À ^U Ah. Agriculture Handbook No. 1 26

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TEST METHODS

with plant-regulating chemicals

By JOHN W. MITCHELL • GEORGE A. LIVINGSTON • PAUL C. MARTH
TEST METHODS

with plant-regulating chemicals

By JOHN W. MITCHELL, Principal Physiologist,

Agricultural Research Service,

United States Department of Agriculture

GEORGE A. LIVINGSTON, Research Associate,

Chemical Biological Coordination Center,

National Research Council

PAUL C. MARTH, Senior Physiologist,

Agricultural Research Service,

United States Department of Agriculture

Agriculture Handbook No. 126

UNITED STATES DEPARTMENT OF AGRICULTURE
CONTENTS

Page

Part I. Methods of applying regulating compounds and of studying

responses they induce 1

Abscission 1

Absorption and translocation 6

Angle and elongation of branches 9

Cell division 10

Cell elongation 11

Cell growth 16

Effect of one compound on the absorption and translccation of another 21

Exudation of regulators from roots 22

Form or growth modification 23

Fruit set 26

Fruit size 30

Fruit thinning 31

Growth inhibition or stimulation 33

Growth modification (aquatic plant) 34

Growth regulation 36

Root growth 37

Root induction 41

Seed formation 43

Part II. Methods of detecting regulating compounds 45

Detection of plant-growth regulators in animals 45

Penetration of soil by regulators 46

Residue of regulators in soil 47

Translocation of radioactively tagged compounds 49

Volatility of plant regulators 51

Detection of regulators in water 53

Part III. Techniques in general use 55

Supplementary information 65

Sources of plant-regulating compounds 65

Measures and equivalents 65

Temperature conversions 66

Methods of making nutrient solutions 66

Descriptive index of methods 67

Washington, D. C. Issued January 1958

For sale by the Superintendent of Documents, U. S. Government Printing Office

Washington 25, D. C. Price 40 cents

II
TEST METHODS

Y\^— *'*•! plant-regulating chemicals

INTRODUCTION

Organic compounds are being used in increasing amounts to control the growth and behavior

of crop and ornamental plants. Not only have

many different compounds been found to possess

growth-regulating properties, but various kinds

of plants and plant parts have been found to respond in widely different ways to these interesting and useful chemicals. For example, 2,4,5-

trichlorophenoxyacetic acid can be used to

retard the formation of an abscission zone in

the stem of an apple. Applied to a rhododendron cutting, it stimulates root formation. 2,4-

Dichlorophenoxyacetic acid often suppresses

growth of pods when applied to snap bean

plants. In contrast, 4-chlorophenoxyacetic acid

has been used experimentally to accelerate

growth of snap bean pods, to increase their succulence, and their vitamin C content.

Plant scientists are searching for and finding

not only a variety of new regulating compounds,

but also new ways in which plants can be made

to behave through the use of these chemicals.

In this publication an attempt has been made to

make the methods used by these scientists easily

available, and thus facilitate research of this

kind.

Many useful methods for studying plantregulating properties of compounds have been

devised. These methods result from the efforts

of many scientists working on a variety of research problems in various countries. Without

drawing the published methods together, it is

difficult to fully utilize them in teaching and

research since their publication occurs in a wide

variety of journals. Furthermore, some of the

methods have not yet been published.

It is hoped that this compilation will be useful

in several ways : First, by affording those experienced in this field of research an opportunity

to compare the ways in which scientists have

tested chemicals for plant-regulating properties, and how they have recorded and evaluated

plant responses obtained with these compounds.

This information should lead to the development

of even more suitable methods. Secondly, the

compilation will afford the experienced worker

and the novice in this field ready access to many

of the methods that have been used. Third, it

may aid in the teaching of college courses where

laboratory exercises are used to demonstrate

the effects of regulating chemicals. Some of the

methods described may also be useful as laboratory exercises in the teaching of high school

courses in biology and plant science.

The compilation is divided into three parts.

The first section includes methods designed to

detect regulating properties of organic compounds and measure the response of plants to

these compounds. The second section includes

methods designed to detect and, in some instances, to measure the amounts of regulating

compounds present in plants, in water, and to

some extent in animals. The third section describes pictorially some techniques that are of

general use in experimenting with plantregulating compounds.

The methods are described step by step in the

manner of a cookbook with the hope that they

will be readily understandable and easy to use.

Descriptive titles have been assigned to each

method indicating the use suggested by the

author. In some instances additional uses have

been suggested by those compiling the methods.

A few well-known and very useful tests, such as

the oat coleoptile test, have not been included

since these tests are widely used and descriptions of them are easily obtainable. It is hoped

that scientists will find the methods described

in the present compilation useful in many other

ways besides those suggested here.

Ill
PART I. METHODS OF APPLYING REGULATING COMPOUNDS AND OF

STUDYING RESPONSES THEY INDUCE

Abscission

APPLE PETIOLE TEST

Method based on research by:

L. J. Edgerton; L. J. Edgerton and M. B.

Hoffman.

Description of test

The rate of abscission of petioles of debladed

leaves on apple water sprouts following treatment with various chemicals is used as an indication of the effectiveness of the compounds in

retarding harvest drop of apples.

This test can also be used to evaluate the

effectiveness of plant regulators, other compounds, or combinations of them, in accelerating

or reducing the rate of leaf abscission.

Suggested plant material

Rapidly growing vegetative branches or shoots

(commonly called water sprouts) from the main

branches or trunks of apple trees (Malus

sylvestris Mill., also called Pyrus mains L.) —

Mclntosh, Winesap, Stayman, or other varieties.

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Hand-sprayer (250 ml.) or

nasal atomizer (15-30 ml.)

25-200 mg. of each compound to

be evaluated

500 mg. of alpha-naphthaleneacetic acid or its sodium

salt for standard treatment

Solvent such as 95 % ethanol

Wetting agent such as Santomerse,

Dreft, or Tween 20

Preparation and sélection of plant material

In midsummer tag apple trees that have produced an abundance of water sprouts during the

current season. New sprout growth can be induced to develop 1 to 2 months earlier than usual

by selecting experimental trees prior to winter

pruning and cutting back the existing water

sprouts, leaving stubs 1-3 in. (3-8 cm.) in

length. These stubs will then produce long,

vegetative shoots early during the next growing

season. One- to two-year-old potted trees may

be used during winter and spring in the greenhouse. Select shoots of uniform diameter, each

having 15 to 25 leaves.

Method of conducting test

Prepare the spray solutions by dissolving the

desired weight of the chemical to be evaluated

in a minimum of solvent (1-2 ml. of 95%

ethanol) ; add enough wetting agent to make a

final concentration of 0.02-0.2% of the wetting

agent. Pour this mixture, while stirring, into

sufficient tap water to make a final concentration of 10-30 p.p.m. of the chemical being tested.

Apply the spray solution with the hand sprayer

to 5 or 6 water sprouts, thoroughly wetting the

leaf blades, petioles, and twigs. Within 2 to 3

days after treatment remove 10 or more leaf

blades from each shoot, leaving the entire

petioles attached to the twigs. To make the

results more nearly uniform, do not use the 4 or

5 basal leaves or the young, partially expanded

leaves near the tip of the shoots. Remove

blades from 10 or more leaves on comparable

unsprayed sprouts of each test tree and

designate these as controls.

Method of taking results

Within 10 to 11 days after treatment, and at

frequent intervals thereafter for the duration

of the test, count the petioles that remain attached in each experimental lot. The final percentage of petioles attached is an index of the

effectiveness of the compound when used for

retarding fruit abscission.
AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Suggested standard treatment for

comparison

10, 20, or 30 p.p.m. concentration of alphanaphthaleneacetic acid or its sodium salt.

Edgerton, L. J. A Method for Evaluating the Effectiveness of Growth Substances in Delaying Apple Abscission. Amer. Soc. Hort. Sei. Proc. 49: 42-44. 1947.

Edgerton, L. J. and Hoffman, M. B. The Effect of Some

Growth Substances on Leaf Petiole Abscission and

Preharvest Fruit Drop of Several Apple Varieties.

Amer. Soc. Hort. Sei. Proc. 62: 159-166. 1953.

ATTACHED PETIOLE TEST

Method based on research by:

J. W. Mitchell, P. Marsh, and R. Bender.

Description of test

The test compound is placed on debladed

petioles, and a known pressure is then applied

to the petioles each day to determine the length

of time required for the petioles to abscise compared with that for untreated petioles.

Suggested plant material

Young cotton plants (Gossypium hirsutum

L.), Deltapine variety, and young bean plants

(Phaseollis vulgaris L.) of the Pinto or other

available varieties.

Apparatus, chemicals, and other materials

Constant-pressure apparatus

(see fig.l)

Razor blades or sharp knife

Lanolin

Endothal acid, monohydrate,

1-2 g.

Phenylmercuric acetate 1-2 g.

Analytical balance or pharmaceutical balance

Vials

Medical applicator sticks

or toothpicks

Tween 20

Compounds to be evaluated as

growth regulators

Preparation and selection of plant material

Plant cotton seeds in composted soil contained

in small clay pots, using several seeds per pot.

After the plants have developed cotyledons

about 1.5 inches across (3-5 cm.), and the first

true leaf is beginning to expand from the terminal bud (10-12 days after planting), select a

group of test plants of uniform size and thin

them to one plant per pot. Arrange the plants

uniformly in rows of 5 plants each on a greenhouse bench that is evenly illuminated. Remove the blade of one cotyledon of each plant by

severing the petiole where it joins the blade.

Method of conducting test

Dissolve 12.5 mg. of the compound to be tested

in approximately 0.25 g. of Tween 20 (7 drops

from a 10 ml. pipette) ; then add 1 g. of melted

lanolin. Stir the mixture thoroughly. Dilute

one-half of this 1% mixture with an equal

amount of the Tween 20-lanolin carrier, and

continue the dilution process with the mixtures

obtained until a suitable range of dosage levels

has been obtained.

Treat the debladed petioles of one row of

plants by applying with disposable applicator

sticks a portion of the 1% mixture about the

size of a wheat seed as a band about 2 mm. wide

around the cotyledonary stump of each plant.

Apply the paste 2-3 mm. from the stems. Apply equal portions of the pastes containing progressively smaller amounts of the test compound

to debladed petioles in corresponding rows in a

similar manner.

To determine when the petioles readily abscise, apply pressure once each day in a downward direction against the upper surface of

each debladed cotyledonary petiole. Accomplish this by pressing the thin metal spring of

the pressure applicator against the petiole until

tension equal to approximately 5 g. develops, at

which time the light on the tension applicator

will flash (fig. 1). Always apply pressure to the

petiole at a predetermined distance (8-10 mm.)

from the stem. The pressure applicator may be

calibrated prior to use by pressing the spring on

a balance with a 5 g. weight on the opposite pan.

Method of taking results

Record the number of petioles that abscise

each day and calculate the number that fall from

the treated plants in terms of the number that

fall from plants treated with the Tween 20-lanolin carrier alone.

Alternative method

Use young bean plants in the same manner

except remove one primary leaf blade of each

plant, leaving the petiole attached. Selectplants

with primary leaves that are approximately 2-3

in. (3-5 cm.) wide, and with the first trifoliate

leaf just beginning to unfold from the terminal

bud.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 3

Figure 1. This pressure apparatus is used to determine whether or not a chemical will make leaves fall from a plant. First, remove the blade

from one leaf on each of several plants, leaving the petioles attached. Apply the chemical to some of the debladed petioles. Use the

apparatus daily to apply a measured amount of pressure to both the treated and the untreated petioles, as illustrated. Place the end

of the spring against the upper side of the petiole. Hold the apparatus in a horizontal position and exert pressure downward until

the spring and adjustable pin make contact and the light bulb is activated; then quickly release the pressure. Determine if treated

petioles fall sooner or later than those not treated, or at the same time. (XEG. px-292)

Suggested standard treatment for

comparison

Endothal acid monohydrate, phenylmercuric

acetate, or naphthyl maleimide for cotton; endothal acid monohydrate, or phenylmercuric

acetate for bean plants.

Mitchell, J. W., Marsh, P. and Bender, R. Unpublished

data.

EXPIANT TEST

Method based on research by:

F. T. Addicott, R. S. Lynch, G. A. Livingston,

and J. K. Hunter.

Description of test

Small sections of leaves (expiants) containing an abscission zone are treated with compounds, and the effect on abscission observed.

Suggested plant material

Citrus leaves or leaves of other kinds of plants

that have abscission zones near a leaf blade.

Apparatus, chemicals, and other materials

Scalpel

Forceps

Filter paper
AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Petri dishes

Dark chamber maintained at

about 77° F. (25° C.)

Analytical balance or pharmaceutical balance

Closed bell jar or desiccator

0.5-1 g. of each compound to be

evaluated

For standard treatment :

ethylene gas or

0.5-1 g. 3-indoleacetic acid

Agar

Preparation and selection of plant material

Excise for use as test material section of

leaves containing the abscission zone and the

adjacent tissues (see fig. 2.) These sections

are called expiants. Trim off all but the midrib of the blade portion of the leaves in preparing the expiants. If the petioles are winged,

trim the wing from them. Always leave a

longer piece of tissue, consistently either distal

or proximal to the abscission zone, so that identification of the blade or petiole tissue can be

made readily. Prepare 10 of these expiants for

each concentration of each treatment, and an

additional 20 or 40 to serve as controls. After

cutting, and before treatment, store the expiants

on moist paper in a Petri dish. In preparation

of the expiants, utilize leaves that have attained

their full size and hard texture but still have a

dark green color.

Method of conducting test

Prepare 4% agar in water, pour it into Petri

dishes, and allow it to cool and set. The depth

of the agar in the dish should be about 14 in-

(6 mm.). After the agar has set, remove a strip

of it, about 1 in. (3 cm.) wide, along the diameter of the plate, leaving a narrow bridge of

agar across one end to hold the agar in place and

to act as a marker (see fig. 2).

Prepare 10-25 ml. of each concentration of

Figure 2. This test is used to learn whether or not a chemical makes leaves drop. Cut segments of petioles from orange or bean leaves,

including the abscission zone in each section, as illustrated. Treat some of the segments with the compound; then place the treated

sections along with untreated sections on agar, as illustrated. Replace the lid of the Petri dish and observe how long it takes abscission

to occur in the treated and untreated segments. (NEO rN'-293)
TEST METHODS WITH PLANT-REGULATING CHEMICALS

each chemical to be tested. The suggested concentration range is 1-1,000 p.p.m. Treat explants (10 per treatment) by immersing them

in the test solution for 30 to 60 seconds. Remove the treated expiants from the solution

and mount them on the agar of a Petri dish so

that the shorter part and the abscission zone

overhang the central channel cut in the agar.

The compound may also be administered by including it in the agar support or by application

of small droplets to the cut petiole or blade surface of the expiant.

Prepare dishes containing untreated expiants

to serve as controls. Keep the Petri dishes containing the expiants in the dark at about 77° F.

(25° C.) except for the short daily period of

examination.

Method of taking results

Observe for evidence of abscission by examining the expiants with the dishes closed. To

determine if abscission is complete but not evident, raise the dish cover and lift the expiants

slightly by pressing the tips of forceps upward

under their free ends. The period during which

the expiants are kept under observation is usually 6 or 7 days. With some experience, various ''stages" of abscission can be identified.

Maintain daily records, listing the percentage

of abscission in each treatment and, if desired,

the ''stage" of abscission which each expiant exhibits at each time of observation.

Suggested standard treatment for

comparison

To initiate or accelerate abscission, place

Petri dishes containing the expiants inside an

air-tight container and add ethylene to make a

concentration of 20-50 p.p.m. Use 0.001-100

p.p.m. of 3-indoleacetic acid (30-60 seconds dip)

as a standard for delay of abscission.

Addicott, F. T., Lynch, R. S., Livingston, G. A., and

Hunter, J. K. A Method for the Study of Foliar Abscission In Vitro. Plant Physiolog-y 24 (3) : 537-539.

1949.

LANOLIN PASTE TEST

Method based on research by:

P. C. Marth, W. H. Preston, Jr., and J. W.

Mitchell.

Description of test and some other uses

The abscission of apple fruits following application of a chemical in lanolin paste around the

stem of the individual fruits prior to the usual

harvest date indicates the effectiveness of the

chemical in retarding fruit abscission.

With slight modifications this test is applicable to studies of abscission rates of a wide

variety of fruits.

Suggested plant material

Apple trees (Malus sylvestris Mill., also called

Pyrus malus L.) bearing an abundance of fruit

readily accessible from the ground — Grimes,

Mclntosh, York, or other varieties.

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Small shell vials with stoppers

Wooden board 6-8 in. wide x

1-2 ft. long X 1 in. thick

15 X 30 X 3 cm.) having I/2 in. (1 cm.)

diameter holes drilled % in. (2 cm.) deep

and 2 in. (5 cm.) apart for carrying

the vials in the field

Toothpicks or disposable wooden

applicators without cotton

Small labels with string or wire attached

25-100 mg. of each compound to be evaluated

0.5 g. of alpha-naphthaleneacetic acid

for standard treatment

Ethanol

Lanolin

Tween 20

Preparation and selection of plant material

Select individual fruits that are readily accessible from the ground and located around the

perimeter of each tree used for the experiment.

The fruits should be of uniform size and free of

blemishes. Make the fruit selection and apply

the treatments about 30 days prior to the estimated, or usual, harvest date for the variety.

Method of conducting test

Prepare lanolin-Tween 20 mixtures containing 0.1 and 1% concentrations of the respective compounds. To prepare the 1% mixture,

place 25 mg. of the compound to be tested in a

vial and add 14 drops of Tween 20. Stir to dissolve the chemical, and add 2 g. of lanolin. Melt

the lanolin by placing the vial in warm water

(not over 55° C.) for a few minutes. Remove

the vial and stir the mixture thoroughly until it

reaches room temperature and becomes semisolid. To prepare the 0.1% mixture, weigh

0.2 g. of the 1% mixture previously prepared
AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

and mix this with 1.8 g. of the lanolin-Tween 20

carrier (the carrier made by mixing 2 g. of lanolin with 14 drops of Tween 20).

Apply a small portion, the size of a wheat

seed, of the mixture as a band about 3 mm. wide

around the stem of each of 10 fruits near the

junction of stem and branch. As each fruit is

treated, attach a label designating the treatment. Repeat, using an additional 10 fruits for

each mixture. Fruits used for an individual

mixture should be distributed evenly around the

perimeter of the tree. Remove all untreated

fruits except 10 which are left as controls.

Method of taking results

One week after treatment and at weekly or biweekly intervals thereafter (depending upon the

prevalence of wind or hail), record the number

of fruits remaining attached in each treatment.

Record data regarding advance or retardation

in maturity, color changes, cracking, or other

responses for 30 days or more after the usual

harvest date for the variety. The percentage of

treated fruits remaining attached throughout

the course of the experiment compared with the

percentage of untreated fruits remaining attached serves as a measure for evaluating the

effect of individual compounds on fruit abscission.

Suggested standard treatment for

comparison

Lanolin paste mixture containing either 0.1 or

1 Jo alpha-naphthaleneacetic acid.

Marth, P. C, Preston, W. H., Jr., and Mitchell, J. W.

Relative EfFectiveness of the Mono-, Di- and TriChlorophenoxyacetic Acids in Retarding the Abscission of Mature Apples. Bot. Gaz. 117: 51-55. 1955.

Absorption and Translocation

BEAN ROOT TEST

Method based on research by:

T. J. Muzik.

Description of test and some other uses

Test plants are arranged with one-half of

their root systems in the test solution and the

other half in tap water ; then the roots that have

been in the test solution are cut off at different

time intervals. The resulting growth modifications or injurious effects of the chemicals can

be observed, and the rapidity of absorption and

translocation of the test compound by the roots

can be determined.

This method can be utilized to study the effects of environmental factors, such as light,

temperature, hydrogen ion concentration, mineral nutrition, and oxygen supply, on the ability

of roots to absorb and translocate regulating

chemicals.

Sufigested plant material

Plants with root systems that can be arranged

in two portions of about the same size — beans

(Phaseolus vulgaris L,) oi similar plants.

Apparatus, chemicals, and other materials

Clay pots 3-6 in. (8-15 cm.)

Composted soil

200- to 500-ml. glass containers

Supports to hold a plant with one-half of its

root system in one container, the other half

in another container

Analytical balance or pharmaceutical balance

Compressed air for aerating

nutrient solutions

Greenhouse space with controlled temperature, humidity, and light intensity, or

devices for recording temperature,

humidity, and light intensity

2-5 g. of each compound to be evaluated

3-( para-Chlorophenyl) -1,1-dimethylurea

for standard treatment

For nutrient solution :

124g.of MgS04 • TH^O

83 g. of CaCl2 • 6 H^O

68 g. of KH2PO4

85g. of Ca(N03)2

Preparation and selection of plant material

Grow bean plants in soil or other suitable

medium. After the plants have developed one

partially expanded trifoliate leaf, select the desired number of plants of uniform size and carefully wash them free of soil. Support the plants

with their roots in nutrient solution aerated by

means of compressed air, and allow them to

grow 3 to 5 days to condition them for the test.

For the nutrient solution, use stock solutions of

MgS04, CaCl2, KH2PO4, and Ca(N03)2 made by

dissolving the quantities listed above, each in a

separate liter of tap water. To 500 ml. of tap

water add 9 ml. each of the MgS04, CaCls, and
TEST METHODS WITH PLANT-REGULATING CHEMICALS

KH2PO4 stock solutions, and 18 ml. of the

Ca (NO3) 2 stock solution. Add suiRcient water

to make 1 liter of nutrient solution.

Method of conducting test

Arrange pairs of containers for the test plants

and fill one container of each pair with a water

solution of the compound to be tested (25-200

p.p.m.). Fill the remaining container of each

pair with tap water. For the control plants fill

both containers with tap water.

Select plants of uniform size and vigor from

those placed in nutrient solution earlier. Support each test plant over a pair of containers

and arrange the roots so that one-half of the

root system is immersed in the test solution and

the other half in tap water. Arrange control

plants with each half of the root system growing

in tap water. In this manner, use 6 to 12 plants

for each test solution and an equal number of

plants with their roots in tap water for untreated controls. Allow the plants to remain in

a greenhouse and record the prevailing temperature, humidity, and light intensity, or grow

them under controlled conditions.

After 30 minutes, cut from one-third of the

treated plants the roots immersed in the test solution but leave on these plants the roots growing in tap water. Also cut one-half of the roots

from each of one-third of the control plants.

After another 30 minutes repeat the procedure,

using the second third of the treated and control plants. Finally, repeat the procedure after

an additional 30 minutes, using the remaining

treated and control plants. Replace the tap

water with nutrient solution that is continuously aerated, and allow the treated and untreated plants to grow for a week or two with

their remaining roots in the nutrient solution.

Method of taking results

Epinasty, formative effects (leaf modification) , discoloration of leaves, or gall formation

observed on the treated plants, but not on untreated controls, indicate absorption and translocation of the test compound. In addition, the

time required for this to occur through the roots

can be determined by comparing the treated and

untreated plants which have had their roots removed at different intervals.

Suggested standard treatment for

comparison

25-200 p.p.m. of 3-(para-chlorophenyl)-l,ldimethylurea.

Unpublished method submitted by Thomas J. Muzik,

Federal Experiment Station, Mayaguez, Puerto Rico.

BEAN STEM CURVATURE TEST

Method based on research by:

A. S. Crafts; B. E. Day ; J. W. Mitchell, W. M.

Dugger, Jr., and H. G. Gauch.

Description of test and some other uses

The compound to be tested is placed on one

leaf of a plant; then absorption and translocation of the compound (or of a metabolite of it)

are evaluated on the basis of stem curvature

that develops. This test is applicable only to

compounds that induce or retard cell elongation.

The test may be used under controlled conditions to compare the relative effectiveness of

different compounds that are known to cause

stem curvatures. With some modifications the

test can also be used to detect the effects of adjuvants on absorption and translocation of a

regulator, or metabolite of it, that induces stem

curvature at a distance from the area to which

the regulator is applied.

Suggested plant material

Young bean plants (Phaseolus vulgaris L.)—

Black Valentine, Red Kidney, Pinto, or other

varieties that germinate and grow uniformly.

Apparatus, chemicals, and other materials

Clay pots 3-6 in. (8-10 cm.)

Composted soil

Greenhouse space maintained

at about 75^-85° F. (24°-29° C.)

Analytical balance or pharmaceutical balance

Several 0.1-ml. pipettes graduated in

0.01-ml. divisions

250 ml. beakers for preparing solutions

Glass applicator (about 15 cm. long)

with one end drawn out to make a narrow

portion of about 2-mm. diameter and

7-cm. length

Facial tissue or paper towels

Source of fluorescent white light

Protractor

Adjustable bevel square

About 100 mg. of each compound to be

evaluated

100 mg. of 2,4-dichlorophenoxyacetic acid

for standard treatment

Tween 20, Glim, Joy, or other solvent

of the liquid detergent type

Distilled water
8 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Preparation and selection of plant material '^:

Plant 3 or 4 seeds per pot in composted soil.

After the plants have developed partially expanded but still wrinkled primary leaves, and

the trifoliate leaves are still tightly folded in

the terminal buds, select one plant in each pot

for similarity of size. Sever the stems of the

remaining plants in each pot at the soil level and

discard them. Place the selected plants in a

well-lighted greenhouse with the pots widely

spaced so that the plants will receive adequate

illumination. As soon as the primary leaves of

the majority of the plants have expanded so that

they are no longer wrinkled, usually about 48

hours after the first selection, reselect the plants

for size and uniformity, and place them in rows

of 8 to 10 plants each. Water the soil surrounding the roots of each plant before conducting the test.

Method of conducting test

Dissolve in Tween 20 or other suitable solvent

a weighed portion of the compound to be tested

and add this mixture to warm, distilled water

so that 0.02 ml. of the final mixture will contain

1-5 /.ig. of the chemical and sufficient Tween 20

to make a final concentration of 0.1% of this

solvent.

