METHODS OF STUDYING

PLANT HORMONES

and

GROWTH-REGULATING

SUBSTANCES

Agriculture Handbook No. 336

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE
METHODS OF STUDYING

PLANT HORMONES

AND GROWTH-REGULATING SUBSTANCES

By John W. Mitchell,

Research Plant Physiologist,

Crops Research Division

and

George A. Livingston,

Senior Staff Associate,

Institutional Relations,

National Science Foundation

Agriculture Handbook No. 336

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE
ACKNOWLEDGMENTS

The authors greatly appreciate efforts by all who contributed to this

book. The assistance given by the late Paul C. Marth, who was coauthor of

the ñrst edition, is gratefully acknowledged.

While it is impracticable to provide a complete list of dealers offering

chemicals, apparatus, and materials mentioned in this compilation, a few

sources are given to aid the reader in obtaining items that may be difficult

to purchase. Trade names and the names of commercial companies are used

solely to provide specific information. Mention of a trade product or manufacturer does not constitute a guarantee or warranty of the product by the

U.S. Department of Agriculture or an endorsement by the Department over

other products not mentioned.

This handbook supersedes Agriculture Handbook No. 126,

'Test Methods With Plant-Regulating Chemicals"

Extra care should be exercised in experimenting with new chemicals

until it has been determined that they are not harmful to man or

animals. The uses of chemicals mentioned in this book are for experimental purposes. These chemicals are not recommended, on the basis

of these experimental methods, for application to plants to be used as

food.

V^ashington, D.C. Issued February 1968

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

Washington, D.C. 20402 — Price 55 cents
CONTENTS

Page

Introduction , 1

Biological methods 1

Abscission 1

Apple petiole method 1

Attached petiole method 2

Coleus petiole method 4

Expiant method 5

Lanolin paste method 7

Absorption and translocation 8

Agar block polar transport method 8

Bean root method 10

Bean stem curvature method 11

Angle and elongation of branches 13

Lanolin method 13

Cell division or multiplication 13

Endosperm method 13

Microculture method 15

Root culture method 16

Single isolated cell method 18

Soybean callus tissue method 19

Stem tissue culture method 20

Submerged cultivation method 21

Tobacco pith method (micro) 22

Cell elongation 23

Oat coleoptile section method 23

Bean hypocotyl method 25

Bean second internode method 26

Oat first internode method 29

Pea stem section method 30

Radish cotyledon method 31

Split pea stem method 32

Tomato hypocotyl method 34

Wheat coleoptile straight growth method 36

Young wheat coleoptile method 40

Cell enlargement 41

Citrus petal method 41

Leaf disk enlargement method 42

Chlorophyll retention 43

Barley leaf method 43

Cocklebur leaf disk method 44

Dock leaf disk method 45

Wheat leaf senescence method 46

Effect of one compound on the absorption and translocation of another 47

Bean stem curvature method 47

Enzyme production 48

Barley endosperm method 48

Exudation of regulators from roots 49

Bean method 49

Root exudation method 52

Flowering 54

Biennial or perennial plant method 54

Form or growth modification 55

Bean leaf method 55

Lanolin paste method 56

Underground growth method 57

Fruit growth 58

Ovary culture method 58

Fruit set 60

Aerosol method 60

Grape method 62

Tomato method 62

Fruit size 64

Grape method 64

Fruit thinning 64

Apple method 64

Peach and prune method 65

Page

Biological methods—Continued

Growth modification 66

Sterile nutrient method 66

Water culture method 67

Field method 68

Growth retardation 68

Vegetative sprout method 68

Wheat seedling method 69

Root growth 72

Agar slant method 72

Cone container method 73

Impregnated filter paper method 74

Nutrient mist method 75

Root induction 76

Rooting cofactor method 76

Root primordia method 78

Stem-cutting method 79

Seed formation 81

Lanolin paste method 81

Detection of exogenous growth regulators in plants— 82

Graft method 82

Detection of exogenous plant growth regulators in

animals 82

Bean stem method 82

Detection of regulators in water 84

Root growth method 84

Penetration of soil by regulators 84

Seed method- 84

Residue of regulators in soil 86

Successive crop method 86

Some physical methods 87

Introduction 87

Radioautographs of entire plants 87

Radioautograph method 87

Translocation of radioactively tagged compounds

(See also ''Absorption and translocation/' p. 8.)-- 89

Ground tissue method 89

Volatility of plant regulators 90

Disposable container method 90

Germinating seed method 91

Activated charcoal method 92

References to other physical methods 93

Chromatography 94

Supplementary techniques 94

Treating plants 94

Absorption cell for introducing compounds into

limited portions of roots 94

Absorption through a leaf flap 95

Agar block preparation and use 96

Application of aqueous mixtures quantitatively to

stems 97

Application of a measured amount of regulating

chemical to a known area of leaf surface 98

Application of regulators as a part of a coating on

seeds 98

Application of growth substances with capillary

tubes 99

Easy method of impregnating dust carriers with

regulating chemicals 100

Convenient method of dispersing relatively insoluble organic compounds in water 100

Segment cutters 101

Device that delivers measured amounts of regulating chemical 102

Direct measurement of volatility 102

Fractionated lanolin as a carrier for growth-regulating compounds 104

m
Page

Supplementary techniques—Continued

Treating plants—Continued

Injecting a regulating substance or hormone into a single, living cell 104

Injecting substances into trees 105

Introduction of chemicals directly into xylem elements in stems of herbaceous plants 105

Lanolin as a carrier for regulating chemicals 106

Movement of regulating substances through plant

membranes or tissues 106

Plastic culture vessel (^'plasticon") for small plants. 107

Simulated spray-droplets 109

Technique for studying absorption and translocation

of regulating substances by aquatic plants 109

Use of dwarf corn to detect and identify gibberellins

and gibberellin-like substances 111

(îrowing plants 112

Highly sensitive test plants 112

References to methods of m.iintaining constant

relative humidity 113

Microisolation device 113

Suggestions concerning the production of greenhouse

plants for experiments with regulating chemicals _ 114

Storage of seeds used as a plant source for the study

of hormones and regulators...^ 1 116

Observing and measuring plants 117

Aphid honeydew technique for study of sieve tube

content _' 117

Aphid stylet technique for studying regulating

substances in sieve tubes 118

Extraction of hormones and growth-regulating

substances from plants 118

iv

Page

Supplementary techniques—Continued

Observing and measuring plants—Continued

Formative effects of regulating chemicals 119

Interferometer method of detecting minute growth

movements 120

Freehand sections for studying cell responses to

regulators 120

Measurement of root response 121

Radioautographs of sectioned plant material 121

Separation of leaf areas treated with a regulating

chemical from the remaining untreated portions

of the leaf 123

Slide chamber for the study of living cells 123

Terminal growth measurements 124

Observation chamber for continuous applicati(m of

hormones or growth regulators to living cells 124

Motion detector for recording plant growth 124

Supplementary information 128

Measures, equivalents, and abbreviations 128

Temperature conversions 128

Short-cut method of converting from Centigrade to

Fahrenheit degrees 128

Conventional method of converting from Centigrade

to Fahrenheit degrees 128

Conventional method of C(mverting from Fahrenheit

to Centigrade degrees 128

Met hods of making nutrient solut ions 129

Nutrient solution made with tapwater 129

Nutrient solution made with distilled water 129

Potassium phosphate buffer mixtures 129

Descriptive index 131
METHODS OF STUDYING PLANT HORMONES

AND GROWTH-REGULATING SUBSTANCES

INTRODUCTION

The purposes of this compilation are, first, to

provide scientists with ready access to methods

useful in the study of plant hormones and

growth-regulating substances, and, second, as

our knowledge in this field advances, to stimulate scientists to devise new methods that will be

necessary to understand more fully how hormones and regulators control the behavior of

plants.

Information in this handbook is presented in

three sections: biological methods that are essential in the study of hormones, some very

useful physical methods, and many supplementary techniques that facilitate research in this

field. A few very useful methods, such as the

well-known oat coleoptile method, have not been

included, since these are widely used and descriptions of them are easily obtained.

Methods in this handbook are described step

by step in the manner of a cookbook, with the

hope that they will be readily understood and

easy to use. Descriptive titles have been assigned to facilitate reference to each method.

The index is arranged so methods can be

found by approaching the handbook from different viewpoints. For example, from a chemical or biochemical viewpoint, methods useful for

detecting gibberellic acid, gibberellin-like compounds, kinetin, or auxin can be readily found.

From the viewpoint of the response induced by

a hormone or regulator, methods useful in detecting substances that cause cell elongation,

abscission, growth retardation, cell division, and

other responses can be located. Methods can be

found readily that involve the use of specific

plant parts or that help accomplish a specific

task, such as injecting a regulator into a living

cell.

Progress in the accumulation of new scientific knowledge depends upon methods with

which the information can be gained. Although

the methods described here are useful and serve

a definite purpose, new methods must be developed continually to make the most rapid

progress in research.

BIOLOGICAL METHODS

ABSCISSION

APPLE PETIOLE METHOD

Based on research by L. J. Edgerton; L. J.

Edgerton and M. B. Hoffman.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Analytical balance

Hand sprayer, 250 ml., or nasal atomizer,

15-30 ml.

25-200 mg. of each compound to be evaluated

500 mg. of alpha-naphthalene-acetic acid or

its sodium salt for standard treatment

Solvent such as 95% ethanol

Wetting agent such as Santomerse, Dreft, or

Tween 20

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 malus L.)—Mclntosh, Winesap, Stayman, or other varieties.

Preparation and selection of plant material—In

midsummer tag apple trees that have produced
AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

an abundance of water sprouts during the current season. New sprout growth can be induced

to develop 1-2 months earlier than usual by

selecting experimental trees before winter

pruning and cutting back the existing water

sprouts, leaving stubs 3-8 cm. long. These stubs

will then produce long, vegetative shoots early

during the next growing season. One- to twoyear-old potted trees may be used during winter and spring in the greenhouse. Select shoots

of uniform diameter, each having 15-25 leaves.

Earlier abscission of the control petioles and

more uniform results with various test compounds are obtained if the shoots are still growing actively at the time the application is made.

Procedure—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 tapwater 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 2-3 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 10-11 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.

Suggested standard for comparison—10, 20, or

30 p.p.m. concentration of alpha-naphthaleneacetic acid or its sodium salt.

References

Edgerton, L. J. A Method for Evaluating the EflPectiveness 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 METHOD

Based on research by J. W. Mitchell, P. B.

Marsh, and Robert Bender.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Constant-pressure apparatus (fig. 1)

Razor blades or sharp knife

Lanolin

1-2 g. endothal acid, monohydrate

1-2 g. phenylmercuric acetate

Analytical balance

Vials

Medical applicator sticks or toothpicks

Tween 20

Compounds to be evaluated as growth regulators

Suggested plant material—Young cotton plants

(Gossypium hirsutuvi L.)—Deltapine variety,

and young bean plants (Phaseolus vulgaris L.)

of the Pinto or other varieties.

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 4 cm. across, 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.

Procedure—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 mixture 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
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES

PN-292

FIGURE 1.—Pressure applicator used to determine whether chemical will make leaves fall from plant by applying

measured amount of pressure to both treated and untreated petioles.

equal portions of the pastes containing progressively smaller amounts of the test compound to debladed petioles in corresponding

rows in a similar manner. Apply an equal

amount of the lanolin-Tween 20 mixture without a chemical added to one more row of petioles

and designate these as treated controls.

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.

For an alternate method, use young bean

plants in the same manner except remove one

primary leaf blade of each plant, leaving the

petiole attached. Select plants with primary

leaves that are approximately 3-5 cm. wide, and

with the first trifoliolate leaf just beginning to

unfold from the terminal bud.

Suggested standard for comparison—Endothal

acid monohydrate, phenylmercuric acetate, or
AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

naphthyl maleimide for cotton plants; endothal

acid monohydrate or phenylmercuric acetate for

bean plants.

Reference

Mitchell, J. W., Marsh, P. B., and Bender, Robert. [Unpublished data.]

COLEUS PETIOLE METHOD

Based on research by L. C. Luckwill.

Description of method—Small filter paper disks

impregnated with the compound being tested

are applied to the cut ends of petioles of excised

nodes of coleus. Delay in abscission due to the

presence of the chemical is measured by comparing the time required for treated and untreated petioles to abscise. Within limits, this

delay in abscission is proportional to the

logarithm of the quantity of growth substance

applied. The method is believed to be specific

for auxins, with the possible exception of

kmetin, and it is particularly convenient for

the detection of auxins on paper chromatograms. Under some conditions this method can

be used to detect growth-accelerating substances

even in the presence of inhibitors that would, in

other methods, mask the accelerating effects.

Apparatus, Chemicals, and Other Materials

Analytical balance

Razor blade

Filter paper

Sheets of glass

Dishes larger than the sheets of glass

Incubator maintained at 25° C.

Needle attached to wooden handle

Disks of filter paper, Whatman No. 1, 0.5 cm.

in diameter, made with a paper punch

Straight pins

Modeling clay

Pipette, 0.1 ml., graduated in 0.01-ml. divisions

Microsyringe

Test tubes

Corks to fit test tubes, or Parafilm

50 mg. of compound to be tested

0.25 g. of alpha-naphthaleneacetic or indole3-acetic acid for standard treatment

Distilled water

Suggested plant material—Coleus {Coleus hlumei Benth.)—green-leaved variety Corunna;

or other ornamental forms.

Preparation and selection of plant material—

Grow the plants with a moderate or low light

intensity and short days so that long internodes

are developed. Avoid excessive nitrogen fertilization since this delays petiole abscission. Use

controlled growth conditions to obtain the most

uniform experimental plants. Allow the plants

to grow until shoots about 30 cm. long with 8

pairs of expanded leaves can be obtained.

Procedure—With a razor blade, cut the required number of vegetative shoots, each about

30 cm. long. Use only 4-7 pairs of leaves

(counting from the apex) for the experiments.

Excise the 4-7 nodes with attached leaves by

severing the stem 1 cm. above and below each

node (fig. 2, A). Remove the laminae and axillary buds. Cut each petiole in a plane parallel

to and at a point 4 mm. from the stem (fig. 2,

B). Split the stem piece vertically (fig. 2, C)\

Place the 2 half-stems with their attached

petiolar stubs on moist filter paper (fig. 2, D)

supported on glass in a dish. Impale one of the

disks of filter paper on the point of a pin and

support the pin in an inverted position by pressing the head into a block of modeling clay. Impregnate the disk with the compound to be

tested by adding to each disk a measured drop

of alcoholic solution delivered from a microsyringe. The alcohol should contain a measured

amount of the compound being tested. Dry the

disks, place them in corked tubes, and store

them m a cool, dark place. Just before applying

the disks to the petiole, dip them momentarily,

and one at a time into distilled water; or support them on a pin and add to each one slightly

more than the amount of water required to

saturate the disk, using a 0.1-ml. pipette graduated m 0.01-ml. divisions. Immediately place the

disks m contact with the freshly cut surface of

FIGURE 2 —Preparing coleus nodes to determine eflFect

A ^^^^}^^^^^ substances on abscission. Steps include:

A, Node with attached leaves; B, axillary buds removed petioles cut to 4 mm.; C, stem piece split

vertically; D, filter paper disk containing auxin or

plant extract applied to cut surface of petiolar stump

(Diagram—courtesy of Journal of Horticultural Sci- ence.)
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES

the petiolar stump (fig. 2 D). After the required

number of petioles have been treated, cover the

dish with a sheet of glass with a moist sheet of

filter paper on the underside to maintain relatively high humidity within the chamber. Store

the dish containing the expiants in darkness at

25° C.

This method is particularly well adapted for

detecting and measuring auxins on paper

chromatograms. To utilize the test in this way,

use a paper punch to punch out disks 0.5 cm. in

diameter along the length of the chromatogram

and record the Rf position of each disk. Moisten

the disks individually with the required amount

of distilled water using pins and the method

described above. Immediately place each disk

separately on one of the coleus petioles, prepared as described above. If a single chromatogram contains insufiScient auxin to give a response, place 2 or more disks, punched from

comparable positions on an adjacent chromatogram, on a single petiole.

Method of taking results—Record daily the

number of petioles that abscise after pressing

each petiole lightly with a needle or apply a

uniform pressure to determine whether abscission has occurred. (See ^'Attached petiole

method,'^ p. 2.)

Suggested standard for comparison—Alphanaphthaleneacetic acid, 0.1-1.0 microgram per

petiole.

Reference

Luckwill, L. C. Two Methods for the Bioassay of Auxins

in the Presence of Growth Inhibitors. Jour. Hort. Sei.

(London) 31: 89-98. 1956.

EXPLANT METHOD

Based on research by F. T. Addicott, R. S.

Lynch, G. A. Livingston, and J. K. Hunter;

variations by F. T. Addicott, R. S. Lynch, G. A.

Livmgston, and J. K. Hunter; and by F. T.

Addicott, H. R. Cams, J. L. Lyon, 0. E. Smith,

and J. L. McMeans.

Description of method—Small segments of

leaves (expiants) containing an abscission zone

are treated with compounds, and the effect on

abscission is observed.

Apparatus, Chemicals, and Other Materials

Scalpel

Forceps

Filter paper

Petri dishes

Dark chamber maintained at about 25° C.

Analytical 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. indole-3-acetic

acid

Agar

Suggested plant material—Citrus spp. leaves or

leaves of other kinds of plants that have abscission zones near a leaf blade.

Preparation and selection of plant material—Excise for use as test material segments of leaves

containing the abscission zone and the adjacent

tissues (ñg. 3). These segments are called explants. 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 preparing the expiants, utilize

leaves that have attained their full size and hard

texture but still are dark green in color.

Procedure—Prepare 4% agar in water, pour

it into petri dishes, and allow it to cool and set.

The agar in the dish should be about 6 mm.

deep. After the agar has set, remove a strip of

it, about 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 (fig. 3).

Prepare 10-25 ml. of each concentration of

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 30-60 seconds. Remove

the treated expiants from the solution and

mount them on the agar of a petri dish so 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 applying 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 25° C.

except for the short daily period of examination.

To use a somewhat different method of supporting the expiants, construct a holder by

supporting a Pyrex glass rod horizontally on a
6 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

PN-29S

FIGURE 3.—Test used to study physiology of abscission: Treated and untreated segments of leaves, each including an

abscission zone, are placed on agar in petri dishes and the late of abscission is observed.

curved foot at each end of the rod. Add a row

of fine glass pins projecting from each side of

the rod (fig. 4). Place the holder in a petri dish

and pour 2 % agar around the feet of the holder

to prevent displacement and to maintain a high

relative humidity within the closed petri dish.

Mount the expiants on the holder by forcing the

cut end of each expiant over one of the glass

pins.

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 that

each expiant exhibits at each time of observation.

If the objective of any test or series of tests

is to determine the effectiveness of compounds

in producing the abscission of organs, the only

fully reliable tests are those conducted with

whole plants. Furthermore, if the compounds

are being tested for potential field use, testing

under a variety of field environmental conditions is essential.

Suggested standard for comparison—To initiate

or accelerate abscission, place petri dishes containing the expiants inside an airtight container

and add ethylene to make a concentration of

20-50 p.p.m. To delay abscission, use 0.001-100

p.p.m. of indole-3-acetic acid (30-60 seconds

dip) as a standard.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES

I CM.

FIGURE 4.—Pyrex glass expiant holder, 2 views. (Photo

—courtesy of Plant Physiology.)

Variation used by Addicott, Cams, Lyon, Smith,

and McMeans—Another variation, involving the

use of young cotton plants, is shown diagrammatically in fig. 5. Using strips of sheet stainless steel, form legs designed to raise the main

part of the strip above the bottom of the petri

dish. Drill 2 rows of 5 holes each in the horizontal part of the strip. Place the stainless steel

expiant holder in the dish. Excise cotyledonary

nodes from cotton seedlings 2 or more weeks

old. Make expiants consisting of two 3-mm.

petiole stumps with a 3-mm. stem stump and

10 mm. of hypocotyl attached. Place these in

the stainless steel holders in the petri dishes.

Apply the test chemical mixed in 1% agar as

5-microliter droplets to the cut surface of the

petioles. During the next 4 days apply daily a

known pressure to each petiole stump using an

abscissor described by Addicott, Lynch, Livingston, and Hunter (paper cited below) ; or

apply a known pressure using a pressure applicator. (See ''Attached petiole method,'' p.

2.) Record the number of petioles that abscise.

References

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

Hunter, J. K. A Method for the Study of Foliar Abscission In Vitro. Plant Physiol. 24: 537-539. 1949.

Addicott, F. T., Garns, H. R., Lyon, J. L., Smith, 0. E.,

and McMeans, J. L. On the Physiology of Abscisins.

Régulateurs Naturels de la Croissance Végétale 123:

687-703. 1963.

LANOLIN PASTE METHOD

Based on research by P. C. Marth, W. H.

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

Description of method—The abscission of apple

fruits following application of a chemical in

lanolin paste around the stem of the individual

fruits before 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.

EXPIANTS

IN

HOLDER

TEST FOR

ABSCISSION

FIGURE 5.—Variation of expiant method using cotton

plants to investigate abscission. (Diagram—courtesy

of Régulateurs Naturels de la Croissance Végétale.)
AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Apparatus, Chemicals, and Other Materials

Analytical balance

Small shell vials with stoppers

Wooden board about 15 cm. wide X 30-60

cm. long X 3 cm. thick having 1-cm. diameter holes drilled 2 cm. deep and 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

oughly until it reaches room temperature and

becomes semisolid. To prepare the 0.1% mixture, weigh 0.2 g. of the 1% mixture previously

prepared and mix this with 1.8 g. of the lanolinTween 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 that are left as controls.

Suggested plant material—Apple trees (Malus

sylvestris Mill., also called Fyrus mains L.)

bearing an abundance of fruit readily accessible

from the ground—Grimes, Mclntosh, York, or

other varieties.

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 before the estimated, or usual, harvest date for the variety.

Procedure—Prepare lanolin-Tween 20 mixtures containing 0.1 and 1^/c concentrations of

the respective compounds. To prepare the IÇf

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 thorMethod 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 fruit remaining attached serves as a

measure for evaluating the effect of individual

compounds on fruit abscission.

Suggested standard for comparison—Lanolin

paste mixture containing either 0.1 or 1%

alpha-naphthaleneacetic acid.

Reference

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

AGAR BLOCK POLAR TRANSPORT METHOD

Based on research by J. B. Zaerr. (Original

method by F. W. Went and H. G. van der Weij.)

Description of method—A segment of stem,

branch, or petiole is placed either upright or in

an inverted position on a receiver block of agar.

A donor block of agar containing a known

amount of radioactively tagged regulator is

then placed on the upper end of the segment.

