched as compared to sections of non-
misted leaves of the same physiological age.

9. Evidence of the transport of reserve substances from the
leaves of cuttings under mist to the stem tissues and also evidence of
early maturation was observed in the hist010gi'cal sections of apple and
cherry cuttings which remained under mist for twenty-five days, as com-
pared to sections of non-misted plant materials of similar physiological

age.

 

 

 

 

70.

LITERATURE CITED
1. Arens, K. Die kutikulare Excretion des Laubblattes. Jb. wiss. Bot. 80:
248. 1934.

2. Biddulph, O. The Translocation of Minerals in Plants. Mineral Nutrition
of Plants. The University of Wisconsin Press. 261-275. 1951.

3. Brandstaetter, Marie. Mist Propagation Reviewed during Lake County
School. American Nurseryman 101 (7): 52-59. 1955.

4. Comar, C. L. Radioisotopes in Biology and Agriculture. McGraw-Hill
Book Company, Inc. 6-10. 1955.

 

5. Dalbro, S. Leaching of Nutrients from Apple Foliage. Proc. XIV Inter-
national Hort. Cong. (Paris). In Press. 1955.

6. Evans, H. Investigations on the Propagation of Cacao. Tropical Agri-
culture 28: 147-203. 1951.

7. Frey-Wyssling, A. Stoffwechsel der Pﬂanzen. (Quoted by Mes,
Ref. 17). 1948.

8. Hess, C. E. , and W. E. Snyder. A Simple and Inexpensive Time Clock
for Regulating Mist in Plant Propagation Procedures. Proc. Third
Annual Meeting, Plant Propagators Society. 56-60. 1953.

9. Hess, C. E., and W. E. Snyder. Interrupted Mist Found Sllperior to
Constant Mist in Tests with Cuttings. American Nurseryman
100(12): 11, 12, 82. 1954.

10. Hoagland, D. R., and D. I. Arnon. The Water-culture Method for
Growing Plants Without Soil. Univ. Calif. Agri. Expt. Sta. Cir.
347. 1938.

11. Kent, D. A., and G. E. Patrick. When to Cut Corn. Iowa State Expt.
Sta. Bul. 21. 1893.

12. Kobel, F. Lehrbuch des Obstbau. Springer-Verlag. Berlin. 75-80.
1954.

 

 

 

 

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

71.

Lausberg, Thusnelda. Quantitative Untersuchungen uber die kutikulare
Exlcretion des Laubblattes. Jb. wiss. Bot. 81: 769-806. 1935.

LeClerc, J. A. , and J. F. Breazeale. Plant Food Removed from Grow-
ing Plants by Rain or Dew. U. S. Dept. of Agr. Yearbook: 389-
402. 1908.

Lees, A. H. Factors Governing Fruit Bud Formation. Long Ashton
Res. Station Rpt., Univ. of Bristol. 42-59. 1926.

Mann, C. E. T. , and T. Wallace. The Effects of Leaching with Cold
Water on the Foliage of Apple. Long Ashton Res. Sta. Rpt. ,
Univ. of Bristol. 17—24. 1925.

Mes, Margaretha G. Excretion (Recretion) of Phosphorus and Other
Mineral Elements by Leaves under the Inﬂuence of Rain. So.
African Jour. Sci. 50: 167-172. 1954.

 

O'Rourke, F. L. Mist Humidification and the Rooting of Cuttings
(Progress Report). Mich. Agr. Exp. Sta. Quart. Bul. 32(2):
245-249. .

Raines, M. A. Some Uses of a. Spray Chamber in Experimentation
with Plants. Amer. Jour. Bot. 27: 18. 1940.

Snyder, W. E. Possibilities with Mist Propagation. Proc. Fourth
Annual Meeting, Plant Propagators Society. 89-101. 1954.

Strugger, S. Fluoreszenzmilcroskopische Untersuchungen uber die
Speicherung und Vt'anderung des Fluoreszeinkaliums in plfanzlischen
Geweben. Flora (Jena), 132, 253. 1938. (Quoted by Mes, Ref. 17).

Tamm, C. 0. Removal of Plant Nutrients from Tree Crowns by Rain.
Phys. Plant., 4: 184-188. 1951.

Toth, S. J. , A. L. Prince, and D. S. Mikkelsen. Rapid Quantitative
Determination of Eight Mineral Elements in Plant Tissue by a
Systematic Procedure Involving the Use of a Flame Photometer.
Soil Sci. , 66: 459-466. 1948.

Watkins, J. V. Propagation of Ornamental Plants. Fla. Agr. Ext.
Bul. 150: 1-56. 1955.

 

 

 

 

72.

25. Wells, J. S. Plant Propagation Practices. The MacMillan Company,
New York. 115-134. 1955.

26. Wilfarth, H., H. Romer, and G. Wimmer.. Uber die Nahrstoffaufnahme
der Pﬂanzen in verschiedenen Zeiten ihres Wachstums. Landw.
Versuchst., LXIII,,1. 1905. (Quoted by Mes, Ref. 17).

27. Will, G. M. Removal of Mineral Nutrients from Tree Crowns by Rain.
Nature 176: 1180. 1955.

 

 

 

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