Fill a pipette with the solution; then drain

exactly 0.02 ml. out so that it adheres as a drop

on the tip of the pipette. Touch the tip of the

pipette near one edge of one primary leaf blade

and move the tip across the leaf, thus applying

the mixture as a narrow band extending from

one side of the leaf blade to the other (fig. 3,

upper photo). Immediately support the blade

by pressing a folded pad of facial tissue or

paper towel under the leaf, place the applicator

in the mixture, and move the applicator slowly

toward the tip of the leaf, thus spreading the

liquid evenly over the surface of the leaf blade

(except for an area near the end to which the

petiole is attached, fig. 3, lower photo.) Apply

other 0.02 ml. portions to one primary leaf of

each of the remaining plants in the row.

Make certain that the treated leaves are oriented on the same side of each row. Arrange

as controls a comparable row of untreated

plants, and a third row treated similarly with

water and Tween 20, or other solvent. Allow

the plants (8 to 10 per treatment) to grow in a

greenhouse, or place them in the fluorescent

light so that they receive illumination of at

least 700-foot-candle intensity.

Figure 3. An easy method of applying minute, measured amounts

of growth-regulating chemicals to leaves. Dissolve a measured

amount of the regulating compound in water and add a wetting agent. Apply a measured volume of the mixture as a

band across the upper surface of a young leaf (upper photo);

then, resting the leaf on a folded paper towel, spread the

mixture evenly over the entire upper surface of the leaf with

a glass rod (lower photo). (XEG. PX-204 A AND B)

Method of taking results

Observe the plants at 30-minute or hourly intervals. Measure the angle of stem curvature
TEST METHODS WITH PLANT-REGULATING CHEMICALS

resulting from the presence of the chemical by

lining up one leg of the adjustable square with

the hypocotyl and lower half of the first internode, and adjusting the other leg of the square

so that it is in line with the upper half of the

first internode and second internode above the

curved section of stem. Compare average readings from the control plants with those from

plants that received the compounds being tested.

The method is applicable to compounds which

induce or retard cell elongation.

Suggested standard treatment for

comparison

1.0 fjig, of 2,4-dichlorophenoxyacetic acid on

one primary leaf of each of 8 to 10 plants.

Crafts, A. S. Herbicides, Their Absorption and Translocation. Agr. and Food Chem. 1: 51-55. 1953.

Day, B. E. The Absorption and Translocation of 2,4-

Dichlorophenoxyacetic Acid by Bean Plants. Plant

Phys. 27: 143-152. 1952.

Mitchell, J. W., Dug-ger, W. M., Jr., and Gauch, H. G.

Increased Translocation of Plant Growth-Modifying

Substances Due to Application of Boron. Sei. 118:

354-355. 1953.

Angle and Elongation of Branches

LANOLIN TEST

Method based on research by:

A. P. Preston and H. W. B. Barlow; L. Verner.

Description of test

A piece of flexible tubing is ñlled with paste

containing the test chemical, slipped onto the

stump of a decapitated branch of a young tree,

and the effects on branch-trunk angle and

growth of the branch are noted.

Suggested plant material

Young trees in whip stage (no lateral

branches) or trees with 1-year-old lateral shoots

—apple (Malus sylvestris Mill., also called Pyrus malus L.), pear (Pyrus communis L.), plum

(Prunus spp.), willow (Salix spp.), or others.

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Several ft. (about 1 m.) of i4-in. (6-mm.)

flexible rubber tubing or sheets of thin,

transparent celluloid

Protractor

Test tubes

1 g. of each compound to be evaluated

About 2 g. of indolebutyric acid for

standard treatment

Lanolin

Preparation and selection of plant material

Select trees of uniform size and stage of development. Use 10 trees for each compound or

concentration to be tested.

Method of conducting test

Prepare lanolin mixtures, 3 g. of lanolin per

mixture, containing an amount of the chemicals

to be tested covering a range of 0.1-1% of the

weight of lanolin used. Apply lanolin mixtures

either to the unbranched trunk or to a vigorous

1-year-old lateral shoot. Cut off the terminal

portion of the shoot to be treated, usually a segment 4-8 in. (10-20 cm.) in length. Cut off a

11/2-in. (4-cm.) length of the rubber tubing and

half-fill it with the paste containing the compound to be tested. Slip the end of the tube

containing the paste over the decapitated shoot

for a distance of about % in. (1 cm.). Double

the empty end of tubing over and tie it in this

position with a string to protect the lanolin mixture. Repeat this procedure, using different

shoots with lanolin alone and also with lanolin

plus 3-indolebutyric acid (0.1%), and designate

these as a control and a standard treatment.

In an alternate method, flexible sheet plastic

or similar material is employed in place of the

rubber tube. Cut strips of the flexible material,

8x6 cm. ; wrap a strip around the end of each

cut branch, and secure it with a rubber band.

Melt the lanolin mixture and put about 5 ml. of

it into the tube formed by the strip. Slip a test

tube over the open end of the tube to protect the

lanolin.

Method of taking results

After the lateral shoots on the controls have

attained an average length of 6-8 in. (15-20

cm.), determine the average length of the first

3 or 4 lateral shoots nearest the treated portion

on each tree. Compare this average with the

averages obtained from trees treated with the

3-indolebutyric acid mixture, and with lanolini

alone.

Measure with a protractor the angle formed

by the trunk of each plant and the first 3 or 4

lateral shoots nearest the treated portion.

Compare these measurements with similar

measurements on plants given the standard

treatment, and on control plants.
10 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Suggested standard treatment for

comparison

A O.l^i laiiolin mixture of 3-inclolebutyric

acid.

Preston, A. P., and Barlow, H. W. B. The Use of

Growth Substances to Widen Crotch Angles. East

Mailing Res. Sta. Ann. Rept. 1950: 76-79 (pub.

1%1).

Verner, L. Effect of a Plant Growth Substance on

Crotch Angles in Young Apple Trees. Amer. Soc.

Hort. Sei. Proc. 36: 415-422. 1939.

Verner, L. Unpublished method. University of Idaho,

Moscow, Idaho.

Ceil Division

CAMBIUM TEST'

Method based on research by:

W. C. Cooper, S. Tayloe, and N. Maxwell;

W. C. Cooper, A. Peynado, and G. Otey.

Description of test

A knife cut is made through the bark in a

ring around the trunk of young evergreen fruit

trees, and growth-regulating chemicals are applied to test for inhibition of cambial activity.

The degree of tightness of the bark that develops subsequent to treatment indicates the

amount of reduction in cambial activity.

Suggested plant material

Young semi-tropical evergreen fruit trees—

orange (Citrus sinensis [L.] Osbeck), grapefruit (Citrus paradisi Macfad.), avocado (Persea americana Mill., also called Persea gratissima Gaertn.), or others.

Apparatus, chemicals, and other materials

Pocket knife

Analytical balance or pharmaceutical balance

Small yials

Applicator sticks with a wad of cotton

on one end

2 mg. of each compound to be evaluated

50% ethanol for compounds

insoluble in water

Distilled water

Preparation and selection of plant material

Use seedlings or budded trees about 2 years

old, preferably growing in the nursery row.

Select trees with trunks free of lateral branches

^ This test is useful in selecting chemicals that might

temporarily reduce vegetative growth of semi-tropical

fruit trees and thus increase their resistance to low temperatures.

and with bark that peels readily. Prepare the

trees for testing by making a knife cut just

through the bark and extending as a ring

around the trunk at a point about 6 in. (15 cm.)

from the ground.

Method of conducting test

The 2-mg. sample of each chemical to be

tested is placed in a separate small vial and dissolved in 2 ml. of distilled water, or in 2 ml. of

50% ethanol if the compound is not water soluble. Using a separate applicator for each

solution, moisten the cotton with the test solution and swab over the knife-cut ring and over

the adjacent trunk surface for a distance of 1

in. (3 cm.) above and below the ring. Use a

separate lot of trees (3 to 6) for each solution.

Method of taking results

At weekly intervals following the treatment,

estimate cambial activity by determining the

ease with which bark can be peeled from the

trees. In this test make two parallel longitudinal cuts through the bark about l^ in. (1 cm.)

long and about % in- (^ mm.) apart. Join these

cuts at the bottom by a transverse cut. Insert

a knife blade under the transverse cut, and peel

the bark from the sapwood by pulling in an upward direction. The force required to remove

the bark is given a rating. The bark-peeling

tests should be made both above and below the

ring and both within and outside the treated

area.

Suggested standard treatment for

comparison

No standard treatment suggested.

Cooper, W. C, Tayloe, S., and Maxwell, N. Preliminary

Studies on Cold Hardiness in Citrus as Related to

Cambial Activity and Bud Growth. Rio Grande Valley Hort. Inst. Ann. Proc. 9: 1-15. 1955.

Cooper, W. C, Peynado, A., and Otey, G. Effects of

Plant Regulators on Dormancy, Cold Hardiness, and

Leaf Form of Grapefruit Trees. Amer. Soc. Hort.

Sei. Proc. 66: 100-110. 1955.
TEST METHODS WITH PLANT-REGULATING CHEMICALS

Cell Elongation

11

BEAN HYPOCOTYL TEST

Method based on research by:

R. L. Weintraub, J. W. Brown, J. A. Throne,

and J. N. Yeatman.

Description of test

A single droplet containing a known amount

of a growth regulator is placed on one side of a

decapitated bean hypocotyl and after 5 hours

the degree of curvature is measured and compared with curvature induced by a standard

compound such as 2,4-dichlorophenoxyacetic

acid.

Suggested plant material

Young bean plants (Phaseolus vulgaris L.)—

Black Valentine, Pinto, Red Kidney, or other

varieties.

Apparatus, chemicals, and other materials

Vermiculite (No. 3)

Galvanized iron pans, 20 in. wide x 26 in.

long X 3 in. deep (51 x 66 x 8 cm.) perforated for drainage

Light-tight room at 87° db 1° F.

(31° G.) with relative humidity of

60 ± 10%

40-w. red fluorescent lamps

Sharp knife or razor blade

Board % x % x 17 in. (2 x 2 x 43 cm.) with

row of V-shaped notches along one edge

0.25-ml. tuberculin-type glass syringe

No. 27 stainless-steel hypodermic needle

with end cut off at right angle

Micrometer caliper with anvil removed and

frame modified to hold syringe securely or

dial-type micrometer modified to hold

syringe, syringe-micrometer assembly

calibrated to 0.0001-ml. units with mercury

60-w. Mazda lamp with Wratten OA filter

Pan 4 in. wide x 16 in. long x 2 in. deep

(10 x 41 X 5 cm.)

Bromide photographic paper

Movable-arm protractor

Analytical balance or pharmaceutical balance

Ghemicals to be evaluated for cell-elongating

activity

Relatively pure 2,4-dichlorophenoxyacetic

acid for standard treatment

95% ethanol

Photographic developer

Petroleum jelly

Preparation and selection of plant material

Germinate the seeds and grow the seedlings

in pans containing vermiculite until the plants

are uniformly 8-13 cm. tall. This requires

about 4 days, and is done in a light-tight room

at 87° ± 1° F. (31° C.) with relative humidity

of 60 dt 10%. Suspend red fluorescent lamps

(40-w.) 4-8 ft. (1-2 m.) above the developing

plants to induce straight stem growth.

Remove plants from vermiculite carefully so

as not to cause serious root damage. Select

plants of uniform size and, with a sharp knife,

remove tops at the base of the hypocotyl crook.

Arrange 20 decapitated plants so that a portion

of the hypocotyl just above the roots is in a

notch of the board support. Place a dab of

petroleum jelly over each to hold it in place.

Rest the ends of the board on the edges of the

shallow pan so that the roots are submerged in

tap water for 2 hours prior to the test.

Method of conducting test

Dissolve the chemical to be tested for cellelongating activity in 95% ethanol. This solution should contain 0.065 ¡x^. of the chemical

per 0.0015 ml. of alcohol if comparison is to be

made with the cell-elongating effect of 2,4-dichlorophenoxyacetic acid. Load the syringe

with the growth-regulator-ethanol solution and

apply a single 0.0015-ml. droplet to one side of

each bean stem 5 mm. below the cut surface.

Treat each of the 20 stems per board ; then repeat the operation for each dosage level of each

compound to be tested. Always apply the treatment in the same relative position on each stem.

Leave one lot of stems untreated and treat another with the solvent only (95% ethanol) for

comparison. Use a 60-watt Mazda lamp fitted

with a Wratten OA filter to furnish light during application of treatments and for a 5-hour

test period thereafter.

Method of taking results

Five hours after treatment, excise the 20 hypocotyl stumps just above the support and lay

them on a sheet of glass over a piece of bromide

paper. Orient the stumps so that the treated

side is always on the left Briefly expose them

to a white light from a source directly above

to produce a shadowgraph. Develop the bromide paper and measure the angle of curvature

of the hypocotyl stumps with the protractor.

The average degree of curvature of treated

stumps in comparison with that of the un-
12 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

treated stumps indicates the relative cell-elongating property of the chemical.

Suggested standard treatment for

comparison

2,4-dichlorophenoxyacetic acid applied in

95% ethanol so that each 0.0015-ml. droplet

contains 0.065 fj.g, of the acid.

Weintraub, R. L., Brown, J. W., Throne, J. A., and Yeatman, J. N. A Method for Measuring Cell-Elongation-Promoting Activity of Plant-Growth Regulators.

Amer. Jour. Bot. 38: 435-440. 1951.

EPIDERMAL ROOT-CELL TEST

Method based on research by:

Hans Burström; Kersin Lexander.

Description of test and some other uses

Germinated wheat seeds are grown in a nutrient solution containing the chemical being

tested, and its effect on the elongation of epidermal cells is observed with a microscope.

This test can be adapted for the study of environmental factors that influence the ability of

root cells to absorb and respond to plant-regulating chemicals.

Suggested plant material

Roots of youijig wheat plants (Triticum aestivum L., also called T. sativum Lam. and T. vulgäre Vill.)—varieties of Durham or soft red

winter types.

Nutrient solution containing the following

concentrations of compounds computed on

an anhydrous basis :

KNO3 (2.0xlO-*M)

KH2PO4 (3.0xlO-^M)

Ca(N03)2 (4.0xlO-^M)

MgS04 (2.0xlO-*M)

MnCl2 (8.0xlO-«M)

ZnS04 (6.2xlO-«M)

H3BO3 (3.2xlO-^M)

H2M0O4 (1.24xlO-«M)

Fe2(S04)3 (2.5xlO-«M)

Copper-free distilled water

Preparation and selection of plant material

Sterilize seeds and all solutions, equipment,

etc., as follows: soak seeds in 0.1% formaldehyde solution for 15-20 minutes ; then rinse well

with sterile distilled water, and dry. Heat

Petri dishes, pipettes, and tweezers in oven at

about 300° F. (150° C.) for 2 hours. Autoclave

nutrient solution for 10 minutes and distilled

copper-free water for 20 minutes at 20-lb. pressure per sq. in. (9 kg. per 6.5 sq. cm.) at 250°-

260° F. (121°-127° C).

Place 25 seeds in each of 12 flasks and cover

them with 0.1% hydrogen peroxide. Stopper

the flasks and allow them to stand at room temperature, about 65°-68° F. (18°-20° C), for

6 hours.

Rinse seeds four times with sterile distilled

water. Place them in Petri dishes, with embryos up, on filter paper moistened with sterile

distilled water. Put dishes in darkened constant-temperature room at about 72° F. (22°

C.) for 2 days for the seeds to develop primary

roots about 1-2 cm. long.

Apparatus, chemicals, and other materials Method of conducting test

Petri dishes

Filter paper.

Pipettes

Tweezers

Sterilizing oven

Autoclave

50-ml. conical flasks

Constant-temperature chamber or room

controlled at about 72° F. (22° C.)

Analytical balance or pharmaceutical balance

Microscope with ocular micrometer

Slides

Cover glasses

Room relatively free of dust

2 mg. of each compound to be evaluated for

its effect on cell elongation

^3-Indoleacetic acid for standard treatment

Formaldehyde

Hydrogen peroxide

Prepare growth-regulator test solutions 10

times the final desired concentrations (10"^—

10-^M) by dissolving the regulators in sterile

distilled water. A range of three concentrations can thus be prepared for each compound

under study.

Add 4.5 ml. of nutrient solution to each of 24

flasks; stopper, and autoclave for 10 minutes.

Add 0.5 ml. of one growth regulator to each of 5

of these flasks. Repeat with the other two concentrations of this growth regulator. Finally,

add 0.5 ml. of sterile distilled water to 5 control

flasks. Select about 90 wheat plants for uniformity of root length. Place one pair of

plants in each of the 20 flasks. Use the remaining 4 flasks, each representing a control

and one concentration of the growth regulator

under study, for measuring pH, and then discard them. Measure the root length of about
TEST METHODS WITH PLANT-REGULATING CHEMICALS 13

èo of the remaining plants in millimeters.

Place the flasks containing the stérile plants in

the darkened constant-temperature room for 24

hours.

Method of taking results

Record the pH of the solutions and the final

root length of plants given the 4 treatments.

Determine root growth by subtracting initial

root length from final root length. Cut sections

about 5 mm. long from the newly formed part

of 20 roots that received one treatment. Gently

squeeze the sections between a slide and a cover

glass. Keep the sections continuously bathed

in the nutrient solution. Measure under a microscope the lengths of at least 240 fully elongated epidermal cells from the different sections.

Measure in the same way cells from sections of

roots given the other treatments. Compare average lengths of cells of treated roots with those

of the control.

Suggested standard treatment for

comparison

10-8—lO-^M concentration of 3-indoleacetic

acid.

Burström, Hans. Studies on Growth and Metabolism

of Roots. IX. Cell Elongation and Water Absorption. PhysioL Plant. (Copenhagen) 6: 2, 262-276.

1953.

Lexander, Kersin. Growth-Regulating Substances m

Roots o^ Wheat. Physiol. Plant. (Copenhagen) 6:

2, 406-411. 1953.

PEA STEM SECTION TEST

Method based on research by:

G. S. Christiansen and K. V. Thimann.

Description of test

The effect of a compound on stem elongation

is measured by floating sections from etiolated

pea stems on the surface of a solution containing the compound, and measuring the response

in terms of increase in length or weight of the

pieces.

Suggested plant material

Etiolated stems of peas (Pisum sativum L.).

Apparatus, chemicals, and other materials

Dark room with temperature controlled at

about 80°-85° F. (27°-29° C.) and

humidity controlled at 85-90%

Analytical balance or pharmaceutical balance

Glass or metal containers to hold sand in

which to germinate seeds

Corning Signal Red filter

Quartz sand

Razor blades mounted 20 mm. apart on

a block of wood

500-ml. beakers

Filter paper for blotting water from seeds

Petri dishes

Sodium hydroxide for adjusting pH of

solutions and M/1000 potassium chloride

1 g. of 3-indoleacetic acid

250-500 mg. of each compound to be tested

Preparation and selection of plant material

Soak the seeds in tap water for 6 to 8 hours ;

plant them in sand moistened with tap water,

and allow the plants to grow in darkness. All

observations and manipulations should be made

in a minimum of light from an incandescent

lamp passed through the Corning filter. After

7 days, select 30 to 50 plants of uniform size

from which to make stem sections.

Method of conducting test

Do not use plants that have developed fourth

internodes longer than 3 mm. Cut 20-mm. sections (exactly measured) from the third internodes. Immediately place the sections in tap

water, wash, and divide them into groups of 15

sections each. Blot dry with filter paper and

weigh the groups separately to the nearest mg.

Keep sections moistened. / "

Prepare aqueous solutions (25-100 ml.) of

the compounds to be tested, adjusting their pH

to 6.0 with the NaOH, and add the KCl to each

to avoid variation in potassium content of the

solutions. Store the solutions in a refrigerator

if they are to be used repeatedly and make up

new ones every few weeks if they are to be used

for an extended period. Concentrations ranging from 0.1 to 2.0 mg. per liter of distilled

water are suggested.

Place the solution containing the compound

to be tested in a Petri dish and float a weighed

group of 15 sections on the surface of the liquid

for a period of 24 hours in darkness.

Method of taking results

At the end of the 24-hour growth period remove the sections, blot with filter paper, weigh

them as a group to the nearest mg., and measure their length individually to the nearest mm.

Compare the weight increase and average

length increase of these sections (from each
14 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

concentration of the test chemical) with comparable measurements of sections treated with

distilled water only, and others treated with distilled water containing different concentrations

of 3-indoleacetic acid.

Suggested standard treatment for

comparison

Solution containing 0.1, 0.5, 1.0, and 2.0 mg.

3-indoleacetic acid per liter.

Christiansen, G. S., and Thimann, K. V. The Metabolism of Stem Tissue During Growth and Its Inhibition. I. Carbohydrates. Arch. Biochem. 25: 230-

247. 1950.

Emendations submitted by K. V. Thimann.

SPLIT PEA STEM TEST

Method based on research by:

Martha Kent and W. A. Gortner; K. V. Thimann and C. L. Schneider; F. W. Went and

K. V. Thimann.

Description of test and some other uses

The angle of curvature that develops when

split pea stems are immersed in an aqueous solution of a compound indicates the influence of

the compound on cell elongation.

This test has been carefully standardized and

widely used in the measurement of cell-elongating properties of synthetically prepared regulators and regulating compounds obtained from

plant material.

Suggested plant material

Stem sections of etiolated pea plants (Pisum

sativum L.)—Alaska variety.

Apparatus, chemicals, and other materials

Quartz sand

Completely darkened room with temperature

controlled at 80^-85° F. (27°-29° C.)

Ruby glass incandescent lamp

Time clock

Razor blades

Analytical balance or pharmaceutical balance

Petri dishes (10 cm.)

Several 500-ml. beakers

Protractor

Photographic paper and developer,

if shadowgraphs are to be made to record

stem curvatures

200-500 mg. of each compound to be

evaluated

500-1,000 mg. of 3-indoIeacetic acid for

standard treatment

Sodium hydroxide

Potassium chloride

Distilled water

Preparation and selection of plant material

Soak pea seeds in water for 6 hours; then

plant them in moist sand in a container which

allows for drainage, and keep them in complete

darkness. After 7 days the plants should have

developed 2 nodes, each bearing a scale, and one

nearer the top bearing a leaf. Using diffused

white light (about 5- to 10-foot candles), select

plants that have developed between this leaf and

the terminal bud an internode less than 5 mm.

in length.

The sensitivity of the test plants can be increased by exposing them to light. This can be

accomplished in either of two ways: First, 32

hours prior to the start of the tests, expose the

plants for 4 hours to light from a 60-w. ruby

glass incandescent lamp, 2 ft. (61 cm.) away;

second, expose the plants for 10 minutes of

every 12 hours during the period in which they

are grown in sand to light from a 25-w. ruby

glass incandescent lamp 2 ft. (61 cm.) away.

Control the exposure time by means of a time

clock and make the exposures in late afternoon

and early morning. (5ut off the top of each of

5-8 plants 5 mm. from the terminal bud and

discard these parts. Split the stems centrally

lengthwise with a razor blade for a distance of

about 3 cm. Cut off the split sections a few

millimeters below the split portion and wash

the split sections for several hours in tap water.

Method of conducting test

Prepare several dilutions (distilled water) of

the compound to be tested (10"^—lO^M) and

place 10 ml. of each dilution in a separate Petri

dish. These solutions should not be more acid

than pH 4. Transfer 6 or 8 of the split sections to each dish and leave them for 6 to 24

hours in darkness. Curvature will be complete at the end of 6 hours and will not change.

In an alternative test, split oat coleoptiles are

used in place of split pea stems. Grow the

plants (Victory variety), as described, for 76

hours. Decapitate the coleoptiles at a distance

of 2-4 mm. from their tips, and split each one

lengthwise with a razor blade for a distance of

20 mm. from the cut surface. Cut the coleoptile off a few millimeters below the split and

wash the split sections in tap water. Discard

the seeds and roots.

Place not more than 8 of the sections in 20

ml. of the solution to be tested. Use Petri
TEST METHODS WITH PLANT-REGULATING CHEMICALS 15

dishes to contain the test solution and adjust

its pH to 5.5 with sodium hydroxide. Add

0.001 M potassium chloride solution, which is

known to increase slightly the effect of auxin.

Method of taking results

Measure the angles A on each section with

the protractor (fig. 4) and average the measurements. If curves are needed to compare

observations, plot the average values against

the logarithms of the concentration. A straight

line will be obtained within certain limits of

concentration. Use the slope of the line to

compare the activity of different compounds by

the formula :

a: : K log Co

Where a is the mean observed curvature

C the concentration (moles per liter)

Co the concentration at which the

curvature is 0

K the slope of the line

Use at least 3 points to characterize the slope

of the line.

If split coleoptile sections are used, measure

the angles that develop during the 24-hour

period immediately following the immersion of

the sections in the test solution and compare the

activity of different compounds.

Suggested standard treatment for

comparison

10-^ 10-^ and 10-^ M 3-indoleacetic acid.

Kent, Martha, and Gortner, W. A. Effect of Pre-illumination of the Seedlings to Red Light. Bot. Gaz.

112: 307-311. March 1951.

Thimann, K. V., and Schneider, C. L. Differential

Growth in Plant Tissues. Amer. Jour. Bot. 25: 627-

641. 1938.

Went, F. W., and Thimann, K. V. Phytohormones.

Macmillan Co. New York. 1937.

WHEAT COLEOPTILE SECTION TEST

Method based on research by:

F. Wightman.

Description of test

The effects of compounds on cell elongation

are measured by floating sections of wheat coleoptiles on aqueous solutions of the chemical and

comparing the length of these with the length

of sections floated on distilled water.

Figure 4. Shadowgraph of a split section of pea stem showing curvature that developed upon exposure to a growth-regulating

chemical, and method of measuring the angle of curvature.

Lay the stem sections on photographic paper (protect the

paper from moisture with a piece of glass), expose the sections

and paper to light, and develop the image on the paper using

usual photographic methods. Draw a line through the center

of each stem and bisect this line with another line parallel

with the tip of the curved, split portion of the section. Measure

the angle A with a protractor. Draw a similiar line using the

opposite segment, and measure the angle that corresponds

to A. Most regulating compounds having cell-elongating properties induce q decrease in the angle indicated compared

with that formed by the section when placed in distilled water.

The angle measurement illustrated is one of several that have

been utilized. For a general discussion of angle measurements

and their relation to the amount and kind of regulating chemical used, see references listed at the end of this method.

(NEG. PN-295>
16 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Suggested plant material

Germinating seeds of wheat (Triticum aestivvrm L., also called T. sativum Lam., and T, vulgäre Vill.)—Eclipse variety.