After an appropriate period of time, the amount

of radioactivity translocated from the donor to

the receiver block is determined by extracting

the receiver block and measuring the amount of

radioactivity obtained. Identity of the translocated material is established chromatographically. Polar transport is measured by comparing the amount of growth regulator translocated

from the donor to the receiver block when the

segment is upright with respect to its usual

position of growth and when it is inverted with

respect to this position.

Apparatus, Chemicals, and Other Materials

Clay pots, 7.5 cm.

Composted soil

Common cutter that can be adjusted to cut
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 9

5-mm. and 2-mm. segments (See ''Segment

cutters," p. 101.)

Forceps for handlmg segments

Glass microscope slides

Spatula for manipulating agar blocks

Petri dishes

Glass tube, 15-30 cm. long, 5 mm. inside

diameter

Solid glass rod, about 30 cm. long, 4 mm.

diameter

Filter paper

Aluminum foil

Paper chromatography equipment

Constant temperature chamber—24°-27° C.

X-ray (no-screen type) film for autoradiographs of chromatographed extracts

Glass vials for extractmg agar blocks, 1-2 ml.

capacity

Concentric ring planchets for measuring

radioactivity

Disposable pipettes for handling agar extracts

Gas flow counter

Sealer

Analytical balance

Radioactively tagged compounds to be evaluated

95% ethanol

Isopropanol, ammonia, or other solvents suitable for chromatographing the growth regulators involved

Agar (ionagar)

Indole-3-acetic-l-C^^ acid for standard treatment

Suggested plant material—Segments of petioles, internodes, or hypocotyls obtained from

young succulent plants, such as bean (Phaseolus vulgaris L.), cucumber (Cucumis sativus

L.), and tomato (Lycopersicon esculentum

Mill.).

Preparation and selection of plant material—

Using bean stem segments as an example of test

plant material, sow 3-5 seeds in each of 36 clay

pots containing composted soil. Moisten the soil

and germinate the seeds. When the primary

leaves are still wrinkled and the terminal bud is

2-3 mm. long, select a group of plants with

straight hypocotyls of uniform size and stage

of development. Cut 5-mm. segments, one from

each hypocotyl, beginning 5 mm. below the

cotyledons. Use the segments immediately.

Procedure—Make 1 % agar by mixing together

and bringing to a boil 0.1 g. of the agar and

10 ml. of water. Pipette this hot agar into the

long glass tube. Hold a finger over the open

lower end to keep the agar within the tube.

When the tube is full, place another finger over

the upper end of the tube and hold the entire

tube under a stream of cold running water.

When the agar has solidified (1-2 minutes),

tap the tube endwise gently on a hard surface

to loosen the agar from the inside wall. Force

the agar from tñe tube with the solid glass rod.

Repeat this process until sufficient molded agar

IS obtained. Cut tñe agar rods into cylindrical

blocks 2 mm. long using the cutter. (See ''Segment cutters,'' p. 101.) Place agar blocks on a

glass slide inside a petri dish lined with moist

filter paper. Store at 5° C. until used. To prepare donor blocks, soak plain agar blocks overnight in an aqueous solution of the growth

regulator to be tested. Remove these blocks from

the solution and store as described above.

Place a receiver agar block (plain block) on

a glass microscope slide. Cut a 5-mm. segment from the plant and place this segment

upright (lower cut surface down) on the receiver block. Place a donor block (containing

the growth regulator) on the upper cut surface

(fig. 6). Repeat this procedure, inverting the

plant segment (apical end resting on the receiver block and basal end supporting the donor

block). Replicate both positions as many times

as desired. Place the glass slides in petri dishes

lined with moist filter paper, wrap the dishes in

aluminum foil, and store at a constant temperature of about 25° C. for the desired transport

period (the time interval during which segments are in contact with the donor and receiver

blocks), which varies from several hours to

several days, depending upon the growth regulator involved.

Method of taking results—At the end of the

transport period, remove and discard the donor

blocks and plant segments. Extract each receiver block separately, using 0.3 ml. of distilled water. Soak each block at least 1/2 hr. for

each extraction and extract 4 times, using fresh

water each time. Combine the extracts from

each block, place each combined extract on a

concentric ring planchet, and evaporate to dryness. Measure the radioactivity present on each

planchet with a gas-flow counter. Compare the

amount of radioactivity that passed through upright segments with the quantity that passed

through inverted segments. Compare the substance collected in the receiver blocks with the

substance initially contained in the donor blocks

by means of autoradiographs of chromatographed extracts from donor and receiver

blocks.

Suggested standard for comparison—Indole-3-

acetic-l-C^^ acid, specific activity about 30 microcuries per micromole, concentration 1 mg./

liter, transport period 8 hr.
10 AGRICULTURE HANDBOOK 336, U.S. 1>EPARTMENT OF AGRICULTURE

FIGURE 6.—Hypocotyl segment used to measure polar

transport showing agar blocks in place: Upper, donor

block containing regulator; lower, receiver block with

no regulator.

Reference

Method submitted by J. B. Zaerr, Forest Research Laboratory, Oregon State University, Corvallis. See also

"Agar block preparation and use," p. 96.

ture, hydrogen ion concentration, mineral nutrition, and oxygen supply) on the ability of roots

to absorb and translocate regulating chemicals.

Apparatus, Chemicals, and Other Materials

Clay pots, 8-15 cm.

Composted soil

Glass containers, 200-500 ml.

Supports to hold a plant with one-half of its

root system in one container, the other half

in another container

Analytical balance

Compressed air for aerating nutrient solutions

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

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 :

124 g. of MgSOi • 7 H2O

83 g. of CaCl2 • 6 H2O

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 partly

expanded trifoliolate 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 compressed

air, and allow them to grow 3-5 days to condition them for the test. For the nutrient solution, use stock solutions of MgS04, CaCb,

KH2PO4, and Ca(N0,i)2 made by dissolving the

quantities listed above, each in a separate liter

of tapwater. To 500 ml. of tapwater add 9 ml.

each of the MgS04, CaCk., and KH2PO4 stock

solutions, and 18 ml. of the Ca(N03)2 stock

solution. Add sufficient water to make 1 liter

of nutrient solution.

BEAN ROOT METHOD

Based on research by T. J. Muzik.

Description of method—Test plants are ai"-

ranged with one-half of their root systems in

the test solution and the other half in tapwater ;

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 used to study the effects of environmental factors (light, temperaSuggested plant material—Plants, such as beans

(Phaseolus vulgaris L.), with root systems that

can be arranged in two portions of about the

same size.

Procedure—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 tapwater. For the control plants fill both containers with tapwater.

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
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 11

root system is immersed in the test solution and

one-half in tapwater. Arrange control plants

with each half of the root system growmg in

tapwater. In this manner, use 6-12 plants for

each test solution and an equal number of plants

with their roots in tapwater 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 tapwater. Also cut one-half of the roots

from each of one-third of the control plants.

After another 30 minutes repeat this 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 tapwater 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

that have had their roots removed at different

intervals.

Suggested standard for comparison—25-200

p.p.m. of 3-(para-chlorophenyl)-l,l-dimethylurea.

Reference

Unpublished method submitted by T. J. Muzik, Department of Agronomy, Agricultural Experiment Station,

Washington State University, Pullman.

BEAN STEM CURVATURE METHOD

Based on research by A. S. Crafts ; B. E. Day ;

J. W. Mitchell, W. M. Dugger, Jr., and H. G.

Gauch.

Description of method—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 can 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.

Apparatus, Chemicals, and Other Materials

Clay pots, 8-15 cm.

Composted soil

Greenhouse space maintained at about 24°-

29° C.

Analytical balance

Several 0.1-ml. pipettes graduated in 0.01-ml.

divisions

Beakers for preparing solutions, 250 ml.

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

Suggested plant material—Young bean plants

(Phaseolus vulgaris L.)—Black Valentine, Red

Kidney, Pinto, or other varieties that germinate

and grow uniformly.

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 trifoliolate 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 and

are no longer wrinkled, usually about 48 hr.

after the first selection, reselect the plants for

size and uniformity, and place them in rows

of 8-10 plants each. Water the soil surrounding

the roots of each plant before conducting the

test.
12 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Procedure—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 0.02 ml. of the final mixture will

contain 1-5 jug. of the chemical and sufficient

Tween 20 to make a final concentration of 0.19f

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. 7,

left). 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. 7, right). 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 (8-10 per treatment) to grow in a

greenhouse, or place them in the fluorescent

light so they receive illumination of at least

700-foot-candle intensity.

Method of taking results—Observe the plants at

30-minute or hourly intervals. Measure the

angle of stem curvature 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.

Suggested standard for comparison—1.0 /xg. of

2,4-dichlorophenoxyacetic acid on one primary

leaf of each of 8-10 plants.

References

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-

Dichloi-ophenoxyacetic Acid by Bean Plants. Plant

Physiol. 27: 143-152. 1952.

Mitchell, J. W., Dugger, W. M., Jr., and Gauch, H. G.

Increased Translocation of Plant Growth-Modifying

Substances Due to Application of Boron. Science 118:

354-355. 1953.

PN-294, A and B

FiGiiRE 7.—Applying minute, measured amount of growth-regulating chemicals to leaves: Left, applying with a

pipette a measured volume of mixture as a band across the upper surface of young leaf ; right, spreading mixture

evenly over upper surface of leaf with a glass rod applicator.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 13

ANGLE AND ELONGATION OF BRANCHES

LANOLIN METHOD

Based on research by A. P. Preston and H. W.

B. Barlow; Leif Verner.

Description of method—A piece of flexible tubing is half filled with paste containing the test

chemical and slipped onto the stump of a decapitated branch of a young tree. The effects on

branch-trunk angle and growth of the branch

are noted.

Apparatus, Chemicals, and Other Materials

Analytical balance

About 1 m. of 6-mm. flexible rubber tubing or

sheets of thin, transparent celluloid

Protractor

Vials

1 g. of each compound to be evaluated

About 2 g. of 3-indolebutyric acid for standard treatment

Lanolin

Suggested plant material—Young trees in whip

stage (no lateral branches) such as apple

{Malus sylvestris Mill., also called Pyrus malus

L.), pear {Pyrus communis L.), plum {Prunus

spp.), willow {Salix spp.).

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.

Procedure—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 10-20 cm. long. Cut

off a 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 tubing

containing the paste over the decapitated shoot

for a distance of about 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.

Ik an alternate method, use flexible sheet

plastic or similar material 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,

l^elt the lanolin mixture and put about 5 ml. of

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

vial over the open end of the tube to protect the

lanolin, leaving uncovered the buds immediately below the point of treatment.

Method of taking results—After the lateral

shoots on the controls are 15-20 cm. long, 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 lanolin

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.

Suggested standard for comparison—0.1% lanolin mixture of 3-indolebutyric acid.

References

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

Substances to Widen Crotch Angles. East Mailing Res.

Sta. Ann. Rpt. 1950: 76-79. 1951.

Verner, Leif. Effect of a Plant Growth Substance on

Crotch Angles in Young Apple Trees. Amer. Soc.

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

Verner, Leif. Unpublished method. University of Idaho,

Moscow.

CELL DIVISION OR MULTIPLICATION

ENDOSPERM METHOD

Based on research by W. T. Jackson, G. P.

Pollitt, and H. Sato.

Description of method—Endosperm cells from

immature seeds are spread on an agar surface

containing glucose and the desired concentration of the plant growth regulator. Division in

these cells following treatment is observed by

phase contrast and polarization microscopy. Abnormalities of mitosis, as recorded by means of

photography and a rating system, indicate the

effect of the test compound on cell division.
14 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Apparatus, Chemicals, and Other Materials

High humidity room (85-90%) at 20°-25°

C. for preparation of chambers

Microscope slides, 50 X 75 mm.

Cover glasses, 24 X 40 mm., No. 11/2

Razor blades

Petri dishes

Flasks

Alcohol lamp

Spatulas

Forceps

Hot plates

Filter paper

Glassware washing facilities

Analytical balance

Phase contrast microscope

Rectified polarizing microscope

35-mm. camera

16-mm. time-lapse movie camera with adapters for photomicrography

{Note: Effective heat absorbers must be

inserted between illuminators and microscopes.)

Monochromatic green light used for illumination of specimen

Tape recorder to record comments of investigator during period of observation

1-1,000 p.p.m. of growth-regulating chemical

to be tested. (20 p.p.m. of colchicine gives

''typicar' C-mitosis; 0.1 p.p.m. of 3-chloroisopropyl N-phenylcarbamate is effective)

Petroleum jelly-lanolin-paraffin sealing compound

Glucose

Glass-distilled water

Suggested plant material—Liquid endosperm

from a variety of plants (including important

crop plants) is suitable. However, the African

blood lily {Haemanthus katherinae Baker), a

member of the Amaryllidaceae family, has thus

far proved the most useful.

Preparation and selection of plant material—Mature bulbs of the African blood lily usually

flower during the late spring or early summer.

The immature fruits are at the proper stage for

study during late summer. Select fruits that

contain seeds about 6 mm. long. Such seeds

possess a liquid endosperm. The cells suspended

in the endosperm fluid do not possess a cell wall,

\^ill flatten within 30 minutes when placed on

an agar surface, and will complete mitosis

within 2-4 hr. Each plant, if fruit set is good,

will provide nearly 100 fruits suitable for study

over a 3-wk. period. Three to 6 years is required

to produce a mature bulb from seed or an offshoot, but mature bulbs can be obtained commercially in limited quantities.

Procedure—Select a fruit of proper size, remove the seed, and measure it. If it is 5.5-6.5

mm. long, it will contam several hundred cells

in the endosperm. Many of these will be undergoing mitosis. Carry out all operations involved

m preparmg the material for microscopic examination m a room maintained at 85-90%

relative humidity and 20°-25° C. Spread evenly

a thin film of liquid 0.5% agar containing 3.5%

glucose plus the desired concentration of growth

regulator in glass-distilled water on the cover

glass. Store this cover glass immediately in a

moist chamber. Coat one of the microscope

slides in the center with a thin layer of the

agar solution to match the size of the cover

glass. Make a filter paper ''gasket'' by cutting

the center out of a piece of 24 X 40 mm. filter

paper leaving a border 1-2 mm. wide. Place the

filter paper ''gasket'' over the coated area of the

slide. Wet the filter paper with the agar solution. Store the microscope slide in a moist chamber (petri dish lined with moist filter paper).

Cut off the end of the seed. Blot off the first

drop of liquid (containing few cells) and gently

squeeze the remaining liquid onto the agar on

the cover glass. Quickly invert the cover glass

and place it on the filter paper "gasket" on the

microscope slide. Melt a small amount of 1:1:1

petroleum jelly-lanolin-paraffin sealing compound on a spatula over an alcohol lamp. Working rapidly, apply the melted sealing compound

to the edge of the raised cover glass, thus sealing

it to the microscope slide. Invert the chamber

formed by the slide and cover glass and place it

so it is slightly inclined for 15 minutes. The excess liquid drains into the filter paper and the

remaining cells flatten to the agar-covered surface of the cover glass due to surface tension.

Examine the cells microscopically.

Method of taking results—It is convenient to

divide mitosis arbitrarily into 18 stages or

events. Select no more than 12 fields of view for

study choosing "typical" cells undergoing mitosis. Have an assistant read the stage coordinates, keep track of time for readings, and

operate a tape recorder while the investigator

gives data verbally concerning the stage of

mitosis for each cell. Record by photography or

by a rating system, or by both systems, at 10-

minute intervals for 4 hr. the progress of mitosis in the cells in each field. Repeat this procedure 5 or 6 times for each concentration.

Make time-lapse 16-mm. movies of individual

cells to record detailed eflfects of the plant

growth regulator that may be missed by visual

examination or with still-photography techniques. For example, details of chromosome

movement can be analyzed with time-lapse

photography.
METHODS OF STUDYINCÍ PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 15

Suggested standard for comparison—Compare

behavior of cells during mitosis with behavior

of untreated cells and cells treated with 20

p.p.m. of colchicine.

Reference

Jackson, W. T., Pollitt, G. P., and Sato, H. Use of Endosperm of the African Blood Lily {HaemcuiUim katlierinae BJ for Studying Mitosis in Living Cells in Both

Teaching and Research. (Abstract) Plant Physiol. 39:

Sup. Ixiii. 1964.

MICROCULTURE METHOD

Based on research by L. E. Jones, A. C. Hildebrandt, A. J. Riker, and J. H. Wu.

Description of method—A microchamber is used

to grow cells aseptically in a droplet of medium

that contains a measured amount of the test

compound. The rate of growth and other characteristics of the treated cells are studied microscopically and compared w^ith those of comparable cells grown in the medium without the test

compound. The method is suggested as a means

of detecting and measuring substances that influence cell division other than those regulating

compounds used as nutrients in this method.

Apparatus, Chemicals, and Other Materials

Prescription bottles, 6 oz.

Reciprocal shaker (60 cycles/minute)

Analytical balance

Microchambers (See ''Procedure" for construction.)

Standard microscope slides

Cover slips, 22-mm. square, No. 1

Paraffin oil (U.S.P. heavy paraffin oil)

Honed or flattened teasing needles

Pasteur pipettes

Dissecting microscope

Reagents needed for the preparation of nutrient solution (See ''Preparation and selection of plant material.")

500 mg. of indole-3-acetic acid for standard

treatment

Suggested plant material—Cells from plant tissue cultures of tobacco (Nicotiana tabacum L.),

marigold (Tagetes spp.) ^ tomato (Lycopersicon

esculentum Mill.), carrot (Dauciis carota L.),

endive (CichoriMm endivia L.), lettuce (Lactuca

sativa L.), potato (Solanum tuberosum L.), or

bean (Phaseolus vulgaris L.) grown with the

liquid medium shake culture method. (See

"Preparation and selection of plant material.")

Preparation and selection of plant material—To

obtain single cells, groups of cells, and nutrient

medium for use in the microchamber, isolate

stem callus cells from tobacco or other species.

Grow these in 39 ml. of liquid medium contained

in the prescription bottles using the reciprocal

shaker. Prepare several cultures in this way

and place tñem together in the shaker. Make

the medium by dissolving the designated

amounts of the following compounds in 850 ml.

of water : ^

800.0 mg. of Na2S04

400.0 mg. of Ca (NO3) 2 • 4H2O

180.0 mg. of MgSOé • 7H2O

80.0 mg. of KNO3

65.0 mg. of KCl

33.0 mg. of NaH2P04 • H2O

40.0mg. of Fe2(C4H40G)3

4.5 mg. of MnS04 • 4H2O

6.0 mg. of ZnS04 • 7H2O

0.375 mg. of H3BO3

3.0 mg. of KI

3.0 mg. of glycine

0.1 mg. of thiamine

20.0 g. of sucrose

6.0 g. of agar

Dissolve each of the following in the 850 ml.

of nutrient solution :

150.0 of coconut milk

2.5 mg. of calcium pantothenate

0.1 mg. of naphthaleneacetic acid

6.0 mg. of 2,4-dichlorophenoxyacetic acid

Procedure—Prepare a microchamber by placing a drop of the paraffin oil near each end of a

microscope slide (fig. 8). Lower a cover slip

onto each droplet to form risers for a central

chamber. Cover the rectangular space on the

slide between the 2 risers with the oil. Make

certain that the oil also covers the inner edge

of each riser. Using a third cover slip, place a

droplet of liquid medium (obtained from that

employed to produce the original shake-culture

if single cells are used) near the center of the

cover slip. With a dissecting microscope and

teasing needles, remove a small group of cells

(2-30) from the shake-culture, or remove cells

from the liquid shake-culture with a Pasteur

pipette, and place these in the droplet of medium. Invert the cover slip onto the rectangle of

oil between the 2 risers in such a manner that

the oil surrounds the liquid medium with its

enclosed cells and the ends of the top cover slip

rest on the inner edge of each riser (fig. 8). Use

extreme care to prevent tearing, bending, overheating, or contaminating the cells.

1 If sunflower tissue is used, for composition of the

nutrient medium consult: Hildebrandt, A. C, Riker, A.

J., and Dug^ar, B. M. The Influence of the Composition

of the Medium on Growth in Vitro of Excised Tobacco

and Sunflower Tissue Cultures. Amer. Jour. Bot. 33:

591-597. 1946.
16 AGRICULTURE HANDBOOK 3:iÜ, U.S. DEPARTMENT OF AGRICULTURE

1 i I I I '- Liquid medium with cells

> I I , L Paraffin oil

I I , L Cover slip 22 X 22 mm.

I I u Cover slip riser 22 X 22 mm.

I u Paraffin oil

I Glass slide 25 x 75 mm.

F'IGURE 8.—Microchamber for culture of single cells or

groups of higher plant cells. (Diagram—coui'tesy of

American Journal of Botany.)

Use the above method to prepare cultures of

untreated cells for comparison with cells grown

in nutrient containing the test chemical.

Prepare additional microcultures containing

the test compound as follows : to obtain a stock

solution containing 1 microgram of the test

chemical per ml. of culture medium, dissolve

completely 250 micrograms of the test chemical

in 25 ml. of culture medium. If single cells are

involved, use shake-culture medium that has

previously supported the growth of cells. If the

test compound is relatively insoluble, place the

weighed crystals in the medium and pulverize

them by crushing them against the bottom of

the flask with the flat end of a glass rod. Stir

the mixture until the crystals are completely

dissolved.

Prepare an exploratory series of dilutions of

the test chemical by pipetting 0.2 ml. of the

original mixture into a vial containing 0.8 ml.

of the culture medium. Mix the diluted compound thoroughly and continue this procedure

of dilution until a series of concentrations is obtained, each mixture being one-fifth the concentration of the next highest. Proceed as described

above by placing approximately the same number of cells or groups of cells from the shakeculture into a droplet of medium from each of

the various levels of dilution. Enclose each droplet in a corresponding microchamber.

Prepare a duplicate series of microcultures

containing the various dosages of the test chemical. Place the culture chambers at room temperature or in a controlled temperature chamber

at about 25° C. Compare the behavior of the

cells in the control nutrient medium with the

behavior of cells in medium containing the

various amounts of the test compound at w^eekly intervals for about 1 month. (See ''Method

of taking results" for responses that might

occur.)

Use this preliminary series of dosage levels

to indicate a more suitable dosage range for

studying the effect of the test compound in detail. After determining the appropriate dosage

range, repeat the experiment with the proper

amounts of the test chemical and record cellular

responses as indicated below.

Method of taking results—Observe the microcultures with a microscope using interference,

phase, and bright-field illumination. Record the

rate of cell division, rate of differentiation (formation of thin-walled elements resembling

fibers, formation of scalariform tracheids or

giant cells), prevalence of mitochondria, presence or absence of starch or oil droplets, and

other detectable changes. Use Janus Green B as

a means of dififerentially staining the mitochondria so they can be identified and their

numbers estimated.

Suggested standard for comparison—To compare

unknowns, determine the response to known

amounts of regulating substances other than

those in the nutrient medium, such as indole-3-

acetic acid.