Apparatus, chemicals, and other materials

Filter paper (10 cm.)

Petri dishes (10 cm.)

Table space in a room with the temperature

controlled at 76^-78° F. (24^-26^ C.)

Constant humidity boxes with glass covers

Kodak Ruby Signal light filter or other

similar filter

Razor blades

Analytical balance or pharmaceutical balance

250-500 mg. of each compound to be

evaluated

500-1,000 mg. of sodium 2,4-dichlorophenoxyacetate for standard treatment

Distilled water

Preparation and selection of plant material

Place wheat seeds with embryos facing upward on moist filter paper in open Petri dishes

(30-40 seeds per dish) and leave them for 72

hours in a humid atmosphere at 76°-78° F.

(24°-26° C). Maintain a humid atmosphere

(80-85% relative humidity) around the seeds

by placing the germination dishes in constant

humidity boxes. During the first 60 hours expose the seeds to light from an incandescent

lamp (25-w.) passed through the red filter.

Place lamp about 2 ft. (61 cm.) from the seeds.

Allow subsequent growth of the plants to take

place in darkness. About 72 hours after placement of the seeds on the moistened paper, select

plants with coleoptiles 17-18 mm. long. With

a razor blade, or with 2 razor blades fastened

on a wooden block 15 mm. apart, cut one section exactly 15 mm. long from each coleoptile.

Place the sections in tap water.

Method of conducting test

Prepare solutions representing a dilution

series of the compound to be tested by dissolving different amounts of it in distilled water;

suggested range of concentrations 0.01, 0.1, 1,

10, and 100 p.p.m. Place about 20 ml. of each

concentration in respective Petri dishes and

float 15 coleoptile sections on the surface of the

liquid in each dish. Allow the sections to grow

for 24 hours in darkness at 76°-78° F. (24°-

26° C). Float additional sections on distilled

water and use these as controls.

Method of taking results

Remove the sections and blot dry. Measure

each section to the nearest millimeter. Compare the average elongation of sections in the

various solutions with that of sections in distilled water. Differences in the average length

of control and treated sections indicate the effect of the compound on cell elongation.

Suggested standard treatment for

comparison

Solution containing 1 p.p.m. of sodium salt of

2,4-dichlorophenoxyacetic acid.

Method submitted by F. Wightman, Agricultural Research Council Unit on Plant Growth Substances and

Systemic Fungicides, Wye College, University of London, London, England.

Cell Growth

OVARY TISSUE CULTURE TEST

Method based on research by:

J. P. Nitsch.

Description of test

Excised ovaries of various plants are grown

in vitro on synthetic nutrient medium. Regulating or other chemicals are added, and their

effects on growth of the fruits are determined.

Suggested plant material

Commercial varieties of crop plants—tomato

(Lycopersicum esculentum Mill.), bean (Phaseolus vulgaris L.), tobacco, (Nicotiana tabacum

L.), strawberry (Fragaria spp.), cucumber

(Cucunds sativus L.), common varieties in

flower.

Apparatus, chemicals, and other materials

Scalpel

Table space, relatively dust free for use in

ovary preparations, transfers, etc.

Jars

Petri dishes

Filter paper

Test tubes

Forceps

Non-absorbent cotton for test tube plugs

Room exposed to indirect sunlight with tem-
TEST METHODS WITH PLANT-REGULATING CHEMICALS 17

peratures of about 73° F. (23° C.) and night

temperatures of about 68° F. (17° C.)

Calcium hypochlorite (5%, C. P. in water)

Salts for nutrient solution :

Ig. of Ca (N03)2 • 4H2O

1 g. of KNO3

1 g. of Mg SO4 • 7 H2O

1 g. of KH2PO4

Chemicals for trace-element solution :

5 ml. of H2SO4, sp. gr. 1.83

5 g. of MnS04 • 4 H^O

1 g. of ZnS04 • 7 H2O

1 g. of H3BO3

1 g. of CUSO4 • 5 H2O

1 g. of Na2Mo04 • 2 H2O

Ferric citrate solution :

10 g. of FeCeOsH, • 5 H^O

Paraffin

Water redistilled in Pyrex

Agar

100 g. sucrose

Analytical balance or pharmaceutical balance

Preparation and selection of plant material

Use healthy greenhouse plants. At flowering, hand-pollinate strawberry and cucumber

flowers in order to insure pollination. Shake

tomato, bean, and tobacco plants occasionally to

insure pollination.

Cut the flowers from the plants 2 days after

pollination and dip the cut ends into melted paraffin to seal them. Place these flowers into sterile jars and conduct all subsequent operations

in a room free from dust, using sterile utensils

and materials.

Pour a decanted or filtered b% calcium hypochlorite solution into the jar containing the

flowers. After about 10 minutes, pour the calcium hypochlorite solution off and wash twice

with sterile water. Transfer the flowers to

Petri dishes containing a double thickness of

filter paper to absorb the excess water.

Method of conducting test

Prepare the following solutions :

Mineral salt solution

Salt Amount per liter of

final solution

Ca(N03)2 • 4H2O 500 mg.

KNO3 125 mg.

MgS04 • 7 H2O 125 mg.

KH2PO4 125 mg.

Add sufficient water redistilled in Pyrex to

make 1 liter of final solution.

Trace-element solution

Amount per liter of

finxd solution

0.5 ml.

3,000 mg.

500 mg.

500 mg.

25 mg.

25 mg.

Component

H2SO4, sp. gr. 1.83

MnS04 • 4 H2O

ZnS04 • 7 H2O

H3BO3

CUSO4 • 5 H2O

Na2Mo04 • 2H2O

Add sufficient water redistilled in Pyrex to

make 1 liter of final solution.

Ferric citrate solution

10 g. of FeCcOgHr • 5 H2O diluted to 1 liter

of water redistilled in Pyrex.

To 1 liter of the mineral salt solution add 1

ml. of the ferric citrate solution, 1 ml. of the

trace-element solution, and 50 g. of sucrose.

Divide the medium into separate aliquots and

add required amounts of the test chemicals.

Three treatments and a control, each having 10

replicates, can be run by dividing 1 liter of nutrient medium into four 250-ml. portions and

supplying each ovary with 25 ml. of nutrient

medium in separate tubes. To support the

ovaries, use filter-paper discs having a diameter

of about twice that of the tubes. Make a hole

in the center of each disc slightly larger than

the diameter of the pedicel. Form a paper cup

with each disc by pressing it against the edge

of a tube. Push one cup inside of each tube,

edges downward, so that it stays at least 2

inches above the level of the liquid. Plug the

tubes with nonabsorbent cotton. Autoclave the

tubes 20 minutes at 15 lbs. pressure. When

planting the sterile ovaries, push the paper down

with the forceps until the pedicel reaches the

liquid.

Avariant of this technique uses no filter paper

but solidifies the media with 1% agar. Suggested concentrations of the chemical to be tested

are 0.001-100 mg. per liter in serial dilutions.

Due to possible breakdown at high temperatures it is sometimes advisable to avoid autoclaving solutions that contain organic compounds.

Addition of test chemicals aseptically to the

nutrient medium can be done as follows : Autoclave the four 250-ml. portions of nutrient

medium mentioned previously and the plugged

tubes containing the filter-paper discs, separately. Sterilize a metal spoon by dipping it

into alcohol, and allow it to dry. Weigh the required amount of test chemical, place it in the

spoon, and add sufficient ethyl alcohol to barely

cover the compound. Allow the alcohol to evaporate; then immerse the bowl of the spoon containing the chemical in one of the sterile portions

of nutrient medium and stir until the compound
18 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

is uniformly dispersed. Remove the cotton

plugs and the filter-paper discs from the tubes

with sterile forceps and pour approximately 25

ml. of the mixture directly into each tube. Replace the filter-paper discs and cotton plugs.

Cut off the stamens, petals, two-thirds of the

sepals, and most of the pedicel from the sterilized flowers. Cut the pedicel off above the

abscission zone (at least 1 mm. below base of

flower) to eliminate the abscission zone and

prevent the abscission phenomenon from interfering with the growth of the ovary. Care must

be taken with tomatoes to avoid any film of

liquid remaining between the sepals and the

ovary, since this seems to cause the ovary to turn

brown and die. Support the sterile, trimmed

flower on the disc of filter paper provided with

a central hole so that only the pedicel extends

into the solution.

Place the tubes containing the ovaries on

shelves where they are exposed to indirect sunlight in a room with a day temperature of about

73° F. (23° C.) and a night temperature of

about 63° F. (17° C).

Method of taking results

At intervals measure length and diameter

with a small translucent metric rule through

the walls of the test tubes. At conclusion of

experiment, remove each ovary and accurately

measure it. Prepare a table of length and

diameter dimensions with corresponding volumes for each specific variety of plant. Use

these data for comparing growth rates of treated

and untreated fruits.

Suggested standard treatment for

comparison

No standard treatment suggested.

Nitsch, J. p. Growth and Development in vitro of Excised Ovaries. Amer. Jour. Bot. 38 (7) : 566-577.

1951.

Nitsch, J. P. Test Tube Fruits: A New Technique in

Fruit Physiology. Report of the Thirteenth International Hort. Congress, pp. 263-266. 1952.

ROOT TISSUE CULTURE TEST

Method based on research by

Philip R. White.

Description of test

Root tips from tomato seedlings or cuttings

cultured in vitro are treated with various regulators or other chemicals, and their effect on

growth of root cells is determined.

Suggested plant material

Ripe tomato fruits, or tomato plants (Lycopersicum esculentum Mill.) from which cuttings

can be made.

Apparatus, chemicals, and other materials

Scalpel

Forceps

Petri dishes

Filter paper (No. 1 Whatman)

Dark chamber maintained at room

temperature of 70°-75° F.

(21^-24° C.)

Wide-mouth glass jars

Blotting paper

Heavy, paraiRn-coated cardboard

Analytical balance or pharmaceutical balance

10-liter bottle

2,500-ml. Pyrex beakers

Test tubes

Rubber test tube stoppers

Refrigerator space

125-ml. Erlenmeyer flasks

Absorbent cotton

Gauze

50-ml. beakers or paper cups

Autoclave

Salts for nutrient solution :

25g.ofCa(N03)2

25 g. of Na2S04

10 g. of KCl

2 g. of NaH^PO^

1 g. of MnS04

1 g. of ZnS04

1 g. of H3BO3

1 g. of KI

50 g. of MgS04

Vitamins :

0.5-1 g. of glycine

0.5 g. of nicotinic acid

0.5 g. of thiamine

0,5 g. of pyridoxine

Sugar-iron solution :

50 g. of C.P. sucrose

lg.ofFe2(S04)3

Compounds to be evaluated as

growth regulators

Sodium hypochlorite solution,

such as laundry bleach

Distilled water

Preparation and selection of plant material

Seed Method: Select sound, ripe tomato

fruits; wash and dry them carefully. Sterile

equipment and aseptic conditions should be

utilized throughout the experiment. With a
TEST METHODS WITH PLANT-REGULATING CHEMICALS 19

scalpel, divide the fruit into quarters by making cuts into the fruit deep enough so that it

can be broken open easily. Start the incisions

about Vè in. (1 cm.) from the stem and cut to

the stylar end. Break open the fruit and expose the seeds carefully so that the seeds do

not come in contact with fingers or with the

outer, nonsterile surface of the fruit. Remove

25 mature seeds that have not been touched

with the scalpel, with sterile forceps, and place

them in 5 Petri dishes (5 seeds per dish) fitted

with filter paper moistened with the sterile

water. Keep the Petri dishes in a dark place

maintained at a temperature of about 70°-75°

F. (21°^24° C.) for a few days until the seeds

germinate and the roots reach a length of

2-3 cm.

Cutting Method: Grow tomato plants in a

greenhouse or field plot. Take 6 straight stem

cuttings about 20 cm. long from a healthy

tomato plant and remove the leaves. If all

these cuttings are from the same plant, genetic

variability will be reduced. Wash thoroughly

with the 5% hypochlorite disinfectant diluted

1 to 10 parts of water. Rinse with sterile

water and shake off the surplus, tine the

wide-mouth glass jars with sterile blotting

paper and add sterile water to a depth of about

3 cm. Cover each jar with a sheet of heavy,

parafíin-coated cardboard a little larger than

the mouth of the jar. Make a hole in the cardboard and insert the basal end of a tomato cutting through it so that about 15 cm. of stem

extends into the moist air above the water surface. Keep the jars containing the cuttings in

a dark place at a temperature of about 70°-75°

F. (21^-24° C.) for about 10 days, or until new

roots develop to a length of 3-5 cm.

Method of conducting test

Prepare a stock salt solution, using the best

grades of analytical chemicals, by dissolving

20 g. of Ca(N03)2, 20 g. of Na2S04, 8 g. of KCl,

1.65 g. of NaH2P04, 0.45 g. of MnS04, 0.15 g.

of ZnS04, 0.15 g. of H3BO3 and 0.075 g. of KI

one at a time in 8 liters of water, redistilled in

Pyrex. Also use water redistilled in Pyrex in

preparing the following solutions. If hydrated

salts are used, it is essential to make weight

corrections. Dissolve 36 g. of MgS04 separately in 2 liters of water. Mix the two solutions slowly in a 10-liter bottle. Store this

stock solution, which is 10 times the concentration needed, in the dark or in a black bottle.

Prepare a second stock solution of vitamins

by dissolving 300 mg. of glycine, 50 mg. of

nicotinic acid, 10 mg. of thiamine, and 10 mg.

of pyridoxine in 100 ml. of water. This solution is 100 times the concentration needed.

Pour 20 ml aliquots of this vitamin stock solution into test tubes, stopper, and store in a

refrigerator.

Sucrose is used as a carbon source for the

tissue cultures. Dissolve 40 g. of C. P. sucrose

in 1 liter of water. Dissolve 10 mg. of

Fe2(804)3 in 100 ml. of water, discard half the

quantity, and add the remainder to the sugar

solution. (Diluting and discarding half the

ferric sulfate solution eliminates the necessity

of weighing accurately a quantity of the salt

less than 10 mg.)

Mix the sugar-ferric sulfate solution with 200

ml. of the stock salt solution and 20 ml. of the

vitamin stock solution, and add sufficient water

to make 1,600 ml. Distribute 40 ml. of this complete nutrient solution to each of the desired

number of the 125-ml. Erlenmeyer flasks. Plug

each flask with cotton covered with gauze and

cap with a 50-ml. beaker or small paper cup to

keep out dust while allowing air exchange.

After being autoclaved (15 lbs. pressure per

sq. in. for 20 minutes) and allowed to cool, the

nutrient in each flask is ready for the addition

of 10 ml. of sterile distilled water containing

the test compound at 5 times the final concentration desired. For control flasks, add 10 ml.

of the sterile water.

Select 10 roots, 2-3 cm. long, from the germinated seeds ; sever them with a sharp scalpel,

and transfer each root to a flask of nutrient.

In order to obtain a suflScient number of root

tips from a single source, culture these roots

for a week ; then cut 1-cm. tips from the main

root and well-established branch roots, using

scissors. With a sterile bacteriological loop,

insert each root tip into a culture flask containing fresh nutrient. If rooted cuttings are used,

cut 1-cm. root tips and insert into culture flasks

in the same manner. To reduce the possibility

of genetic effects influencing the results, use

root tips derived originally from a single seed

or from cuttings from a single plant. Or, if a

larger amount of material is needed for a more

extensive experiment, label and distribute such

genetically uniform roots so that one or more

is used for each treatment.

Treat the excised roots with plant regulators

by adding these in the required amounts aseptically (i.e., using sterile water and utensils)

to the nutrients as described above. A range

of final concentrations from 10"^^ to 10-^ g. per

liter, prepared by serial dilution is suggested.

Do not autoclave the aqueous solutions of regulating chemicals. Some regulating chemicals

are relatively insoluble in water in the acid,

ester, or amide form. Employ a salt form of

these compounds to facilitate their use in this

method. Use a group of untreated solutions,

each brought to a volume of 50 ml. with sterile

distilled water as controls. Place the culture
20 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

flasks in a dark place at 70^-75" F. (21°-

24° C).

Method of taking results

Since roots grown in culture do not undergo

secondary thickening, and therefore the dianieter remains constant to within a few millimeters of the growing point, length measurements are an accurate measure of volume.

Without removing the root from the culture

flask, make day-by-day measurements from the

outside using a flexible celluloid rule, either by

bending the rule to approximate the root curvature or by washing the root up onto the wall

of the flask so that it lies straight. Also measure growth at the end of the week by removing

the root tissue and obtaining wet or dry weights.

Suggested standard treatment for

comparison

No standard treatment suggested.

White, Philip R. The Cultivation of Animal and Plant

Cells. 239 pp. Ronald Press Co., New York. 1954.

STEM TISSUE CULTURE TEST

Method based on research by

A. C. Hildebrandt and A. J. Riker; B. E.

Struckmeyer, A. C. Hildebrandt, and A. J.

Riker; P. R. White.

Description of test

Squares of phloem tissue from willow are

grown on nutrient agar containing regulating

chemicals and effects of the chemicals on

growth and tissue development determined.

Suggested plant material

Young willow tree (Salix nigra L. or S,

caprea L.).

Apparatus, chemicals, and other materials

Sharp knife

Scalpel

Tweezers

Cork or soft pine board

5-liter bottle

125-ml. Erlenmeyer flasks

Test tubes

Rubber test tube stoppers

Refrigerator space

Cotton

Gauze

50-ml. beakers or paper cups

Autoclave

Chemicals for complete nutrient

solution (see page 18)

Compounds to be evaluated as

growth regulators

Agar (Difco "Noble" brand)

Distilled water

Analytical balance or pharmaceutical balance

Preparation and selection of plant material

Cut from the young willow tree a piece of

branch or stem about 1 ft. (30 cm.) long and

3-10 mm. in diameter. Take the branch into

the laboratory. Surface-sterilize by first scrubbing the surface with soap and water. Dip the

branch into 95% alcohol several times and burn

the alcohol off each time after the branch is

dipped.

Method of conducting test

Prepare a standard salt solution and a vitamin stock solution, as described on page 19, but

use only half the volume of water. Use sterile

water redistilled in Pyrex for all solutions.

Dissolve 40 g. of C. P. sucrose in 400 ml. of

water. Dissolve 10 mg. of Fe2(S04)3 in 100

ml. of water, discard half the quantity, and add

the remaining 50 ml. to the sugar solution.

Prepare a 1 % agar solution by dissolving 10 g.

of Difco "Noble" agar (or its equivalent in

thoroughly leached agar) in 1 liter of hot water.

Combine the agar, the sugar-ferric sulfate solution, 100 ml. of stock salt solution, and 10 ml.

of vitamin stock solution, and add sufficient

water to make 1,600 ml. Mix together thoroughly and divide the hot nutrient-agar solution

into aliquots, each containing 60 ml., and autoclave. To a 60-ml. aliquot of the hot agar solution, add 15 ml. of warm aqueous solution of

the test compound. This solution should be

prepared with sterile distilled water in sterile

utensils and should be 5 times the desired final

concentration. It is desirable to use salt forms

of the growth regulator. While the agarchemical mixture is still hot, distribute equal

volumes of it among 5 sterile test tubes. Repeat, using other concentrations and regulators,

and prepare one set, using distilled water in

place of the regulator, as controls. A range of

concentrations of the regulator from 10-^^-10"^

g. per liter is suggested. Plug the test tubes

with cotton, and cap them with 50-ml. beakers

or paper cups to keep out dust. If the nutrient-agar medium does not solidify satisfactorily on cooling, adjust the acidity to a pH not

lower than 5.5 with 0.1 N sodium hydroxide.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 21

Lay the sterile branch on a sterile block and,

with a sterile scalpel, make a series of transverse cuts through the branch, about 1 mm.

apart. In this way, obtain 10 or 15 branch

sections or discs. If the branch is 5 mm. or

more in diameter, divide the discs into several

sectors. If a smaller branch is used, merely

cut the discs in half. Cut off and discard outer

cortical tissue (bark) and much of the wood.

The remaining blocks should be rectangular

with the long axes tangential to the original

surface and transversed by the cambium.

After the nutrient agar has cooled and solidified, transfer one of the prepared blocks to each

tube using sterile tweezers. Push the blocks

part way into the agar so that they are left half

exposed and half submerged. Stopper the tubes

with cotton and set them aside in the dark at a

room temperature of 70°-75° F. (21°-24° C).

Method of taking results

After 4 to 6 weeks, compare the growth of

tissue on nutrient agar containing various concentrations of growth regulators with growth

on nutrient agar alone. Proliferation should

occur at or just above the agar level, the new

growth spreading out over the agar in the control tubes. Study the effects of the growth

regulators on development of the treated tissue

during a 4- to 6-weeks' observation period.

After this period, remove the samples of tissue

grown on nutrient agar and on nutrient agar

plus regulating chemical, and compare their

wet or dry weights. In addition, anatomical

effects, such as total number of cells, number

of trachéal elements per unit area, and gross

morphological effects, should be recorded.

Suggested standard treatment

No standard treatment suggested.

Hildebrandt, A. C, and Riker, A. J. Influence of Some

Growth-Regulating Substances on Sunflower and Tobacco Tissue in vitro. Amer. Jour. Bot. 34: 421-427.

1947.

Struckmeyer, B. E., Hildebrandt, A. C, and Riker, A. J.

Histological Effects of Growth-Regulating Substances

on Sunflower Tissue of Crown-Gall Origin Growth in

vitro. Amer. Jour. Bot. 36: 491-495. 1949.

White, P. R. The Cultivation of Animal and Plant Cells.

239 pp. Ronald Press Co., New York. 1954.

Effect of One Compound on the Absorption and Translocation of Another

BEAN STEM CURVATURE TEST

Method based on research by:

A. S. Crafts; J. W. Mitchell, W. M. Dugger,

Jr., and H. G. Gauch.

Description of test

The magnitude of stem curvature induced by

a known amount of a growth-modifying substance is compared with that induced by the

same amount of the substance plus an adjuvant.

Suggested plant material

See page 7.

Method of conducting test

Using the bean stem curvature test, page 8,

determine the amount of ammonium 2,4-

dichlorophenoxyacetate or other water-soluJDle

salts of the acid required to induce a stem curvature of about 5°-10° in 21/2-3 hours. In

these preliminary tests, dissolve the salt directly in distilled water and use a concentration

range that includes 0.2, 0.4, 0.6, 0.8, 1.0, and

1.2 fig. of the salt per 0.02 ml. of solution.

After the threshold concentration has been

determined, prepare 500 ml. of this concentration. Divide it into the required number of

50-ml. portions. Reserve one portion to be

applied as a control, and mix each adjuvant to

be tested with a 50-ml. portion. Apply to test

plants as described on page 8.

Apparatus, chemicals, and other materials Method of taking results

See page 7.

Compounds to be tested as adjuvants are also

required.

Preparation and selection of plant material

See page 8.

Determine the effect of the adjuvants on the

magnitude of stem curvature induced in 21/4 to

3 hours by comparing the average curvature

developed by plants treated with the salt alone

with that developed by plants treated with the

salt plus the adjuvant.
22 AGRICULTURE HANDBOOK 126, U.S. DEPT. OP AGRICULTURE

Suggested standard treatment for

comparison

0.8, 0.9, or 1.0 ¡Jig, of ammonium 2,4-dichlorophenoxyacetate or other water-soluble salt

applied in distilled water alone.

Crafts, A. S. Herbicides, Their Absorption and Translocation. Agr. and Food Chem. 1: 51-55. 1953.

Mitchell, J. W., Dug-grer, W. M., Jr., and Gauch, H. G.

Increased Translocation of Plant Growth-Modifying

Substances Due to Application of Boron. Sei. 118:

354-355. 1953.

Exudation of Regulators from Roots

BEAN TEST

Method based on research by:

W. H. Preston, Jr., J. W. Mitchell, and W.

Reeve.

Description of test

Two young plants, one treated by placing a

compound on its stem or leaves, are grown in

soil contained in a pot. Formative or other

effects that become apparent as the untreated

plants develop indicate transfer of the compound from one plant to the other through the

root systems.

Suggested plant material

Young herbaceous dicotyledonous plants

known to respond morphologically to the compounds to be tested—snap bean (Phaseolus vulgaris L.), cucumber (Cucumis sativus L.),

tomato (Lycopersicum esculentum Mill.), squash

(Cucúrbita spp.), sunflower (Helianthus annuus

L.).

Apparatus, chemicals, and other materials

Clay pots 3,4, or 5 in. (8,10,

or 13 cm.)

Composted soil

Greenhouse space maintained at

70^-85° F. (21°-29° C), slightly

cooler at night

Shell vials (about 10-ml. capacity)

Analytical balance or pharmaceutical balance

Small glass or wooden rods used

as applicators (1- to 2-mm.

diameter, 5-10 cm. long)

250-500 mg. of each compound to be

evaluated

Alpha-methoxyphenylacetic acid for

standard treatment ^

About 50 ml. of Tween 20

About 50 g. of lanolin

2-400 ml. beakers

Source of compressed air

Preparation and selection of plant material

Plant 3-4 seeds per pot in composted soil if

a single species is used. If two species are

employed, plant 3-4 seeds of each per pot.

After germination, allow the plants to grow

until the first leaf formed above the cotyledons

is partially expanded. While the next leaves

are still tightly folded in the terminal buds,

select in each pot two vigorously growing plants

that are the same species or of two different

species. Discard all plants except these two

by severing their stems near' the soil level.

Arrange 3-5 pots (each containing a pair of

plants) in a row to be used for each compound

tested.

Method of conducting test

Prepare a 1% lanolin mixture of the compound being tested, as described on pages 5-6.

With an applicator, apply a portion of the paste

about the size of a wheat seed to one plant in

each pot in one row. Leave the other plant in

each pot untreated. Apply the paste as a band

2-3 mm. wide around the first internode of each

plant midway between the first and the second

node (or around the upper portion of the hypocotyl if cucumber or sunflower is used). Allow

the treated and untreated plants to grow in a

greenhouse for a week or more.

For an alternative method, carefully wash

the roots of at least three of the young plants

free from soil. Support the plants with their

roots immersed in about 200 ml. of tap water

in the beaker. Aerate the water by passing

compressed air as fine bubbles through it.

Treat the stems of all of the plants as described.

Arrange a second set of plants in another

beaker of water without treatment as controls.