Reference

Jones, L. E., Hildebrandt, A. C, Riker, A. J., and Wu,

J. H. Growth of Somatic Tobacco Cells in Microcultui-e. Amer. Joui". Bot. 47: 468-475. 1960.

ROOT CULTURE METHOD

Based on research by P. R. White; S. M.

Caplin.

Description of method—Root tips from tomato

seedlings or cuttings cultured in vitro are

treated by incorporating regulators or other

chemicals into the culture medium or into an oil

overlay and the effects of these compounds on

growth of root cells are determined.

Apparatus, Chemicals, and Other Materials

Scalpel

Forceps

Petri dishes

Filter paper (No. 1 Whatman)

Dark chamber maintained at room temperature of 21°-24° C.

Wide-mouth glass jars

Blotting paper

Heavy, paraffin-coated cardboard

Analytical balance

Bottle, 4 liters

Test tubes
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 17

Rubber test tube stoppers

Refrigerator space

Erlenmeyer flasks, 125 ml.

Aluminum foil

Beakers or paper cups, 50 ml.

Autoclave

Salts for nutrient solution :

25g.of Ca(N03)2

25 g. of Na2S04

10 g. of KCl

2 g. of NaH2P04

1 g. of MnS04

1 g. of ZnSOé

1 g. of HsBOs

1 g. of KI

50 g. of MgSOé

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

Ig. of Fe2(S04)3

Compounds to be evaluated as growth regulators

Sodium hypochlorite solution, such as laundry

bleach

Distilled water

Suggested plant material—Ripe tomato fruits,

or tomato plants (Lycopersicon esculentimi

Mill.) from which cuttings can be made.

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 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 1 cm. from the stem

and cut to the stylar end. Break open the fruit

and expose the seeds carefully so they do not

come in contact with fingers or with the outer,

nonsterile surface of the fruit. With sterile forceps, remove 25 mature seeds that have not been

touched with the scalpel, 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 at a temperature of about 21°-24° C. for a few days until the

seeds germinate and the roots are 2-3 cm. long.

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 with 1:10 water.

Rinse w^ith sterile water and shake off the surplus. Line 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, paraffin-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

the dark at a temperature of about 21°-24° C.

for about 10 days, or until new roots develop to

a length of 3-5 cm.

Procedure—Prepare 4 stock salt solutions,

using the best grades of analytical chemicals.

Dissolve :

(1) 15.0 g. Ca(N03)2 • 4H2O, 4.0 g. KNO3,

3.2 g. KCl, 1,000 ml. H2O.

(2) 37.5 g. MgS04 • 7H2O, 10.0 g. Na2S04,

1,000 ml. H2O.

(3) 0.95 g. NaH2P04 • H2O, 1,000 ml. H2O.

(4) 0.25 g. MnS04 • 4H2O, 0.15 g. ZnS04 •

7H2O, 0.075 g. H3BO3, 0.04 g. KI, 1,000

ml. H2O.

When each stock salt solution is fully dissolved, mix the 4 together slowly. Store this

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

Prepare a 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 the tubes, and store them 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(S04)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. Cover

with squares of foil, flaming the outside to insure sterility. Cap with a 50-ml. beaker or small

paper cup to keep out dust while allowing air

exchange. After being autoclaved (15 lb. 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 con-
18 AGRICULTURE HANDBOOK 336^ U.S. DEPARTMENT OF AGRICULTURE

taining 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.

To obtam a sufficient number of root tips from

a single source, culture these roots for a week;

then with scissors cut 1-cm. tips from the main

root and well-established branch roots. 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 them 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

are used for each treatment.

Treat the excised roots by adding plant regulators in the required amounts aseptically (i.e.,

using sterile solutions 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 flasks in the dark at 21°-

24° C.

Method of taking results—^Since roots grown in

culture do not undergo secondary thickening,

and therefore the diameter 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 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.

Variation used by Caplin—For prolonging the

time between transfers of tissues grown on culture, S. M. Caplin suggests that tissue expiants

of uniform size of carrot secondary phloem can

be grown on agar culture medium containing

White's basal nutrients plus coconut milk (or

other growth-promoting substances) and overlayered with mineral oil. The resultant slower

growth of the cultures under mineral oil prolongs the subculture period from the usual 4-6

wk. to 4-6 mo., with attendant savings in effort,

time, and materials. The fact that plant tissue

cultures grow under mineral oil suggests a

method to test the efi'ectiveness of growth-regulating substances that are relatively insoluble

in water, for example, esters of growth regulators. Also suggested is the possibility of uniform

lateral application to a culture tissue of a drop

of oil containing the test material.

Suggested standard for comparison—Roots

grown in a control solution to which only the

equivalent amount of sterile, distilled water has

been added serve as a standard of comparison.

References

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

Ed. 2 (rev.), 228 pp. Ronald Press Co., New York.

1D()3.

Caplin, S. M. Mineral Oil Overlay for Conservation of

Plant Tissue Cultures. Amer. Jour. Bot. 46: 324-329.

1959.

Caplin, S. M. Effect of Initial Size on Growth of Plant

Tissue Cultures. Amer. Jour. Bot. 50: 91-94. 1963.

SINGLE ISOLATED CELL METHOD

Based on research by A. C. Hildebrandt; W.

H. Muir, A. C. Hildebrandt, and A. J. Riker.

Description of method—Single cells obtained

from tissue of marigold or tobacco grown in

liquid culture are placed on filter paper sheets

that have been resting on tissue culture hosts.

After several weeks, uniform young cultures of

tissue that develop from the single cells are

transferred to agar medium containing the compound to be tested. Comparable cultures are

transferred to agar without the compounds

added and these serve as controls. Responses are

detected by microscopic examination of the cultures at an early stage of their development or

later by microscopic examination. Growth of the

cultures on the medium containing the test compound is compared with that of cultures on the

control medium. Acceleration or suppression of

growth or differentiation of cells compared with

that of the control cultures is a measure of the

growth-regulating properties of the compound

tested.

Apparatus, Chemicals, and Other Materials

Analytical balance

Scalpel

Reciprocal shaker

Dissecting microscope
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 19

Micromanipulator or ''biscuit cutter'' type

isolator (See ''Microisolation device/' p.

113.)

Flattened needle

Forceps

Sterile filter paper, Reeve Angel, crepe surface. No. 202, 8X8 mm.

Petri dishes

Mm. rule

Liquid culture medium

Agar medium (See "Stem tissue culture

method," p. 21.)

Suggested plant material—Tissue cultures from

crown galls of marigold (Tagetes erecta L. var.

Sunset Giant) of crown gall origin or from the

normal stem of tobacco (Nicotiana tabacum L.

var. Havana 38).

Preparation and selection of plant material—

Prepare tissue cultures from crown gall of

marigold or from the normal stem tissue of tobacco. Grow these cells in liquid culture on a

reciprocal shaker. (See p. 15.)

Procedure—Remove suspensions of cells from

the liquid culture and disperse them over a firm

agar medium in a petri dish. Locate single cells

under a dissecting microscope and use the "biscuit cutter" type isolator or micromanipulator

to remove them individually with a flattened

needle under aseptic conditions. Place each cell

on the square sheet of sterile filter paper that

has been resting for 2 or more days on the top

surface of a young tissue culture (about 7 mm.

in diameter) of marigold or tobacco growing

on an agar medium.

Return each filter paper square to the upper

surface of the "host" culture. These operations

require speed to avoid exposing the single-cell

isolates to excessive light and desiccation. The

upper surface of the filter paper should not

come into direct contact with the underlying

host tissue. Either marigold or sunflower tissue

cultures may be used as hosts for single marigold cells. Use tobacco tissue cultures as hosts

for single tobacco cells.

As the host culture becomes senescent, the

young culture it supports will also lose vigor.

The filter paper with its growing tissue must,

therefore, be transferred to a fresh host one or

more times during the test to maintain an

optimum rate of growth.

As a matter of convenience, one single-cell

culture has been referred to in describing this

method. Actually, many of these cultures are

necessary in order to determine the growthregulating properties of a chemical by this

means. The number of replications needed and

the total cultures required must be determined

for each experiment involved. Stocks of singlecell origin may be carried through 4 or more

agar medium transfers, the period between each

transfer being approximately 5 wk. The cultures from single cells may be subdivided into

portions approximately 4 mm. in diameter without diminution in growth rate as a result of

this division. In this way a sufficient number of

replicate cultures can be obtained, all of which

came from the same single cell.

To test the compound, transfer the cultures

resulting from a single cell directly to agar

medium that contains appropriate amounts of

the regulator to be tested.

Method of taking results—Acceleration or suppression of growth of the tissue developed from

a single cell, compared with that of comparable

cultures on untreated agar, indicates response

to the chemical used. Differentiation of cells of

cultures supported by agar medium containing

the test chemical also indicates response to the

compound under test. This response is detected

by macroscopic examination or by examination

of the culture through a dissecting microscope.

Suggested standard for comparison—No standard treatment is suggested.

References

Hildebrandt, A. C. Stimulation or Inhibition of Virus-Infected and Insect-Gall Tissues and Single-Cell Clones.

Nat. Acad. Sei. Proc. 44: 354-363. 1958.

Muir, W. H., Hildebrandt, A. C, and Riker, A. J. Plant

Tissue Cultures Produced from Single Isolated Cells.

Science 119: 877-878. 1954.

SOYBEAN CALLUS TISSUE METHOD

Based on research by C. 0. Miller.

Description of method—Soybean callus tissue

previously grown on a nutrient medium containing kinetin is transferred to a medium without kinetin. Compounds or extracts from plant

tissues to be tested for kinetin-like properties

are added and the resultant increase in cell

division is used as a measure of the activity of

the compounds or extracts tested.

Apparatus, Chemicals, and Other Materials

Erlenmeyer flasks, 125 ml.

Razor blades

Space illuminated by fluorescent light (25

foot-candles) and maintained at 27° C.

Autoclave

Mercuric chloride

Kinetin

Basal medium for soybean assay :
20 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Concentration

Component mg./liter

Ca(N03)2 347.0

KNO3 1,000.0

NH4NO3 1,000.0

KH2PO4 300.0

MgS04 35.0

KCl 65.0

Sodium ferric ethylenediaminetetraacetate 32.0

MnSOé 4.4

ZnS04 1.5

H3BO3 1.6

KI 0.8

Glycine 2.0

Nicotinic acid 0.5

Thiamin • HCl 0.1

Pyridoxine • HCl 0.1

Sucrose 30,000.0

Agar 10,000.0

Adjust pH to 5.8 with NaOH solution

Suggested plant material—Seeds of soybean

(Glycine max (L.) Merrill variety Acme).

Preparation and selection of plant material—

Sterilize soybean seeds by soaking them in 0.1%

mercuric chloride for 15 minutes. Rinse the

seeds 4 times with sterile distilled water. Plant

3 seeds in each of a number of Erlenmeyer

flasks containing 50 ml. of the medium described above.

After the seeds have germinated, remove the

cotyledons and cut them into blocks 4 X 4 X

2 mm. Place 1 block per flask on the medium

shown above to which 0.5 mg./liter of kinetin

has been added. After about 3 wk., subculture

the wound callus, placing it on the same kind of

medium. Continue to subculture the tissue until

a sufficient quantity of stock culture has been

accumulated to initiate tests.

Procedure—Using stock tissue cultures that

have turned slightly yellow while being grown

on nutrient medium containing kinetin, place 3

small blocks of this tissue (approximately 2 X

2X2 mm.) on 50 ml. of the medium without

the kinetin contained in a flask. Use 4 such

plantings for each treatment and retain 4 of

these flasks as controls. Prepare other cultures

of tissue in the same way, but add to the nutrient the compound or extracts to be tested. Keep

the flasks under fluorescent light of about 25

foot-candles and at a temperature of 27° C.

Method of taking results—After 4 or 5 days,

examine the cultures to determine in a preliminary way which of the compounds being tested

accelerated cell division, as evidenced by a

whiter and larger appearance of the tissue

pieces. Weigh the tissue pieces at the end of 2

wk., or at the end of a longer period if larger

weight differences are desired.

Response in terms of fresh weight is directly

proportional to the logarithm of the kinetin

concentration over a range of about 0.004-10

mg./liter.

Suggested standard for comparison—Kinetin at

various concentrations added to the nutrient described above. Range of concentrations 1

through 1,000 X 10"^ M.

Reference

Miller, C. 0. Kinetin and Kinetin-Like Compounds. Mod.

Methods of Plant Anal. 6: 194-202. 1963.

STEM TISSUE CULTURE 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 method—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.

Apparatus, Chemicals, and Other Materials

Sharp knife

Scalpel

Tweezers

Cork or soft pine board

Bottle, 5 liters

Erlenmeyer flasks, 125 ml.

Test tubes

Rubber test tube stoppers

Refrigerator space

Cotton

Gauze

Beakers or paper cups, 50 ml.

Autoclave

Analytical balance

95% alcohol

Chemicals for complete nutrient solution (See

p. 17.)

Compounds to be evaluated as growth regulators

Agar (Difco "Noble" brand)

Distilled water

Suggested plant material—Young willow tree

(Salix nigra L. or S. caprea L.).

Preparation and selection of plant material—Cut

from the young willow tree a piece of branch or

stem about 30 cm. long and 3-10 mm. in diam-
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 21

eter. 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.

Procedure—Prepare a standard salt solution

and a vitamin stock salt solution, as described

on page 21, 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 other half 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 sugarferric 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 nutrientagar 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. Use salt forms of

the growth regulator. While the agar-chemical

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"i^-10"i 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.

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, to obtain 10 or 15 branch sections or

disks. If the branch is 5 mm. or more in diameter, cut the disks into several sectors. If a

smaller branch is used, merely cut the disks 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 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 21°-24°C.

Method of taking results—After 4-6 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-week 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, record

anatomical effects, such as total number of cells,

number of trachéal elements per unit area, and

gross morphological effects.

Suggested standard for comparison—No standard suggested.

References

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.

Historical 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.

SUBMERGED CULTIVATION METHOD

Based on research by L. G. Nickell.

Description of method—A culture is maintained

in the form of single plant cells, small clumps of

cells, or both, submerged in a liquid medium.

Growth measurement is determined by first

measuring the number or volume of cells in an

aliquot of culture medium immediately after

inoculating the medium with a cell suspension

and then measuring the number or volume of

cells again at the end of the test. The number or

volume increase of cells in medium containing

the chemical to be tested is compared with that

which occurs in the control culture medium.

Apparatus, Chemicals, and Other Materials

Analytical balance

Erlenmeyer flasks, 300-ml.

Scalpel

Autoclave

Rotary shaker, 230 r.p.m.

Pipettes, 1 and 10 ml.

Inoculating loop

Centrifuge

Graduated centrifuge tubes, Kolmer type or

equivalent
22 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Hemacytometer counting chamber

White's medium (See p. 17.) with addition

of:

2,4-dichlorophenoxyacetic acid (0.6 p.p.m.)

Coconut milk (18% of final volume) or

mixture of yeast and malt extracts

(0.1% each)

Agar

500 mg. of 2,4-dichlorophenoxyacetic acid,

gibberellic acid As, or indole-3-acetic acid

for standard treatment

Suggested plant material—Pole bean (Phaseolus

vulgaris L.) ; century plant {Agave tourneyana

Trel.) ; rose {Rosa spp.) Paul's Scarlet variety;

holly {Ilex aquifolium L.) ; sugarcane {Saccharum officinale).

Preparation and selection of plant material—

Sterilize several seeds of the plant desired, place

them on a nutrient medium (1% agar added to

nutrient described under ''Root culture

method,'' p. 17) in flasks and allow the seeds

to germinate and develop into plants. Aseptically excise leaves, stems, roots, and other organs and place them again on the agar medium

to induce callus formation. Continue this type

of culture until a sufliicient number of cultures

are obtained. When the callus is 0.5-1 cm. in

diameter, make subcultures.

Procedure—After making several subcultures,

transfer tissue masses to White's medium without agar. To do this, dispense 50 ml. of the

liquid medium into the flasks and autoclave 15

minutes at about 15 Ib./sq. in. pressure before

the transfer is made. Then transfer tissue

masses from agar cultures to this sterile medium after it has cooled. Shake flasks on rotary

shaker at 230 r.p.m. or use such methods as

unidirectional shaking, propeller stirring, or

alternating rotary shaking with baflles. Continuous shaking of the culture for several days

reduces the culture tissue mass to a submerged

living cell suspension.

Method of taking results—Determine the number of cells per unit volume by microscopic

examination immediately after initiation of the

test and compare with that in an equal volume

obtained at the end of the culture period before

the next subculture. The increase in number of

cells indicates rate of growth.

Also determine pack-cell volume by centrifuging cells contained in a 5-ml. portion of the

culture at the beginning and end of growth

periods. Centrifuge the 5-ml. cell suspensions in

the Kolmer-type centrifuge tubes or equivalent

at 1,500 r.p.m. for 2-3 minutes until cells are

tightly compacted. Read the volume of cells

directly from the calibrated centrifuge tube.

An alternative method of determining increase in cell numbers, which also yields data

concerning changes in the structure of cells, is

microscopic examination. Take the required volume of the suspension for a hemacytometer

counting chamber and then make counts as

directed. At the same time, compare the structure of cells grown in medium containing the

test compound with the structure of those

grown in the nutrient medium alone.

Suggested standard for comparison—0.6 p.p.m.

of 2,4-dichlorophenoxyacetic acid compared

with absence of the acid or presence of gibberellic acid, indole-3-acetic acid, or other

growth regulators.

References

Nickell, L. G. The Continuous Submerged Cultivation of

Plant Tissue as Single Cells. Nati. Acad. Sei. Proc.

42: 848-850. 1956.

Nickell, L. G. Growth Effects of Antibiotics on Plant

Cells and Their Reaction to a Modified Gram Stain.

Phyton 11: 93-96. 1958.

Nickell, L. G., and Tulecke, W. Submerged Growth of

Cells of Higher Plants. Jour. Biochem. and MicrobioL,

Technol. and Engn. 2: 287-297. 1960.

Nickell. L. G. Submerged Growth of Plant Cells. Adv.

AppL MicrobioL 4: 213-236. 1962.

Nickell, L. G. Tissue and Cell Cultures of Sugarcane—

Another Research Tool. Hawaiian Planters' Rec. 57*

223-229.1964.

TOBACCO PITH METHOD (MICRO)

Based on research by W. Bottomley, N. P.

Kefford, J. A. Zwar, and P. L. Goldacre.

Description of method—Blocks of tobacco pith

are placed aseptically on a basal nutrient solution containing indole-3-acetic acid. Addition of

kmetin or plant extracts with kinetin-like substances induces the formation of neoplasm on

the pith blocks. Increase in the number of these

neoplastic cells is an indication of the amount of

kinetin or kinetin-like substance present in the

test material.

Apparatus, Chemicals, and Other Materials

Petri dishes

Scalpel

Test tubes, 7.5 X 1.2 cm., fitted with aluminum caps

Filter paper strips, Whatman No. 1, 4 X 0.9

cm.

Autoclave

Dark chamber maintained at a high humidity

and 25° C.

Dissecting microscope, 10 X magnification

Distilled water

Sodium hypochlorite solution

250 mg. kinetin for standard of comparison
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 23

Make a basal stock nutrient solution as follows and adjust it to pH 5.8:

0.3 g. of kH.'P04

1.0 g. of KNO:-.

1.0 g. of NH4NO:;

0.5 g. of Ca(NO:;)i>-4H20

0.035 g. of MgSOi

0.065 g. of KCl

0.8 mg. of KI

4.4 mg. of MnSO.

1.5 mg. of ZnSOi

1.6 mg. of H;;BO:í

0.1 mg. of Thiamin • CHI

0.5 mg. of Nicotinic acid

0.1 mg. of Pyridoxine • HCl

1.75 mg. of lndole-3-acetic acid

20.0 g. of Sucrose

Add sufficient distilled water to make 1 liter

of solution.

Suggested plant material—Stem pith tissue of

tobacco {Nicotiana iabacum L.) cultivar Wisconsin Havana 38.

Selection and preparation of plant material—

Grow the tobacco plants in soil under greenhouse conditions using approximately 30° C.

day temperature and 25° C. night temperature.

Use high mineral nutrition and subject the

plants to natural light of high intensity to produce thick, easily w^orked stems.

Cut pieces of stem 15 cm. long and sterilize

them by soaking them in sodium hypochlorite

solution for 30 minutes or by aseptically peeling

off the outer layers of tissue. Cut the stem pieces

transversely into cylinders 10 mm. long. With

longitudinal cuts, make blocks about 10 X 5 X

3 mm., taking care to avoid including any vascular tissue. Store the blocks in petri dishes containing a small amount of the sterile basal medium before transferring the blocks to test

tubes.

Procedure—Prepare a dilution series by placing the substance to be tested in the basal medium and using this mxcdium as a diluent. Place

a filter paper strip in one of the test tubes. Add

1.0 ml. of one of the prepared solutions to the

test tube. Close the test tube and prepare additional test tubes with paper and solution to give

3 replicates for each dilution. Sterilize the tubes

at a pressure of 18 Ib./sq. in. for 5 minutes.

Transfer aseptically one pith block to each tube

so the block contacts the medium only through

the filter paper. Close the tubes and place them

in a rack so that they lie at an angle of about

20° from horizontal. Place all cultures in darkness at high humidity and at 25° C.

Method of taking results—Inspect the cultures

in light at the end of 14 and 21 days using the

dissecting microscope. Cytokinin activity can be

estimated by assessing the area of the block that

is covered with neoplasm. This varies from no

cells, through a few small clusters of tiny cells,

to the stage where the whole block is covered

with new cells. The new cells are readily recognized, since they are smaller than the cells of

the original pith tissue, round, and opaque. If

a dilution series is used, an index of cytokinin

activity can be obtained by taking the reciprocal of the lowest concentration that causes neoplasm to form.

Suggested standard for comparison—Response

can be compared with the increase in cell division induced when a dilution series of kinetin is

added to the basal medium.

Reference

Bottomley, W., Kefford, N. P., Zwar, J. A., and Goldacre,

P. L. Kinin Activity from Plant Extracts. I. Biological

Assay and Sources of Activity. Austral. Jour, of Biol.

Sei. 16: 395-406. 1963.

CELL ELONGATION

The first test of this kind to be accepted for

wide use in phytohormone research was the

Avena coleoptile curvature test. This has been

described in great detail by F. W. Went and

K. V. Thimann, Phijtohormones, Macmillan Co.,

New York, 1937, and, for this reason, is not

described here.

Description of method—Segments- of oat coleoptiles of uniform length are incubated in solutions of the test compounds. A comparison of

length measurements of the segments maintained in the test solutions with those of comparable segments maintained in a standard solution indicates the extent to which the growth

rate is affected by each compound.

OAT COLEOPTILE SECTION METHOD

Based on research by D. H. McRae and James

Bonner; Par Fransson; J. C. Sirois.

2 The term "segment" is generally used in this compilation instead of ^'section" since the term "section" often

refers to a thin slice viewed with a microscope.
24 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Apparatus, Chemicals, and Other Materials

Stainless steel, glass, plastic, or enamel-coated

pans about 35 X 25 X 5 cm.

Standard Avena room (or growth chamber)

maintained at 25°-26" C. and 90% relative humidity. (If a growth chamber is

used instead of a standard Avena room, the

chamber should be conveniently near a dark

room where manipulations can be made.)

Red bulb, 10 w.

Analytical balance

Centimeter rule graduated to 0.1 mm. or a

calibrated ocular micrometer to use with

dissecting microscope

Dissecting microscope

Microscope slides

Forceps

Double-bladed cutting tool constructed of

razor blades held parallel to each other exactly 5 mm. apart (See ''Segment cutters,"

p. 101.)

Petri dishes

1% sodium hypochlorite solution

0.0025 M potassium maléate buffer solution

containing 3% sucrose prepared with

water distilled in Pyrex

0.01-200 mg. of each compound to be tested

175 mg. of indole-3-acetic acid for standard

treatment

Detergent

Distilled water

Vermiculite or perlite

Suggested plant material—Seeds of oat (Avena

sativa L.) Siegeshafer or Victory variety.

Seeds of the victory variety can be obtained

from the Department of Genetics, University of

Alberta, Edmonton, Alberta, Canada, as No.

C.A.N. 426, or from the Plant Products Division, Production and Marketing Branch, Canada Department of Agriculture, Ottawa, Ontario, as Victory oats.

Preparation and selection of plant material—

Sort the seeds eliminating any that are shriveled

or diseased. Sterilize the selected seeds for 10

minutes in the 1 % sodium hypochlorite solution

containing a small amount of detergent, and

then soak the sterilized seeds in distilled water

for 1 hr. Fill the pans with vermiculite (or

perlite) to a depth of approximately 4 cm. Wet

the vermiculite thoroughly with distilled water

and allow it to equilibrate overnight. Pour off

excess water from one corner of the pan. Remove about 0.6 cm. of vermiculite. Firm and

smooth the remaining vermiculite but do not

pack it; then sow the seeds on the surface of

this prepared bed. Cover with the previously removed vermiculite; smooth and firm it.

Fransson suggested a variation of this

method of obtaining plant material that is more

time consuming but results in a higher number

of coleoptiles with straight growth. Plant the

soaked seeds in vermiculite with their husks

removed, spacing each seed about 1 cm. apart.

Insert the seeds into the vermiculite with the

embryo ends downward to such a depth that the

upper ends of the seeds are 0.5 cm. below the

surface. Cover the entire seedbed with a thin

layer of vermiculite. Place the pans in the

growth chambers at about 25° C. and allow the

seeds to germinate under continuing low-intensity red light (10-w. red bulb 1-2 m. from

trays).

Eighty-two to 84 hr. after sowing the seeds,

select for use in the tests plants of uniform

length (2.8-3.3 cm.) with uniformly straight

coleoptiles. This and the further operations may

be carried out in the growth room under phototropically inactive orange (longer than 550

millimicrons) light. Corning Filters 243 or 348

are satisfactory. Sprinkle distilled water over

the seedlings approximately 24 hr. later, and

again about 12 hr. before harvesting. This

facilitates the handling of the coleoptiles and

increases the sensitivity of the isolated segments

to added auxins, particularly to indole-3-acetic

acid.

Procedure—Excise with the double-bladed cutting tool one 5-mm. segment from each selected

coleoptile. The upper cut should be made no

closer than 2-3 mm. from the coleoptile tip and

all segments should be cut at a uniform distance

from the tip. The piece of leaf contained within

the coleoptile segment may be allowed to remain

in place. Immediately after cutting the segments, place them in groups of 20 for about 1

hr. in a portion of basal medium consisting of

water distilled in Pyrex, 3% sucrose, and

0.0025 M potassium maléate buffer (pH 4.5).'

This will bring all segments into equilibrium

and wash out the endogenous auxin from the

segments. In this way limited growth of the

controls is obtained. After this pretreatment

period, place one set of 20 segments in the

fresh basal medium to serve as controls. Place

another set of 20 in fresh basal medium to which

the test compound has been added and the final

mixture adjusted to pH 4.5. Suggested range of

concentrations to be tested is lO'^-lO'^ M solutions. Prepare as many replicates of 20-segment sets as needed (usually a total of 2 or 3

sets). Incubate the segments in the solutions

in the dark for 12 hr. at 25°-26° C.

Method of taking results—At the end of the incubation period, transfer the segments (20 at a

test) to a microscope slide, placing them side
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 25

by side, parallel, in a row. Add 1 or 2 drops of

the solution in which they have been incuDated

to keep them from drying out. Transfer the

slide to the stage of the microscope and measure

and record the length of each segment to the

nearest 0.1 mm., using a graduated cm. rule or

ocular micrometer. Compare the final length of

the segments incubated in the basal medium

plus test compound with the length of those

incubated in basal medium alone. Of particular

value is information concerning the concentration of test compound that elicits one-half the

maximal growth effect and the growth effect

elicited by the test compound when applied in

optimal concentrations.

Suggested standard for comparison—Basic solution containing 0.175 p.p.m. (10 *^ M) of indole3-acetic acid.

Sirois suggested a modification of the Coleoptile Section Method that makes possible direct

reading of auxin concentrations and the use of

two segments from each coleoptile, thus doubling the number of assays hitherto possible

with a given number of coleoptiles. The range,

accuracy, and sensitivity of the modified assay

are comparable to those of the standard Avena

curvature test.

Surface sterilize oat seeds of the Brighton

variety with 2% sodium hypochlorite solution.

Rinse the seeds in tapwater for 1 hr. and soak

them in distilled water for 2 hr. Germinate the

seeds on tissue paper in distilled water under

red light for 20 hr. at 24° C. and in darkness

for 48 hr. at 26° C. Select coleoptiles 20 mm. ±z

2 mm. long and decapitate them 3 mm. from the

apex. (See '^Segment cutters,'' p. 101.) Excise

two 5-mm. segments from each coleoptile and

place each in distilled water contained in separate petri dishes for 3 hr.

Place a 2.5-cm.- piece of Chromatographie

paper in a petri dish and add 2 ml. of 0.005 M

K2HPO4-O.OO25 M citric acid buffer at pH 4.8.

Place 10 coleoptile segments on the paper. Prepare as many dishes in this manner as required,

adding to some the desired amounts of indole-3-

acetic acid or test compound. Incubate the dishes

containing the coleoptiles in darkness at 26° C.

for 20 hr. and then measure the length of the

segments under 10 X magnification. Elongation

of the coleoptile segments is directly proportional to the concentration rather than to the

logarithm of the concentration of indole-3-

acetic acid.

References

Modification suggested by Par Fransson, Institute of

Plant Physiology, Lund, Sweden.

McRae, D. H. and Bonner, James. Chemical Structure

and Antiauxin Activity. Physiol. Plant. 6: 485-510.

1953.

Sirois, J. C. Studies on Growth Regulators. I. Improved

Avena Coleoptile Elongation Test for Auxin. Plant

Physiol. 41: 1308-1312. 1966. [Plant Research Institute, Research Erancn, Canada Department of Agriculture, Ottawa, Ontario.)

BEAN HYPOCOTYL METHOD

Based on research by R. L. Weintraub, J. W.

Brown, J. A. Throne, and J. N. Yeatman.

Description of method—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.

Apparatus, Chemicais, and Other Materials

Vermiculite, No. 3

Galvanized iron pans, 51 cm. wide X 66 cm.

long X 8 cm. deep perforated for drainage

Light-tight room at 31° C. with relative humidity of 60 ± 10%

Red fluorescent lamps, 40 w.

Sharp knife or razor blade

Board 2 X 2 X 43 cm. with row of V-shaped

notches along one edge

Tuberculin-type glass syringe, 0.25 ml.

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 10 cm. wide X 41 cm. long X 5 cm. deep

Bromide protographic paper

Movable-arm protractor

Analytical balance

Chemicals to be evaluated for cell-elongating

activity

Relatively pure 2,4-dichlorophenoxyacetic

acid for standard treatment

95% ethanol

Photographic developer

Petroleum jelly

Suggested plant material—Young bean plants

(Phaseolus vulgaris L.)—Black Valentine,

Pinto, Red Kidney, or other varieties.

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
26 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

at 31° C. with relative humidity of 60 ± 10%.

Suspend red fluorescent lamps (40-w.) 1-2 m.

above the developing plants to induce straight

stem growth.

Remove plants from vermiculite carefully to

prevent 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 portion to hold it in place. Rest

the ends of the board on the edges of the shallow

pan so the roots are submerged in tapwater for

2 hr. before the test.

Procedure—Dissolve the chemical to be tested

for cell-elongating activity in 95% ethanol. This

solution should contain 0.065 fig. 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-hr.

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

Suggested standard for comparison—2,4-dichlorophenoxyacetic acid applied in 95% ethanol so

that each 0.0015-ml. droplet contains 0.065 /^g.

of the acid.

Reference

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

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

BEAN SECOND INTERNODE METHOD

Based on research by J. W. Mitchell and

Greta D. York.

Description of method—In this micro method,

the compound or extract to be tested is uniformly dispersed in 250 micrograms of fractionated lanolin. The mixture is applied with the

aid of a dissecting microscope to the second

internode of a bean stem when the internode is

about 0.7-1.0 mm. long. Within limits, elongation of the internode is proportional to the

amount of cell-elongating compound present.

This method, which involves intact plants, is

also useful for studying effects of extremely

small amounts of compounds of the gibberellin

type (fig. 9). It can also be used to detect compounds that retard elongation and those that inriuence cell multiplication in stem tissues.

Apparatus, Chemicals, and Other Materials

Analytical balance

Tweezers

Dissecting microscope

Shell vials

Fractionated lanolin

Water bath with temperature control

Standard microscope slides

Melting-point tubes

Aquarium pump or compressed air

Razor blades

Glass rod pointed to about 0.5 mm. diameter

Room with 700-foot-candle illumination from

Slimline fluorescent tubes, photoperiod

control, and temperature of 22°-24° C.

Gibberellic acid for standard treatment

Suggested plant material—Young bean plants

(Phaseolus vulgaris L., Pinto var.) viiih primary leaves only partly unfolded and with terminal buds having the tip of the center leaflet

in the first trifoliolate leaf slightly separated

from the 2 lateral leaflets. The second internode

should not be more than 1 mm. long at this

stage of development. Discard plants with second internodes longer than 1 mm.

Preparation and selection of plant material—

Select plants of uniform size and in the required

stage of development. With pointed tweezers,

remove the bract that subtends the dorsal side

of the center leaflet in the terminal bud so that

a smooth, well-defined scar is left and one side

of the second internode is thus exposed. Measure

the distance from the lower edge of the scar to

the base of the bracts on the third node (fig.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 27

BN-28960

FIGURE 9.—Comparison of length of bean second internode (a) treated with 250 micrograms of fractionated lanolin

with length of second internode (6) treated with an equal amount of fractionated lanolin containing 63 trillionths of a gram of gibberellic acid As. Photo taken 4 days after treatment.

10). Record this as the initial length of the internode before treatment.

Procedure—After eluting a segment of a paper

chromatogram or extracting the powder from a

thin-layer plate containing the chromatographed

compound, place the eluate in a small shell vial,

evaporate the solvent, and add 1 ml. of ether

containing 250 micrograms of fractionated lanolin. (See "Fractionated lanolin as a carrier for

growth-regulating compounds," p. 104.) Dissolve the solids in the vial by stirring and

slightly warming the mixture, if necessary.

Transfer the ether mixture from the vial to a

small area on the surface of a microscope slide.

To make this transfer, dip a capillary meltingpoint tube into the ether mixture and allow the

liquid to rise in the tube by capillarity. Use the

tube as a delivery pipette and place the ether

mixture dropwise on the slide in an area directly under a stream of gently flowing air. In

this way evaporate the ether leaving the compound to be tested thoroughly mixed with the

fractionated lanolin.

Remove the lanolin mixture quantitatively

from the slide with a razor blade in a way that

leaves the paste piled on the beveled edge at one

end of the blade. With the aid of a dissecting

microscope, use the narrow glass rod to remove

the lanolin mixture from the blade and place it

on the second internode of one test plant (fig.

11).

If the cell-elongating properties of the chemical are to be studied, grow the plants in 700 footcandles of light from Slimline fluorescent tubes

for 9 hr. daily followed by darkness. Maintain

the plants at 20°-23° C. during both the light

and dark periods. If growth inhibition is to be

studied, add about 50 foot-candles of illumination from Mazda lamps and increase the temperature to make the test plants elongate the

desired amount. If maximum stem elongation is

required, use about 50 foot-candles of light intensity from Mazda lamps for 2 hr. in the

middle of the 15-hr. dark period.

Method of taking results—At the end of 4 days,

measure the length of the second internode of

each plant as described under "Preparation and

selection of plant material." Subtract the initial

length of each internode and compare the

growth with that of plants treated with the

fractionated lanolin alone.

Since the test plants are exposed to greenhouse conditions during their early growth,

their sensitivity to cell-elongating substances
28 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-28933

FIGURE 10.—Terminal bud of young bean plant showing

scar left after biact has been removed and the distance measured to determine length of second internode (A).

varies from experiment to experiment, depending on the prevailing light intensity. For example, plants grown during partly cloudy

weather are generally more responsive than are

those grown in bright sunlight during clear

weather. However, even the relatively insensitive ones are capable of responding to extremely

small amounts of gibberellic acid and other cellelongating substances.

Suggested standard for comparison—Gibberellic

acid applied at a dosage of 0.001 microgram or

less per second internode.

A convenient way to obtain 0.001 microgram

of gibberellic acid (1 billionth of a gram) is as

follows: dissolve 2 mg. of gibberellic acid As in

200 ml. of ethyl alcohol and designa*:e this Solution I. Mix 1 ml. of Solution I witii 93 ml. of

ethyl alcohol to make Solution II. Finally, mix

0.5 ml. of Solution JI with 2 ml. of ethyl alcohol

to make Solution III. Pbce 0.0-5 ml. of Solution

III (which contains 0.001 microgj-ams of gibberellic acid) in a shell vial. Evaporate the

alcohol in a water bath at 50° C. Add 1 ml. of

ethyl ether containing 250 micrograms of lanolin fraction. (See "Fractionated lanolin as a

carrier for growth-regulating compounds," p.

104.) Stir until the gibberellic acid is thoroughly

dissolved. Transfer the lanolin fraction-gibberellic acid mixture to the surface of a clean slide

using a melting-point tube pipette and a gentle

stream of air to evaporate the ether as described

above. Apply the residual lanolin mixture of

gibberellic acid to second internodes of bean

plants as described in the above assay. If less

than 0.001 microgram of gibberellic acid is desirable as a standard, extend the dilution series

described and carry out the second internode

test. When gibberellic acid is to be used repeatedly as a standard, prepare a large number of

slides containing the lanolin fraction-gibberellic

acid mixture as described above, and store these

in a slide box at 0° C. for use when needed.

Reference

Method submitted by J. W. Mitchell and Greta D. York,

Agricultural Research Service, U.S. Dept. Agr.

BN-28947

FIGURE 11.—Mixture of 250 micrograms of fractionated

lanolin and a plant extract (a) applied to second

internode of a test plant after bract at base of internode had been removed.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 29

OAT FIRST INTERNODE METHOD

Based on research by J. P. Nitsch and Colette

Nitsch; D. G. Crosby, R. V. Berthold, and Roy

Spencer, Jr.

Description of method—Segments 4 mm. long of

first internodes of etiolated oats are incubated

20-24 hr. with the solution to be assayed. The

increase in length is proportional to the logarithm of the concentration, at least between limits. The test is sensitive to 0.001 microgram of

indole-3-acetic acid or gibberellic acid As. This

method is also useful for assaying paper chromatograms.

Apparatus, Chemicals, and Other Materials

Plastic containers, about 30 X 20 X 10 cm.,

with lids

Vermiculite or clean sawdust

Incubator at 25° C. ± 0.5° C.

Dark chamber with green light (about 540

millimicrons)

Cutter with 2 parallel razor blades and a

guide set to cut segments 4 mm. ± 0.05

mm. long (See ''Segment cutters," p. 101.)

Analytical balance

Petri dish with gauze or muslin stretched

over the top

Paper towels

Forceps

Fine scissors

Test tubes, about 13 mm. diameter

Roller-tube apparatus (rotating at 1 r.p.m.)

Binocular microscope (lOX) with ocular

micrometer

Stainless steel spatula

A fraction of a milligram of each compound

to be evaluated

250 mg. gibberellic acid As or indole-3-acetic

acid for standard treatment

Suggested plant material—Seeds of oat (Avena

sativa L., Brighton var.). These can be obtained for research from: (1) Cereals 'Department, Canada Department of Agriculture,

Experimental Farm, Scott, Saskatchewan, Canada; (2) 0. Gelin, Plant Breeding Institution,

Weibullsholm, Landskona, Sweden; (3) Small

Grains Collection, Cereal Crops Research

Branch, U.S. Department of Agriculture, Plant

Industry Station, Beltsville, Md. 20705.

Preparation and selection of plant material—

Place a 2.5-cm. layer of moist vermiculite

or sawdust in plastic boxes. Sprinkle over

it the Brighton oat seeds. Cover with another 2.5-cm. layer of the planting medium.

Close the lid tightly as an increase in CO2 enhances the growth of the first internodes. Place

the boxes in the incubator at 25° C. and keep

them in total darkness. (Neither seedlings nor

segments should ever be exposed to anything

more than the minimum amount of dim green

light required for perception.) The seedlings

are ready for use 64 hr. later.

Procedure—Prepare the following buffer solution:

K2HPO4:1.794 g./liter

citric acid monohydrate: 1.019 g./liter

sucrose:20 g./liter

Into each test tube, place 0.5 ml. of this solution in which may be incorporated the extracts

or chemicals to be tested.

The method is especially suitable for assaying

paper chromatograms. Use paper approximately

2 cm. wide to develop the chromatogram. Cut

the chromatogram horizontally into 1-cm.

strips. Make certain that each strip is numbered

starting with the segment nearest the origin of

the chromatogram. Place each piece of the chromatogram separately into a test tube containing

0.5 ml. of the buffer solution, leaving 2 tubes

with paper segments cut below the starting line

and above the front of the chromatogram to

serve as a control.

In a dark room with the dim green light,

harvest the seedlings, breaking them at the

seed. With fine scissors, remove the coleoptile

by cutting exactly at the node. Place the first

internodes on wet paper towels, orienting the

apical (nodal) ends uniformly.

With the cutter using parallel razor blades,

cut from each first internode one 4-mm. segment 2 mm. below the node. (See "Segment cutters," p. 101.) Wash the segments by placing

them for 1 hr. on a piece of muslin stretched

over a petri dish filled with glass-distilled water.

The segments are partially immersed in this

way but are exposed continuously to air. The

muslin prevents the segments from sinking into

the water.

Transfer 10 segments to each of the test

tubes using a thin spatula. Place tubes in the

roller-tube apparatus and rotate them at 1 r.p.m.

for 20-24 hr. in darkness at 25° C. and in an

atmosphere where the humidity is near saturation. If a high humidity cannot be obtained,

seal the tubes with Parafilm.

Method of taking results—After the incubation

period, remove the segments from the tubes

with a wire loop and place them on microscope

slides. Measure the length of the segments to the

nearest 0.1 mm. under a binocular microscope
30 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

with an ocular micrometer. Compare the average length of each of the 10 segments in each

tube with curves based on response to different

amounts of the standard compounds, plotted on

semi-logarithmic paper.

Suggested standard for comparison—Either indole-3-acetic acid or gibberellic acid As at concentrations of 1, 3, 10, 30, and 100 micrograms/

liter.

Other compounds active when used in this

method are: auxins, such as the amide, nitrile,

and esters of indole-3-acetic acid; cis-cinnamic

acid (above 300 micrograms/liter) ; various

growth-regulating substances; gibberellins Ai,

A2, A4, As, Ae, AT, AS (above 10« M), A9, but

not steviol; helminthosporol ; helminthosporic

acid ; and dehydrohelminthosporic acid. The last

3 compounds have a relatively low activity.

Variation used by Crosby, Berthold, and Spencer

—First internode segments from 7 varieties of

oats were compared with respect to sensitivity

and uniformity of elongation response to different growth-regulating substances. The

method used in these comparisons was essentially the same as that described above. The

Forkedeer variety was most suitable for obtaining first internode segments with which to

measure the effect of regulating substances and

extracted plant hormones on elongation of segments from oat first internodes. Small amounts

of Forkedeer seed oats to be used for scientific

investigations are obtainable from: (1) Farmers Supply Co., Smithville, Tennessee 37166;

(2) D. R. Mayo Seed Co., Box 10247, Knoxville,

Tennessee 37919; and (3) J. M. McCalla, Jr.,

Covington, Tennessee 38019.

References

Crosby, D. G., Berthold, R. V., and Spencer, Roy, Jr.

Naturally Occurring Growth Substances. II. An Improved Straight Growth Test and Its Applications.

Plant Physiol. 36: 48-51. 1961.

Nitsch, J. P., and Nitsch, Colette. Studies on the Growth

of Coleoptile and First Internode Sections. A New,

Sensitive, Straight-Growth Test for Auxins. Plant

Physiol. 31: 94-111. 1956.

Nitsch, J. P., and Nitsch, Colette. Comparison of Activities of Nine Gibberellins by Three Biological Tests.

Ann. Physiol. Vég. 4: 85-97. 1962.

PEA STEM SECTION METHOD

Based on research by G. S. Christiansen and

K. V. Thimann.

Description of method—The effect of a compound on stem elongation is measured by floating segments from etiolated stems of pea on the

surface of a solution containing the compound,

and measuring the response in terms of increase

in length or weight of the segments.

Apparatus, Chemicals, and Other Materials

Dark room with temperature controlled at

.about 27°-29° C. and humidity controlled

at 85-90%

Analytical balance

Glass or metal containers

Corning Signal Red niter and 60-w. lamp

Quartz sand

Razor blades mounted 20 mm. apart on a

block of wood (See ''Segment cutters/' p.

101.)

Beakers, 500 ml.

Filter paper

Petri dishes

Sodium hydroxide

1 g. of indole-3-acetic acid

250-500 mg. of each compound to be tested

Suggested plant material—Etiolated stems of

pea {Pisum sativum L.)

Preparation and selection of plant material—Soak

the seeds in tapwater for 6-8 hr. ; plant them in

sand moistened with tapwater, and allow the

plants to grow in darkness. All observations

and manipulations should be made in a minimum of light from a 60-w. incandescent lamp

passed through the Corning filter. After 7 days,

select 30-50 plants of uniform size from which

to make stem segments.