Allow all of the plants to grow for 3 days, replacing any water lost. Then remove and discard all of the plants. Introduce an equal

number of young untreated plants and allow

them to grow for several days with their roots

in the continuously aerated water that previously supported growth of the treated and

untreated plants.

Method of taking results

In the case of plants with their roots in soil

contained in the same pot, observe in the un-

- Obtainable from The Monroe Chemical Company,

P.O. Box 388, Rockland, Mass.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 23

treated plants any growth modification such as

deviation in size or shape of newly expanded

leaves (formative effects). Make a similar

comparison for the alternative test using

aerated water.

Supplementary test — Determination of the

path of movement of the compound

In the case of volatile compounds there is a

possibility that the compound may pass from

the treated plant to the untreated one through

air. To determine the path of movement, select 5 pots of test plants as previously described

with 2 plants of uniform size growing in each.

Also select 10 other plants, each growing individually in a pot. Cut out 10 squares (5 cm.)

of cardboard and punch 2 holes, 1 cm. in diameter and 15 mm. apart, in each square. Make

a narrow slit from the edge of the cardboard

to each hole. Using the pots containing 2

plants each, slip the cardboard around the stem

of each plant so that the stems protrude through

the holes and are thus held about 15 mm. apart.

Place the pots containing individual plants in

pairs and slip a cardboard piece around the

stems of each pair of plants in the same manner.

Apply the compound to be tested to the stem

of 1 plant of each pair. Allow the plants to

grow in a greenhouse until the next 2 leaves

are partially expanded. Leaf modification in

both plants with their roots in separate pots

indicates that the compound moved through the

air. Modification of leaf shape in both plants

growing in soil contained in the same pot, but

not in the untreated plants of pairs growing

with their roots in separate pots, indicates that

the compound passed from one plant to the

other through their root systems.

Suggested standard treatment for

comparison

Alpha-methoxyphenylacetic acid, 1 % lanolinTween 20 paste.

Preston, W. H., Jr., Mitchell, J. W., and Reeve, W.

Movement of Alpha-methoxyphenylacetic Acid from

One Plant to Another Through Their Root Systems.

Science 119: 437-438. 1954.

Form or Growth Modification

BEAN LEAF TEST

Method based on research by:

J. W. Brown and R. L. Weintraub.

Description of test and some other uses

The growth-regulating chemical in a small

droplet applied to the terminal bud of bean

plants suppresses the vegetative growth of the

young leaves, which serves as an index of

growth-suppressing activity.

With slight modification, this test can be

used for studying the effect of regulating chemicals on the formation of abscission layers near

the terminal buds of succulent plants.

Suggested plant material

Young bean plants (Phaseolus vulgaris L.)

—Black Valentine, Red Kidney, Bountiful

varieties. ' ';

Apparatus, chemicals, and other materials

Composted soil

4-in. (10-cm.) clay pots or

other suitable containers in

which to grow plants

Greenhouse or, for more precise

experiments, use an artificially

lighted room (about 700-foot

candles) equipped with temperature

and humidity controls

Analytical balance or pharmaceutical balance

5-lambda pipette

Photographic paper, printer,

and developer

200 mg. of each compound to

be evaluated

200 mg. of 2,4-dichlorophenoxyacetic acid for

standard treatment

95% ethanol

Tween 20 or a similar wetting

agent

Sulfuric acid

Preparation and selection of plant material

Plant 3 or 4 bean seeds in soil in each of the

containers and grow the plants under greenhouse conditions. If the plants are grown

under controlled environmental conditions, employ a day length of approximately 14-16 hours,

and a minimum of 700-foot candles of light, a

day temperature of about 75° F. (24° C), a

night temperature of about 72° F. (22° C.)

and, if possible, a relative humidity of 50-70%.

Select test plants for uniformity (1 plant per
24 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

pot) after 6-8 days when the second internode

is 3-7 mm. in length. Cut off and discard all

other plants.

Method of conducting test

Dissolve a weighed amount of chemical to be

tested in a measured volume of solvent mixture

composed of 95% ethanol and 1% Tween 20.

With the lambda pipette, apply a 5-lambda

droplet to the terminal bud of each of 10 to 20

test plants. Repeat the procedure, using additional plants for each desired dosage level.

Apply equal amounts of the solvent mixture to

additional plants for comparison, and leave

others untreated for controls. Replace all

plants in the greenhouse or under the uniform,

controlled environmental conditions described

until the control plants have developed a partially expanded, trifoliate leaf (5-13 days).

Method of taking results

At the end of this growth period, remove the

first trifoliate leaf from each plant, and determine its fresh weight and area. Leaf area

may be obtained by tracing the outline of the

leaf on paper and cutting out the image. Weigh

a piece of the paper that is exactly 10 cm.

square. Determine the weight of the leaf

image and calculate its area from the known

weight of 1 square cm. of paper. A contact

shadowgraph of the leaf made on photographic

paper can be substituted for the tracing as used

above. Calculate the effect of the chemical on

leaf area as percentage increase or decrease in

comparison with controls. Record any other

differences between leaves on treated and control plants.

Description of test

Compounds are applied in a paste carrier to

stems of young plants, and the effect on their

subsequent growth and development is observed.

Suggested plant material

Young herbaceous plants—bean (Phaseolus

vulgaris L.), cucumber (Cucumis sativus L.),

sunflower {Helianthus annuus L.).

Apparatus, chemicals, and other materials

4-in. (10-cm.) clay pots

Composted soil

Greenhouse space with temperature controlled at 75°-85° F.

(24°-29° C.)

Shell vials (about 10-ml.

capacity)

Wooden applicators that can be

used once and discarded (obtainable without cotton from

drug- or hospital-supply store)

Analytical balance or pharmaceutical balance

100-200 mg. of each chemical

to be evaluated

For standard treatment :

50 mg. of 2,4-dichlorophenoxyacetic acid, or 50 mg. of

(4-hydroxy-5-isopropyl-2-methylphenyl) trimethyl ammonium

chloride, 1-piperidine-carboxylate

(Amo-1618), or 50 mg. of 3-indoleacetic acid

Tween 20

Lanolin

Suggested standard treatment for

comparison

Use standard dose of 0.015 ¡ig, of 2,4-dichlorophenoxyacetic acid per 5-lambda droplet

per plant. This induces approximately 48%

suppression in leaf area.

Brown, J. W., and Weintraub, R. L. A Leaf-repression

Method for Evaluation of Formative Activity of Plant

Growth-Regulating- Chemicals. Bot. Gaz. Ill: 448-

456. 1950.

Preparation and selection of plant material

Plant 3 or 4 seeds of one of the species per

pot in the composted soil. Germinate the seeds

at about 85° F. (29'' C.) and as soon as the

plants appear above the soil, reduce the temperature 5°-10° F. (9°-18° C). After the

plants are erect and about 2-3 in. (5-8 cm.)

tall, select one in each pot that is of average

size. Pinch the remaining plants oif at the soil

level and discard them.

LANOLIN PASTE TEST

Method based on research by:

P. C. Marth, W. H. Preston, Jr., and J. W.

Mitchell.

Method of conducting test

Arrange each species of plant in a group with

3 to 5 plants per row. Prepare lanolin paste

as described on page 5. With an applicator

stick, apply a portion of the paste about the

size of a wheat seed in the form of a band 3-6
TEST METHODS WITH PLANT-REGULATING CHEMICALS 25

mm. wide around the first internode midway

between the first and second nodes of each bean

plant in one row. Use the same procedure if

cucumber and sunflower plants are employed

except place the band on the hypocotyls of these

plants just below the cotyledons. Allow the

plants to grow foi* 7-12 days along with several

rows of untreated plants. Additional plants

should be treated in a similar manner with the

lanolin-Tween 20 paste alone to study the effect

of the carriers.

As a modification, using additional plants,

apply an equal amount of the paste containing

the chemical to be tested to an area about 4 mm.

wide and 10 mm. long on the upper surface of

one primary leaf of each bean plant and on the

upper surface of the first true leaf of the cucumber and sunflower plants. For the most

effective treatment, cover an area starting at

the junction of the petiole and leaf blade and

extending toward the tip of the blade. Cover

the midrib and an area extending about 2 mm.

on each side of it with the paste.

Method of taking results

For bean and sunflower plants, measure the

terminal growth that occurs during the week

following treatment by placing the end of a

ruler at the node immediately above the treated

section of the stem and determining the distance from this point to the tip of the terminal

bud. Measure the untreated plants in a similar manner. In the case of cucumber plants,

place the end of the ruler at the cotyledonary

node and measure the distance from this point

to the tip of the first true leaf, since during the

test period the measurable growth occurs almost entirely in this leaf. Measure corresponding control plants in the manner described.

Average the measurements in each test and

compute the percentage of inhibition or stimulation of terminal growth of bean and sunflower and leaf growth of cucumber by the

following formula :

Untreated control ^ minus treated

Untreated control

-X 100 = % inhibition

(plus value) or

stimulation

(minus value)

During the test, note any differences in the

shape of leaves, stems, or other parts that develop, the development of galls indicating cell

enlargement or cell proliferation, initiation of

roots, color changes, and other modifications of

' Control plants treated as described with the lanolinTween 20 paste alone should also be included if the effect

of such substances on the test plants is not known.

growth that result from presence of the chemical in the plant. Note the position of the

affected parts in relation to the treated area as

an indication of transportability of the compound.

Suggested standard treatment for

comparison

On the basis of knowledge gained from the

literature or previous experience, select several

compounds known to be relatively effective in inducing specific responses, such as 2,4-dichlorophenoxyacetic acid for gall formation, translocation, and inhibition of terminal growth;

Amo-1618 for suppression of stem growth;

3-indoleacetic acid for gall formation and induction of root primordia on stems. Apply

these to the selected test plants as examples of

compounds that are relatively effective in inducing these various types of growth modifications.

Marth, P. C, Preston, W. H., Jr., and Mitchell, J. W.

Growth-Controlling Effects of Some Quaternary Ammonium Compounds on Various Species of Plants.

Bot. Gaz. 115: 200-204. 1953.

UNDERGROUND GROWTH TEST

Method based on research by:

T. J. Muzik and H. J. Cruzado.

Description of test

Plants are grown in soil in boxes constructed

with glass sides in order to observe the effects

of chemicals on growth and development of the

underground parts without disturbing the

plants.

Suggested plant material

Plants or plant parts whose underground

growth is to be observed, such as seeds, stem

pieces, root pieces, bulbs, corms, tubers, and

rhizomes.

Apparatus, chemicals, and other materials

Wooden or metal boxes 9 in. wide at

the top X 30 in. long x 17 in.

deep (23 x 76 x 43 cm.) with glass

sides slanting inward at an angle

of about 60 "" and with removable

wooden, tar paper, metal, or cloth

shutters to exclude light

Composted soil

Greenhouse or other space suitable

for growing test plants
26 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Small hand sprayer

1-5 g-. of each compound to be

evaluated

For standard treatment :

1-2 g. of 2,4-dichlorophenoxyacetic

acid, maleic hydrazide, or 3-indolebutyric acid depending upon type of

response to be studied

Tween 20 or other suitable solvents

and wetting agents for preparing and

applying sprays

Lanolin

Analytical balance or pharmaceutical balance

Preparation and selection of plant material

Select uniform plant parts such as seeds,

stem pieces, chains of nutgrass nutlets (Cyperus rotundus), rhizomes, and bindweed {Convolvulus arvensis L.), or Canada thistle

(Circium arvense Scop.), and plant them in

composted soil against the sloping glass sides

of the boxes.

Method of conducting test

Apply the cheniical to be tested to the plant

parts before planting, after emergence, or to

the soil surface before emergence. Use a foliage spray application (1,000 p.p.m. suggested),

a dust or spray application (range of 0.1-16 lb.

per acre suggested) (1 and 7 kg. per 4,047 sq.

m.), or as a paste (0.1-1% in lanolin) applied

to the leaves or stems covering areas of 1-4 sq.

cm. Leave some entire boxes of plants untreated as controls. Maintain the plants at

optimum moisture, light, and temperature conditions for growth of the test species employed.

Method of taking results

Observe the underground parts of treated and

untreated plants periodically by removing the

shutters. Periodically, record data on root initiation and elongation, death of roots, rate of

tuber formation, bud or stem growth, discoloration, and other responses. Comparison of

these results with those from the control plants

indicates the effectiveness of the compounds for

inducing the type of response involved.

Suggested standard treatment for

comparison

For standard treatment, compound used will

depend upon type of plant response involved.

For example, growth suppression, 2,4-dichlorophenoxyacetic acid, maleic hydrazide ; root initiation on stem or root pieces, 3-indolebutyric

acid.

Muzik, T. J., and H. J. Cruzado. The Effect of 2,4-D on

Sprout Formation in Cyperus Rotundus. Amer. Jour

Bot. 40: 507-512. 1953.

Fruit Set

AEROSOL METHOD

Method based on research by:

L. D. Goodhue; C. L. Hamner, and H. A.

Schomer; P. C. Marth, and E. M. Header.

Description of test

Regulating or other chemicals are applied to

plants or plant parts in the form of mists produced by use of liquefied gases.

Suggested plant material

Tomato plants (Lyc&persicum esculentum L.)

that have developed two open flowers in the first

cluster.

Apparatus, chemicals, and other materials

Test-tube-type apparatus (fig. 5) for

small-scale experiments or screening tests

Metal (steel) containers of 0.5-1 lb.

(.23-.45 kg.) capacity developed for

applying insecticidal liquefied gas aerosol

mixtures and fitted with 1/2-in (1.27 cm.)

refrigerant valves

Flexible rubber pressure tubing for

transferring liquefied gases

Brass fittings for connecting valves

and tubing

Analytical balance or pharmaceutical balance

Suction pump or air vacuum line

50-mm. metal funnel soldered into i4-in.

(.5 cm.) threaded brass valve fitting

Spring scales or balance with a capacity

of 1,000 g.

Spray nozzles (standard oil burner nozzles

or capillary metal tube soldered into

14-in. [.5-cm.] thread fitting)

25 mg.-l g. of each compound to be tested

5.0 g. beta-naphthoxyacetic acid for

standard treatment

Dimethyl ether

100-200 ml. of solvents such as cyclohexanone, acetone, or carbitol that are
TEST METHODS WITH PLANT-REGULATING CHEMICALS 27

miscible with liquefied gases and which

will dissolve chemicals to be tested

5-25 lb. (2.3-11.3 kg.) supply tank of

methyl chloride (boiling point 10.5° F.

[-11.9° C.]), or dichlorodifluoromethane

(Freon) (boiling point 21.7° F. [-6° C.})

or dimethyl ether (boiling point 9.6° F.

[-12.4° C.})

c

Figure 5. Views of unassembled and assembled glass-walled aerosol bomb used in experimentally preparing aerosol mixtures of regulating

chemicals. (NEG. PX-296 A,B)
28 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Preparation and selection of plant material

Tomato plants (Lycopersicum esculentum L.)

of varieties such as Marglobe, Globe, Rutgers,

or others that can readily be grown in a greenhouse.

Method of conducting test

Preparing and loading aerosol mixtures

A. For test-tube-type apparatus shown in figure 5 : Prepare 25 g. of aerosol mixture containing a 1% concentration of beta-naphthoxyacetic

acid as follows : Weigh out 2.5 g. of this acid

and place in bottom of the test tube ; dissolve

this in 2.5 ml. of cyclohexanone (or acetone) ;

place the test tube in holder and seal tightly

with basal plug. Connect the valve with a suction pump or vacuum line by means of the

pressure tubing and evacuate as much air as

possible from the test tube. Close the test tube

valve tightly, thus maintaining a partial vacuum. Connect the valve opening of the test tube

to the supply tank of methyl chloride or dimethyl ether with pressure tubing. The supply

tank must be supported in an inverted position to obtain the liquefied gas. Warm the supply tank gently, using hot water or a small hot

plate. Now, open the valve on the test tube ;

then slowly open the valve on the supply tank,

and allow the liquefied gas to flow out and completely fill the pressure tubing with only a drop

or two overflowing into the test tube. Close

the test tube valve and weigh the test tube with

the pressure tubing still attached to the test

tube and the supply tank. Open the valve of

the test tube and allow 22-25 g. of the liquefied gas to flow into the test tube. Close the

valve of the test tube and the reserve supply

tank ; then disconnect the pressure tubing frr

the test tube, and attach the spray nozzle in

place of the tubing. Mark level of the liquid

on the test tube for reloading in the future;

invert the test tube, and apply the aerosol spray

to clusters of tomato flowers. Repeat procedure for each compound to be evaluated. Count

the number of flowers and the number of fruit

that set (remain attached to the plant) per

cluster within 14-20 days after treatment, or

before the plants become excessively pot bound

or the above-soil parts crowded. Determine

the percentage of fruit set by the treated and

the untreated clusters.

B. For large (0.5—1.0 lb. or larger) aerosol

cylinders: Evacuate the air from the cylinder

with a suction pump ; close valve ; attach funnel

to valve and fill with solvent plant-regulator

solution prepared as in "A" above; open valve

and allow partial vacuum to suck contents of

the funnel into cylinder (retain as much partial

vacuum as possible within the cyHnder), close

the valve, and load with liquefied gas, as in "A"

above. Conduct test with tomato plants as

described. Do not use Freon as most acids are

not soluble in it. Freon can be used for dispersing esters and other oil-soluble compounds

since they are soluble in it.

Suggested standard treatment for

comparison

Dimethyl ether aerosol mixture containing

Ifo beta-naphthoxyacetic acid and 10% cyclohexanone solvent for tomato fruit-set test.

Precautions: Methyl chloride gas is toxic to

animals and humans, and should not be inhaled.

As a safety precaution, prepare mixtures under

a ventilated hood, or use a gas mask.

Dimethyl ether forms an explosive, inflammatory mixture with air at about 18% concentration. Load tubes only in well-ventilated

rooms and in the absence of open flames.

Freon is non-inflammable and relatively nontoxic to plants and animals.

Goodhue, L. D. Insecticidal Aerosol Production. Spray

Solutions in Liquefied Gases. Ind. Eng. Chem. 34:

1456-1459. 1942.

Hamner, C. L., Schomer, H. A., and Marth, P. C. Application of Growth-Regulating Chemicals in Aerosol

Form with Special Reference to Fruit-Set in Tomato.

Bot. Gaz. 106: 108-123. 1944.

Marth, P. C. and Header, E. M. Influence of GrowthRegulating Chemicals on Blackberry Fruit Development. Amer. Soc. for Hort. Sei. Proc. 45: 293-299.

1944.

GRAPE TEST

Method based on research by:

R. J. Weaver.

Description of test and some other uses

Various compounds may be evaluated as

growth regulators by dipping individual clusters of grape flowers or the immature fruits in

solutions of the compound and recording improvement in fruit set and size.

This test is also applicable for the study of

fruit set of many kinds of berries.

Suggested plant material

Grape vines bearing numerous clusters of

flower buds—Black Corinth, White Corinth

(Vitis vinifera L.), or other varieties that ordinarily set relatively few berries.

Apparatus, chemicals, and other materials

Containers of suitable size, such as discarded
TEST METHODS WITH PLANT-REGULATING CHEMICALS 29

but clean 1-qt. (.95-liter) tin cans, to hold

solutions so that the flower clusters can be

dipped without bruising or damaging them

Analytical balance or pharmaceutical balance

Small labels with string or wire attached

25-100 mg. of each compound to be evaluated

About 0.5 g. of para-chlorophenoxyacetic acid

for standard treatment

Wetting agent such as Dreft, Tween 20,

Santomerse

Solvent such as 95% ethanol

Preparation and selection of plant material

Select clusters of uniform size having about

the same number of flowers that have attained

the same stage of development. Tag the selected clusters and prepare to apply treatments

when the flowers are in full bloom or within 10

days thereafter.

TOMATO TEST

Method based on research by:

D. D. Hemphill; F. S. Howlett; A. E. Murneek, S. H. Wittwer, and D. D. Hemphill; R.

H. Roberts and B. E. Struckmeyer.

Description of test and some other uses

Compounds are applied in liquid or paste

carriers to flower clusters of greenhouse plants,

and the effectiveness of the substances in preventing flower or fruit drop is evaluated.

This method is also adapted to studies of the

effect of regulating compounds on seed development and the morphological deîvelopment of

fruits. It is also useful in studying the eflfect

of regulators on the number of flowers produced.

Method of conducting test

Prepare aqueous solutions of each compound

at concentrations of 5, 15, and 50 p.p.m. by

first dissolving the required amount of the regulator in 0.5-1 ml. of alcohol ; then adding the

alcoholic solution to the required amount of

water. Add sufficient wetting agent to make a

final concentration of 0.05-0.1% of the detergent.

Dip the clusters of flowers when they are in

full bloom or within 10 days thereafter. Use

10 clusters per treatment and leave comparable

labeled clusters untreated for controls. Additional clusters should be dipped in a solution

containing the solvents and the wetting agent

to determine the effect of these constituents.

Method of taking results

Determine the percentage of fruit set by first

counting the number of flowers immediately

after dipping them. After the fruits are relatively mature, count the number of berries present. Calculate the percentage of fruits that developed from the flowers. Compare this figure

with that of the controls.

Suggested standard treatment for

comparison

5, 15, and 50 p.p.m. of para-chlorophenoxyacetic acid.

Weaver, R. J. Response of Certain Varieties of Grapes

to Plant Growth Regulators. Bot. Gaz. 113: 75-85.

1951.

Suggested plant material

Tomato plants (Lycopersicum esculentum L.)

of varieties such as Marglobe, Globe, Rutgers,

or others that can be readily grown in a greenhouse.

Apparatus, chemicals, and other materials

Clay pots 4-10 in. (10-23 cm.) or discarded

1 qt. (.95 liter) tin cans with perforated

bottoms

Composted soil

Space in greenhouse maintained at about

70°-80° F. (21°-27° C.) by day and at

60^-70° F. (15°-21° C.) by night

Small vials (20-25 ml.)

Clean containers of 100-200 ml. capacity

Toothpicks or wooden medicinal applicators

or a nasal spray atomizer (15-30 ml.)

Analytical balance or pharmaceutical balance

25-100 mg. of each compound to be evaluated

About 0.5 g. para-chlorophenoxyacetic acid

Lanolin

Solvents such as ethanol, methanol

Wetting agents such as Tween 20, Carbowax

1500

Preparation and selection of plant material

A. For tests of limited duration ( 14—20 days) :

Grow the plants in 4- to 6-in. (10- to 15-

cm.) pots set side by side in rows 4 inches

(10 cm.) apart, and select those of uniform size which have developed two open

flowers on the first cluster.
30 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

B. For prolonged tests involving maturation

of the fruit (35-45 days): Grow the

plants in 10- to 12-inch (25- to 30-cm.)

pots, or grow them in ground beds. In

either case select uniform plants that have

developed 2 open flowers in the first cluster. The plants should be at least 12 in.

(30 cm.) apart, in rows 2 ft. (61 cm.)

apart.

Method of conducting test

A. Paste Method: Prepare a lanolin-Tween

20 paste containing 1% of the chemical

(see pages 5, 6), and apply a narrow

band of the mixture around the stalk

(peduncle) of the first flower cluster and

about 1-2 cm. from the main stem of the

plant. To obtain reliable data, apply one

treatment to an individual row of plants

extending across the bench or soil bed.

Repeat the procedure using additional

treatments and corresponding rows.

Leave an additional row of plants untreated to serve as controls. When all

of the treatments have been applied and

the control row designated, an experimental block has been completed. Repeat

the procedure making a second, third, and

fourth block, randomizing the relative positions of each treatment within each

block.

B. Spray Method: Prepare aqueous mixtures

to obtain a concentration range of 10, 20,

40, or 80 p.p.m. of the compound to be

tested (see pages 8, 28-29). Use the above

experimental plan and spray the mixture

on the flower cluster with a nasal atomizer until the liquid drips from each flower.

Spray flowers of control plants in a similar way, using water and the co-solvent

only.

Method of taking results

A. Plants used in tests of limited duration :

Count the number of flowers and the number of fruit that set (remain attached

to the plant) per cluster within 14 to 20

days after treatment, or before the plants

become excessively pot bound or the abovesoil parts crowded. Determine the percentage of fruit that set. Cut the green

fruits midway and at right angles to their

axes, and record the relative amount of

gelatinous pulp and the relative numbers

of seeds present.

B. Plants used in prolonged tests: Record data

as described above and, in addition, record

the number of days required for the fruit

to develop a pink or red color.

If needed, use the second and third

flower clusters on each plant to repeat the

earlier treatments. Plants in ground beds

may develop 5 or 6 flower clusters that

are suitable for treatment. In all instances, apply only one concentration of

one chemical to a plant.

Suggested standard treatment for

comparison

A paste of 0.1% lanolin-Tween 20 or a spray

mixture containing 30 p.p.m. of para-chlorophenoxyacetic acid.

Hemphill, D. D. The Effects of Plant Growth-Regulating Substances on Flower Bud Development and Set.

Mo. Agr. Expt. Sta. Res. Bui. 434. 1949.

Howlett, F. S. Experiments Concerning the Practicability of Certain Chemicals as a Means of Inducing

Fruit Setting in the Tomato. Amer. Soc. Hort. Sei.

Proc. 37: 886-890. 1939.

Murneek, A. E., Wittwer, S. H., and Hemphill, D. D.

Supplementary "Hormone" Sprays for Greenhouse

Tomatoes. Amer. Soc. Hort. Sei. Proc. 45: 371-381.

1944.

Roberts, R. H. and Struckmeyer, B. E. The Use of

Sprays To Set Greenhouse Tomatoes. Amer. Soc.

Hort. Sei. Proc. 44: 417-427. 1944.

Fruit Size

GRAPE TEST

Method based on research by:

R. J. Weaver.

Description of test and some other uses

Various compounds may be evaluated as

growth regulators by dipping individual clusters of grape flowers or the immature fruits in

solutions of the compound, and recording improvement in fruit size.

This test is useful in studying the effect of

plant regulators on the growth of a variety of

different kinds of fruits. It is also a useful

means of studying the effect of regulating chemicals on the production of parthenocarpic fruit,

such as tomatoes and blackberries.

Suggested plant material

Grape vines bearing numerous clusters of

flower buds—Thompson Seedless, Monukka,

Sultana {Vitis vinifera L.), or other varieties

that set numerous fruits.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 31

Apparatus, chemicals, and other materials

See page 28.