Procedure—Do not use plants that have developed fourth internodes longer than 3 mm.

Cut 20-mm. segments (exactly measured) from

the third internodes. In cutting segments, first

decide which portion of the stem is most suitable for the test at hand. Purves and Hillman

showed that segments from an area farthest

from the apex respond least to gibberellic acid

and sucrose, but these segments show a relatively great diff'erence in response to indole-3-

acetic acid as compared with gibberellic acid.

Short segments taken from the stem close to the

apex are relatively responsive to gibberellins.

Therefore, select an appropriate portion of the

stem from which to obtain segments and, in

reporting results, indicate its location.

Immediately place the segments in tapwater,

wash, and divide them into groups of 15 segments each. Blot dry with filter paper and

weigh the groups separately to the nearest milligram. Keep segments moistened.

Prepare aqueous solutions (25-100 ml.) of

the compounds to be tested, adjusting their pH
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 31

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 0.1-2.0 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 segments on the surface of the

liquid for 24 hr. in darkness.

Method of taking results—At the end of 24 hr.

remove the segments, blot them with filter

paper, weigh them as a group to the nearest

milligram, and measure their length individually to the nearest millimeter. Compare the

weight increase and average length increase of

these segments (from each concentration of the

test chemical) with comparable measurements

of segments treated with distilled water only,

and of others treated with distilled water containing different concentrations of indole-3-

acetic acid.

Variations of the above method and the effect

of various experimental conditions on the response of pea stem segments to growth substances were studied in detail by Galston and

Hand.

Suggested standard for comparison—Solution

containing 0.1, 0.5, 1.0, and 2.0 mg. of indole-3-

acetic acid per liter.

References

Christiansen, G. S., and Thimann, K. V. The Metabolism

of Stem Tissue During- Growth and Its Inhibition. I.

Carbohydrates. Arch. Biochem. 26: 230-247. 1950.

[Emendations submitted by K. V. Thimann.]

Galston, A. W. and Hand, Margery E. Studies on the

Physiology of Light Action. I. Auxin and the Light Inhibition of Growth. Amer. Jour. Bot. 36: 85-94. 1949.

Phillips, I. D. J., and Jones, R. L. Gibberellin-like Activity in Bleeding-Sap of Root Systems of Heliantlius

annuus Detected by a New Dwarf Pea Epicotyl Assay

and Other Methods. Planta 63: 269-278. 1964.

Purves, W. K., and Hillman, W. S. Response of Pea

Stem Sections to Indoleacetic Acid, Gibberellic Acid,

and Sucrose as Affected by Length and Distance from

Apex. Physiol. Plant. 11: 29-35. 1958.

RADISH COTYLEDON METHOD

Based on research by Tetsuo Takematsu.

Description of method—The increased angle

that develops when cotyledon and cotyledonary

petiole are sprayed with an aqueous solution of

a compound indicates the influence of the compound on cell elongation.

Apparatus, Chemicals^ and Other Materials

Composted soil

Clay pots, 9 cm. in diameter

Scissors or razor blade

Small glass tubes or pipettes

Analytical balance

Atomizer

Protractor

Photographic paper and developer

Glass plate

Waterproof cellophane

Distilled water

50 mg. of compound to be tested

5 mg. of alpha-naphthaleneacetic acid for

standard treatment

Suggested plant material—Seedlings of Japanese radish (Raphanus sativus L.)—Tokinashi

Daikon variety, or any other variety that develops relatively large and persistent cotyledons. The variety suggested is especially sensitive to growth-regulating substances; the size

and shape of the cotyledon and cotyledonary

petiole form angles that are easy to measure;

and the cotyledon is dark green and vigorous,

providing uniform plant material throughout

the year. Other varieties with comparable characteristics can also be used, but it may be necessary to determine the responsiveness of these

under the conditions available.

Preparation and selection of plant material—Select mature, round, fresh seeds. Make 10 holes

0.5 cm. in diameter and 1 cm. deep in the soil

in each pot. Plant 2 seeds in each hole. Place the

pots in sunlight or bright light and maintain the

humidity at about 60-807^. Select 20 seedlings

of uniform size 4-8 days after germination. Use

10 plants for the application of each compound

to be tested and 10 as controls.

Procedure—Dissolve each compound in distilled water using the following concentrations :

1, 10, 50, 100, and 200 p.p.m. Spray 1-5 ml. of

the solution to be tested on the whole upper

surface of the 10 seedlings. Spray 10 control

seedlings with a corresponding amount of distilled water. Place the plants in a dark room for

24 hr. After the cotyledons and cotyledonary

petioles have developed curvature, sever the

aboveground portion of the plants at the base

of the stem with scissors or a razor blade. Cut

off the cotyledons at the point where they join

the cotyledonary petioles. Make shadowgraphs

of petioles and stem segments to record the

curvature of the petioles. Blueprint paper or

other light-sensitive paper can be used to make

shadowgraphs. Place a sheet of waterproof cellophane over the sensitive paper and arrange
32 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

the pieces of plant to be shadowgraphed on the

cellophane. Lay a glass plate on the plant samples so that undistorted shadowgraphs can be

made by means of the proper light exposure

and then measure the angle y (fig. 12).

For an alternate method place the petiole and

stem segment on heavy paper; with pins mark

the crossing point (fig. 12, h) of the cotyledonary petioles and the 2 points at which the

cotyledonary petioles begin to bend downward

(fig. 12, a and c). A line drawn to connect these

3 pinholes indicates angle y.

Method of taking results—Measure the angle y

on treated and untreated petiole and stem segments. The difiierence of the angle between

treated and untreated segments indicates the

cell-elongating property of the compound applied. In general, this angle on untreated segments is about 20°, depending on the humidity

of the soil and the amount of light. It is important to subject both the plants to be treated

and those to be used as controls to the same

growing conditions.

Suggested standard for comparison—0.1-50

p.p.m. solution of alpha-naphthaleneacetic acid

or 2,4-dichlorophenoxyacetic acid.

Reference

Takematsu, Tetsuo. New Methods for the Qualitative

and Quantitative Assays of Growth Substances; Research on the Raphanus Test and Its Application.

Utsonomiya University, College of Agriculture. Gakujutsu Hokoku Tokushu. Spec. Bui. 12, 193 pp. 1961.

[In Japanese.]

FIGURE 12.—Method of measuring angle ij, formed by

intersecting lines drawn through points a, b, and c on

the stem and petioles of radish seedling to show response to growth substance. (Diagram by Tetsuo

Takematsu.)

SPLIT PEA STEM METHOD

Based 07i research by Martha Kent and W. A.

Gortner; K. V. Thimann and C. L. Schneider;

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

Description of method—The angle of curvature

that develops when split stems of pea are immersed in an aqueous solution of a compound

indicates the inñuence of the compound on cell

elongation.

This test has been carefully standardized and

widely used in measuring cell-elongation properties of synthetically prepared regulators and

regulating compounds obtained from plant

material.

Apparatus, Chemicals, and Other Materials

Quartz sand

Completely darkened room with temperature

controlled at 27^-29° C.

Ruby glass incandescent lamp

Time clock

Razor blades or segment cutter (See ''Segment cutters,'' p. 101.)

Analytical balance

Petri dishes, 10 cm.

Several beakers, 500-ml.

Protractor

Photographic paper and developer, if shadowgraphs are to be made to record stem curvatures

20-50 mg. of each compound to be evaluated

20 mg. of indole-3-acetic acid for standard

treatment

Sodium hydroxide

Potassium chloride

Distilled water

Suggested plant material—Stem segments of

etiolated pea plants (Pisum sativum L.) —

Alaska variety.

Preparation and selection of plant material—Soak

pea seeds in water for 6 hr. ; then plant them in

moist sand in a container that allows for drainage, and keep them in complete darkness. Increase sensitivity by daily exposure to 4 hr. of

red light from a 25-w. ruby bulb about 61 cm.

away. If an automatic timer for the light is not

available, use a single exposure to red light 32

hr. before harvest.

Harvest the plants under ordinary room light

after 7 or 8 days, when the fourth internode is

less than 5 mm. long. Use plants with a third

internode 4-7 cm. long and cut a 3.5-cm. segment 5 mm. below the top of the third internode. With a razor blade, split the segments
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 33

centrally for three-fourths of the length downward and drop the segments into water. The

segments may be washed for several hours in

tapwater but some investigators have found

this unnecessary. Discard the seeds and roots.

Procedure—Prepare several dilutions (distilled water) of the compound to be tested

(10"'-10~^ 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 segments to each dish and leave them

for 6-24 hr. in darkness. Curvature will be

complete at the end of 6 hr. 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 peas, for

76 hr. 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 segments in tapwater. Discard the

seeds and roots.

Place not more than 8 of the segments in 20

ml. of the solution to be tested. Use petri 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.

Some researchers find improved response

using a pH 7.0 phosphate buffer included at

3 X 10"^ M in each dilution tested. If compounds

are difficult to dissolve, 0.15 ml. of 2.5% sodium

hydroxide or 0.5 ml. of 95% ethanol may be

added to a 20-mg. sample before making to

volume.

To obtain the response, place 5 pea segments

in 25 ml. of test solution in each petri dish

overnight in the dark at room temperature.

Method of taking results—Lay the stem segments on photographic paper (protect the paper

from moisture with a piece of glass), expose

the segments and paper to light, and develop

the image on the paper using usual photographic

methods. Draw lines parallel with the tip and

base of one curved, split portion of segment.

Measure the angle a with a protractor. Draw

similar lines using the opposite segment, and

measure the angle that corresponds to a. Most

regulating compounds having cell-elongating

properties induce a decrease in the angle indicated compared with that formed by the seg- ,

ment 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 chemicals used,

see references listed at the end of this method.

Measure the angle a on each segment with the

protractor (fig. 13) 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:

C

"" Klo^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 segments are used, measure

the angles that develop during the 24-hr. period

immediately following the immersion of the segments in the test solution and compare the activity of different compounds.

The percent relative activity is obtained by

comparison of curvatures to a reference standard such as indole-3-acetic acid or alpha-naphthaleneacetic acid at the corresponding concentration. The percent relative activity is calculated by dividing the average curvature of the

test compound by that of the reference compound and multiplying by 100. The average

curvature for each compound is found by measuring both arms of 5 split segments and dividing by 10. The angle of curvature is measured

at the point of inflection between the inward

and outward curvature (angle a in fig. 13).

FIGURE 13.—Shadowgraph of split segment of pea stem

showing curvature that developed upon exposure to

growth-regulating chemical and method of measuring

angle of curvature (a). (Diagram suggested by F. W.

Went.)
34 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

The concentrations most suitable for the testing

of synthetic growth regulators are 10 and 100

p.p.m. or 10 and 200 p.p.m. Compounds vary as

to the concentration for optimum curvature and

toxicity is evidenced by limpness of the split

segments.

Suggested standard for comparison—10"^-10~^ M

indole-3-acetic acid.

References

Kent, Martha, and Gortner, W. A. Effect of Pre-illumination of the Seedlings to Red Light. Bot. Gaz. 112:

307-311. 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.

TOMATO HYPOCOTYL METHOD

Based on research by G. F. Pegg; I. D. J.

Phillips and R. L. Jones.

Description of method—Segments of etiolated

tomato hypocotyls are incubated with solutions

of compounds to be tested while revolving

slowly on a clinostat. Growth inhibition is assessed by comparing the linear extension of

the test segments with that of the controls.

This test can also be modified to measure activity of gibberellins by incubating the hypocotyl tissue in white light during the assay.

Apparatus, Chemicals, and Other Materials

Glass petri dishes, 10 cm. in diameter

Seed germination disks, 10 cm.

Humidity box with standing grill over free

water surface, approximate relative humidity 90%

Light-proof incubator with uniform temperature of 25° C.

Dark room

Red light source, Osram 6 R.K. 60-w. tungsten lamp

Forceps

Sheet of glass marked with 1-cm. divisions

Magnifying lens or bench binocular microscope

Single-edge razor blades

CameFs-hair brush

Glass combustion tubes with internal diameter less than 10 mm., 75 X 8 mm.

Plastic or rubber caps

Strips of Whatman No. 1 Chromatographie

paper, 64 X 13 mm.

Automatic pipette delivering 1.5 ml. solution

Analytical balance

Clinostat with drums to hold tubes

Constant temperature room with continuous

light at 700 foot-candles (warm white

80-w. fluorescent tubes)

Glass graticule with 1-mm. divisions

pH meter

Deionized water

250 mg. of 2,4-dinitrophenol, coumarin, or

gibberellic acid As for standard treatment

M/200 potassium hydrogen phosphate, citric

acid buffer at pH 7.0

1.0 g. of 2.4-dinitrophenol

0.5 g. of coumarin

0.5 g. of gibberellic acid As

Suggested plant material—Seeds of tomato

(Lycopersicon esculenfum Mill, cultivars Potentate and Ailsa Craig.)

Preparation and selection of plant material—Soak

virus-free tomato seeds for 3 hr. at 25° C. in

deionized water. Sow 80-90 seeds on a seedgermination disk in a petri dish. Sow seeds in as

many of these dishes as are required. Irrigate

each germination disk with 1.5 ml. deionized

water. Place the dishes in a humidity box maintained at about 90% relative humidity and

incubated at 25° C. for 116 hr. in total darkness. The etiolated seedlings will then be 12-15

mm. high. While using red light, remove seedlings of uniform size from the dishes with forceps and place them on a sheet of wet glass.

Use a magnifying lens or a bench binocular

microscope and a razor blade to remove carefully the apical meristem and cotyledons. Take

a 1-cm. segment, measured from the apex of

the hypocotyledonary arch to the taproot.'This

leaves a crozier-shaped segment (fig. 14) with

the terminal portion of the hook about 3 mm.

long. Float the segments on distilled water in

the dark for not more than 2 hr. until the test

is ready to be set up.

Procedure—Using red light, remove washed

segments from the distilled water with a paint

brush. Place 2 segments in a glass combustion

tube containing a strip of Whatman No. 1

Chromatographie paper washed with acetone

and then thoroughly dried. The filter paper

strip is included to reduce variables when chromatographed plant extracts and known regulating substances are compared. Pipette automatically into the tube 1.5 ml. of the solution

to be tested, made up in 200 M potassium hydrogen phosphate-citric acid buffer at pH 7.0. (See

buffer table, p. 129.) Place a plastic or rubber
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 35

cap over the end of the tube. Place the tubes in

racks and revolve them at 2 r.p.m. on a clinostat in a dark incubator for 48 hr. In a similar

way, set up segments for controls in plain buffer solution.

When working with crude plant extracts,

Chromatograph once or twice, the second time

with solvent ascending on Whatman No. 1 papers

25.4 cm. square. Cut the paper into 20 strips

of equal width (12.7 mm.), cutting parallel to

the line on which the samples were spotted.

Number each strip, starting at the end where

the samples were spotted. Divide each strip into

4 segments each 63.5 mm. long and number

these lA, IB, IC, ID, etc.; 2A, 2B, 2C, 2D, etc.,

making a total of 80 segments of the chromatogram. Fit each of these into a combustion tube

(described above). Add 1.5 ml. of the pH 7.0

buffer solution and 2 of the plant segments to

each tube. In this way the chromatographed

substances are eluted into the buffer solution

during rotation on the clinostat simultaneously

affecting the growth response of the hypocotyl

segments.

It is imperative that all pi'eparatory tvork

and subsequent incubation be carried out in air

cm.

FIGURE 14.—Etiolated tomato seedling (a) 48 hr. after

germination, showing only part of the taproot;

hypocotyl segment (b) prepared for bioassay with

root and cotyledons removed. Shaded portion denotes

meristematic region. (After diagram by G. F. Pegg.)

that is not contaminated with fuel gas, i,e,, in a

room free from gas fittings.

Where the test is designed to measure

growth-promoting activity oí solutions of giboerellms or of chromatographed plant extracts

containing gibberellm-like substances, set up

the plant segments as described, but incubate

them on the clinostat under continuous white

light of approximately 700 foot-candles using

warm white fluorescent tubes. A disadvantage

of this particular assay is that naturally occuring inhibitors must be removed first or they

will mask any gibberellin growth response.

Method of taking results—Carefully remove the

elongated segments from the tubes with forceps.

Place them on blotting paper and measure to the

nearest 0.5 mm. against a glass graticule. Calculate inhibition or stimulation as follows:

Final length of test segment .

Final length of control segment

= % growth inhibition or stimulation

The results may be conveniently expressed as

a histogram.

Suggested standard for comparison—Standard

treatment for growth inhibition, for example,

between 1 X 10' and 1 X 10^ M. 2,4-Dinitrophenol or 1 X 10"'-1 X 10- M coumarin. For

growth promotion, use 1 X lO""* M gibberellic

acid A-á. Noninhibited control segments usually

undergo a threefold extension in buffer solution.

Variation used by Phillips and Jones—Soak

dwarf pea seeds. Meteor variety, for 8-12 hr.

in tapwater and then sow them 6-8 cm. deep in

damp vermiculite contained in boxes. After

germinating the plants for 4 days at 25° C. in

darkness, sever the epicotyls from the cotyledons

and roots and wash the epicotyls with distilled

water. Pour 4 ml. of the test solution into each

of a number of small glass vials. Stand 10 epicotyls upright in each of the vials. Use a comparable setup with distilled water instead of

test solution for controls. Illuminate the epicotyl

segments with light from Daylite fluorescent

tubes at 20° C. for 3 days. Determine the mean

length of the epicotyls above the first node for

segments in each individual tube. Regard the

initial length of each epicotyl above the first

node as zero and use the final length above the

node as a measure of absolute growth over the

3-day incubation period.

This test is sensitive to a concentration of

10"^ mg. of gibberellic acid/liter of water. Under these conditions, the segments are insensitive to indole-3-acetic acid and there is no additive or synergistic interaction between indole3-acetic acid and gibberellic acid.
36 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

If paper chromatography is used in conjunction with this test, elute the desired segments

of the paper with water and subject the eluate

to the pea epicotyl test without placing the

paper segment in the tube. If thin-layer chromatography is used, place the silica gel containing a separated component in the tube with

water and the epicotyls.

The difference in the length of the treated

and control epicotyls at the end of 3 days is

used as a measure of activity of the sample in

terms of cell elongation.

References

Pegg, G. F. A New Method for Growth Assays: The

Extension Growth of Segments of Etiolated Tomato

Seedling Hypocotyls. Ann. Bot. (n.s.) 26: 207-218.

1962.

Pegg, G. F. The Effect of Selected Growth-Promoting

and Growth-Inhibiting Substances on the Extension of

Segments of Tomato Seedling Hypocotyls. Ann. Bot.

(n.s.) 26: 219-232. 1962.

Phillips, I. D. J., and Jones, R. L. Gibberellin-like Activity in Bleedmg-Sap of Koot Systems of Helianthu^

annuus Detected by a New Dwarf Pea Epicotyl Assay

and Other Methods. Planta 63: 269-278. 1964.

WHEAT COLEOPTILE STRAIGHT GROWTH METHOD

Based on research by F. Wightman; variations by C. R. Hancock, H. W. B. Barlow, and

H. J. Lacey; G. F. Pegg; and J. van Overbeek

and L. Dowding.

Description of method—The effects of compounds on cell elongation are measured by floating segments of wheat coleoptiles on aqueous

solutions of the chemical and comparing the

length of these with the length of segments

floated on distilled water.

Apparatus, Chemicals, and Other Materials

Filter paper, Whatman No. 3, 10 cm. in

diameter

Petri dishes, 10 cm. in diameter

Table space in a room with the temperature

controlled at 24°-26° C.

Constant humidity boxes with glass covers

Kodak Ruby Signal light filter or red incandescent lamp (50 w.)

Razor blades and cutting instrument. (See

''Segment cutters,'' p. 101.)

Small forceps or paint brush

Needle

Analytical balance

50-100 mg. of each compound to be evaluated

50-100 mg. of sodium 2,4-dichlorophenoxyacetate for standard treatment

Glass-distilled water

Suggested plant material—Germinating seeds of

wheat {Triticum aestivum L., also called T.

sativum Lam., and T. vulgäre Vill.) Eclipse

variety. Other varieties of wheat may also be

used for this test.

Preparation and selection of plant material—Soak

wheat seeds in distilled water for 2 hr. and then

place them with embryos facing upward on

moist filter paper m open petri dishes (30-40

seeds per dish). Allow the seeds to germinate

for about 66 hr. at 24^-26° C. Maintain a humid

atmosphere (80-90%) around the seeds by

placing the petri dishes in a constant humidity

box (fig. 15). Place the open dishes on a perforated metal shelf resting on the upper edges

of a metal water-containing tray in the bottom

of the box. During the first 50 hr. of growth,

expose the seeds to light from the red incandescent lamp or to light passed through a ruby

red filter. The red light should be about 60 cm.

from the seeds. Allow subsequent growth of the

plants to take place in darkness.

Approximately Q^ hr. after placing the seeds

on the moistened filter paper, select plants with

coleoptiles 17-20 mm. long. Then, with the aid

of the cutting instrument excise 1 segment

10 mm. long from the upper-middle region of

each coleoptile. Use the segment cutter designed

so the apical 3-mm. segment of each coleoptile

is removed and left in the instrument, while the

next 10-mm. segment of the coleoptile is excised

by the razor blades and forms the test segment.

Remove the primary leaf by threading the segments onto fine glass capillaries, 2 segments per

capillary, and then allow the segments to float

on distilled water in a petri dish until required

for the experiment. This pretreatment period

should not exceed 3 hr.

In other methods, the 15-mm. segment used

as the test segment is measured, starting 2 mm.

from the tip. The tip segment is again removed

to eliminate, as far as possible, the effects of

endogenous auxin.

Procedure—Prepare solutions representing a

dilution series of the compound to be tested by

diluting a concentrated stock solution of the

compound with distilled water; suggested concentrations are 0.01, 0.1, 1, 10, and 100 p.p.m.

Place about 20 ml. of each concentration in

respective petri dishes and float 10 coleoptile

segments on the surface of the liquid in each

dish. Allow the segments to grow for 24 hr. in

darkness at 24°-26° C. Float additional segments on distilled water and use these as

controls.

Method of taking results—Remove segments

from the petri dishes with small forceps or
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 37

i JIH ■■^■■■■l

iiixîooooooQr

IIM^I■■ p

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'i^Kf#C X j( )( j( JC tC ML^^r' -

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BN-28931

FIGURE 15.—Constant humidity box containing germinating wheat seeds in open petri dishes. Note water-containing

tray at bottom of box and strips of soft rubber around upper edges of box to provide good seal with hinged

plate-glass top. All inside surfaces of box are coated with several layers of paraffin wax. (Photo—courtesy of

F. Wightman.)

paint brush and blot them dry. Measure each

segment to the nearest 0.5 mm. under a glass

graticule, or place these segments on a slide in

a photographic enlarger and measure the projected shadow. Compare the mean elongation

of segments in the different solutions with that

of the control segments in distilled water. Express the results as a percentage. Thus,

final length of treated segments (mm. X 100)

final length of water control (mm.)