Preparation and selection of plant material

Select clusters of uniform size having about

the same number of flowers. Tag these to be

used after the flowers are fully open and before

any berries have abscised.

Method of conducting test

Prepare aqueous solutions of each compound

at concentrations of 5, 15, and 50 p.p.m. using

alcohol as a solvent, and a wetting agent (see

page 29). Dip the flower clusters in the solutions soon after the flowers are fully open. Use

10 clusters per treatment and leave comparable

labeled clusters untreated for controls. Additional clusters should be dipped in a solution

containing the solvents and wetting agent to

determine the effect of these constituents.

Method of taking results

Determine the effect of treatment on berry

size by removing the berries from the plants

after they are mature, and obtaining the fresh

weight of 200 fruits selected at random from

each treatment. Compare this weight with the

results of similar measurements using control

fruits. Note any difference in time of maturity

or other effects due to treatments.

Suggested standard treatment for

comparison

5, 15, and 50 p.p.m. of para-chlorophenoxyacetic acid.

Weaver, R. J. Response of Certain Varieties of Grapes

to Plant Growth-Regulators. Bot. Gaz. 113: 75-85.

1951.

Fruit Thinning

APPLE BLOSSOM TEST

Method based on research by:

C. P. Harley, H. H. Moon, and L. 0. Regeimbal.

Description of test and some other uses

By applying regulating chemicals to selected

limbs of trees when in blossom, the compounds

are evaluated as a means of thinning the fruit.

By modifying the dosage, the method may be

useful in entirely removing undesirable fruits

of such trees as the Gingko, mulberry, and hickory without injury to the trees.

Suggested plant material

Apple trees (Malus sylvestris Mill., also

called Pyrus maltts L.), with the majority of

the blossoms fully open and with a few petals

falling—Jonathan, Wealthy, Golden Delicious,

Grimes Golden, Baldwin, or other varieties.

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

For whole-tree experiments use standard

orchard-spray equipment such as is used

for applying insecticidal or fungicidal

sprays under high pressure (400-800 lb.

pressure per sq. in. [181-363 kg. per 6.5 sq.

cm.])

Sprayers having a large tank divided into

several 50- or 100-gal. (189- or 379-liter)

compartments facilitate the work

3-gal. (11-liter) hand-pressure sprayer can

be used if individual limbs are to be

sprayed

Hand tally registers (2 or 3 per person)

needed to record data

5-10 g. of each compound to be evaluated

10-20 g. of alpha-naphthalene-acetamide for

standard treatment

Tween 20 or other wetting agent

Preparation and selection of plant material

For whole-tree experiments, select and tag

trees of uniform size that have produced about

the same number of flowers per tree. Five

trees are suggested for each treatment.

If individual limbs are to be used, select on

comparable trees outer limbs of about the same

size and bearing about the same number of

blossoms at the desired stage of development.

Apply each treatment to a single limb selected

at random from each of five or more trees. In

this way several treatments can be used on

each tree.

Method of conducting test

Prepare aqueous sprays containing several

concentrations of each chemical (see page 29)

(10, 30, and 60 p.p.m. are suggested). Apply

sprays within a few days after about 75 per^
32 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

cent of the petals have fallen, so as to wet thoroughly all parts of the tree or branch. About

1 gal. (3.8 liters) for each year of age is required for spraying entire trees. Leave unsprayed trees or branches as controls, and

hand-thin other trees or branches so as to have

branches or trees thinned an optimum amount

for comparison.

Method of taking results

Determine the number of fruits thinned

chemically after June drop has occurred. With

the aid of two tally registers, one in each hand,

record separately the total number of spurs

that blossomed and the number that set fruits.

Spurs from which fruits have dropped are

easily identified by the pedicel scars. Use a

third tally register to record vegetative spurs

if desired. Compare the number of spurs that

bear fruits with the number from which fruits

have fallen. Compare ratios thus obtained with

those of the untreated and the hand-thinned

controls.

Suggested standard treatment for

comparison

50 p.p.m. spray concentration of alphanaphthalene-acetamide.

Harley, C. P., Moon, H. H., and Regeimbal, L. O. A

Modified Method of Obtaining Fruit-Set Records for

Apples. Amer. Soc. Hort. Sei. Proc. 63: 146-148.

1954.

PEACH AND PRUNE FRUIT TEST

Method based on research by:

P. C. Marth and V. E. Prince.

Description of test and some other uses

A method of evaluating the fruit-thinning

effects of chemicals applied as sprays to limbs

or entire trees is described.

By modifying the dosage used, these methods

may be useful in entirely removing undesirable

fruits of such trees as Gingko, mulberry, and

hickory, without injury to the trees.

Suggested plant material

Peach or plum trees bearing young fruits

(30-45 days after full bloom)—Elberta, Halehaven, Hiley, or other varieties of peach

(Prunus pérsica [L.] Batsch) or Stanley, Italian, or German varieties of plum (Prunus

domestica L.).

Apparatus, chemicals, and other materials

Same as on page 31 except that 50-100 g. of

isopropyl N-(3-chlorophenyl) carbamate are

required for standard treatment.

Preparation and selection of plant material

See page 31.

Method of conducting test

Prepare aqueous sprays containing several

concentrations of each chemical (see page 29)

(125, 250, and 500 p.p.m. suggested). Apply

sprays about 30-45 days after full bloom or

10-20 days after shucks (dried flower parts)

have fallen. Wet all parts of the tree or branch

thoroughly. At the time the fruits are sprayed

they should be about 1-2 cm. in diameter.

About 1 gal. (3.8 liters) for each year of age is

required for spraying entire trees. Leave unsprayed trees or branches as controls; also

hand-thin other trees or branches so as to leave

an optimum number of fruits for further comparison.

Method of taking results

Within a day or two after applying the

sprays, determine the number of fruits that remain attached. After June drop has occurred,

determine the number of fruits thinned by comparing the number on sprayed, unsprayed, and

hand-thinned branches or trees. At the usual

harvest date record the number, size, the degree

of coloration of fruits, and the effect of treatment on time of maturation. Compare these

data to evaluate effectiveness of the chemical

treatment.

Suggested standard treatment for

comparison

Spray concentration of isopropyl N-(3-

chlorophenyl) carbamate, 200 p.p.m.

Marth, P. C, and Prince, V. E. Effect of 3-Chloro-isopropyl-N-phenyl Carbamate on Abscission of Young

Fruits of Peach. Science 117: 497-498. 1953.
TEST METHODS WITH PLANT-REGULATING CHEMICALS

Growth Inhibition or Stimulation

33

VEGETATIVE SPROUT TEST

Method based on research by:

P. C. Marth and E. S. Schultz; P. C. Marth.

Description of test and some other uses

Pieces of carrots, potato tubers, and small

onions are dipped in a solution of the test compound and placed in darkness. Measurement of

sprouts indicates the effectiveness of the compound as a sprout inhibitor.

This test may also be adapted for a study of

the growth and development of buds on any

vegetative reproductive organ.

Suggested plant material

Unsprouted vegetable storage organs such as

carrots {Daucus carota L.), onions (Allium

cepa L.), and potato tubers {Solarium tuberosum L.) that have experienced a rest period.

Apparatus, chemicals, and other materials

i/2-in. (1-cm.) cutting spoon such as is used

for making melon balls for salad

Analytical balance or pharmaceutical balance

Sphagnum, peat moss, or fine quartz sand

Petri dishes or other shallow containers about

4 in. (10 cm.) in diameter

Space in darkened room with temperature

controlled at 65°-75° F. (18°-24° C.)

0.1-0.5 g. of each compound to be evaluated

0.5 g. of methyl ester of alpha-naphthaleneacetic acid for standard treatment

Preparation and selection of plant material

Carrot: Select carrots of about medium size

for uniformity. Sever petioles so that about

1/4 in. (1 cm.) of each petiole remains attached

to each root. Remove and discard the lower

part of the root saving about 1 in. (2.5 cm.) of

the upper portion. Place the pieces in humid

air at 40°-50° F. (4°-10° C.) for 3-4 days or

until they become suberized.

Onion: Small onions about 1/2 in. (1 cm.) in

diameter and of uniform shape are prepared for

treatment by removing the loose outer scales.

Potato tuber: Select tubers of medium size

and, with a cutting spoon, remove pieces of the

tubers so that each piece contains an eye (fig.

6). Place the pieces in humid air at 40°-50° F.

(4°-10° C.) for severaldays until they become

suberized.

Figure 6. A melon bail cutting spoon provides a means of easily

removing buds from potato tubers, (above). Cut around each

eye with the instrument and place the pieces along with small

onion sets and pieces of carrot roots on moistened sphagnum

moss contained in a Petri dish (below). (N'EG. p.\-!>97 A. B)
34 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Method of conducting test

Select 10 of the prepared carrot pieces. Dip

these momentarily in a tap-water mixture containing the desired amount of the chemical being tested (1,000 p.p.m. suggested). Allow

pieces to drain, and place equal numbers of

them upright on the moist sphagnum, peat

moss, or sand contained in dishes about 4 in.

(10 cm.) in diameter and without covers (fig.

6). Dip comparable batches of carrot pieces

in tap water and place them on sphagnum or

sand in the manner described to serve as controls. Keep the sphagnum or sand moist, and

allow the plant material to grow in darkness

at room temperature, 65^-75° F. (18°-24° C).

Apply the chemical to the onion sets and the

potato pieces, and culture them in the same

manner as described.

Method of taking results

After the controls have developed a measurable amount of new vegetative growth, measure

the length of each sprout and measure the fresh

weight of the detached sprouts. Compare these

measurements with similar ones for the treated

material.

Suggested standard treatment for

comparison

1,000 p.p.m. of methyl ester of alpha-naphthaleneacetic acid.

Marth, P. C, and Schultz, E. S. Effect of Growth Regulators on Sprouting of Stored Table Stock Potatoes

and on Waste Piles for Control of Diseases. Amer.

Potato Jour. 27: 23-32. 1950.

Marth, P. C. Unpublished data, Plant Industry Station, Beltsville, Md.

Growth Modification (Aquatic Plant)

STERILE NUTRIENT TEST

Method based on research by:

L. G. Nickell and A. C. Finley.

Description of test

Lemna minor L. (duckweed) is grown under

aseptic and rigidly controlled conditions in a

nutrient solution. Regulating chemicals (including antibiotics) are added to the nutrient,

and their effect on growth of the plant studied.

Suggested plant material

Duckweed plants (Lemna minor L.).

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Petri dishes

Tank in which to maintain a stock supply of

the test plant. (Lemna minor will grow

vigorously through the year in dilute nutrient solutions at a pH of 5-6 or in tap

water over a small amount of sandy soil.)

Table space in a room with constant light

intensity (700- to 1,000-foot candles for 12

hr. per day) and the temperature controlled at 75^-80° F. (24°--27° C.)

0.2 g. of each compound to be evaluated

0.5-1 g. of 2,4-dichlorophenoxyacetic acid in

water-soluble salt form for standard treatment

Basic nutrient medium containing the ingredients hsted :

KNO, 0.002M Thiamine lOOy/1.

CaiNOa), 0.003M Pyridoxine! 800y/l.

KH2PO4 O.OOIM Nicotinamide 800y/l.

MgSO, O.OOIM B 0.1 p.p.m.

CaCl. 0.003M Mn 0.1 p.p.m.

KCI 0.002M Zn 0.3 p.p.m.

MgCl. O.OOIM Cu 0.1 p.p.m.

Sucrose 2% Mo 0.1 p.p.m.

Fe 0.5 p.p.m.

Dilute sodium hydroxide and hydrochloric

acid solutions

Glass or other type filters with which solutions can be filtered free of contaminating

organisms

Suction flasks with adapters for filters

Erlenmeyer flasks (125 ml.)

Tweezers

Cotton

Bacitracin, Penicillin G, or isonicotinic

hydrazide for standard solutions

Solution of mercuric chloride (1:2000 of

water) for sterilizing plants

O.T. Clear Aerosol or comparable detergent

Preparation and selection of plant material

Select a vigorously growing supply of Lemna,

Sterilize this material by immersing rosettes

(3-4 fronds) in the mercuric chloride solution

(1:2000 water). Add one drop of a 1:20 dilution of O.T. Clear Aerosol to each 100 ml. of

the mercuric solution. Place several rosettes

in the solution and remove one at 5-minute intervals so that a series of treated rosettes result

which have been exposed from 5 to 30 minutes
TEST METHODS WITH PLANT-REGULATING CHEMICALS 35

to the mercuric chloride mixture. As each

rosette is removed, rinse several times in sterile

water and finally place it in a flask containing

sterile nutrient medium. Allow all of the

plants to grow in the flasks for a week to determine which are aseptic. Uncontaminated cultures are used for further experimentation.

^ Method of conducting test

i The pH of the basic medium should be about

5.0. Adjust any variation from this with dilute acid or alkali solutions. Prepare 50-100

ml. of an aqueous stock solution of the regulator

to be tested so that it contains 300 p.p.m. of

the chemical. Remove contaminating organ-

' isms in this solution by passing it through the

i sintered glass filter. Place 50 ml. of the sterile

nutrient medium aseptically into each of 5

flasks of 125 ml. capacity. Introduce one sterile duckweed plant into each flask and designate

these as controls. Repeat the procedure, but

before placing a plant in the nutrient, add a

' suflicient volume of the sterile stock solution

_ containing the regulating chemical to make a

final concentration of 1 p.p.m. Prepare other

concentrations of the test chemical in additional

aliquots of the nutrient (5, 10, 20, 30 p.p.m.

etc.). For each concentration level, use at

least 5 replicate flasks. Place one of the sterile,

tr selected, duckweed plants in each flask asep-

^ tically. Stopper the flasks with cotton and

place them under controlled environmental conditions, 22.5° C. with light from white or daylite fluorescent tubes having a constant intensity of at least 700-foot candles.

Method of taking results

After the required period of growth (1-2

wks.), remove the test plants, blot them free of

water, for each flask record the number of plants

and the number of fronds on each, and immediately obtain the fresh weight of all plants

from each flask, weighing to the nearest milligram. Compare any increase or decrease in

growth of control and treated plants in terms

of number of plants, fronds, and average

weight per plant.

Suggested standard treatment for

comparison

Bacitracin, Penicillin G, or isonicotinic hydrazide at 20 p.p.m. concentration.

Nickell, L. G., and Finlay, A. C. Growth Modifiers,

Antibiotics, and Their Effect on Plant Growth.

Agr. and Food Chemistry 2: 17-182. 1954.

WATER CULTURE TEST

Method based on research by:

H. R. Oflford.

Description of test

An aquatic plant, duckweed (Lemna minor

L.), is grown in water containing a regulating

compound, and the growth-modifying effect of

the chemical is observed.

Suggested plant material

Duckweed plants (Lemna minor L.).

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Petri dishes

Tank in whi^h to maintain a stock supply of

the test plant. (Lemna minor will grow

vigorously through the year in dilute nutrient solutions at a pH of 5-6 or in tap

water over a small amount of sandy soil.)

Table space in a room with constant light

intensity (700- to 1,000-foot candleg for 12

hr. per day) and the temperature controlled at 75°-80° F. (24°-27° C.)

0.2 g. of each compound to be evaluated

0.5-1 g. of 2,4-dichlorophenoxyacetic acid in

water-soluble salt form for standard treatment

Dilute solution of potassium hydroxide

Dilute solution of sulfuric acid

Preparation and selection of plant material

Select relatively large, uniformly green

plants that are growing vigorously.

Method of conducting test

Prepare tap water solutions (150 ml.) of each

compound in a range of concentrations (0.001-

1,000 p.p.m. suggested). Place 50 ml. of each

concentration in each of 3 Petri dishes. Place

the same number of Lemna plants in each test

solution, using 10 or more for each. Include

some dishes that contain plants in tap water as

controls. Record the number of plants, the

total number of fronds, and the time of immersion for each culture. Using dilute sulfuric

acid or potassium hydroxide, adjust the pH of

all solutions to approximately the same level

(about 5-6), since excess acid or alkali exerts

a toxic action on Lemna. Maintain the tem-
36 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

perature of the solutions at 75°-80° F. (24°-

27° C), and the light intensity constant so that

tests carried out at different times of the year

can be compared.

Method of taking results

At regular intervals, record any visible differences in the plants such as change in color,

presence or absence of vegetative division, and

presence or absence of new fronds. Evaluate

the relative effectiveness of each concentration

of each compound tested, as follows :

Marked inhibitory effect: Color change becomes steadily more apparent until a uniform

discoloration of the fronds is evident. The

typical color of fronds in this category is reddish brown to pale yellow or white. There is

no change in total area of fronds or in number

of plants.

Moderate inhibitory effect: Plants divide

and form single or double fronds before discoloration becomes noticeable. Most or all of

the plants split into single fronds. Chlorosis

occurs slowly, but uniformly, on all fronds, and

uniform discoloration results. Typical color of

the plants is pale or yellowish green. There is

no change in the total area of the fronds.

Slight inhibitory effect: Symptoms appear

slowly. Single fronds from parent plants may

be unevenly discolored, but fronds of the parent

plants remain fairly green. No change is apparent in the total area of the fronds.

No inhibitory effect: Plants are divided into

single or double fronds and are dark green in

color. There is an apparent increase in the

number and in the total estimated area of the

fronds.

Suggested standard treatment for

comparison

Determine by trial the minimum concentration of 2,4-dichlorophenoxyacetic acid which

induces marked inhibitory effect. No standard

suggested for stimulation of growth.

Oiford, H. R. Rapid Estimation of the Phytocidal

Action of Chemicals. Science 103: 474-476. 1946.

Growth Regulation

FIELD TEST

Method based on research by:

J. R. Havis; P. C. Marth and R. E. Wester.

Description of test

Test chemicals are sprayed on soil in which

seeds have been planted or on leaves and stems

of plants growing under field conditions, and

their effect on growth and productivity of the

plants is measured.

Suggested plant material

Pieces of stem of sugarcane (Saccharum

officinarum L.) such as are ordinarily used for

planting this crop, or seeds of crop plants such

as corn (Zea mays L.), wheat (Triticum aestivum L., also called T. sativum Lam. and T. vulgäre Vill.), bean (Phaseolus vulgaris L.), soybean (Glycine max [L.] Merr.), lima bean

(Phaseolus lunatus L.), pea (Pisum sativum

L.), or others.

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Knapsack sprayer, to be operated at 30 lb.

pressure per sq. in. (14 kg. per 6 sq. cm.)

5-50 g. of each compound to be evaluated as

growth regulator >^

5 g. of 2,4,5-trichlorophenoxyacetic acid for ^

standard treatment ^

Solvent for preparing concentrated solutions

of chemicals to be evaluated (alcohol, acetone, or other organic solvent)

Preparation and selection of plant material ^'

\

Plant seeds of crop plants or uniform stem

pieces of sugarcane in the usual manner. For

tall-growing crops such as corn or sugarcane,

use four or more plots of 1/100 acre (40 sq. m.)

for each chemical or concentration tested.

Space the plants 18-24 in. (46-60 cm.) apart in ^

rows 40-42 in. (101-106 cm.) apart. For shortgrowing crops such as soybeans and peas, space

the plants 6 to 8 in. apart in rows 30-36 in. ^^

(76-91 cm.) apart. Use two or more replications of rows 5-10 ft. (1.5-3 m.) long for each

chemical or concentration tested. It is sometimes advantageous to select uniform plants 2 ^

ft. (60 cm.) or more apart; mark these and use

10 of them for each chemical or concentration *^

tested. A,

V

Method of conducting test

For pre-emergence application, apply the test ^^
TEST METHODS WITH PLANT-REGULATING CHEMICALS 37

•chemicals within a few days after planting,

using suggested rates of 1, 2, 4, and 16 lb. per

acre (0.5, 1, 2, and 7 kg. per 4,047 sq. m.).

For post-emergence treatment, apply the sprays

(0.001-1,000 p.p.m.) at any selected stage of

development after emergence.

Method of taking results

Obtain growth and yield data by making linear measurements of at least 10 individual

plants in each treatment during the growing

period. Also obtain yield of fruits or seeds as

they mature. In pre-emergence experiments.

plants near the center of each plot should be

measured.

Suggested standard treatment for

comparison

Aqueous sprays (0.001-1,000 p.p.m. of 2,4,5-

trichlorophenoxyacetic acid or 2,4-dichlorophenoxyacetic acid) if applied post-emergence,

or 1, 2, 4, and 16 lbs. per acre (0.5,1, 2, and 7 kg.

per 4,047 sq. m.) if applied pre-emergence.

Havis, J. R. Effect of 2,4-D Sprays on the Growth of

Sug-arcane. Weeds 2: 148-154. 1953.

Marth, P. C, and Wester, R. E. Effect of 2,4,5-Trichlorophenoxy-Acetic Acid on Flowering and Vegetative Growth of Fordhook 242 Bush Lima Beans.

Amer. Soc. Hort. Sei. Proc. 63: 325-328. 1954.

AGAR SLANT TEST

Root Growth

Method of conducting test

Method based on research by:

R. L. Jones, T. P. Metcalfe, and W. A. Sexton.

Description of test and some other uses

Seeds of rape and oats are germinated on

agar slants containing a test chemical, and the

root growth studied.

This method is also useful in studying the

root growth of a wide variety of other kinds of

germinating seeds.

Suggested plant material

Seeds of a crop and a weed plant, such as oat

(Avena sativa L.) and rape (Brassica napus

L.).

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Test tubes 1/2 x 6 in. (1 x 15 cm.)

Test tube racks

Light-proof chamber at room temperature of

70°-75° F. (21^-24° C.)

About 0.1 g. of each compound to be evaluated

For standard treatment:

250-500 mg. of 2,4-dichlorophenoxyacetic

acid

250-500 mg. of isopropyl-N-phenylcarbamate

Agar

Preparation and selection of plant material

Sort the seeds, eliminating any that are

shriveled or diseased.

Prepare about 4% agar in water in an

amount sufficient to provide 500 ml. of agar

mixture for each concentration of each chemical to be evaluated plus 1,500 ml. to be utilized

for an untreated control and for standard treatments with 2,4-dichlorophenoxyacetic acid and

isopropyl-N-phenylcarbamate. Dissolve 50 mg.

of each test compound in 500 ml. portions of

the melted agar mixture.

Pour the agar-water and agar-water-chemical mixtures into separate, labeled test tubes,

10 ml. per tube. Slant the tubes so that the

agar extends about 5 in. (13 cm.) up the side

of the tube and allow the agar to solidify with

the tube held in this position. Approximately

46 tubes of each agar-water-chemical medium

plus an equal number of plain agar-water tubes

will then be available for testing. Use at least

12 replicates of each treatment for statistically

reliable results when high concentrations of the

chemicals, such as 50 and 100 p.p.m., are employed. Eighteen replicates are sufficient for

lower concentrations.

Using half the test tubes of each treatment,

place two oat seeds on the agar near the top of

the slant in each tube. Removal of the husks

(glumes) facilitates germination. Place 3 rape

seeds on the agar near the top of the slant of

each of the remaining test tubes. It may be

necessary to apply a slight pressure to partially

embed the seeds in the agar to hold them in

place.

Place the tubes in an upright position and

store them in racks in a dark chamber at room

temperature of 70°-75° F. (21°-24° C.) for

the duration of the test. Maintain a high relative humidity in the storage chamber to prevent the agar from drying out and to favor

uniform growth of the plants. Drying of the
38 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

agar must be avoided in order to prevent a

corresponding increase in concentration of the

test chemical.

Method of taking results

After 5-7 days, or when the rape roots in the

untreated control tubes have attained a length

of 5-6 cm., measure and record the lengths of

the rape roots in each tube. Eight to 10 days

after initiation of the test, or when the oat

roots in the untreated control tubes have attained a length of 6-7 cm., measure and record

the lengths of the oat roots in each tube. Also

record the number of seeds in each tube that

failed to germinate.

Calculate the average length of the roots in

the control tubes, but do not include the zero

root growth of the non-germinated seeds in the

calculation of these averages. Determine the

percentage of growth that the roots of each

plant in each treated tube exhibit as compared

with the average root length of the untreated

control plants.

If one-half or more of the test plants of one

species exhibit 50 per cent or greater reduction

in root growth in a treatment, the applied chemical is considered to be very active under the

conditions of the test. If one-half or more of

the test plants of one species exhibit 20-50%

reduction in root growth in a treatment, the

applied chemical is considered to be active under the conditions of the test. If less than onehalf of the test plants of one species exhibit

greater than 20 per cent reduction in root

growth in a treatment, the applied chemical is

considered to be inactive under the conditions

of the test. If a compound is shown to be active or very active at the initial concentrations

tested, conduct tests at lower concentrations

and repeat until the minimum dosage required

to produce an effect classified as active is determined.

Suggested standard treatment for

comparison

Use 2,4-dichlorophenoxyacetic acid at 0.1

p.p.m. concentration as a standard for rape, and

isopropyl-N-phenylcarbamate at 0.1 p.p.m. as a

standard for oats. Isopropyl-N-phenylcarbamate and 2,4-dichlorophenoxyacetic acid at 0.1

p.p.m. are rated as very active by this test when

applied to oat and rape plants as described.

Jones, R. L., Metcalfe, T. P., and Sexton, W. A. The

Relationship Between the Constitution and the Effect

of Chemical Compounds on Plant Growth. I. 2-

Phenoxyethylamine Derivatives. Biochem. Jour. 45:

143-149. 1949.

CONE CONTAINER TEST

Method based on research by:

T. K. Pavlychenko.

Description of test and some other uses

Seeds of barley or other grass plants are

dipped in dilute concentrations of a regulating

chemical and germinated in soil held in coneshaped containers. Growth of roots and other

parts of the plants is studied in detail by removing the containers and washing away the

soil from some of the plants at intervals as the

plants mature.

This method can be modified to study the effect of application of different regulating chemicals to either the tops of the plants or to the

soil in which the plants are grown.

Suggested plant material

Barley seeds (Hordeum sativum L.), well

matured (stored for 8-9 months after harvest),

or other grass sseds.

Apparatus, chemicals, and other materials

Cone-shaped galvanized iron cylinders, 8 in.

diameter at top, 10 in. at bottom, 24 in. or

more high (20, 25, 61 cm.) fitted with perforated wooden or metal removable

bottoms

Analytical balance or pharmaceutical balance

Sandy loam soil free from undecomposed

organic material, uniformly mixed and

well packed into cone-shaped cylinders

Greenhouse maintained at about 65°-80° F.