Invert the slide of coleoptiles on the blades

of the cutter (fig. 16), keeping the tips against

the guide block, and then exerting a gentle

downward pressure. (See also "Barlow cutter,"

p. 101.) Remove the unwanted 3-mm. tips thus

cut from the coleoptiles by passing a needle

along the outside of the 2 blades. Lift off the

10-mm. segments by inserting the needle between the blades and lifting with the slide. Retain only the cut segments that are completely

filled with leaf tissue. Place the segments in

bundles of 3 or 5 on moist filter paper in closed

petri dishes, in chronological order, and store

at 5° C.

Place 3 or 5 coleoptile segments and 0.5 ml.

of solution to be tested in a specimen tube 50 X

8 mm., again using the coleoptiles in chronological order. Place these tubes in a rack (fig.

17). Place one or several of these racks filled

with tubes in a clinostat (fig. 18). Rotate the

tubes horizontally about their long axis for 17-

20 hr. at 20° C. and at about 180 r.p.h. It is

preferable to leave the tubes uncorked, in which

case the open ends must be prevented from

touching the ends of the frame or the base of

the next rack. Remove racks of tubes from
38 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OP AGRICULTURE

clinostat in the constant-temperature room to

the laboratory. Shake the coleoptile segments

or draw them up with a needle onto the side of

the tube, clear of the solution or Chromatographie paper if present. Measure them directly

through the wall of the tube with a ruler to the

nearest 0.5 mm. and record the increase over

the original length.

Van Overbeek and Dowding followed this

same method of taking results but used segments of oat leaf bases for assaying gibberellins. The tubes are rotated for 48 hr. and the

solutions preferably contain 2% sucrose.

Suggested standard for comparison—Solution

containing 1 p.p.m. of sodium 2,4-dichlorophenoxyacetate.

Variation used by Hancock, Barlow, and Lacey—

Obtain feldspar chips of a size that will pass

through a 2.5-mm. mesh screen but will be retained by a 1-mm. mesh screen. Wash the feldspar and ovendry it. Soak the seeds for 1 hr.

in a shallow layer of tapwater at 20° C. Place

a measured amount of the feldspar in an aluminum dish, add a measured quantity of seeds.

cover these with a measured amount of the

feldspar to make a layer about 3 times the

width of the seeds, and add a predetermined

volume of water sufficient to moisten the feldspar. Cover dishes with glass sheets and maintain .them at 20° C. for 80 hr. in darkness. Sow

in white light but, for further manipulations,

use phototropically inactive safe lights (60-w.

bulb behind a Kodak filter OA or OB).

Remove the glass covers when the coleoptiles

are just showing through the feldspar. Add sufficient water at room temperature to replace

that evaporated, and then let dishes stand about

17 hr. until the coleoptiles are about 20-30 mm.

long. Sever coleoptiles from the seeds with a

scalpel and place them in rows on wet microscope slides, keeping them in the chronological

order in which they were cut. Arrange coleoptiles at right angles to, and with their tips

coincident with, one edge of the slide. Place the

slide against the zero line of an aluminum

grader scribed at 5-mm. intervals. Place coleoptiles with bases falling between any 2 adjacent

lines in one length grade, maintaining chronological order, and "line them up" as before to

cut a 10-mm. segment from them.

BN-28932

FIGURE 16.—Segments of coleoptiles have been cut by pressing slide carrying 20 coleoptiles against 2 razor blades.

Tips of the coleoptiles rest against the stainless steel guide block. (Photo—courtesy of Journal of Experimental

Botany.)
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 39

BN-28980

FIGURE 17.—Rack to cany 100 tubes. Note polythene above middle rigid sheet, by which the tubes are gripped when

being used uncorked. (Photo—courtesy of Journal of Experimental Botany.)

BN-28929

FIGURE 18.—Clinostat in which 4 racks of tubes are rotated. Driving motor is at right; adjustable balancing arm at

left. (Photo—courtesy of Journal of Experimental Botany.)
40 AGRICULTURE HANDBOOK 380, U.S. DEPARTMENT OF AGRICULTURE

Variation used by Pegg—Place 2 coleoptile segments in a glass tube 80 X 15 mm. containing

1 ml. of solution to be tested. Seal with a closefitting plastic or rubber cap. Place the tubes

horizontally on a clinostat or circular drum revolving at 2 r.p.m. in total darkness at 25° C.

Allow the segments to grow for 24 hr. Prepare

10 tubes for each concentration to be tested and

10 tubes containing distilled water and control

segments. Measure responses as described by

Wightman.

References

Hancock, C. R., Barlow, H. W. B., and Lacey, H. J. The

East Mallinie Coleoptile Straight Growth Test Method.

Jour, of Expt. Bot. 15: 166-176. 1964.

Variation submitted by G. F. Pegg, Department of Botany, Wye College, University of London, near Ashford, Kent, England.

Van Overbeek, J., and Dow^ding, L. Inhibition of Gibberellin Action by Auxin. Plant Growth Regulation,

pp. 657-663. Iowa State Univ. Press. 1961.

Method submitted by F. Wightman, Department of

Biology, Carleton University, Ottawa, Canada.

Clinostat designed to hold test tubes rotating

at 1 r.p.m. at 25° C. in darkness

Volumetric flasks, 100 ml.

Test tubes, 7.5 X 1.3 cm.

Analytical balance

3-100 mg. of the substance to be evaluated

250 mg. of kinetin or gibberellic acid Aa for

standard treatment

Distilled water

Suggested plant material—Germinating wheat

seeds (Triticum vulgäre Vill. Eclipse variety).

Preparation and selection of plant material—Soak

wheat seeds in distilled water for 2 hr. Sow

them on moist seed-testing paper in petri dishes,

keeping the embryos oriented uppermost. The

seeds should be sown at a density of 50 per dish

and placed in a constant humidity box (80-85%

relative humidity) for 24 hr. at 23° C. in darkness.

YOUNG WHEAT COLEOPTILE METHOD

Based on research Inj S. T. C. Wright.

Description of method—Young, whole coleoptiles are excised from germinating wheat seeds

24 hr. after sowing. The coleoptiles are floated

on aqueous solutions of the compounds to be

tested and their length is measured before and

after treatment. The growth of the treated coleoptiles is compared with that of untreated ones

floating on distilled water. Cell elongation using

this method is stimulated by gibberellins and

cytokinins, but not by auxins.

Apparatus, Chemicals, and Other Materials

Petri dishes, 10 cm. in diameter

Constant humidity box in a room with the

temperature controlled at 23^-24° C.

Forceps

Scalpel

Binocular dissecting microscope, low power,

10 X magnification

Whatman Seed Testing Papers, 9 cm. in diameter

Microscope slides

Eyepiece micrometer, 10 mm. with 100 divisions

CameFs-hair brushes

Pieces of filter paper, Whatman No. 1, 5 X

2.5 cm.

Parafilm

Procedure—Using fig. 19 as a guide, excise the

coleoptiles with a scalpel under a binocular

dissectmg microscope. Place the coleoptiles, with

their enclosed primary leaves, on filter paper

moistened with distilled water until a sufficient

number of them have been cut for the experiment. Arrange the coleoptiles in groups of 5

on microscope slides and measure them under

the binocular dissecting microscope with a

micrometer eyepiece. Carefully transfer each

group of 5 coleoptiles with a camel's-hair brush

to a strip of filter paper and insert them into a

test tube containing 0.6 ml. of the test solution.

The purpose of the filter paper is to prevent

drying of the coleoptiles on the sides of the

tubes. Use 5 replicates of 5 coleoptiles per treatment, including the water controls. Seal the

tubes with Parafilm and rotate them in a horiFiGURE 19.—Germinating wheat seed showing coleoptile

(c) and plane of incision (broken lines) used to remove coleoptile. (After diagram by S. T. C. Wright.)
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 41

zontal position at 1 r.p.m. on a clinostat in

darkness at 25° C. for 24 hr. Remove the coleoptiles from the tubes and measure them again.

Method of taking results—Calculate for each

tube the mean length of the 5 coleoptiles before

and after treatment and from these 2 figures

obtain the percentage increase in the mean

length of the 5 coleoptiles. Determine the average of these percentages for the 5 replicate

tubes. Compare the average growth stimulation

produced by the substance under evaluation

with the average growth of the coleoptiles in

the test tubes containing distilled water.

Suggested standard for comparison—Gibberellic

acid at a concentration of 10"^"* M (i.e., 3.46 mg./

liter) and kinetin at a concentration of 10"^ M

(i.e., 21.5 mg./liter). Dosage response curves

may be prepared using the following concentrations: for gibberellic acid—10"*\ 10 ^ 10% 10 ^

10-, 10-^ M; for kinetin—10 \ 10 ", 10 ^ 10 •,

10 ^ and 3.16 X 10"^ M.

Reference

Wright, S. T. C. A Sequential Growth Response to Gibberellic Acid, Kinetin and Indolyl-3-Acetic Acid in the

Wheat Coleoptile (Triticum vulgäre). Nature 190:

699-700. 1961.

CELL ENLARGEMENT

CITRUS PETAL METHOD

Based on research by E. E, Goldschmidt and

S. P. Monselise.

Description of method—The degree of curvature of detached citrus petals is used to measure

the indole-3-acetic acid content of the agar medium in which the petals are implanted. This

method can also be used to detect and measure

other synthetically prepared growth-regulating

substances and has been used to assay extracts

of citrus tissue.

Apparatus, Chemicals, and Other Materials

Petri dishes, 5 cm. in diameter

Glass bell jars or other watertight containers

to enclose dishes and maintain humid atmosphere

Constant temperature chamber, about 20° C.

Razor blade

Protractor or other device to measure angle

of curvature

Analytical balance

0.2-1.0 g. of indole-3-acetic acid for standard

treatment

Agar

Sucrose

Suggested plant material—Citrus flowers, collected just before the petals open. Different species and varieties of the Citms genus may be

used.

Preparation and selection of plant material—After the agar preparations and equipment are

ready to use, collect citrus flowers at the stage

at which the petals would normally open within

a few hours if the buds were left on the tree.

This stage of development is indicated by the

separation of one of the petals from the others

(fig. 20, &).

Sever the petals of several flowers at their

juncture with the floral axis. Make a random

sample of the petals collected by mixing them

thoroughly.

Procedure—Prepare 1% agar. Add an amount

of sucrose equal to 3% of the weight of the

agar mixture. Place 5.0-6.0 ml. of this agar

mixture in each of 5 petri dishes. Use this as

the control medium to measure variation in the

behavior of untreated petals. Prepare appropriate concentrations of the compound or plant

extract to be tested by dissolving the required

amount of the compound or extract in measured

amounts of the agar-sucrose mixture. Use 5

replicate dishes for each concentration.

An alternative method is to pour 1 ml. of

solution containing the compound or extract to

be tested over the surface of a petri dish after

the agar-sucrose mixture has solidified in the

dish. Rotate the dish so that the added solution

wets the entire surface of the agar. Repeat to

make 5 replications of each treatment.

Implant 6 petals into the agar in each dish so

that their bases are embedded to a depth of

about 2 mm. and the petals are held upright

(fig. 20, a). Arrange the petals with their

ventral side facing the center of the dish so

they can curve freely. Place the dishes in a

humid atmosphere at 20° C. in either darkness

or continuous light for 22-24 hr.

Method of taking results—After 22-24 hr. under the conditions described above, measure the

degree of curvature (fig. 21, angle a) with a

protractor. The curvature produced is proportional, within limits, to the concentration of the

growth-regulating compound in the agar.
42 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-28928

FIGURE 20.—Arrangement of citrus blossom petals Implanted in agar in a petri dish (a) and citrus blossom (6) at

stage of development suitable for collection of petals.

\

\

FIGURE 21.—Angle (a) used to measure curvature response of citrus petals to growth substances.

Suggested standard for comparison—Solutions of

indole-3-acetic acid at a concentration of 0.02-

200 p.p.m. used at the rate of 1 ml. per dish. If

the test compound is mixed with the agar during preparation of the agar-sucrose medium,

use equivalent amounts of the test compound.

Reference

Goldschmidt, E. E., and Monselise, S. P., Citrus Petal

Bioassay Based on the Indolyl-3-acetic Acid Effect on

Flower Opening. Nature 212: 1064-1065. 1966.

LEAF DISK ENLARGEMENT METHOD

Based on research by C. 0. Miller.

Description of method—Etiolated disks from

bean leaves are placed on filter paper moistened

with nutrient solution in which the compound

to be tested is dissolved. Increase in the diameter of the disks when these are kept in darkness is used as a basis for measurement of

growth-regulating activity. This method is used

to detect kinetin and cytokinins.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 43

Apparatus; Chemicals, and Other Materials

Coarse sand

Dark room with temperature controlled at

about 25° C.

Cork borer, 5 mm. in diameter

Analytical balance

Green safelight of low intensity

Petri dishes

Filter paper disks, Whatman No. 1, 9 cm.

Dissecting microscope with eyepiece micrometer

KNOs

250 mg. of kinetin or gibberellic acid A:; for

standard treatment

D-glucose

Distilled water

Suggested plant material—Dwarf stringless

greenpod beans {Phaseohis vulgaris L.).

Preparation and selection of plant material—

Plant the seeds in coarse sand and germinate

them in a dark room at about 25' C. for 7-9

days until the primary leaves are about 2 cm.

sq. in area. Cut disks 5 mm. in diameter with a

cork borer from the 2 primary leaves of the

etiolated plants. Cut 2 disks from each leaf, one

from the basal section on each side of the midvein. A section of a main lateral vein approximately bisects each disk. Perform all manipulations involving the disks in dim green light.

Procedure—Make a basal nutrient solution that

contains 3% D-glucose by weight and 0.08 M

KNO3. The pH of this solution should be approximately 5.6. Add 5 ml. of the test solution

to a petri dish containing 3 disks of the filter

paper. Place 10 of the leaf disks, lower epidermis up, on the pad of filter paper, wetted

with the test solution. Maintain the petri dishes

containing the leaf disks in a dark cabinet at

24°-26" C.

Marked disk expansion is induced by exposing leaf disks on nutrient solution only to 200

foot-candles of light from an incandescent filament for 15 minutes before placing the disks

in a dark cabinet. Short exposures to red light

are also very eñ'ective; such exposures are reversed by far red. Co(NO;])2 • 6H2O will also

accelerate leaf disk expansion when the salt is

added to the nutrient solution in Co'" concentrations of 2-5 p.p.m. (about 25 mg./liter of

Co(N03)2»6H20).

Method of taking results—Three days after beginning the experiment, measure the diameter

of each disk. Measure at right angles to the

heavy veins that bisect each disk. Compare the

increase in diameter of the disks on the paper

impregnated with nutrient containing the test

solution with disks on paper impregnated with

the nutrient solution only. Increase in diameter

of disks indicates amount of growth regulator

activity.

Suggested standard for comparison—Kinetin and

gibberellic acid (at about 5 mg./liter).

References

Miller, C. O. Promoting? Effect of Cobaltous and Nickelous Ions on Expansion of Etiolated Bean Leaf Disks.

Arch. Biochem. and Biophys. 32: 216-218. 1951.

Miller, C. O. Similarity of Some Kinetin and Red Light

Effects. Plant Physiol. 31: 318-319. 1956.

CHLOROPHYLL RETENTION

BARLEY LEAF METHOD

Based on research by H. Kende.

Description of method—Segments from leaf

blades of young barley plants are conditioned

and then floated on the solution being tested for

the presence of substances with kinetin-like activity. The segments are then extracted with

ethanol and the chlorophyll lost during the storage period is measured in terms of optical

density. Retention of chlorophyll by the leaf

segments compared with loss of chlorophyll

from leaf segments floated on water represents

the kinetin activity in the sample of the solution being tested.

Apparatus, Chemicals, and Other Materials

Razor blade

Beakers, 500 ml.

Screw-cap vials

Densiometer

Analytical balance

250 mg. of kinetin for standard treatment

Penicillin G

80% ethanol

Suggested plant material—Barley plants (Hordeum distichon L.) about 2 wk. old.

Preparation and selection of plant material—Germinate barley seeds in soil at 20° C. under continuous illumination from a fluorescent light for
44 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

13 days. Select plants for uniformity and cut a

segment from each first leaf. Take a 1-cm. segment from the leaf, severing the blade so the

distal end of the segment is about 3 cm. from

the tip of the leaf. Age the segments for 24 hr.

while ñoating them on distilled water at 25° C.

in darkness.

Procedure—Remove the leaf segments from

the aging bath, blot them, and transfer 4 segments to each of 3 vials, each vial containing

1 ml. of test solution and 250 units of penicillin G.

Method of taking results—At the end of 48 hr.

of incubation at 25° C. in darkness, remove

each group of 4 segments from each vial separately.

Extract each group with 5 ml. of 80% ethanol. Measure the optical density of each extract

using a wavelength of 665 millimicrons. Compare the optical densities obtained with those

of a standard curve made by subjecting barley

leaves under comparable conditions to amounts

of kinetin varying from 0.003-3.0 micrograms/

ml. Express the results in terms of equivalent

amounts of kinetin.

Suggested standard for comparison—Kinetin in

concentrations ranging from 0.003 to 3.0 micrograms/ml.

Reference

Kende, H. Preservation of ChlorophyH in Leaf Sections

by Substances Obtained from Root Exúdate. Science

145: 1066-1067. 1964.

COCKLEBUR LEAF DISK METHOD

Based on research by Daphne J. Osborne and

D. R. McCalla.

Description of method—Disks cut from leaves

at a specific stage of senescence are placed on

filter paper. Known amounts of kinetin are

added to the filter paper as standards. Comparable disks are placed on filter paper moistened with the solution being tested or on a

moistened segment of chromatogram containing

separated components of an extract. The presence of kinetin-like compounds is indicated by a

suppression of yellowing due to the retardation

of chlorophyll degradation in the leaf disks. The

quantity of these compounds present is estimated by comparing the loss of chlorophyll in

disks in contact with the unknown sample with

the loss in disks in contact with known amounts

of kinetin. The test is quantitative over the

range from 0.05-5.0 micrograms of kinetin. It

is especially useful in testing zones cut from

paper chromatograms and may also be used for

materials in solution.

Apparatus, Chemicals, and Other Materials

Temperature-controlled room

Means of supplementing daylight with incandescent light to obtain a 20-hr. photoperiod

Plexiglas boxes for storage of leaves

Means of supplying leaves with light from

incandescent lamp (50 foot-candles)

Filter paper disks, Whatman No. 1, 4.25 cm.

in diameter

Petri dishes, 5 cm. in diameter

Cork borer, 12 mm. in diameter

Graduate cylinder, 100 ml.

Large petri dish in which 5-cm. petri dishes

can be stored

Enamel trays in which large petri dishes can

be stacked

Aluminum foil

Dark area at 24° C.

Graduated centrifuge tubes

Water bath

Beckman spectrophotometer. Model B

250 mg. of kinetin for standard treatment

Ethyl acetate

Acetic acid

80% ethanol

Suggested plant material—Cocklebur (Xanthium pennsylvanicum Wall.). Other kinds of

senescent leaf tissues may be used satisfactorily

in this method as an assay for cytokinins, for

example, radish (Raphanus sativus L.), wheat

(Triticum sativum Lam.), and barley (Hordeum vulgäre L.), but these are less sensitive

than cocklebur.

Preparation of plant material—Grow the cocklebur plants from seed in the greenhouse at temperatures from 20°-30° C. Supplement daylight

with incandescent light to give a photoperiod of

20 hr. in order to maintain plants in the vegetative condition. After the plants have produced

10 leaves, remove the fifth fully expanded leaf

(counting back from the apex) leaving the petiole attached. Store the detached leaves with only

their petioles under water at low light intensity

(50 foot-candles from an incandescent lamp).

Enclose the leaves in Plexiglas boxes during this

storage period and maintain a temperature of

20° C. After 3 days under these conditions, during which the first stages of senescence take

place, select for experimental use those leaves

whose blades are uniformly pale green.

Procedure—Prepare kinetin standards at a

concentration of 0.063-16 mg./liter. Apply 0.5

ml. of each of these concentrations to the cor-
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 45

responding disks of filter paper in the petri

dishes. Prepare control filter papers in similar

dishes and add 0.5 ml. of distihed watex to

each dish.

To test chromatographed plant extracts, segment the chromatogram into strips not less than

12 mm. in the narrow dimension. Cut the long

dimension of the strips to 4.25 cm. and place

each chromatographed segment in a petri dish.

Moisten each segment of the chromatogram by

adding 0.5 ml. of distilled water to each dish.

To avoid toxic materials, make sure that Chromatographie paper used for separating components of plant extracts is washed thoroughly

before the separation. Use ethyl acetate, acetic

acid, water (10:5:2, v/v) for this preliminary

wash and glass-distilled water for the second

wash.

Cut uniform disks 12 mm. in diameter from

interveinal blade tissue of the detached leaves

previously aged for 3 days as described above.

(Sixty disks may be obtained from each suitable leaf.) Place 4 of these disks, or a larger

number of smaller disks, in each petri dish so

they are resting with their abaxial surfaces in

contact with the filter paper disks or the chromatogram segments.

Extract duplicate sets of 4 (or more) leaf

disks from those originally obtained by boiling

them in 80% ethanol. Retain the extract in the

dark for use in determining the original chlorophyll content of the disk.

Place each 5-cm. petri dish with its enclosed

papers and disks on 2 layers of damp filter

paper in a larger petri dish. Close the larger

dishes and stack them in enamel trays lined

with damp filter paper and cover the entire

assembly with aluminum foil. Maintain them in

darkness for 48 hr. at 24° C. At the end of this

period, examine the leaf disks. The control leaf

disks should be yellow or yellow green, and the

chlorophyll almost completely degraded; disks

in the higher concentrations of kinetin should

still be green. If this difference does not exist,

prolong the storage period.

Drop each group of disks from the various

dishes into corresponding graduated centrifuge

tubes with each tube containing a 5-6 ml. of hot

80% ethanol. Boil the ethanol in each tube

gently on a water bath until the chlorophyll has

been extracted. Cool the tubes and adjust the

volume to 10 ml., using 80% ethanol. Measure

the optical density of each solution with a Beckman spectrophotometer Model B (or other suitable instrument) at 665 millimicrons, the absorption maximum in the red region for chlorophyll a.

Method of taking results—Compare graphically

the loss of chlorophyll in leaf disks exposed to

various concentrations of kinetin with the loss

of chlorophyll from leaf disks exposed to the

unknown components of an extract or paper

chromatogram. The retention of chlorophyll

bears a hnear relation to the logarithm of the

kinetin concentration over a range of approximately 0.05-5.0 micrograms (0.1-10 mg./liter).

Indole-3-acetic acid and other synthetic regulating compounds tested so far have not retarded chlorophyll loss in this test.

High concentrations of gibberellin Aa show

slight activity.

Suggested standard for comparison—Kinetin in

concentrations ranging from 0.063-16 mg./

liter.

Reference

Osborne, Daphne J., and McCalla, D. R. Rapid Bioassay

for Kinetin and Kinins Using Senescing Leaf Tissue.

Plant Physiol. 36: 219-221. 1961.

DOCK LEAF DISK METHOD

Based on research by Pamela Whyte and L. C.

Luckwill; R. A. Fletcher and Daphne J. Osborne.

Description of method—To test for the presence

of gibberellin-like hormones, disks cut from

dock leaves are placed on filter paper moistened

with a solution of the compound to be tested.

Comparable disks are placed on paper moistened

with distilled water and all the disks are kept

in darkness for 4 or 5 days. The chlorophyll content of the treated disks is then compared with

that of the control disks. The amount of chlorophyll retained by the treated disks is proportional, within certain limits, to the logarithm

of the amount of gibberellin-like substance present. This test is extremely sensitive to small

amounts of gibberellic acid As.

Apparatus, Chemicals, and Other Materials

Temperature-controlled room, 25° C.

Clay pots

Greenhouse space

Filter paper

Small glass dishes

Petri dishes

Spectrophotometer

Cork borers, 2.5 cm. and 7 mm. in diameter

Pipettes, 0.1-ml. graduations

100-500 mg. of gibberellic acid A.i for standard treatment

Methanol or acetone

Distilled water
46 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Suggested plant material—Broadleaf dock

(Rumex obtusifolius L.).

Preparation and selection of plant material—

Propagate, clonally, dock plants in soil contained in clay pots under greenhouse conditions.

Select the oldest leaves that are still uniformly

green. Detach these leaves and immerse their

petioles in water for 24 hr. in darkness.

Procedure—Cut disks 2.5 cm. in diameter from

filter paper. Place each of these in a small

glass dish. Moisten each filter paper disk with

0.3 ml. of the test solution. Cut 4 leaf disks, 7

mm. in diameter, each from a separate leaf.

Place these 4 leaf disks on one filter paper disk,

the abaxial surfaces down. Repeat the procedure, using separate dishes and distilled water

rather than the test solution to moisten the

filter paper disks. Make 4 replications of this

arrangement. Place the small glass dishes containing the filter paper and leaf disks in petri

dishes lined with moistened filter paper and

incubate at 25° C. in darkness. Place the small

dishes containing the treated disks and those

containing the control disks in separate petri

dishes.

Method of taking results—When the chlorophyll

has almost disappeared from the control leaf

disks—usually after 4 or 5 days—place the 4

leaf disks of each replicate separately in 6 ml.

of methanol or acetone. Extract the chlorophyll

at room temperature overnight and determine

the optical density of the resulting solutions at

665 millimicrons (chlorophyll a) with the spectrophotometer. Express the chlorophyll content

of the treated leaf disks as percentage of that

of the control.

Suggested standard for comparison—Gibberellic

acid Aa 0.00001-0.1 microgram/ml.

Variation used by Fletcher and Osborne—Grow

dandelion (Taraxacum) plants under greenhouse conditions or use plants growing outdoors. Detach mature, fully expanded leaves

that are uniformly pale green. If young leaves

are used, place them under a bell jar with petioles in water to ''age." Keep the leaves in this

condition until their glossy green color fades

to a uniformly pale green. Cut interveinal portions of convenient diameter from the leaves

with a cork borer. Extract duplicate sets of

disks at once with hot 80% ethanol. Store this

extract in darkness for subsequent determination of the original chlorophyll content. Place

other matched sets of disks in petri dishes on

filter paper moistened with either water or the

test solution. Also use a graduated series of

gibberellic acid concentrations for comparison.

Do not use too much solution to moisten the

filter paper; e.g., 0.8 ml. of solution per 7-cm.

diameter filter paper is satisfactory. Place the

abaxial surface of the leaf disks in contact with

the moist filter paper. Line the covers of the

petri dishes with moistened filter paper. Place

the closed petri dishes containing the leaf disks

in a plastic box with high humidity and store

in darkness at 25° C. for about 4 days.

Place each set of disks separately into test

tubes containing hot 80% ethanol and boil

gently. Stopper the tubes and transfer them to

an incubator maintained at 37° C. overnight to

complete the extraction of chlorophyll. Less

time is required by boiling the disks in the

ethanol until all the chlorophyll has been extracted. Bring the chlorophyll extracts to a

uniform volume with 80% ethanol and determine the absorbency of each solution and that

of the initial controls at 665 millimicrons with a

suitable colorimeter.

References

Whyte, Pamela, and Luckwill, L. C. A Sensitive Bioassay

for Gibberellins Based on Retardation of Leaf Senescence in Rumex obtusifolius (L.). Nature 210: 1360.

1966.

Fletcher, R, A., and Osborne, Daphne J. A Simple Bioassay for Gibberellic Acid. Nature 211: 743-744. 1966.

WHEAT LEAF SENESCENCE METHOD

Based on research by S. T. C. Wright.

Description of method—Detached wheat leaves

age and turn yellow more quickly when floated

on water than when floated on solutions of cytokinins. The degree of senescence is determined by extracting the leaves with ethanol and

estimating the chlorophyll content spectrophotometrically 4 days after detachment.

Apparatus, Chemicals, and Other Materials

Forceps

Vermiculite, No. 4

Plastic seed trays, 36 cm. long X 23 cm. wide

X 5 cm. deep

Growth room with a 15-hr. photoperiod (500

lumens/sq. ft. from fluorescent tubes giving ^'natural daylight'') at 23° C.

Incubator at 25° C.

Cm. rule

Scissors

Cutting board

Analytical balance

Volumetric flasks, 100 ml.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 47

Petri dishes, 10 cm. in diameter

Pyrex test tubes, 10 X 1.5 cm.

Pipette, 10 ml.

Test tube wire baskets, divided

Thermometer, 0°-100° C.

Suitable vessel for water bath

Glass marbles

Stop watch

Bunsen burner

Black cloth

Spectrophotometer or colorimeter capable of

measuring light absorption at a wavelength of 665 millimicrons

80% ethanol

250 mg. kinetin for standard treatment

3-100 mg. of compound to be evaluated

Distilled water

Suggested plant material—Seedlings of wheat

(Triticum vulgäre Vill.) Eclipse variety.

Preparation and selection of plant material—Soak

wheat seeds in distilled water for 2 hours. Using

forceps, sow the seeds in moist vermiculite in

the seed trays with the embryos oriented uppermost and with about 1.5 cm. between the seeds

and rows. Cover the seeds with a thin layer of

vermiculite and compress the surface with a

firming board. Place the trays in the growth

room for 7 days.

Procedure—Excise the shoots (approximately

11 cm. long) with scissors, cutting just above

the soil level, and line them up on a cutting

board with their tips against a wooden guide.

Sever the shoots 7.5 cm. from the tips. This

severed portion of the shoot comprises mainly the

first leaf but clasped within it are portions of

other leaves. Use only the first leaf ; remove and

discard the others. Weigh the leaves in batches

of 3 to the nearest mg. and ñoat each batch, with

their adaxial surfaces uppermost, on 10 ml. of

test solution contained in a petri dish. Use 3

replicate dishes for each treatment and 3 dishes

containing distilled water as controls. Extract 3

sets of leaves from untreated plants immediately in boiling 80% ethanol (as described be-^

low) and store the extracts in darkness at 2° C.

for determining the original chlorophyll content of the leaves. Maintain the petri dishes at

25° C. for 4 days in darkness.

Method of taking results—At the end of the 4-

day treatment period, transfer the leaves from

each petri dish to a test tube and pipette 10 ml.

of 80% ethanol into the tube. Stopper the test

tubes with glass marbles to prevent evaporation. Place the tubes in a vertical position in a

test tube wire basket. Immerse the basket containing the tubes in a water bath. Bring the

water bath temperature up to 80° C. and hold

at this temperature for 10 minutes. Remove the

tubes containing the boiling ethanol from the

water bath. Since chlorophyll extracts fade

quickly in bright sunlight, cover the tubes with

a black cloth while they are cooling. Measure

the optical density of each solution against 80%

ethanol (using a 0.5-cm. cell) in a spectrophotometer at 665 millimicrons, the absorption

maximum in red light of chlorophyll a. Express

the results as optical density/mg. of leaf.

Suggested standard for comparison—10"^ M solution of kinetin (21.5 mg./liter). A dosage response curve can be prepared by usino: the following concentrations of kinetin: 10"^, 10"^,

10«, 10 ^ 10-4, and 3.16 X 10-4 M.

Reference

Method submitted by S. T. C. Wright, Plant Growth

Substance and Systemic Fungicide Unit, Agricultural

Research Council, Wye College, Near Ashford, Kent,

England.

EFFECT OF ONE COMPOUND ON THE ABSORPTION AND

TRANSLOCATION OF ANOTHER

BEAN STEM CURVATURE METHOD

Based on research by A. S. Crafts; J. W.

Mitchell, W. M. Bugger, Jr., and H. G. Gauch.

Description of method—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.

Apparatus, Chemicals, and Other Materials

See p. 11.

Compounds to be tested as adjuvants are

also required.

500 mg. of ammonium 2,4-dichlorophenoxyacetate for standard treatment

Suggested plant material—See p. 11.
48 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Preparation and selection of plant material—See

p. 11.

Procedure—Using the ''Bean stem curvature

method/' p. 12, determine the amount of ammonium 2,4-dichlorophenoxyacetate or other

water-soluble salts of the acid required to induce a stem curvature of about 5°-10° in 21/9-3

hr. 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 /xg. of the salt per 0.02 ml. of solution.

After determining the threshold concentration, 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 p. 12.

Method of taking results—Determine the effect

of the adjuvants on the magnitude of stem curvature induced in 2i/^-3 hr. 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.

Suggested standard for comparison—0.8, 0.9, or

1.0 fjLg. of ammonium 2,4-dichlorophenoxyacetate or other water-soluble salt applied in distilled water alone.

References

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

Mitchell, J. W., Dugger, V^. M., Jr., and Gauch, H. G.

Increased Translocation of Plant Growth-Modifying

Substances Due to Application of Boron. Science 118:

354-355. 1953.

ENZYME PRODUCTION

BARLEY ENDOSPERM METHOD

Based on research by J. E. Varner and G. R.

Chandra ; R. L. Jones and J. "E. Varner.

Description of method—Endosperm of barley

seeds releases alpha amylase and other enzymes

in the presence of gibberellic acid As and some

gibberellin-like substances. The amount of enzymes released is measured in terms of amylolytic activity, which is proportional, within limits, to the logarithm of the concentration of

gibberellic acid As or gibberellin-like substances

present.

Apparatus, Chemicals, and Other Materials

Razor blade

Petri dishes, 10-cm.

Sand

Erlenmeyer flasks, 25-ml.

Pipettes, 1- and 10-ml. volume

Autoclave

Shaker apparatus

Centrifuge

Centrifuge tubes, 100 X 7.5 mm.

Spectrophotometer

Sodium hypochlorite, 1% (commercial

bleach)

500 ml. of acetate buffer (pH 4.8)

10 g. of calcium chloride

1 g. of chloramphenicol

5 g. of potato starch (nonsolubilized)

10 g. of potassium phosphate

5 g. of potassium iodide

1 oz. of hydrochloric acid

5-10 g. of iodine

1 p.p.m. of gibberellic acid As solution for

standard treatment

Sterile water

Suggested plant material—Seeds of any variety

of barley (Hordeum vulgäre) that contain a

relatively small amount of endogenous gibberellin are suitable. One such cultivar is 'Himalaya.'

Preparation of plant material—Cut 30-40 barley

seeds transversely and discard the portion of

each seed that contains the embryo. Sterilize the

halves containing endosperm by soaking them

in 1% sodium hypochlorite for 20 minutes.

Wash the seed halves three times with sterile

water. Ignite sand to sterilize it and place 100 g.

of it in a petri dish. Moisten the sand with 20

ml. of sterile water. Place the sterilized pieces

of seeds on the sand and cover the petri dish.

Allow the seed pieces to imbibe water for 3 days

at room temperature. Use the remaining seed

pieces in lots of 10 in a comparable way as

replications.

Procedure—Transfer aseptically 10 seed pieces

to an Erlenmeyer flask containing 2 micromoles

of the acetate buffer and 20 micromoles of calcium chloride. Autoclave the calcium chloridebuffer mixture. Add enough of the compound to

be tested or of the extract dissolved in 2 ml. of

sterile water to give a suitable amount of amylolytic activity (determined with exploratory

tests). Add 20 micrograms of chloramphenicol

to each flask. Close the flasks and incubate them
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 49

for 24 hr. at 25° C. with continuous shaking at

40 oscillations/minute. Decant the liquid from

each flask into a centrifuge tube and wash the

seeds with 3 ml. of sterile water. Combine the

wash water with the decántate. Centrifuge the

tubes for 10 minutes at 2,000 times gravity.

Method of taking results—Transfer a suitable

volume of the centrifuged supernatant liquid

(0.02-0.2 ml.) from each tube to a separate

flask, adding enough sterile water to make a

total volume of 1 ml./flask. Add 1 ml. of starch

substrate prepared by boiling a mixture of 150

mg. of the potato starch, 600 mg. of KH2PO4,

and 2 mg. of calcium chloride in 100 ml. of distilled water for one minute. Centrifuge for 10

minutes at 3,000 times gravity. Decant the supernatant liquid for use in the assay. Allow the

enzymatic digestion of starch to proceed for 10

minutes or for the required time previously determined with exploratory tests. Stop the reaction by adding 1 ml. of iodine reagent prepared

by mixing 6 mg. of potassium iodide and 600

mg. of iodine in 100 ml. of water and adding

1 ml. of this stock solution to 0.05 N hydrochloric acid to give a final volume of 100 ml.

Add 5.0 ml. of distilled water to each of the final

mixtures. Determine the optical density of each

mixture at 620 millimicrons. On the basis of exploratory tests, adjust the enzyme volume and

time of hydrolysis so that the optical density of

the starch-iodine complex is about half that of

the zero time control. Convert the optical density values to micrograms of alpha-amylase

using a factor obtained for each starch sample

by standardization with a pure amylase preparation.

Calculate the results arbitrarily as units of

alpha-amylase :

—rr y/ yf == 1 unit 01 alpha-amylase

AOD = absorbency of zero time control minus

absorbency of sample after hydrolysis

for time (T) measured in minutes

V — total volume of enzyme solution expressed as ml.

V = volume of enzyme solution taken for

assay expressed as ml.

Suggested standard for comparison—Gibberellic

acid Aa, 0.1-1.0 microgram/ml.

References

Jones, R. L., and Varner, J. E. The Bioassay of Gibberellins. Planta [Berlin] 72: 155-161. 1967.

Varner, J. E., and Chandra, G. R. Hormonal Control of

Enzyme Synthesis in Barley Endosperm. Nati. Acad.

Sei. Proc. 52: 100-106. 1964.

EXUDATION OF REGULATORS FROM ROOTS

BEAN METHOD

Based on research by W. H. Preston, Jr., J.

W. Mitchell, and W. Reeve; P. J. Linder, J. W.

Mitchell, and Greta D. Freeman.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Clay pots, 8, 10, or 13 cm.

Composted soil

Greenhouse space maintained at 21°-29° C,

slightly cooler at night

Shell vials, about 10-ml. capacity

Analytical 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

Beakers, 200-400 ml.

Source of compressed air

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 {Lijcopersicon esciilentum Mill.), squash {Cucúrbita spp.), sunflower (Helianthus annuus L.).

Preparation and selection of plant material—

Plant 3 or 4 seeds per pot in composted soil if

a single species is used. If 2 species are used,

plant 3 or 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

2 vigorously growing plants that are the same

species or of 2 different species. Discard all
50 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

plants except these 2 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 to be tested. Use a sheet of

plastic to prevent the aboveground parts of the

treated plant from touching those of the untreated plant (fig. 22).

Procedure—Prepare a 1% lanolin mixture of

the compound being tested, as described on pp.

8,106. 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 two.

BN-28927

FIGURE 22.—Sheet of plastic used to separate aboveground parts of treated plant from aboveground parts

of untreated plant.

Figure 23 illustrates results of a test with 4

plants grown under similar conditions. Alphamethoxyphenylacetic acid was applied (arrow)

to plant a shown at the extreme right. The acid

was absorbed by the plant and then translocated

to the roots where some of the acid was exuded.

Plant b growing in the same pot, with its roots

near those of the treated plant, then absorbed

some of the exuded acid, translocated this to its

terminal bud where the regulating substance

induced growth of malformed leaves. Untreated

plants c and d are shown for comparison.

For an alternative method, carefully wash

the roots of at least 3 of the young plants free

of soil. Support the plants with their roots immersed in about 200 ml. of tapwater in the

beaker. Aerate the water by passing compressed

air as fine bubbles through it. Treat the stems

of all the plants as described. Arrange a set of

untreated plants in another beaker of water as

controls. Allow all the plants to grow for 3 days,

replacing any water lost. Then remove and discard all 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 untreated plants any growth

modification such as deviation in size or shape

of newly expanded leaves (formative effects)

(fig. 23, b). Make a similar comparison for the

alternative test using aerated water. (See "Root

exudation method," p. 52.)

Supplementary tests: 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
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 51

BN-28926

FIGURE 23.—Alpha-methoxyphenylacetic acid applied to stem of plant a was absorbed by this plant, then translocated to its roots where some of the acid was exuded. Plant b, with its roots near those of plant a, absorbed some

of the exuded acid, translocated this to its terminal bud where the regulating substance induced growth of malformed leaves. Untreated plants c and d are shown for comparison.

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.

Another test to determine whether the chemical moves from the lanolin mixture through the

air in sufficient amounts to affect a plant can be

made as shown in fig. 24. Lanolin mixture is

applied to each of 4 glass rods that are placed

near the test plant. The amount used for each

rod and method of application are the same as

those described on p. 50. Cylinders of screen

wire, used as illustrated (fig. 24), keep the mixture in the treated area on each rod from touching the plant. Modification of shape or formative effects in new leaves indicate that a volatile

growth regulator has moved from the glass rods

to the plants in amounts that induce the response.

Still another method of measuring response

is as follows: estimate the amount of the compound that exudes from the roots of the plant

to which the chemical is applied by comparing

the suppression of growth in trifoliolate leaves

of a nearby plant with reduction in trifoliolate

leaves of other plants treated directly with various amounts of the acid. Make these measurements by determining the fresh weight of

growth above the primary leaf node.

Suggested standard for comparison—Alphamethoxyphenylacetic acid, 1% lanolin-Tween

20 paste.
52 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-28951

FIGURE 24.—Testing volatility of compound mixed with

lanolin by applying mixture to glass rods placed near

test plant and then noting growth responses that develop. Screen wire prevents mixture from touching the

plant.

Apparatus, Chemicals, and Other Materials

Clay pots, 8-10 cm.

Composted soil

Razor blade

Glass tubes, 32 mm. inside diameter, 15-cm.

long, to be used as root chambers

Rubber stoppers, 2-hole, No. 12, 1 for each

root chamber

Soft glass tubing to fit the holes of the rubber

stoppers

Glass tubing, about 5 mm. outside diameter

Plastic tubing, approximately 4 mm. and 5

mm. inside diameter

Rubber tubing, 3 mm. inside diameter

Brass needle valves, 1 for each root chamber

Pinch clamps, 1 for each root chamber

Aluminum foil

Cellophane tape

Ring stands, 1 for each pair of root chambers

Sheet metal for constructing plant supports

Aluminum paint

Beakers, 100-ml., 1 for each root chamber

Glass rod, 4 mm. inside diameter, 15 cm. long

Small aquarium pump

Burette clamps, 1 for each root chamber

Analytical balance

Geiger counter

2-10 mg. of the stable or C"-tagged alphamethoxyphenylacetic acid containing 1-5

microcuries of radioactivity/mg. for standard treatment

Distilled water

References

Linder, P. J., Mitchell, J. W., and Freeman, Greta D.

Persistence and Translocation of Exogenous Regulating Compounds That Exude from Roots. Agr. and

Food Chem. 12: 437-438. 1964.

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.

ROOT EXUDATION METHOD

Based on research by J. W. Mitchell, P. J.

Linder, and Melba B. Robinson.

Description of method—The ability of plants to

absorb a regulating compound into their leaves,

translocate it downward, and exude it from

their roots into the surrounding medium is

measured with the use of radioactivity or by

means of a bioassay.

Suggested plant material—Young snap bean

plants {Phaseolus vulgaris L.) with primary

leaves approximately 4-6 cm. wide and with

their trifoliolate leaves still folded in the terminal bud. Other kinds of young plants can also

be used with this method.

Preparation and selection of plant material—

Plant several bean seeds in composted soil in

pots so one plant of uniform size can be selected

in each pot. Sever the stems of the undesirable

plants at the soil surface and discard the aboveground portions. Submerge the pots in water to

the depth of the soil surface for a short period

and then carefully wash the soil from the roots

of the selected plants in running water. Keep

the roots in water until the plants are needed.

Procedure—Construct the apparatus as follows

(fig. 25) : Fit a 2-hole rubber stopper in the

lower end of the 32-mm. diameter glass tubing

(cylinder). Connect one of the outlets in the

stopper, using the soft glass and plastic tubing

(4 mm. inside diameter), with a manifold made

by connecting a series of needle valves with
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 53

BN^2892S

FIGURE 25.—Apparatus for studying exudation of regulating substances applied to leaves of intact plants.

plastic tubing. Make one end of the glass tubing

extend 1 cm. above the stopper into the cylinder.

Use the other opening in the stopper at the

lower end of the cylinder as a drainage outlet

by inserting a piece of glass tubing with a short

length of rubber tubing attached. Close this outlet with a pinch clamp. Cover the cylinder with

aluminum foil leaving an uncovered portion to

serve as a window. Support each pair of cylinders with a single ring stand.

Construct a metal plant support by marking

the surface of a metal sheet (a tin can is satisfactory) using the pattern shown in fig. 26.

Bend the metal into the form illustrated in fig.

27, c. Spray these plant supports with aluminum

paint to prevent rusting and mount them on

cylinders (fig. 27, d). Cut pieces of the rubber

tubing (3 mm. inside diameter) approximately

5 mm. long (fig. 27, a) and pieces of the plastic

tubing (5 mm. inside diameter) approximately

20 mm. long (fig. 27, h). Cut along the side of

each piece of tubing so that it can be opened

and fitted around the stem of the plant.