(18°-27° C.)

Beakers or other containers in which to soak

the seed

50-100 mg. of each chemical to be evaluated

for root growth effects

1-5 g. alpha-naphthaleneacetamide for standard treatment

About 100 ml. 95% ethanol

Preparation and selection of plant material

Discard any diseased, broken, or malformed

seeds.

Method of conducting test

Dip a number of seeds in aqueous mixtures of

each plant regulator at each of three dosage

levels : 0.002, 0.004, and 0.006 per cent are suggested. The chemicals are dispersed in water
TEST METHODS WITH PLANT-REGULATING CHEMICALS 3»

by the aid of a small amount of ethanol. The

alcohol concentration in the final mixture should

not exceed 0.1 per cent. Plant 3 seeds in duplicate cylinders for each chemical and each concentration level. Repeat so as to obtain duplicate sets that can be examined after 3 weeks,

6 weeks, and at maturity. Three days after

emergence, remove all but the one most vigor-

"^ ous plant in each cylinder. Grow plants in a

^ cool 68^-70° F. (20^-21° C.) greenhouse for 2

weeks; then increase the temperature to 75°-

r* SS"" F. (24°-29° C.) thereafter. Dip seeds in

water only and grow them in the same manner

for controls.

Method of taking results

^ Record time of emergence and the height of

^ the plants at 3-day intervals. Harvest a duplicate lot of plants from each treatment and also

from the controls at the end of 3 weeks, 6 weeks,

and at maturity, tapping the cylinders and then

lifting them off over the tops of the plants.

w Separate roots from tops and record the number, fresh and dry weight of leaves and stems,

"^ and the number of tillers. Carefully remove

roots from soil by soaking in water the cylinders

of soil containing them and washing away the

remainder of the soil with a stream of water.

'^ Record depth of root penetration, number and

^ length of roots, and then the dry weight. Compare all of these measurements with those of

•«^ the controls.

Suggested standard treatment for

f comparison

r^ Seeds soaked in 0.002, 0.004, and 0.006%

« alpha-naphthaleneacetamide.

Unpublished method proposed by T. K. Pavlychenko,

American Chemical Paint Co., Ambler, Pa.

IMPREGNATED FILTER PAPER TEST

Method based on research by:

J. W. Brown.

Description of test

Root growth of germinating seeds on filter

paper treated with the test chemicals is

observed.

Suggested plant material

Wide variety of seeds—morning glory (/pomoea spp.), sunñower {Helianthus annuus L.),

pigweed {Afiiaranthus spp.), crabgrass {Digitaria spp.), mustard (Brassica spp.), wheat

(Triticum aestivum L., also called T. sativum

Lam. and T. vulgäre VilL), pea (Pisum sativum L.), bean (Phaseolus vulgaris L.), buckwheat (Fagopyrum esculentum Moench),cotton

(Gossypium hirsutwn L.), cucumber (Cucumis

sativus L.), barley (Hordeum sativum L.), corn

{Zea mays L.), oat (Avena sativa L.), rice

(Oryza sativa L.), and rye (Sécale céréale L.)

have been used.

Apparatus, chemicals, and other materials

Analytical balance or pharmaceutical balance

Pressure-sensitive tape with adhesive on both

sides (Scotch brand No. 400, 1/2 in. [13

mm.] wide)

Filter paper (Whatman No. 1) 3 x 9 in. or

3 X 18 in. (8 X 23 or 8 X 46 cm.) depending

on seed size

Filter paper strips i/^ in. (1 cm.) wide with

14-in. (6-mm.) holes punched with their

centers 1/2—1 in. (1-3 cm.) apart. For

large seeds such as bean, pea, sunflower,

and corn use holes 1 in. apart; for small

seeds, 1/2 in. apart

Paper drinking cups 4 in. (10 cm.) tall

Metal trays 19 in. wide x 22 in. long x 6 in.

deep (48 x 56 x 15 cm.) with false perforated bottoms 1 in. (3 cm.) above the solid

bottom of the tray

Glass plates large enough to cover completely

the top of the metal trays

Constant-temperature chamber maintained

at about 82° F. (28° C.)

10 mg. of each compound to be evaluated

0.5-1 g. of 2,4-dichlorophenoxyacetic acid for

standard treatment

Acetone

Preparation and selection of plant material

Sort the various kinds of seeds, eliminating

any that are shriveled or diseased.

Method of conducting test

Dissolve 10 mg. of the test chemical in 5 ml.

of 100% acetone; then dilute to 1 liter with tap

water, making a 10 p.p.m. solution. Make 1

and 0.1 p.p.m. solutions by diluting aliquots of

this original solution with a 0.5% concentration

of acetone in water.

Place the pressure-sensitive tape along one

edge of the large piece of filter paper, extending the tape i/^ in. (1 cm.) past the edge of the

large paper. Place the filter paper strip with

the evenly spaced holes on top of the tape.
40 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Make the length of paper strip such that it will

leave fifteen l^-in. (6-mm.) discs of adhesive

exposed. Place 15 seeds of the first species to

be tested on the exposed discs, one seed to a disc.

Roll the filter paper with the seeds inside and

hold the roll in place with the exposed pressuresensitive tape. Repeat using additional species.

Stand the rolls upright in the paper cups with

the part containing the seeds uppermost. Pour

70 ml. of a concentration of one chemical over

rolls containing seeds of each plant species to

be tested. Repeat with other concentrations

and chemicals. On one set of rolls containing

seeds pour an equal volume of a 0.5% concentration of acetone in water to serve as controls.

Place the cups containing the treated and

control rolls in the metal trays filled with water

to a depth of i/^ in. (1 cm.) beneath the perforated false bottom. Cover the trays with

the glass plates to maintain a high relative humidity, and place the trays in a chamber maintained at about 82° F. (28° C).

Method of taking results

After 72-96 hours (depending upon the germination rate of the species) open the seed

rolls, and measure the length of the primary

roots to the nearest centimeter. The average

length of the 10 longest roots is used for computing the percent inhibition of root elongation :

Average control root length

(actetone-water solution) minus

average treated root length

Average control root length

(acetone-water solution)

-X 100 = percent

inhibition

In case the root elongation is obviously affected by some factor other than the test compound, such as profuse fungal or bacterial

contamination, or if the control roots average

less than 5 cm. long, the test is rerun.

Suggested standard treatment for

comparison

1 p.p.m. 2,4-dichlorophenoxyacetic acid in

tap water containing a concentration of 0.5%

acetone.

Brown, J. W. Two Tests for Herbicide Screening.

Northeastern Weed Control Conference Proc. (Sup.)

7:71-74. 1953.

NUTRIENT MIST TEST

Method based on research by:

B. T. P. Barker; W. Carter; L. J. Klotz;

M. C. Vyvyan and G. F. Trowell.

Description of test and some other uses

Trees are grown with their roots in a closed

chamber containing air filled with a fine mist

of nutrient solution in which the chemical being

tested has been dissolved or suspended, and

effects of the regulating chemical on growth of

the roots are observed.

This method can be adapted for the study of

growth responses of many different kinds of

plants.

Suggested plant material

Young trees—^maple {Acer spp.), pine (Pinus

spp.), oak (Quercus spp.), apple (Malus sylvestris Mill., also called Pyrus mains L.), peach

{Prunus pérsica [L.} Batsch), and Citrus,

Apparatus, chemicals, and other materials

Mist chamber ^

Analytical balance or pharmaceutical balance

5 g. of each compound to be evaluated

Nutrient solution

Optional materials :

lanolin

solvent, such as 95% ethanol

wetting agent, such as Tween 20

Preparation and selection of plant material

Select young trees at a uniform stage of development and growing at approximately the

same rate.

Method of conducting test

Support trees (2 to 6 of each species) so that

their roots are suspended in the chamber containing air filled with a mist of nutrient solution. Set up duplicate groups of trees for each

additional compound or concentration to be

tested. Add the plant regulator or other chemical to be tested directly to the feed tank containing the nutrient solution (concentrations of

0.01-10 p.p.m. suggested). Maintain comparable sets of trees with their roots in the nutrient

mist only and designate these as controls.

As an alternative method, the chemical to be

tested can be applied to the exposed tops of the

trees as over-all aqueous sprays (1-1,000 p.p.m.

suggested) or in a lanolin-paste mixture as a

narrow band on the stem (100-10,000 p.p.m.

suggested).

If regulating chemicals are to be applied uniformly to all the trees, several trees may be

^ Construction of various types of mist chambers is described in articles listed at the end of this method.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 41

grown in the same chamber. The size of the

chamber may be varied from approximately

1-liter capacity to room size, or large enough

to permit a person to enter.

Method of taking results

After roots of the controls have developed a

measurable amount of new growth, measure

and record the length of all of the roots, and

record the number of those living and dead on

both treated and control trees.

Suggested standard treatment for

comparison

No standard treatment suggested.

Barker, B. T. P. Studies of Root Development. Long

Ashton Res. Sta. Ann. Rpt. 1921: 9-20.

Carter, W. A Method of Growing Plants in Water Vapor to Facilitate Examination of Roots. Phytopathology 32: 623-625. 1942.

Klotz, L. J. A Simplified Method of Growing Plants

with Roots in Nutrient Vapors. Phytopathology 34:

507-508. 1944.

Vyvyan, M. C, and Trowell, G. F. A Method of Growing Trees with Their Roots in a Nutrient Mist. East

Mailing Res. Sta. Ann. Rpt. 1952: 95-98. (Pub.

1953.)

Root Induction

STiM-CUTTING TEST

Method based on research by:

J. W. Mitchell and P. C. Marth; H. B. Tukey ;

J. S. Wells.

Description of test and some other uses

Cuttings are soaked, dusted, or dipped in mixtures containing chemicals, and the effect on

root initiation and growth determined.

Compounds that affect top growth can also

be applied separately or simultaneously with

root-inducing substances.

Suggested plant material

Pieces of stems of woody plants such as privet

(Ligustrum spp.), yew (Taxus spp.)> osmanthus (Osmanthus spp.), and holly (Ilex spp.),

or of herbaceous plants such as chrysanthemum

(Chrysanthemum spp.), gersnniwoa (Geranium

spp.), lantana (Lantana spp.), and carnation

(Dianthus caryophyllus L.).

Apparatus, chemicals, and other materials

Knife with sharp, thin blade 4-6 in. (10-15

cm.) long

Analytical balance or pharmaceutical balance

Containers of suitable size to hold cuttings

upright in solutions

Propagating case with top or sides made of

translucent material, with temperature

controlled at 65^-70° F. (18°-21° C.) and

humidity controlled at 75-95%

Perlite or fine sand washed free of organic

matter, clay, or other foreign material

Clay pots 3 in. (8 cm.) in diameter

Composted soil

100-200 mg. of each compound to be evaluated

500 mg. of 3-indolebutyric acid or alphanaphthaleneacetamide

Solvent such as 95% ethanol

1 lb. (0.5 kg.) of dust carrier such as talc,

fuller's earth, or finely ground clay

Preparation and selection of plant material

Cuttings of woody plants: Select terminal

shoots during late summer or early fall, or a

few weeks after vegetative growth has ceased.

The leaves should be fully developed and the

wood hard. Plant species vary with regard to

the best time of year that cuttings should be

taken in order to obtain optimum results. Remove the cuttings from the plants with a sharp

knife, making one cut on a slant and at the base

of the current season's growth. Cuttings 4-6

in. long (10-15 cm.) or pieces including 3 to 6

nodes are then made from these branches.

Make the basal cut immediately below a node

and the uppermost cut just above a node. Remove the lower leaves, but let 3 or 4 of the uppermost leaves remain attached. Using rubber

bands, fasten cuttings in bundles of 10 to 20

with stems parallel and bases even. Do not

allow cuttings to dry out or the leaves to wilt

at any time during these preparations.

Cuttings from herbaceous plants: Stock

plants may be maintained under greenhouse or

outdoor conditions so that succulent new shoots

are available for cutting material throughout

the year. Prepare bundles of cuttings for

treatment in the same manner as with woody

plant material.
42 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Method of conducting test

Three methods of treating cuttings have

proved suitable for this test :

Soaking method : Prepare aqueous solutions

of each compound to be tested in concentrations

of 25, 50, and 100 p.p.m. Stand a bundle of

cuttings in each dilution so that the bases of

the cuttings are covered with the solution to a

depth of about 1 in. (3 cm.). Vary the size

of the container to suit the size of the batch of

cuttings, and do not allow the leaves to come

in contact with the solution. After 2 to 4 hours

in diffused light, remove the cuttings and insert

the basal ends of the stems to a depth of 2 to 3

in. (5-8 cm.) in the rooting medium contained within the propagating chamber. Pack

the moist rooting medium firmly around each

stem and water thoroughly. Keep the medium

moistened throughout the test (fig. 7).

'igure 7. Steps to follow in testing the effect of regulating

chemicals on the initiation of roots by stem cuttings.

The chemical can also be applied by soaking the cuttings in

a water mixture of the compound. To accomplish this, stand

the cuttings in a mixture containing a known amount of the

regulator so that the basal ends are immersed to a depth of

1 to Vh inches. Place the container with the cuttings in diffused light and allow the cuttings to stand in the mixture for

3 to 6 hours.

After preparing a dust mixture containing a known amount

of the test compound, moisten the end of the cutting with

tap water; then roll the wet end in the dust. Tap the cutting

lightly to remove loose dust.

insert the cuttings in a trench made in moist building sand

(or other suitable inert material) to a depth of about Vs of

their length. Press the sand tightly around the bases of the

cuttings. Shade the cuttings and keep the sand moistened

with water but not overly wet during the next 2 to 4 weeks.

If possible, keep the temperature below 70°-75° F. (21°-

24° C).
TEST METHODS WITH PLANT-REGULATING CHEMICALS 43

Examine several of the cuttings at intervals to determine

whether roots have formed. (XEG. PX-20S AD)

Dust method: Succulent cuttings are usually

treated with a mixture of 1 part of regulating

chemical to 1,000-5,000 parts of dust carrier;

woody cuttings usually require a stronger dust

mixture—1 part regulator in 500-750 parts of

dust carrier. To prepare dust mixtures, first

dissolve the desired weighed amount of the compound in a volatile solvent such as ethanol, then

add the proportionate weighed amount of the

dust carrier. The volume of solvent should be

sufficient to make a thin paste of the regulatordust mixture. Stir the entire mixture thoroughly, then allow the solvent to evaporate at

room temperature, with occasional stirring during this process.

Treat each cutting individually by rolling

about 1 in. (3 cm.) of the basal part in the dust

mixture so that a thin coating of dust adheres

to it. Tap off any loose dust and set the treated

portion of the cutting in the rooting medium.

Do not rub the dust from the cuttings when inserting them in the medium ; place in a trench

wide enough so that the dust remains on the

stem.

Dip method : Prepare a solution of each regulating chemical, using 50-95 % ethanol. Vary

the concentration of each compound in a range

of 100-500 p.p.m. for succulent cuttings and

500-1,000 p.p.m. for woody cuttings. Dip the

bases of one bundle of cuttings to a depth of

1 in. (3 cm.) in one dilution of a compound, and

insert the treated portion of these cuttings individually in the rooting medium. Repeat, using other bundles of cuttings for each concentration of each test chemical.

Method of taking results

After 10-14 days in the case of succulent

cuttings, or 14-30 days in the case of woody

ones, carefully loosen the rooting medium

around the cuttings and remove each one individually from the medium. Sort them into

the following categories : (a) Those that are

dead, (b) those not rooted, (c) those with a few

short roots, (d) those with a moderate number

and fairly long roots, (e) those with many relatively thick, long roots. Note and record the

number of cuttings in each category. Also note

any unusual characteristics of roots in each

category. Pot the cuttings, using soil, and observe their subsequent growth.

Suggested standard treatment for

comparison

Soak method, using 3 concentrations of 3-

indolebutyric acid or alpha-naphthaleneacetamide (25, 50, and 100 p.p.m.) for 2-4 hours.

Mitchell, J. W., and Marth, P. C. Growth Regulators.

Univ. of Chicago Press, Chicago, 111. 129 pp. 1947.

Tukey, H. B., editor. Plant Regulators in Agriculture.

John Wiley and Sons, Inc., New York. 269 pp. 1954.

Wells, J. S. Plant Propagation Practices. Macmillan

Co., New York. 344 pp. 1955.

Seed Formation

LANOLIN PASTE TEST

Method based on research by:

S. L. Emsweller and N. W. Stuart; R. E.

Wester and P. C. Marth.

Description of test

A mixture of lanolin and a regulator is applied to a wound on the petal base or on the

pedicel, and the effect on seed development is

observed.

Suggested plant material

Species of Lilium such as L. longiflorum

(Creole, Croft, or Ace clones), bush lima beans

(Phaseolus lunattis L.), Fordhook, Early Market, or Peerless varieties, or other plants difficult to hybridize.
44 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Apparatus, chemicals, and other materials

Clay pots 6-10 in. (15-25 cm.)

Composted soil

Space in greenhouse maintained at about

70^-85° F. (21°-24° C.) by day and 60°-

75° F. (18°-21° C.) by night

Small vials (25-50 ml.) or other containers

Analytical balance or pharmaceutical balance

250-500 mg. of each compound to be evaluated

1 g. each of alpha-naphthaleneacetamide,

para-chloropenoxyacetic acid and 3-indolebutvric acid for standard treatment

Lanolin

Tween 20

Preparation and selection of plant material

Grow the plants, known to be difficult to

cross, under usual greenhouse conditions.

Method of conducting test

Prepare a range of concentrations of the

chemical to be tested in lanolin, as described

on page 5.

Lilies: Immediately before hand pollinating

the flowers, separate one "petal" (i.e., one mem-,

ber of the outer whorl of the perianth) from the

receptacle, and cover the injured part of the receptacle with lanolin-regulator mixture.

Lima beans: Immediately after cross-pollination has been completed, scratch the base of

the flower stalk (pedicel) lightly with a pin or

dissecting needle. The wound should be made

close to the juncture of the pedicel and peduncle.

With a pin or dissecting needle, place a small

portion of the lanolin-regulator mixture on this

wounded surface. Pollinate additional flowers

as controls.

Method of taking results

Observe and record the number of fruits and

their rate of development. When fruits are

mature, remove the seeds and record the number of mature and immature ones. Determine

the viability of the seeds.

Suggested standard treatment for

comparison

For lima beans, make a lanolin mixture coning sufficient indolebutyric acid to make a concentration of 0.1 %, and a second lanolin mixture

containing sufficient para-chlorophenoxyacetic

acid to make a 0.1% mixture. Combine portions of these two mixtures in a ratio of 4 parts

of the indolebutyric mixture to one part of the

phenoxyacetic acid mixture.

Emsweller, S. L., and Stuart, N. W. Use of GrowthRegulating Substances to Overcome Incompatibilities

in Lilium. Amer. Soc. Hort. Sei. Proc. 51: 581-589.

1948.

Wester, R. E., and Marth, P. C. Some Effects of a

Growth Regulator Mixture in Controlled Cross-Pollination of Lima Bean. Amer. Soc, Hort. Sei. Proc.

53:315-318. 1949.
PART II. METHODS OF DETECTING REGULATING COMPOUNDS

Detection of Plant-Growth Regulators ¡n Animals

BEAN STEM TEST

Method based on research by:

J. W. Mitchell, R. E. Hodgson, and C. F.

Gaetjens.

Description of test and some other uses

Regulating chemicals are extracted from

animal tissues, fluids, secretions, or excrement.

The extract is applied to stems of young bean

plants, and elongation indicates the presence of

plant-regulating compounds in the extract.

The method described here has been used to

estimate the amount of 2,4-.dichlorophenoxyacetic acid (or its salts) in the blood of an animal to which this compound was fed. The

method has also been used to detect the presence

of this chemical in the animal's tissues and milk.

Naturally occurring, cell-elongating compounds

present in urine and feces can also be detected

through the use of this method.

Suggested plant material

Young bean plants (Phaseolus vulgaris L.)

of the Black Valentine or Pinto variety.

Apparatus, chemicals, and other materials

Millimeter rule

Pipette, 0.1 ml. capacity graduated in 0.01 ml.

Fluorescent lights that will supply 800-foot

candles of light intensity at leaf surface

Wiley Mill or suitable means of grinding

samples to 60 mesh

Glass containers for holding samples

Analytical balance or pharmaceutical balance

1-2 g. of sodium or ammonium salt of 2,4-

dichlorophenoxyacetic acid

Centrifuge

Composted soil

Preparation and selection of plant material

Germinate bean seeds in composted soil contained in 3-in. (8-cm.) clay pots, using several

seeds per pot. Allow the plants to grow in a

well-illuminated greenhouse until they are

about 3.5 in. (9 cm.) tall. If the temperature

is maintained near 80° F. (27° C.) during

germination, and adequate sunlight is available,

about 5 or 6 days will be required for the plants

to produce sturdy stems and reach the required

height. Select plants having first internodes

that do not vary more than 1 mm. in length

from the average of the entire group selected.

The average internode length of the selected

plants should, whenever possible, be 13 mm. but

may in some cases be 12 or 14 mm. Select a

sufficient number of plants so that 10 can be

used for each treatment. Arrange the plants

under the fluorescent lights making certain that

the lights extend beyond the edge of the area

covered by the plants so as to obtain light,

solely from the artificial source, of relatively

uniform intensity throughout. Number each

plant, and measure to the nearest mm. the

length of each first internode. Assign treatments in the form of randomized blocks.

Method of conducting test

Prepare four aqueous solutions of sodium

2,4-dichlorophenoxyacetate, the first containing

60 p.p.m. of the salt (6 mg. salt in 100 ml.

water), the second, third, and fourth containing 30, 15, and 7.5 p.p.m., respectively, of the

salt. Make these by diluting the more concentrated solutions with water.

In detecting the regulator in blood, obtain

blood serum from animals that have not injested the chemical under test, and also serum

from animals that have been fed the test compound. Measure out 5 portions of the *'controV' serum, each of 3 ml. volume. Prepare

standard growth-regulator-serum mixtures by

adding none of the aqueous salt solutions to

the first control serum aliquot, 1 ml. of the

60 p.p.m. solution to the second aliquot of

serum, 1 ml. of the 30 p.p.m. solution to the

third aliquot of serum, continuing until each

concentration of salt has been added to a corresponding aliquot of "contror* serum. Measure out several 3-ml. portions of serum from the

animal which was fed the growth regulator.

Evaporate separately all of the serum samples on a steam bath. Grind the residues

quantitatively through a 60-mesh screen and

45
46 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

extract each sample for 15 minutes with 5 ml.

of hot distilled water. Filter the mixtures and

wash the residues with hot water. Evaporate

the filtrates on a steam bath just to dryness,

cool the receptacles, and wash the residues obtained into centrifuge tubes using 1 ml. of

water for each, and centrifuge for 5 to 10 minutes. Decant the centrifúgate and apply 0.01

ml. of each preparation to each of 10 plants.

Place the measured serum preparation on the

first internode of each plant as a narrow band

(approximately 2 mm. wide) extending around

the internode midway between the first and

second node. Deliver the measured volume

directly from the 0.1 ml. pipette onto the stem.

Illuminate the plants, using only the artificial

light source and 12 hours of light followed by

12 hours of darkness.

Method of taking results

Twenty-four hours after treatment, measure

the length of each first internode to the nearest

mm. Calculate the elongation that occurred

during the period. Compare the elongation of

plants which received serum from untreated

animals with that of plants which received serum from the treated animals. Determine if

the differences are statistically significant.

Draw a standard curve showing internode

elongation induced by the known amounts of

2,4-dichlorophenoxyacetate when added to serum from the untreated animal. Using this

standard curve, express the amount of elongation obtained with serum from the treated

animal in terms of parts per million of 2,4-

dichlorophenoxyacetate equivalent.

Use this method for measuring the amount

of the salt in animal organs or in milk.

Suggested standard treatment for

comparison

Sodium or ammonium salt of 2,4-dichlorophenoxyacetic acid used as described above.

Mitchell, J. W., Hodgson, R. E. and Gaetjens, C. F. Tolerance of Farm Animals to Feed Containing 2,4-

Dichlorophenoxyacetic Acid. Jour. Anim. Sei. 5:

226-233. 1946.

Penetration of Soil by Regulators

SEED TEST

Method based on research by:

P. J. Linder.

Description of test and some other uses

Chemicals are applied to the surface of soil

contained in glass tubes. Soil samples are

collected as thin layers from various depths

and bioassayed for the presence of the chemical.

This test is useful in studying the effect of

simulated rainfall on the movement of regulating chemicals through soil, the effect of various adjuvants, types of soil, and the physical

structure of the soil on the rate of movement of

these compounds through soil. It also can be

adapted to the study of persistence of regulating chemicals in soil as affected by factors

such as temperature, moisture, duration of

exposure, and microbial content of the soil.

Suggested plant material

Seeds of mustard (Brassica spp.) or other

small-seeded plants known to be sensitive to

the compounds to be tested.

Apparatus^ chemicals, and other materials

Straight glass tubes ll^ in.

(3 cm.) in diameter x 5i/^ in.

(14 cm.) in length

Cork stoppers to plug one end of the glass

tubes

Fertile soil passed through a screen with

14-in. (0.6-cm.) openings

Cork stoppers with a maximum diameter

slightly less than the inside diameter of

the glass tubes

14-in. (0.6-cm.) dowel 1 ft. (30 cm.) long

Analytical balance or pharmaceutical balance

Hand-sprayer capable of distributing a test

solution accurately over a given area

Razor blade

Shallow tin or plastic cups or screw-type

jar lids of about 2i4-in. (6-cm.) diameter

Dark chamber maintained at about 80° F.

(27° C.) with a high relative humidity

About 5 g. of each compound to be tested

Preparation and selection of plant material

Sort the seeds, eliminating any that are

shrunken or defective.

Method of conducting test

Tightly stopper one end of the glass tube.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 47

Pour 75 g. of air-dry soil into the stoppered

tube. Then place a loose-fitting cork on top

of the soil. Place the end of the dowel against

the loose-fitting cork and subject the other end

of the dowel to a pressure of 2 kg. for the purpose of compressing the soil a measured amount.

Remove the tightly fitting cork stopper from

the tube and, by pressing the dowel against the

loose-fitting stopper, force the soil upward in

the tube until the surface is flush with the open

end of the tube. Mark off an area of about 1 sq.

yd. (1 sq. m.) on the floor. If need be, cover

the area with disposable paper or other material. Stand the tubes to be used for one treatment on end within the marked area so that

the flush and exposed soil surfaces are uppermost.

Apply the compound to be evaluated evenly

over the entire marked area including the exposed soil surfaces. This can be done by spraying the mixture back and forth over the entire

area, first in one direction, and then in a direction at right angles to this, so that the area is

covered uniformly with the mixture.

Using separate sets of tubes of soil, various

amounts of the chemical can be tested. To determine the amount of each chemical to be used,

carry out preliminary experiments with each

compound to find the dosage range necessary to

inhibit shoot extension above the soil from 0 to

100 percent. Use 10 soil tubes for each concentration of each chemical, and treat 5 tubes

with only water and solvent, if a solvent was

used, to serve as solvent-treated controls.

To test for the presence of the applied compound, collect a soil sample by using the dowel

and loosely fitting stopper to push the column

out through the open end of the cylinder for a

distance of 3 mm.; then slice off the ejected

portion with a razor blade. In this way it is

possible to collect a surface layer 3 mm. or

more in thickness, or any lower layer of soil

desired.

To obtain a sufficient volume of soil for

assaying, collect comparable samples from 5

replicates and combine and mix them. Divide

the composite sample into 3 equal parts. Place

each soil sample in the shallow tin or plastic

cup. Sow seeds of the test plants uniformly

in the soil. Add uniformly suflicient water to

bring each soil sample to optimum moisture

content for seed germination. Store the cups

containing the soil and seeds in a dark, moist

place at about 80^ F. (27° C).

Method of taking results

Measure the height (or fresh weight) of the

plants in the various soil samples, including

the prepared standards and the solvent-treated

control. Compare the average amount of

growth with the solvent-treated controls and

with that of the plants grown in cups containing soil samples to which have been applied

known concentrations of the test chemical.

The soil in the tubes can be (1) assayed, as

described, shortly after chemical application to

determine the rate and depth of penetration of

the chemical, or (2) stored under controlled

moisture, temperature, and other conditions

before being assayed to determine the effect

of these on persistence of the chemical, or (3)

subjected to simulated rainfall for leaching

studies, etc., before the assay. The prepared

series of standards may be used also to determine the amount of chemical present in field

plots which have been treated with a test

chemical.

Suggested standard treatment for

comparison

No standard treatment suggested.

Linder, P. J. Movement and Persistence of Herbicides

Following: Their Application to the Soil Surface.

Northeastern Weed Control Conference Proc. 6:

7-11. January 1952.

Residue of Regulators in Soil

REPEATED PLANT TEST

Method based on research by:

A. J. Loustalot and R. Ferrer; W. W. Robbins, A. S. Crafts, and R. N. Raynor.

Description of test and some other uses

Several successive crops of plants are grown

in soil treated once v^ith a chemical in order to

determine hov^ long the chemical remains in

the soil in sufficient amounts to affect plant

growth.

This test is convenient for testing the effect

of cultivation or mixing of the chemical with

the surface or with lower layers of the soil, and

for studying the effect of environmental conditions on the persistence of the chemical in

soil.

Suggested plant material

Use plants known to be highly sensitive to
48 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

the compound under test. If the sensitivity of

plants to the chemical is not known, select a

number of plant species and determine useful

ones on the basis of their sensitivity to direct

application of the chemical to be tested.

Apparatus, chemicals, and other materials

Soil to be tested

Unperforated small containers

(Clean, used No. 2 tin cans have the advantage over clay pots of not absorbing any

of the applied chemicals.)

Analytical balance or pharmaceutical balance

Greenhouse space

About 2-3 g. of each compound to be

evaluated

Preparation and selection of plant material

Sort the seeds, and eliminate any that are defective.

Method of conducting test

The amount of the chemical used in these

tests will depend on the specific compound

tested. (A mixture of 0.25 mg. of a chemical

with 500 g. of a clay loam soil is equivalent to

1 lb. [0.5 kg.] of the chemical applied to an acre

[4,047 sq. m.] of clay loam soil and mixed to a

depth of 7 in. [18 cm.}.)

Insoluble compounds, or compounds that are

dry and of small particle size, can be dry-mixed

with the soil before placing the chemical-soil

mixtures in the containers. Use a total of 500

g. of soil per container.

To estimate the amount of moisture to be

maintained in each container of soil, punch 10

to 12 holes in the bottom of one can with a 10-

penny nail. Place 500 g. of soil in the can and

record the total weight of can plus soil. Immerse the can in water to a distance of 1 in. (3

cm.) and maintain the water at this level until

the surface of the soil becomes moist. Remove

the can containing the soil and allow it to drain

10 to 15 minutes. Record the weight of the

can, water, and soil as the weight indicating the

amount of moisture to be maintained. In conducting the experiment, add enough water each

day or two to maintain this weight. Subtract

the weight of the can plus soil from the weight

of the can, soil, and moisture to obtain the initial weight of water to be added. To distribute

the chemical throughout the soil in each container, dissolve the required amount of soluble

compound in the above-determined weight of

water and pour this evenly over the surface of

the soil.

An alternative method that provides even distribution of the chemical throughout any soil

can be used with any compound that is soluble

in water or suitable organic solvents (acetone,

alcohol, etc.). Dissolve a measured amount of

the test compound in 6 ml. of a suitable solvent

(addition of a wetting agent, such as 0.4 ml. of

Tween 20, may be advantageous). Add 20 g.

of fine, clean quartz sand to this solution and

stir to make a slurry. Dry this mixture at

room temperature. Add the chemically-coated

sand to enough soil to make a total of 500 g. and

mix it thoroughly before placing the treated soil

in the container.

After the seeds have been planted, bring the

soil samples to the correct moisture level. The

number of seeds to be planted in each can will

depend on the size of the plant.

Grow the test plants for 30 days. Then measure the height of the plants in the treated and

untreated containers, cut off the plants at the

soil level, and determine the fresh weight of

the tops. Return the tops to the cans where

they were originally grown and, after a 30-day

drying period, pulverize and replace the soil,

putting the now dry tops beneath the soil. Replant the soil and bring it to the correct moisture level. Six crops a year can be grown in

this manner in order to determine the residual

effects of the compounds.

A variation of this test is as follows : Treat

the soil with the test chemical as above but do

not plant all of the containers at once. Store

some of the containers for i^, 1, and 2 months

under different conditions. Conditions that can

be altered easily are soil temperature and moisture content. Combinations of these conditions

can be used, but it is strongly suggested that

the conditions selected closely approximate the

normal variation found in the field.

Method of taking results

The weight and height measurements taken

after each successive growth period are compared. Comparative studies of residual effects

can be made following use of one chemical and

many soil types, or several chemicals in a single

or a few soil types.

Suggested standard treatment for

comparison

No standard treatment suggested.

Loustalot, A. J., and Ferrer, R. Studies on the Persistence and Movement of Sodium Trichloroacetate in

the Soil. Agronomy Jour. 42: 323-327. 1950.

Robbins, W. W., Crafts, A. S., and Raynor, R. N. Weed

Control, 1st Edition, 503 pp. McGraw, Hill Book Co.,

New York. 1942.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 49

Translocation of Radioactively Tagged Compounds

ISOTOPE COUNTER TEST

Method based on research by:

John W. Mitchell and Paul J. Linder.

Description of test

Translocation of radioactively tagged molecules is measured by placing a tagged compound

on one part of a plant and measuring radioactivity that results from the movement of the

compound, metabolites, or degradation products

of it to other parts of the plant. Radioactivity

in dried, ground, plant tissue is measured directly. Since self-absorption is involved, the

method is only reliable for comparison of radioactivity in comparable tissues ground to approximately the same particle size.

Suggested plant material

Any kind of terrestrial plant.

Apparatus, chemicals, and other materials

Conventional Geiger tube

Q-gas counter (or counter of similar sensitivity if carbon tagged compounds are

used)

Wiley mill

Sealer

Lead tube for shield

Remote control applicator (fig. 8)

Brass rings (inside diameter 25 mm., outside

diameter 31 mm., 3 mm. high)

Cellophane tape, 1.5 in. wide (3 cm.)

Analytical balance or pharmaceutical balance

Radioactively tagged plant regulator

Lanolin

Tween 20

Preparation and selection of plant material

The follov^ing method is designed for use in

studying translocation from leaves of plants

such as bean (Phaseolus vulgaris L.), tomato

(Lycopersicum esculentum L.), or other plants

commonly grown in a greenhouse, but it can be

modified for application to any part of a terrestrial plant. Plant seeds in soil contained in

clay pots and allow several plants to grow in

each pot until the primary leaves or the first

secondary leaves have become photosynthetically active and capable of supplying photosynthate to other parts of the plants. Thin the

plants to one in each pot, retaining only plants

of uniform size.

Figure 8. Diagram of a remote-control applicator useful for applying small volumes of so^uiions containing radioactive regulating chemicals to leaves and stems of plants. Support an

aluminum rod (A), approximately 65 cm. long and 5 mm. in

diameter, by means of two 13-mm. aluminum angles (B).

Thread one end of the rod and screw it through a plate into a

metal box (C) so that it rests against a movable plate (D).

Turn the rod to press the plate (D) against the piece of soft

rubber tubing (E) which is held in place by clamps. Close the

upper end of the tubing with a glass plug. Insert a 0.1 ml.

pipette (F) in the lower end of the tubing, and fasten the

pipette with a clamp (G). Turn the rod and expel the air from

the pipette; then replace it with a solution containing the

radioactive growth regulator. Force a measured portion of

the solution (0.005, 0.01, or 0.Ô2 ml.) out of the pipette by

turning the rod, and apply the drop by touching the tip of the

pipette to the surface of a plant. When necessary, use the

t'p of the pipette to spread the liquid evenly over the plant

surface. (NEG. PX-299)

Method of conducting test

Prepare an aqueous mixture which contains

0.1% of Tween 20 (or other suitable cosolvent)

and 10 /.ig. of the radioactive plant regulator

per 0.01 ml. This may be easily accomplished

by using the proper precautions ^ and weighing

the required number of mg. of the radioactive

substance into a vial, adding the required

amount of distilled water containing the cosolvent. By means of a short piece of glass rod

3-5 mm. in diameter, crush all crystals of the

growth regulator and stir until the very fine

pieces dissolve.

Use a short piece of rubber tubing (6-7 mm.

inside diameter) and stamp a ring of lanolin on

the upper surface of the leaf enclosing an area

in the desired position (fig. 15). By means of

the remote control applicator, place 0.01 ml. of

^ Experiments using radioactive materials should not

be undertaken without a thorough knowledge of the

hazards involved.
50 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

the mixture within the lanolin ring. If the

entire upper surface of the leaf is to be covered

with the mixture, use tne method described on

page 8. Replicate treatment with any one

ragged compound at least five times, using an

equal number of comparable plants.

After application of the radioactive regulator, place the plants in a well-illuminated

greenhouse, or if necessary, illuminate them by

means of ñuorescent tubes so that they receive

at least 700-foot candles of light at the leaf surface. After the desired length of time (from a

few hours to several days) remove the treated

leaves very carefully and place them in a paper

bag to be discarded. Wash the remainder of

the plants free of soil, divide them into roots,

stems, etc. Dry each part in a well-ventilated

oven and grind each sample to 40 mesh, exerting care that one sample does not become contaminated by another.

The size and density of the tissue particles

that make up a sample of ground plant material

control, to some extent, the amount of selfabsorption of radioactivity that occurs within

the sample. It is difficult to grind to comparable particle size tissues from plants grown

under widely different, environmental conditions, or tissues from different parts of plants

grown under comparable conditions. The

amount of fiber and secondary thickening that

develops in stems and leaves is dependent to

some extent upon the environmental conditions

to which the plants are subjected. Plants

grown in relatively high light intensity often

contain proportionally more fiber than do those

grown in shade and the former are, therefore,

more diflScult to grind to a small particle size.

More mature parts of stems generally contain

proportionally more fiber than do relatively immature parts of stems, and samples of the

former are, therefore, diflScult to grind into

particles of relatively small size. Care must

be exercised in the utilization of the present

method so that self-absorption, due to differences in size and density of particles, does not

introduce significant errors.

Method of taking results

To determine the radioactivity in a sample,

place a brass ring on the tacky surface of the

cellophane and trim off the cellophane flush

with the outside of the ring; then determine

the weight of the ring and cellophane. Thoroughly mix the powdered sample and place a

small portion of it on the tacky surface of the

cellophane. By gently tapping the ring, roll

the powdered tissue back and forth until the

entire surface is evenly covered. Shake the

excess of tissue into its original container and

weigh the ring, cellophane, and adhering tissue

to determine the amount of tissue used to measure the radioactivity. Place the ring in the

holder of the counter and determine the number

of counts per minute that emanate from the

sample. Calculate net counts per milligram

per minute by subtracting background counts.

Suggested standard treatment for

comparison

Ten /xg. of 2,4-dichloro-5-iodophenoxyacetic

acid tagged with I^^S one of its salts, or 2,4-

dichlorophenoxyacetic acid tagged with C^^

Mitchell, John W., and Linder, Paul J. Some Methods

Used in Tracing Radioactive Growth-Regulating Substances in Plants. Bot. Gaz. 112: 126-129. 1950.

RADIOAUTOGRAPH TEST

Method based on research by:

A. S. Crafts.

Description of test

Radioactive 2,4-dichlorophenoxyacetic acid is

placed on a leaf of the plant. The plant is

later dried and stored adjacent to an X-ray film

which is then developed. The autographic outline thus obtained is registered with an outline

of the plant on Ozalid paper, and the presence

of the radioactivity in different parts of the

plant observed.

Suggested plant material

Succulent plants that can be readily obtained

in the field or grown in a greenhouse.

Apparatus, chemicals, and other materials

Darkened room

Oven for drying plants

Waxed paper

Dry ice

Dry sheets of newsprint

Blotters of a size that will cover the entire

plant

Kodak No-screen X-ray film

Aluminum foil

Empty metal cartridge cases to be used as

light-tight containers (obtainable from

army supply stores) or other suitable lighttight boxes

Analytical balance or pharmaceutical balance

Ozalid paper
TEST METHODS WITH PLANT-REGULATING CHEMICALS 51

2,4-Dichlorophenoxyacetic acid labeled in the

carboxyl position with carbon ^* and having a specific activity of at least 1 microcurie per mg.

Developer and fixer for use with X-ray film

Wetting agent such as Tween 20, or other

detergents

Preparation and selection of plant material

Select plants that suit the objectives of the

test, generally plants that are growing vigorously and that have developed suitable leaves

located in the same relative position on each

plant. Use only leaves that have reached a

stage in their development at which they are

capable of supplying photosynthate to other

parts of the plant. Very young, rapidly expanding leaves do not translocate regulating

compounds to other parts of the plant.

Method of conducting test

Prepare mixtures containing the radioactive

regulator as described ^ (see page 49). Apply

from 1-10 fig. of the labeled compound to one

leaf of several plants to be used as replications. After 2 or 3 days (or any other desired period of time), wash the plants free of

soil and place them between sheets of waxed

paper, then between blocks of dry ice. The

plants will freeze solid within 2 minutes. After

5 minutes, remove the plants and place them

between clean, dry sheets of newsprint; and

place these, in turn, between blotters. Dry the

blotters, papers, and plants in a well-ventilated

oven at 70°-80° C. for a period of 10 to 12

hours. Place one of the dry plants on a sheet

of the X-ray film (in a darkened room) and

cover this with a blotter to which has been

clipped a sheet of aluminum foil of equal size.

Place another blotter on top of the "pile'', then

another sheet of film, and continue to stack the

plants and sheets until each plant is next to a

sheet of film. Bind all together between suitable light-weight boards and place the entire

"pile*' in a cartridge case. Store the "piles"

for a period of 4 weeks at room temperature;

then develop the film and make an Ozalid print

of each plant. Mark each autograph and Ozalid print so that they can be readily identified.

Method of taking results

Place an autograph over the corresponding

Ozalid print and register the images. Compare

visually the autographic image developed from

the different plants and record any differences

in translocation of radioactive substances.

Suggested standard treatment for

comparison

Ten /xg. of 2-4-dichlorophenoxyacetic acid

(C^*OOH) applied in an aqueous mixture containing 0.1% Tween 20 or other wetting agent.

Specific activity of acid, 1 microcurie per mg.

Unpublished method suggested by A. S. Crafts, Calif.

Agr. Expt. Sta.

Volatility^ of Plant Regulators

mSPOSABLE CONTAINER TEST

Method based on research by:

P. C. Marth and J. W. Mitchell.

Description of test

This test is useful for detecting volatility of

plant-regulating compounds that induce easily

detectable growth responses. It can be used to

detect or measure evaporation of regulating

chemicals from surfaces of plants previously

treated with these compounds, or from surfaces

of other materials to which these substances

have been applied.

Suggested plant material

Determine a suitable test plant by placing a

small amount of the chemical to be tested on

several kinds of young plants; suggested are

snap bean (Phaseolus vulgaris L.), buckwheat

(Fagopyrum esctdentum Moench), cucumber

(Cucumis sativus L.), and tomato (Lycopersù

cum esculentum Mill,),

Apparatus, chemicals, and other materials

3-in. (8-cm.) clay or paper pots or clean discarded tin cans of about 1-lb. (454-g.)

capacity

Analytical balance or pharmaceutical balance

Greenhouse space maintained at 75°-85° F.

*A thorough understanding of hazards involved must

first he obtained. ,,,.,, ^ ^. ' Carelessness in the use or disposal of highly ettectiye

or volatile, regulating chemicals can result in contamination of an entire laboratory and greenhouse, or loss

of valuable plants and use of space contiaminated.
52 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

(24''-29° C.) for growing test plants

Table or bench space in darkened room maintained at 70°-90° F. (27°-32° C.) for

conducting tests

Several 1- and 5-ml. pipettes

20-30 ml. vials with tightly-fitted stoppers

or screw caps

Filter paper No. 1 or other absorbent paper

discs 7-9 cm. in diameter

Cellophane bags 4 in. (10 cm.) square and

16 in. (41 cm.) tall

Adhesive cloth, plastic tape, paper clips or

electric iron for sealing cellophane bags

95% ethanol or acetone for solvent

0.1-1 g. of each pure compound to be tested

1-2 g. of the butyl or isopropyl ester of 2,4-

dichlorophenoxyacetic acid for standard

treatment

Preparation and selection of plant material

Plant several seeds of bean, tomato, or other

test plant in soil contained in disposable pots

or containers. Employ optimum greenhouse

conditions of light, temperature, and moisture

so as to produce rapidly growing seedlings for

the tests. Select bean plants that have primary

leaves that measure 3 cm. across, and with trifoliate leaves still folded in the terminal buds

(about 5-7 days old) ; or tomato plants 2.5-3

in. (6.4-7.6 cm.) tall (about 25 days old).

Thin the plants, leaving a single plant of uniform size in each pot.

Method of conducting test

Prepare an alcoholic or acetone solution of

the pure chemical by placing a weighed amount

of it in a vial and adding sufficient 95% ethanol

or acetone to give the desired concentration.

For example, weigh 5 mg. of the chemical into

a vial and add 25 ml. of solvent to obtain a

solution containing 0.2 mg. of the compound

per ml.

Impregnate a filter paper or other suitable

paper disc with 1 ml. of the alcoholic mixture,

and allow the solvent to evaporate completely

(5-30 minutes). Use 3 or more replications for

each compound tested.

Place a pot containing the selected test plant

upright in the cellophane bag. Fasten the impregnated filter paper with a small piece of

cellophane tape inside the bag and about 2 in.

(5 cm.) from the top. Do not allow the impregnated filter paper to come in contact with

the plant during the test. Close the open ends

of the cellophane bags and fold them downward

at a distance of about 1 in. (2.5 cm.) ; then seal

with a hot iron.

Expose the plants to the vapor from the test

chemical for the desired length of time (24-72

hr.) and temperature; then remove, and allow

them to remain in a greenhouse for later observation. Dispose of the plants, filter paper containing the chemical, bags, etc., so as to avoid

contamination of room or greenhouse.

Method of taking results

The response of the plant (leaf and stem curvature) is due to two separate factors ; first, the

ability of the chemical to induce the response

when in direct contact with the plant; and

secondly, the rate that the chemical evaporates

from the paper and comes in contact with the

plant. Since this method is based both on the

biological effectiveness of the compound and its

ability to evaporate, the method cannot be used

to measure either of these factors separately.

Immediately before removal of the plants

from the bags, record the degree of curvature,

comparing the plants with a diagram or photograph illustrating typical plants that have responded slightly, moderately, or to a marked

degree. For convenience, assign these categories numerical values such as 0, 1, 2, 3, and 4.

Carefully dispose of all contaminated materials

including plants exposed to the vapors.

Suggested standard treatment for

comparison

A filter paper disc that is impregnated with

0.2 mg. of butyl ester of 2,4-dichlorophenoxyacetic acid and the plants exposed to the vapors

for 48 hr. at 80°-90° F. (27°-32° C).

Marth, P. C, and Mitchell, J. W. Comparative Volatility of Various Forms of 2,4-D. Bot. Gaz. 110:

632-636. 1949.

GERMINATING SEED TEST

Method based on research by:

W. P. Anderson.

Description of test

Relative volatility of regulating compounds is

determined by exposing germinating seeds to

vapors of these chemicals in closed containers.

Suggested plant material

Cucumber, buckwheat, corn, or other seeds.
TEST METHODS WITH PLANT-REGULATING CHEMICALS 53

Apparatus, chemicals, and other materials

Petri dishes about 15 cm. diameter x 2 cm.

deep and 5 cm. diameter x 2 cm. deep

Filter paper (No. 1 Whatman) to fit the

Petri dishes

Forceps

Wide-mouth moisture- and air-tight containers of 1- to 2-pint size (0.5-1 liter)

Constant-temperature box or room controlled

at 70^-75° F. (21°-24° C.)

Analytical balance or pharmaceutical balance

250-500 mg. of each compound to be

evaluated

Highly-volatile solvent such as acetone, ether,

or ethanol

Compounds known to have a relatively high

degree of vapor activity, such as isopropyl

ester of 2,4-dichlorophenoxyacetic acid and

isopropyl N-(3-chlorophenyl) carbamate

for standard treatment

Preparation and selection of plant material

Germinate seeds in the large Petri dish containing moistened filter paper at a temperature

of about 70°-75° F. (21^-24° C). When the

seed coat has cracked and the tip of the radical

is visible through this opening, select 5 seeds

at a uniform stage of germination. Place these

on moistened filter paper in the small Petri dish

and then place the dish in the air-tight container. In the case of cucumber and buckwheat

seeds, this stage is reached 17 and 20 hours,

respectively, after placing the seeds in moist

Petri dishes.

Method of conducting test

Impregnate a disc of filter paper (5 cm. diameter) with 50 mg. of the chemical to be tested,

dissolved in highly volatile solvent. After

evaporating the solvent, place the filter paper

in the air-tight container in such a manner as

to avoid direct contact with the Petri dish containing the germinating seeds. Close the container tightly and place it in the constanttemperature box along with others for the same

test. The period of exposure may vary from a

few to 48 hours or more, depending upon the

type of response to be measured and the relative

rates of evaporation of the chemicals.

At the end of the designated period, remove

the Petri dishes from the containers, cover

them, and allow the seedlings to grow for several more days; observe variations in growth

caused by the treatment.

Method of taking results

Make visual comparisons of the response of

the seedlings, noting increase or reduction in

length of the root hair zone, and the relative

number of hairs, increase or decrease of branching along the tap root, and the relative lengths

of the tap and branch roots ; also abnormalities

occurring in the growth of the hypocotyl.

Suggested standard treatment for

comparison

Compounds known to have a relatively high

degree of vapor activity, such as isopropyl ester

of 2,4-dichlorophenoxyacetic acid and isopropyl

N-(3-chlorophenyl) carbamate.

Unpublished method submitted by W. P. Anderson,

American Chemical Paint Company, Ambler, Pa.

Detection of Regulators in Water

ROOT GROWTH TEST

Method based on research by:

D. Ready and V. 0. Grant.

Description of test and some other uses

Small amounts of 2,4-dichlorophenoxyacetic

acid in aqueous solutions are detected by the

inhibiting effect on growth of primary roots of

cucumber.

This test may also be adapted for the detection of other compounds in water solution provided the roots of the test plant used are sufficiently sensitive to the chemical being tested.

Suggested plant material

Cucumber seeds (Cucumis sativus h.).

Apparatus, chemicals, and other materials

Petri dishes 6 in. (15 cm.) in diameter

Filter paper

Analytical balance or pharmaceutical balance

Darkened constant-temperature room maintained at about 82^ í . (28° C.)

1 g. of 2,4-dichlorophenoxyacetic acid for

standard treatment

Distilled water
54 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Preparation and selection of plant material

Use seeds having a high percentage of germination ; sort them, and discard those that are

defective.

Method of conducting test

Place 15 cucumber seeds on filter paper in the

bottom of each Petri dish. Using 5 Petri

dishes for each concentration level of each solution to be tested, moisten the paper in each dish

with 15 ml. of the test solution. A range of 15

concentrations varying in uniform steps from

0.001 p.p.m. to 10 p.p.m. is suggested. Cover

the Petri dishes and place them in a darkened

constant-temperature room maintained at about

82° F. (28° C). Prepare 5 dishes using distilled v^ater instead of the test solution and

maintain in the same manner for controls.

Method of taking results

After 96 hours, measure the length of the

primary root of each seedling in each dish to

the nearest millimeter. Compare these root

measurements with those of the control seedlings and the seedlings treated with a known

range of concentrations of 2,4-dichlorophenoxyacetic acid. The amount of reduction in length

of the primary roots indicates the amount of

the plant regulator in the test solution.

Suggested standard treatment for

comparison

2,4-Dichlorophenoxyacetic acid at concentrations of 0.001, 0.005, 0.025, 0.050, 0.075, 0.10,

0.25, 0.50, 0.75, 1, 2.5, 5, 7.5, and 10 p.p.m.

Ready, D. and Grant, V. 0. A Rapid Sensitive Method

for Determination of Low Concentrations of 2,4-Dichlorophenoxyacetic Acid in Aqueous Solution. Bot.

Gaz. 99: 39-44. 1947.
PART III. TECHNIQUES IN GENERAL USE

Some of the techniques developed by various

scientists are of general use in studying the responses of plants to regulating chemicals. Several of these techniques are described on the

foUov^ing pages with the belief that they may

be of particular interest to those who are undertaking work with regulating chemicals for the

first time. No attempt is made to credit authors of these techniques since these methods

are widely known and used, and because of the

difficulty of determining the exact origin of

each technique.
56 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

FIGURE 9. IXEG. PN-300)

Lanolin as a carrier for regulating chemicals

Lanolin is widely used as a carrier for regulating chemicals (fig. 9). Applied in small

amounts, this semi-solid, fatty substance does

not injure plants. It makes close contact with

the plant surface, and regulators, when mixed

with lanolin, diffuse readily from a thin layer

of the paste into the plant. Place a weighed

amount of lanolin in a vial and immerse the

vial in warm water to melt the lanolin. Weigh

out the finely powdered or liquid regulator and

stir it thoroughly into the lanolin until the mixture becomes semi-solid. Apply the mixture to

stems or leaves with a toothpick or other disposable applicator.
TEST iMETIIO-rS WITH PLANT-REGUi-ATING CHEMICALS Dt

FIGURE 10. (XEG. TN 308 A. B)

Terminal growth measurements

Many regulating chemicals cause an increase

or a decrease in the rate of growth of the main

stems of plants. The effect of a compound on

the length of the stem is therefore one means of

evaluating the regulating properties of the

chemical.

Apply the chemical in a suitable carrier (lanolin or aqueous mixture) to bean plants, for

example, that have developed primary leaves

which are partially expanded (3.5-5 cm. across)

and when the trifoliate leaves are still folded in

the terminal bud (fig. 10, left). Measure the

length of the terminal bud from second node to

tip of bud at the time of tr-îatment. Measure

from the same node (second) to the tip of the

terminal bud about 1 week after the treatment

(fig. 10, right), and compare growth with comparable measurements of untreated plants.
58 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

FIGURE 11. (XEG. PN-302;

Convenient way to measure effect of

regulating chemicals on root growth

Place a disc of blotting paper "^ impregnated

with a water mixture of the regulating chemical

and another impregnated with tap or distilled

water in separate Petri dishes. Place several

cucumber seeds of uniform size on the surface

of each paper. Store the closed dishes at room

temperature in darkness for 24 hours; then

measure growth of the roots repeatedly during

the following 2 or 3 days with the dishes closed

(fig. 11). Compare growth of the roots on the

chemically treated papers with that of roots on

the papers impregnated with water.

A device that delivers measured amounts of

a regulating chemical

This microinjector delivers measured vol-

' Ordinary blotting papers often contain chemicals

which inhibit root growth. Blotting paper especially

prepared for seed tests such as that described can be

obtained from the Standard Paper Manufacturing

Company, Richmond. Va.

umes of lanolin paste containing a regulating

chemical (fig. 12, above). Prepare a mixture

of lanolin and the regulating chemical. While

the mixture is melted, draw the syringe about

one-third full; then allow the mixture in the

syringe to solidify. Fasten the syringe in the

clamp and operate the lever until a uniform

amount of the paste is delivered with each

stroke. Collect portions of desired volume

(regulated by the number of strokes used) on

the end of a toothpick or other disposable applicator (fig. 12, below) and apply the paste

quantitatively to leaves or stems. Determine

the weight of paste delivered per stroke by first

weighing a piece of aluminum foil, then collecting on the foil the amount of paste ejected after

one stroke, and reweighing the paste and foil.

Repeat the procedure 5 to 10 times; then calculate the average number of micrograms of

growth-regulating chemical delivered per

stroke.

Construction of this microinjector is described by C. C. Roan and Shizuko Maeda in
TEST METHODS WITH PLANT-REGULATING CHEMICALS 59

FIGURE T2. (NEG. rX-303 A, B)

"A Microdevice for Rapid Application of Toxi- and Plant Quarantine, United States Departcants to Individual Insects," publication No. ment of Agriculture, 1953.

ET-306 of the former Bureau of Entomology
60 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

FIGURE 13. (NEQ. PN-309)

Simulated spray-droplets

Some investigators use a small glass loop to

place droplets of relatively uniform size on

leaves. With the aid of a flame, draw out a

glass rod so that it forms a thin thread about

0.5-0.75 mm. in diameter. Break the thread

of glass and with a very little heat bend the end

of the small rod so that a loop 2-3 mm. in diameter is formed. Dip the loop into an aqueous

mixture of the regulating chemical. Transfer

the liquid that adheres to the loop onto the surface of a leaf by touching the loop to the desired

area (fig. 13). Droplets of different size and

various patterns of distribution can be obtained

to simulate spray-droplets.

Formative effects of regulating chemicals

Some regulating compounds cause plants to

change their usual pattern of growth, especially their pattern of leaf growth. Very

minute amounts of such compounds as 2,4-

dichlorophenoxyacetic acid on such plants as

cotton, bean, and grape often result in relatively small leaves with narrow curled blades

and sometimes enlarged veins (fig. 14; leaf

from treated plant, right).

FIGURE M. .NEO. P\-30ú)
TEST METHODS WITH PLANT-REGULATING CHEMICALS 61

FIGURE 15. (NEU. P.\-307)

Simplified method of separating areas of leaves treated with a regulating chemical from

the remaining untreated portions of ths leaf

In studying the movement of radioactively

tagged regulating chemicals in leaves, it is often

desirable to remove the treated portion of a

leaf so that the uncontaminated portion which

remains can be assayed for radioactivity. The

following simplified technique is useful since

the equipment required is inexpensive and, if

accidentally contaminated, it can be discarded,

thus minimizing the danger of contaminating

untreated portions of the leaf. Place the detached leaf on a piece of sandpaper (C weight.

3/0-120) or emery cloth. Place the open end

of a vial over the treated portion of the leaf in

such a way that the inside edge of the vial does

not come in contact with the radioactive material on the leaf. Turn the vial back and forth

with slight pressure and thus cut out and remove the treated area of the leaf (fig. 15).

Treated portions of several leaves can be removed without contamination of untreated portions by using only 1 vial and 1 piece of abrasive

material and then discarding the equipment.
62 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

FIGURE 16. ' ^^O- ^^-^"^ *• I*)

Highly sensitive test plants

Some plants respond quickly to regulating

chemicals. Young rapidly growing plants are

generally more sensitive to these chemicals than

are more mature plants. Young bean, cucumber, and sunflower plants are among the more

sensitive, and these are especially useful since

they can be quickly and easily grown to a size

that is useful for tests with regulators. Plant

the seeds in composted soil; keep the soil uniformly moist and at a temperature of not less

than 80° F. (about 26° C.) until the seedlings

appear above the surface of the soil; then reduce the night temperature to 70°-75° F. (about

21°-24° C). About 5 to 8 days are usually

required for such plants to grow to a useful

size. Application of the regulator in a suitable carrier, such as lanolin, to the stem or

leaves of these test plants (fig. 16, above) will

often induce a marked growth response within

a few hours or during the following over-night

period (fig. 16, below).

FIGURE 17. (NEG. PN'-304)

An easy method of impregnating dust

carriers with regulating chemicals

Weigh the desired amount of carrier (diatomaceous earth or other finely ground inert

material) and place in a container. Dissolve

the required amount of regulating chemical in

a sufficient amount of ethyl alcohol to make a

slurry when combined with the dust carrier.

Combine the alcoholic solution with the dust

(fig. 17). Stir the slurry thoroughly and allow

the alcohol to evaporate at room temperature.

Mix the impregnated dust thoroughly before it

is applied to plants.
TEST METHODS WITH PLANT-REGULATING CHEMICALS

Useful concentrations of regulating chemicals in dust carriers often range from 500 to

5000 parts of the chemical in 1,000,000 parts

of carrier. Effectiveness of some regulators

applied in talc dust has been slightly enhanced

experimentally by the addition of small amounts

of hygroscopic agents, such as glycerine, to

the alcoholic mixture, and its final incorporation in the dust preparation. The amount of

hygroscopic agent used should not be so great

as to interfere with the dustability of the

preparation.

Application of a measured amount of

regulating chemical to a known area of

leaf surface

It is sometimes necessary to apply measured

amounts of a regulating chemical to a limited

area on a leaf, especially when radioactively

tagged regulators are used. Slip a short piece

of rubber tubing of appropriate diameter over

the end of a cork borer of suitable size, leaving

about 1 cm. of tubing extending beyond the end

of the cork borer. Calculate the inside crosssectional area of the rubber tube. Spread lanolin over the surface of a glass plate to the

depth of about 1-2 mm. and press the end of the

rubber tubing in the lanolin layer ; then gently

stamp a ring of the lanolin on the leaf surface

(fig. 18, above). Fill a 0.1 ml. pipette (graduated in 0.01 ml. portions) with the aqueous

mixture of the regulating chemical to be used,

touch the tip of the pipette against a piece of

glass or other hard surface to remove excess

mixture, then allow 0.01 ml. of the mixture to

drain from the pipette so that the measured

volume remains suspended at the end of the

pipette. Touch the end of the pipette to the

area enclosed by the lanolin ring and move the

tip gently over the area until the measured

volume of liquid is spread evenly over the

enclosed surface (fig. 18, below). Aqueous mixtures of regulating chemicals spread over leaf

surfaces most readily when a surfactant such

as Tween 20 or Triton X 100 is present in the

mixture at a concentration of 0.05 or 0.1 percent.

FIGURE 18. (NEC. P.\-30f> A. Ii>
64 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

FIGURE 19. (NEG. PN-3:o)

Convenient method of applying aqueous

mixtures of regulating chemicals

quantitatively to stems

Apply two narrow lanolin bands about 1 to 2

cm. apart and extending around the stem of the

selected plant. Allow 0.01 ml. of the aqueous

mixture containing the regulating chemical to

flow from a 0.1 ml. pipette so that the measured drop clings to the tip of the pipette. Hold

the stem in a horizontal position and transfer

the drop from the pipette to the stem surface

between the lanolin bands (fig. 19). Revolve

the plant and spread the mixture over the surface between the lanolin bands with the tip of

the pipette. Keep the stem in a horizontal position and revolve the plant at frequent intervals

so that the liquid remains evenly spread over

the surface while it dries. Return the plant to

an upright position and record growth effects

that develop.
SUPPLEMENTARY INFORMATION

Sources of Plant-Regulating Compounds

Eastman Kodak Co.

Chemical Division,

Rochester 4, N. Y.

Compounds mentioned in this compilation

can be obtained fronl chemical supply houses

such as the following :

E. H. Sargent Co.,

4647 West Foster Avenue,

Chicago 30, 111.

Fisher Scientific Co.,

1458 North Lamon Avenue,

Chicago 51, 111.

Will Corporation of Maryland,

5 North Haven Avenue,

Baltimore, Md.

Measures and Equivalents

WEIGHTS LENGTHS

1 kilogram (kg.) =: 1,000 grams (g.) = 2.2 pounds (lb.)

1 gram (g.) = 1,000 milligrams (mg.) = 0.35 ounce

avoirdupois (oz.)

1 milligram (mg.) =1,000 micrograms (Y or gamma)

1 pound (lb.) =: 16 ounces avoirdupois = 453.6 grams

or 0.45 kilogram

1 ounce avoirdupois (oz.) — 28.35 grams

1 pint (pt.) of water weighs approximately 1 pound

1 gallon (gal.) of water weighs approximately 8.34

pounds

VOLUMES

1 liter (1.) = 1,000 milliliters (ml.) =: 1.057 fluid

quarts (qt.)

1 milliliter (ml.) or cubic centimeter (cc.) == 0.034

fluid ounce

1 milliliter or cubic centimeter of water weighs 1 gram

1 liter of water weighs 1 kilogram

1 gallon (gal.) = 4 quarts = 3.785 liters

1 quart (qt.) =2 pints = 0.946 liter

1 pint (pt.) =r 16 fluid ounces = 0.473 liter

1 fluid ounce (oz.) zr 29.6 milliliters or cubic centimeters

1 gallon (gal.)

1 quart (qt.)

spoons

1 pint (pt.) -

spoons

1 cup = 8 fluid ounces

spoons

1 fluid ounce (oz.)

1 tablespoon (tbsp.)

=: 16 cups = 128 fluid ounces

4 cups = 32 fluid ounces = 64 table2 cups = 16 fluid ounces = 32 table16 tablespoons == 48 tea2 tablespoons = 6 teaspoons

3 teaspoons (tsp.)

While li IS impracticable K> provide a complete list

«of dealers, this partial list is furnished for your information, with the understanding that no discrimination

is intended and no w*uaraDtee of reliability implied.

1 mile (mi.) = 5,280 feet = 1.609 kilometers

1 rod (rd.) ~ 16.5 feet =: 5.029 meters

1 yard (yd.) =3 feet = 0.914 meter

1 foot (ft.) ~ 12 inches =0.305 meter

1 inch (in.) r= 2.540 centimeters

1 kilometer (km.) = 1,000 meters = 0.621 mile

1 meter (m.) = 100 centimeters = 3.281 feet

1 centimeter (cm.) == 10 millimeters = 0.394 inch

1 millimeter (mm.) r= 0.039 inch = 1,000 microns

1 micron (/tí.) = 1,000 millimicrons = 0.0000394 inch

1 millimicron (m/x.) = 10 Angström units

1 square kilometer (sq. km.) = 1,000,000 square meters

= 0.386 square mile

1 square meter (sq. m.) =10,000 square centimeters =

10.764 square feet

1 square centimeter (sq. cm.) = 100 square millimeters

= 0.155 square inch

1 square millimeter (sq. mm.) = 0.002 square inch

AREAS

1 square mile (sq. mi.) = 640 acres == 2.590 square

kilometers

1 acre (A.) = 160 square rods = 4046.873 square

meters = 43,560 square feet

1 square rod (sq. rd.) = 30.25 square yards = 25.293

square meters

1 square yard (sq. yd.) = 9 square feet = 0.836 square

meter

1 square foot (sq. ft.) = 144 square inches = 0.93

square meter

1 square inch (sq. in.) 6.452 square centimeters

65
66 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Temperature Conversions

Short-cut method of converting from

Centigrade to Fahrenheit degrees

bouD.e the Centigrade reading, subtract 10% of this

value, and add 32.'"^

Conventional method of converting from

Centigrade to Fahrenheit degrees

Multiply the Centigrade reading by nine-fifths and

add 32.

("C. X 9/5) +32== °F.

Conventional method of converting from

Fahrenheit to Centigrade degrees

Subtract 32 from the Fahrenheit reading and multiply

by five-ninths.

("F. —32) X 5/9 = °C.

Methods of Making Nutrient Solutions

If plants are to be grown only for a period of

2 to 3 weeks in nutrient solution, a suitable

nutrient can be prepared by adding only macronutrients to tap water. If the plants are to be

grown for a longer period, however, or if distilled water is used, it is generally necessary to

add micro-nutrients.

Nutrient solution made with tap water

Prepare a i/^-molar stock solution of each of

the following macro-nutrients in separate containers using tap water :

KH2PO4 (68.07 g. per liter)

MgS04 • 7H2O (123.25 g. per liter)

CaCl2 • 6H2O (109.54 g. per liter)

NaNOs (42.50 g. per liter)

To make the nutrient solution, add the macronutrients from the stock solutions in the following amounts to 50 liters of tap water :

156 ml. of V2-i^olar solution of KH2PO4

581 ml. of i/^-molar solution of MgSOi

• 7H2O

306 ml. of i/2-i^olar solution of CaClg

• 6H2O

375 ml. of i^-ïïiolar solution of NaNOs

^ Reprinted by permission from Chemical and Engineering News, Nov. 21, 1949.

Nutrient solution made with distilled water

Prepare a V2-iï^olar stock solution of each of

the following macro-nutrients in separate containers using distilled water :

KH2PO4 (68.07 g. per liter)

MgSO^ • 7H2O (123.25 g. per liter)

CaCl2 • 6H2O (109.54 g. per liter)

NaNOs (42.50 g. per liter)

In preparing the macro-nutrient solution,

add the macro-nutrient from the stock solutions

in the following amounts to 50 liters of distilled

water :

156 ml. of 1/2-molar solution of KH2PO4

581 ml. of 1/2-ïnolar solution of MgS04

• 7H2O

306 ml. of 1/2-nniolar solution of CaCls

• 6H2O

375 ml. of 1/2-inolar solution of NaNOa

Prepare micro-nutrient stock solutions :

A. Add the following micro-nutrients to 2

liters of distilled water:

5.72 g. of H3BO4

3.62 g. of MnCl^

0.44g. ofZnS04 • 7H2O

0.16 g. of CUSO4 • 5H2O

0.18 g. of H2M0O4

Add 1 cc. of the micro-nutrient stock

solution per liter of prepared macronutrient solution.

B. Add 1 ml. of 0.5% ferric citrate per liter

of macro-nutrient solution.
DESCRIPTIVE INDEX OF METHODS

Abscission

Apple petiole test

The rate of abscission of petioles of debladed

leaves on apple water sprouts following treatment with various chemicals is used as an indication of the effectiveness of the compounds

in retarding harvest drop of apples 1

Attached petiole test

The test compound is placed on debladed petioles and a known pressure is then applied to

the petioles each day to determine the length of

time required for the petioles to abscise compared with that of untreated ones 2

Expiant test

Small sections of leaves (expiants) containing

an abscission zone are treated with compounds

and the effect on abscission observed 3

Lanolin paste test

The abscission of apple fruits following

application of a chemical in lanolin paste

around the stem of the individual fruits prior

to the usual harvest date indicates the effectiveness of the chemical in retarding fruit

abscission 5

Absorption and translocation of non-radioactive

regulators

Bean root test

Test plants are arranged with one-half of their

root systems in the test solution and the other

half in tap water ; then the roots that have been

in the test solution are cut off at different time

intervals. The resulting growth modifications

or injurious effects of the chemicals can be observed, and the rapidity of absorption of the

test compound and its translocation (or translocation of a metabolite of it) by the roots can

be determined 6

Bean stem curvature test

The compound to be tested is placed on one leaf

of a plant; then absorption and translocation

of the compound (or of a metabolite of it) are

evaluated on the basis of stem curvature that

develops. This test is applicable only to compounds that induce or retard cell elongation 7

Angle and elongation of branches

Lanolin test

A piece of flexible tubing is filled with paste

containing the test chemical, slipped onto the

stump of a decapitated branch of a young tree,

and the effects on branch-trunk angle and

growth of the branch are noted ._ 9

Cell division

Cambium test

A knife cut is made through the bark in a ring

around the trunk of young evergreen fruit trees,

and growth-regulating chemicals are applied

to test for inhibition of cambial activity. The

degree of tightness of the bark that develops

subsequent to treatment indicates the amount

of reduction in cambial activity 10

Cell elongation

Bean hypocotyl test

A single droplet containing a known amount

of a growth regulator is placed on one side of

a decapitated bean hypocotyl and after 5 hours

the degree of curvature is measured and compared with curvature induced bv a standard

compound such as 2,4-dichloropheonoxyacetic

acid 11

Epidermal root-cell test

Germinated wheat seeds are grown in a nutrient solution containing the chemical beingtested and its effect on the elongation of epidermal cells is observed with a microscope 12

Pea stem section test

The effect of a compound on stem elongation is

measured by floating sections from etiolated

pea stems on the surface of a solution containing the compound, and measuring the response

in terms of increase in length or weight of the

pieces 13

Split pea stem test

The angle of curvature that develops when

split pea stems are immersed in an aqueous solution of a compound is used to indicate the

influence of the compound on cell elongation 14

Wheat coleoptile section test

The effects of compounds on cell elongation are

measured by floating sections of wheat coleoptiles on aqueous solutions of the chemical and

comparing the length of these sections with the

length of section floated on distilled water 15

Cell growth

Ovary tissue culture test

Excised ovaries of various plants are grown

in vitro on synthetic nutrient medium. Regulating or other chemicals are added and their

effects on growth of the fruits are determined __ 16

Root tissue culture test

Root tips from tomato seedlings or cuttings

cultured in vitro are treated with various regulators or other chemicals, and their effect on

growth of root cells is determined 18

Stem tissue culture test

Squares of phloem tissue from willow are

grown on nutrient a gar containing regulating

chemicals and effects of the chemicals on

growth and tissue development determined 20

Detection of plant-growth regulators in animals

Bean stem test

Regulating chemicals are extracted from animal tissue«?, fluids, secretions or excrement.

The extract is applied to stems of young bean

plants and cell elongation is used to indicate

presence of plant-regulating compounds in the

extract 45

Effect of one compound on the absorption and

translocation of another

Bean stem curvature test

The magnitude of stem curvature induced by a

known amount of a e:rowth-modifying substance is compared with that induced by the

same amount of the substance plus an adjuvant 21

Exudation of regulators from roots

Bean test

Two young plants, one treated by placing a

compound on its stem or leaves, are grown in

soil contained in a pot. Formative or other

effects that become apparent as the untreated

plants develop indicate transfer of the compound from one plant to the other through

the root systems 22

Form or growth modiflcation

Bean leaf test

The growth-regulating chemical in a small

droplet applied to the terminal bud of bean

plants suppresses the vegetative growth of the

young leaves which serves as an index of

growth-suppressing activity 23

67
68 AGRICULTURE HANDBOOK 126, U.S. DEPT. OF AGRICULTURE

Lanolin paste test

Compounds are applied in a paste carrier to

stems of young plants, and the effect on their

subsequent growth and development is observed 24

Underground growth test

Plants are grown in soil in boxes constructed

with glass sides in order to observe the effects

of chemicals on growth and development of

the underground parts without disturbing the

plants 25

Fruit set

Aerosol method

Regulating or other chemicals are applied to

plants or plant parts in the form of mists

produced by use of liquefied gases 26

Grape test

Various compounds may be evaluated as

growth regulators by dipping individual clusters of grape flowers or the immature fruits

in solutions of the compound and recording

improvement in fruit set and size 28

Tomato test

Compounds are applied in liquid or paste carriers to flower clusters of greenhouse plants

and the effectiveness of the substances in preventing flower or fruit drop is evaluated 29

Fruit size

Grape test

Various compounds may be evaluated as

growth regulators by dipping individual clusters of grape flowers or the immature fruits

in solutions of the compound and recording

improvement in fruit size 30

Fruit thinning

Apple blossom test

By applying regulating chemicals to selected

limbs of trees when in blossom, the compounds

are evaluated as a means of thinning the fruit 31

Peach and prune fruit test

A method of evaluating the fruit-thinning

effects of chemicals applied as sprays to limbs

or entire trees is described 32

Growth inhibition or stimulation

Vegetative sprout test

Pieces of carrots, potato tubers, and small

onions are dipped in a solution of the test

compound and placed in darkness. Measurement of sprouts indicates the effectiveness of

the compound as a sprout inhibitor 33

Growth modification (aquatic plant)

Sterile nutrient test

Lemna minor L. (duckweed) is grown under

aseptic and rigidly controlled conditions in a

nutrient solution. Regulating chemicals (including antibiotics) are added to the nutrient,

and their effect on growth of the plants

studied 35

Water culture test

An aquatic plant (Lemna minor L,) is grown in

water containing a regulating compound, and

the growth-modifying effect of the chemical observed 36

Growth regulation

Field test

Test chemicals are sprayed on soil in which

seeds have been planted, or on leaves and stems

of plants growing under field conditions, and

their effect on growth and productivity of the

plants is measured 46

Penetration of soil by regulators

Seed test

Chemicals are applied to the surface of soil

contained in glass tubes. Soil samples are collected as thin layers from various depths and

bioassayed for the presence of the chemical 46

Residue of regulators in soil

Repeated plant test

Several successive crops of plants are grown

in soil treated once with a chemical in order

to determine how long the chemical remains

in the soil in sufficient amounts to affect plant

growth 47

Root growth

Ag*ar slant test

Seeds of rape and oats are germinated on

agar slants containing a test chemical, and

the root growth studied 37

Cone container test

Seeds of barley or other grass plants are dipped

in dilute concentrations of a regulating chemical and germinated in soil held in cone-shaped

containers. Growth of roots and other parts of

the plants is studied in detail by removing the

containers and washing away the soil from

some of the plants at intervals as the plants

mature 38

Impregnated-filter-paper test

Root growth of germinating seeds on filter paper

treated with the test chemicals is observed 39

Nutrient mist test

Trees are grown with their roots in a closed

chamber containing air filled with a fine mist

of nutrient solution in which the chemical

being tested has been dissolved or suspended,

and effects of the regulating chemical on

growth of the roots are observed 40

Root induction

Stem-cutting test

Cuttings are soaked, dusted, or dipped in mixtures containing chemicals, and the effect on

root initiation and growth determined 41

Seed formation

Lanolin paste test

A mixture of lanolin and a regulator is applied

to a wound on the petal base or on the pedicel,

and the effect on seed development is observed 43

Translocation of radioactively tagged regulators

Isotope counter test

Translocation of radioactively tagged molecules is measured by placing a tagged compound on one part of a plant and measuring

radio activity that results from the movement

of the compound, metabolites, or degradation

products of it to other parts of the plant 49

Radioautograph test

Radioactive 2,4-dichlorophenoxyacetic acid is

placed on a leaf of a plant, the plant later

dried and stored adjacent to an X-ray film,

which is then developed. The autographic outline thus obtained is registered with an outline

of the plant on Ozalid paper and the presence

of the radioactivity in different parts of the

plant observed 50

Volatility of plant regulators

Disposable container test

This test is useful for detecting volatility of

plant-regulating compounds that induce easily

detectable growth responses. It can be used to

detect or measure evaporation of regulating

chemicals from surfaces of plants previously

treated with these compounds or from surfaces

of other materials to which these substances

have been applied 51

Germinating seed test

Relative volatility of regulating compounds is

determined by exposing germinating seeds to

vapors of these chemicals in closed containers 52

Water contaminated with regulator

Root-growth test

Small amounts of 2,4-dichlorophenoxyacetic

acid in aqueous solutions are detected by the

inhibiting effect on growth of primary roots of

cucumber 53

^U. S. GOVERNMENT PRINTING OFFICE.- 1958-429834