Connect the manifold to the aquarium pump

leaving at the end of the manifold an outlet that

can be adjusted by a pinch clamp, and thus

divert the appropriate amount of air through

the glass cylinder. Drill fine holes in a short

piece of the plastic tubing to serve as a "bubbler." Plug one end with a short piece of glass

rod and attach the other end to the 1-cm. length

of glass tubing that extends from the stopper

into the cylinder. The air from the pump will

be forced through the holes and form small

bubbles. Start the aquarium pump and adjust

the manifold outlet needle valve and the other

needle valves, so air is passing through each

cylinder. Introduce 100 ml. of tapwater or nutrient solution into each cylinder. Adjust the

rate of airflow so the number of bubbles passing

gently through each cylinder is about equal.

Carefully place the roots of 1 plant in each cylinder and support the stem of the plant at the

proper height with the plastic and rubber collars and metal supports (fig. 25 and 27).
54 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

FIGURE 26.—Pattern to be used in cutting metal support. (See also fig. 27, c).

Apply 1-100 micrograms of the chemical being tested to one or both primary leaves as illustrated in fig. 7, p. 12.

Method of taking results—Place a 100-ml.

beaker under each cylinder and collect separately the liquid surrounding each root at 12-

hr. intervals for several days. Replace the tapwater or nutrient solution in each cylinder.

Evaporate the liquid collected and determine

the amount of radioactivity in each sample with

a Geiger counter. If a nonradioactive compound

is used, bioassay the samples using the "Bean

second internode method" described on p. 26,

the "Wheat coleoptile straight growth method"

described on p. 36, or other suitable bioassay

method. Estimate the amount of compound exuded by comparing the amount of radioactivity

or the biological response to standard curves

made with known amounts of the compound

involved.

Suggested standard for comparison—10 micrograms of carboxyl-tagged C^* or stable alphamethoxyphenylacetic acid applied to the upper

surface of each primary leaf.

Reference

Mitchell, J. W., Linder, P. J., and Robinson, Melba B.

Mechanism of Root Exudation of a-Methoxyphenylacetic Acid in the Bean Plant. Bot. Gaz. 123: 134-137.

1961.

BN-28924

FIGURE 27.—Plant support : o. Rubber tubing, 3 mm. inside diameter; b, plastic tubing, 5 mm. inside diameter;

c, metal support; d, glass cylinder covered with aluminum foil.

FLOWERING

BIENNIAL OR PERENNIAL PLANT METHOD

Although research on hastening the time of

flowering of biennial and woody plants with

regulators is an important field, the methods

used simply involve the application of a regulator in solution to soil surrounding the roots or

to the aboveground parts as a paste or spray.

The time required for the treated plants to

initiate flowers compared with that required for
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 55

similar untreated plants is a measure of the

effectiveness of the compound. A detailed description of these methods is, therefore, unnecessary here.

References

Batjer, L. P., Williams, M. W., and Martin, G. C. Effects

of n-Dimethyl Amino Succinamic Acid (B-Nine) on

Vegetative and Fruit Characteristics of Apples, Pears,

and Sweet Cherries. Amer. Soc. Hort. Sei. Proc. 85:

11-16. 1964.

Bukovac, M. J., and Wittwer, S. H. Gibberellin and

Higher Plants: II. Induction of Flowering in Biennials. Mich. Agr. Expt. Sta. Quart. Bui. 39: 650-660.

1957.

Cathey, H. M., and Taylor, R. L. Regulating Flowering

of Rhododendron: Light and Growth Retardants. The

Amer. Nurseryman. Jan. 1, 1965.

Clark, H. E., and Kerns, K. R. Control of Flowering

with Phytohormones. Science 95: 536-537. 1942.

Edgerton, L. J. New Growth Retardant for Fruit Trees.

Farm Res. 30: 10-11. 1964.

Gaskill, J. 0. A Preliminary Report on the Use of Gibberellic Acid to Hasten Reproductive Development in

Sugar Beet Seedlings. Amer. Soc. Sugar Beet Technol.

Jour. 9: 521-528. 1957.

Lang, Anton, Sandoval, J. A., and Bedri, Ahmed. Induction of Bolting and Flowering in Hyoscyamus and

Samolus by a Gibberellin-Like Material from Seed

Plants. Nat. Acad. Sei. Proc. 43: 960-964. 1957.

Marth, P. C. Effect of Growth Retardants on Flowering,

Fruiting, and Vegetative Growth of Holly (Ilex).

Amer. Soc. Hort. Sei. Proc. 83: 777-781. 1963.

Stuart, N. W. Stimulation of Flowering in Azaleas and

Camellias. XVI Internati. Hort. Cong. Proc, Brussels,

Belgium. 5: 58-64. 1962.

FORM OR GROWTH MODIFICATION

BEAN LEAF METHOD

Based on research by J. W. Brown and R. L.

Weintraub.

Description of method—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

to study the effect of regulating chemicals on

the formation of abscission layers near the

terminal buds of succulent plants.

Apparatus, Chemicals, and Other Materials

Composted soil

Clay pots or other suitable containers in

which to grow plants, 10 cm.

Greenhouse or, for more precise experiments,

an artificially lighted room (about 700 footcandles) equipped with temperature and

humidity controls

Analytical balance

Pipette, 5 lambda

Photographic paper, printer, and developer

Photometer

Planimeter

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

Suggested plant material—Young bean plants

(Phaseolus vulgaris L.)—Black Valentine, Red

Kidney, Bountiful varieties.

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 hr., a minimum of 700 foot-candles of light, a day temperature of about 24° C, a night temperature of

about 22° C, and, if possible, a relative humidity of 50-70%. Select test plants for uniformity (1 plant per pot) after 6-8 days when the

second internode is 3-7 mm. long. Cut off and

discard all other plants.

Procedure—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 10-20 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 trifoliolate leaf (5-13 days).

Method of taking results—At the end of this

growth period, remove the first trifoliolate leaf

from each plant, and determine its fresh weight

and area. Leaf area can be obtained directly

using an area photometer or indirectly using

a planimeter and tracing an outline of a leaf on

paper. Also, leaf area can be obtained by tracing

the outline of the leaf on paper and cutting out

the image. Weigh a piece of the paper that is
56 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

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 oí 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.

Suggested standard for comparison—Use standard dose of 0.015 ¡ng. of 2,4-dichlorophenoxyacetic acid per 5-lambda droplet per plant. This

induces approximately 48% suppression in leaf

area.

Reference

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.

LANOLIN PASTE METHOD

Based on research by P. C. Marth, W. H.

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

Description of method—Compounds are applied

in a paste carrier to stems of young plants, and

the effect on their subsequent growth and development is observed.

Apparatus, Chemicals, and Other Materials

Clay pots, 10 cm.

Composted soil

Greenhouse space with temperature controlled at 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

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 (Amo1618) ; or 50 mg. of indole-3-acetic acid

Tween 20

Lanolin

Suggested plant material—Young herbaceous

plants—bean (Phaseolus vulgaris L.), cucumber (C?ira7ms sativus L.), sunflower (Helianthus annuits L.).

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 29° C. and, as soon as the plants appear

above the soil, reduce the temperature 2°-5° C.

After the plants are erect and about 5-8 cm.

tall, select one in each pot that is of average

size. Pinch the remaining plants off at the soil

level and discard them.

Procedure—Arrange each species of plant in a

group with 3-5 plants per row. Prepare lanolin

paste as described on p. 8. 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

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 for 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 rule 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. For cucumber

plants, place the end of the rule 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 ;
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 57

Untreated control ^

_ minus treated^^ ^^^ ^ % inhibition

Untreated control (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 mdicating cell

enlargement or cell proliferation, initiation of

roots, changes in color, and other modifications

of 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 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; and

indole-3-acetic 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.

Reference

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

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

Gaz. 115: 200-204. 1953.

3 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.

UNDERGROUND GROWTH METHOD

Based on research by T. J. Muzik and H. J.

Cruzado ; T. J. Muzik and J. W. Whitworth.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Wooden or metal boxes, 23 cm. wide at the

top X 96 cm. long X 43 cm. deep 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

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

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.

Preparation and selection of plant material—Select uniform plant parts such as seeds, stem

pieces, chains of nutgrass nutlets {Cyperus rotundus), rhizomes of bindweed {Convolvulus

arvensis L.) or of Canada thistle {Circium arvense Scop.), and plant them in composted soil

against the sloping glass sides of the boxes.

Procedure—Apply the chemical 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 Ib./acre suggested), 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 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.
58 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Variation used by Muzik and Whitworth—Boxes

of somewhat different shape have also proved

useful. Make glass-faced boxes using marine

plywood approximately % in. thick. A convenient size has inside dimensions of 5 cm. deep X

20 cm. wide X about 1 m. high. Leave one side

of the box open and equip this side with grooves

so that a glass plate can be fitted into the

grooves. Make a close-fitting, removable shutter

to cover the glass plate and fasten this in place

with clips so that the roots can be kept in darkness.

Fill the box with vermiculite, sand, or soil

(preferably vermiculite), and moisten the medium. Place seedlings or rooted cuttings on the

surface of the medium with the part ordinarily

above the soil level extending out of the open

end of the box. Spread the roots out to permit

ready observation and place the glass over them.

Fasten the wooden shutter over the glass and

place the box at an angle of 30°-45° with the

glass side down.

Using a wax pencil, mark the position of the

roots on the glass at frequent intervals to indicate their growth. Use these marks to make

final growth measurements. If it is necessary

to remove the glass plate during the experiment, wait several hours after watering the

plants before doing this because the wet vermiculite adheres to the glass.

This simple technique permits the detailed

study of root growth and development with a

variety of plants under a wide range of experimental conditions.

References

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

Sprout Formation in Cyperus rotundus. Amer. Jour.

Bot. 40: 507-512. 1953.

Muzik, T. J., and Whitworth, J. W. A Technique for the

Periodic Observation of Root Systems in Situ. Aeron.

Jour. 54: 56-57. 1962.

FRUIT GROWTH

OVARY CULTURE METHOD

Based on research by J. P. Nitsch.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Scalpel

Microbiological transfer room

Jars

Petri dishes

Filter paper

Test tubes

Forceps

Nonabsorbent cotton for test tube plugs

Room exposed to indirect sunlight with temperature of about 23° C. and night temperature of about 17° C.

Analytical balance

Calcium hypochlorite, 5%, c.p. in water

Salts for nutrient solution :

Ig.of Ca(N03)2*4H20

1 g. of KNOs

1 g. of MgS04 • 7H2O

1 g. of KH2PO4

Chemicals for trace-element solution :

5 ml. of H2SO4, sp. gr. 1.83

5 g. of MnS04 • 4H2O

1 g. of ZnS04 • 7H2O

1 g. of H3BO3

1 g. of CUSO4 • 5H2O

1 g. of Na2Mo04 • 2H2O

1 g. ferric citrate

5 g. of NaOH

5-10 ml. of HCl

100 g. of sucrose

500 ml. of 70% ethanol

Paraffin

Water redistilled in Pyrex

Agar

Detergent, 10 ml./liter of water

Suggested plant material—Commercial varieties

of crop plants—tomato (Lycopersicon esculenturn Mill.), bean {PMseolus vulgaris L.), tobacco (Nicotiana tabacum L.), strawberry

(Fragaria spp.), cucumber (Cucumis sativus

L.), common varieties of other crop plants in

flower.

Preparation and selection of plant material—Use

healthy greenhouse plants. At flowering, handpolhnate strawberry and cucumber flowers to

msure pollination. Shake tomato, bean, and tobacco plants occasionally to insure pollination.

Cut the flowers from the plants 2 days after

pollmation and dip the cut ends into melted

paraffin to seal them. Place these flowers into

clean jars, sterilized by washing with 70%

ethanol. Pour a detergent solution over the flow-
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 59

ers and then drain the jars. Add a decanted or

filtered 5% calcium hypochlorite solution. From

then on conduct all operations in a microbiological transfer room, using sterile utensils and

materials.

After about 10 minutes, pour the calcium

hypochlorite solution off and wash twice with

sterile water. Transfer the flowers to sterile

petri dishes containing a double thickness of

filter paper to absorb the excess water.

Procedure—Prepare the following solutions:

Mineral salt solution

Salt

Ca(N03)2«4 H2O

KNO3

MgS04 • 7 H2O

KH2PO4

Amount per liter of

final solution

500 mg.

125 mg.

125 mg.

125 mg.

Add sufliicient water redistilled in Pyrex to

make 1 liter of final solution.

Trace-element solution

Component

H2SO4, sp. gr. 1.83

MnSOi • 4 H2O

ZnS04•7 H2O

H3BO3

CuS04•5 H2O

Na2Mo04 • 2 H2O

Amount per liter of

final solution

0.5 ml.

3,000 mg.

500 mg.

500 mg.

25 mg.

25 mg.

Add sufficient water redistilled in Pyrex to

make 1 liter of final solution.

To 1 liter of the mineral salt solution add 10

mg. of ferric citrate, 1 ml. of the trace-element

solution, and 50 g. of sucrose. Adjust the pH to

5.5 with NaOH or HCl.

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 disks having a diameter

about twice that of the tubes. Make a hole in

the center of each disk slightly larger than

the diameter of the pedicel. Form a paper cup

with each disk by pressing it against the edge

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

downward, so it stays at least 5 cm. above the

level of the liquid. Plug the tubes with nonabsorbent cotton. Autoclave the tubes 20 minutes

at 15 lb. pressure. When planting the sterile

ovaries, push the paper down with the forceps

until the pedicel reaches the liquid.

A variant of this technique uses no filter

paper but solidifies the media with 0.8% 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

using agar media: Autoclave the four 250-ml.

portions of nutrient medium mentioned previously and the plugged, but empty tubes. Sterilize a metal spoon by dipping it into alcohol, and

allowing it to dry. Weigh the required amount

of test chemical, place it in the spoon, and add

sufliicient 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 before the agar has solidified and

stir until the compound is uniformly dispersed.

Remove the cotton plugs from the tubes and

pour approximately 25 ml. of the mixture directly into each tube. Replace the cotton plugs.

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

sepals, and most of the pedicel of 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 disk 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

23° C. and a night temperature of about 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 da^-a for comparing growth

rates of treated and untreated fruits.

Suggested standard for comparison—No standard treatment suggested.

References

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. XIII Internatl. Hort. Cong. Pr^r.

1: 263-266. 1952.
60 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

FRUIT SET

AEROSOL METHOD

Based on research by L. D. Goodhue ; C. L.

Hamner, H. A. Schomer, and P. C. Marth; P. C.

Marth and E. M. Header.

Description of method—Regulating or other

chemicals are applied to plants or plant parts

in the form of mists produced by use of liquefied gases.

Apparatus, Chemicals, and Other Materials

Test-tube-type apparatus (fig. 28) for smallscale experiments or screening tests

Steel containers 0.23-0.45 kg. capacity, developed for applying insecticidal liquefied

gas aerosol mixtures and fitted with 1.27-

cm. refrigerant valves

Flexible rubber pressure tubing for transferring liquefied gases

PN-296, A and B

FIGURE 28.—Views of unassembled (upper) and assembled (lower) glass-walled aerosol bomb of the test-tube type

used experimentally to prepare aerosol mixtures of regulating chemicals.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 61

Brass fittings for connecting valves and

tubing

Analytical balance

Suction pump or air vacuum line

50-mm. metal funnel soldered into 0.6-cm.

threaded brass valve fitting

Spring scales or balance, 1,000-g. capacity

Spray nozzles (standard oil burner nozzles or

capillary metal tube soldered into 0.6-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 miscible

with liquefied gases and will dissolve chemicals to be tested

2.3-11.3 kg. supply tank of methyl chloride,

boiling point —11.0° C; or dichlorodifluoromethane (Freon), boiling point —6° C;

or dimethyl ether, boiling point —12.4° C.

Suggested plant material—Tomato plants {Lycopersicon esculentum Mill.) that have developed 2 open flowers in the first cluster.

Preparation and selection of plant material—Tomato plants (Lycopersicon esculentum Mill.)

Marglobe, Globe, Rutgers, or other varieties

that can readily be grown in a greenhouse.

Procedure—Preparing and loading aerosol mixtures—

A. For test-tube-type apparatus shown in figure 28:

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. 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 2 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 from

the test tube, and attach the spray nozzle in

place of the tubing. Mark the 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 potbound

or the above-soil parts become crowded. Determine the percentage of fruit set by the treated

and the untreated clusters.

B. For large (0.23-0.45 kg. 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 cylinder), 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.

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 noninflammable and relatively nontoxic to plants and animals.

Suggested standard for comparison—Dimethyl

ether aerosol mixture containing 1 % beta-naphthoxyacetic acid and 10% cyclohexanone solvent

for tomato fruit-set test.

References

Goodhue, L. D. Insecticidal Aerosol Production. Spray

Solutions in Liquefied Gases. Indus, and Engin. 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.
62 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Marth, P. C, and Header, E. M. Influence of GrowthRegulating Chemicals on Blackberry Fruit Development. Amer. Soc. Hort. Sei. Proc. 45: 293-299. 1944.

GRAPE METHOD

Based on research by R. J. Weaver and W. 0.

Williams; R. J. Weaver; R. J. Weaver and S. B.

McCune.

Description of method—Various compounds can

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.

Apparatus, Chemicals, and Other Materials

Containers of suitable size, such as discarded

but clean tin cans, approximately 1-liter,

to hold solutions so the flower clusters can

be dipped

Analytical 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 or 0.5 g. of gibberellic acid

Wetting agent such as Dreft, Tween 20,

Santomerse

Solvent such as 95 % ethanol

Suggested plant material—Seedless grape varieties, such as Black Corinth (Vitis vinifera L.),

or seeded grapes that ordinarily set few berries, such as Muscat of Alexandria, Pinot Chardonnay.

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.

Procedure—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 and then

adding the alcohol solution to the required

amount of water. Add suflScient 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. Do not

bruise or damage these clusters. 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 for comparison—5, 15, and

50 p.p.m. of para-chlorophenoxyacetic acid, or

10, 50, and 100 p.p.m. of gibberellic acid.

References

Weaver, R. J., and Williams, W. O. Response of Flowers

of Black Corinth and Fruit of Thompson Seedless to

Applications of Plant Growth-Regulators. Bot. Gaz.

11: 477-485. 1950.

Weaver, R. J. Plant Regulators in Grape Production.

Calif. Agr. Expt. Sta. Bui. 752, 26 pp. 1956.

Weaver, R. J. Effect of Gibberellic Acid on Fruit Set

and Berry Enlargement in Seedless Grapes of Vitis

vinifera. Nature 181: 851-852. 1958.

Weaver, R. J., and McCune, S. B. Response of Certain

Varieties of Vitis vinifera to Gibberellin. Hilgardia

28(13) : 297-350. 1959.

TOMATO METHOD

Based on research by D. D. Hemphill; F. S.

Howlett; L. C. Luckwill; A. E. Murneek, S. H.

Wittwer, and D. D. Hemphill; R. H. Roberts

and B. E. Struckmeyer.

Description of method—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 development of

fruits. It is also useful in studying the effect

of regulators on the number of flowers produced.

Apparatus, Chemicals, and Other Materials

Clay pots or discarded tin cans, 0.95 liter with

perforated bottoms

Composted soil
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 63

Space in greenhouse maintained at about

21°-27° C. by day and at 15°-21° C. by

night

Small vials, 20-25 ml.

Clean containers, 100-200 ml. capacity

Toothpicks or wooden medicinal applicators

or a nasal spray atomizer, 15-30 ml.

Analytical balance

25-100 mg. of each compound to be evaluated

About 0.5 g. para-chlorophenoxyacetic acid

or beta-naphthoxyacetic acid for standard

treatment

Lanolin

Solvents such as ethanol, methanol

Wetting agents such as Tween 20, Carbowax

1500

Suggested plant material—Tomato plants {Lycopersicon esculentum Mill.), Marglobe, Globe,

Rutgers, or other varieties that can be readily

grown in a greenhouse.

Preparation and selection of plant material—

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

Grow the plants in 10- to 15-cm. pots set side

by side in rows 10 cm. apart; select plants of

uniform size which have developed 2 open flowers in the first cluster.

B. For prolonged tests involving maturation of the

fruit (35-45 days): Grow the plants in 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 30 cm. apart, in rows

60 cm. apart.

Procedure—

A. Paste Method: Prepare a lanolin-Tween 20

paste containing 1% of the chemical. (See p.

8.) 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 ground bed. Repeat

the procedure using additional treatments and

corresponding rows. Leave an additional row

of plants untreated to serve as controls. When

all the treatments have been applied and the

control row has been 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 p.

100.) 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.

C. Droplet Method: A variation of the spray

method is the application of a droplet onto the

individual ovaries of 2 flowers in the cluster

using a 1-ml. capacity hypodermic syringe fitted

with a No. 17 needle. (Method by L. C. Luckwill.)

Method of taking results—

A. Plants used in tests of limited duration: Count

the number of flowers and the number of fruits

that set (remain attached to the plant) per

cluster within 14-20 days after treatment, or

before the plants become excessively potbound

or the above-soil parts crowded. Determine the

percentage of fruits 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 for comparison—A paste of

0.1% lanolin-Tween 20 or a spray mixture containing 30 p.p.m. of para-chlorophenoxyacetic

acid ; a paste of 0.3% beta-naphthoxyacetic acid,

or a spray at 100 p.p.m.

References

Hemphill, D. D. The Effects of Plant Growth-Re^ulatin^ 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

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

Proc. 37: 886-890. 1939.

Luckwill, L. C. A Method for the Quantitative Estimation of Growth Substances Based on the Response of

Tomato Ovaries to Known Amounts of 2-Naphthoxyacetic Acid. Jour. Hort. Sei. 24: 19-31. 1948.

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,
64 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

FRUIT SIZE

GRAPE METHOD

Based on research by R. J. Weaver and W. 0.

Williams; R. J. Weaver; R. J. Weaver and S. B.

McCune.

Description of methods—Various compounds

may be evaluated as growth regulators by dipping individual clusters of grape flowers at

bloom, or after the shatter of impotent berries

following bloom, in solutions of the compound,

and by 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.

Apparatus, Chemicals, and Other Materials

See p. 62.

Suggested plant material—Seedless grape varieties such as Black Corinth, Thompson Seedless,

Sultana, Black Monukka (Vitis vinifera L.).

Seedless varieties are more responsive than are

seeded ones for this test.

Preparation and selection of plant material—

Select clusters of uniform size having about the

same number of flowers. Tag those to be used

after the calyptras have fallen. The best time

to treat grapes for studies on increasing the

size of the berry is after the shatter of impotent

berries following bloom.

Procedure—Prepare aqueous solutions of each

compound at concentrations of 5, 15, and 50

p.p.m. using alcohol as a solvent and adding a

wetting agent. (See p. 100.) 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. Volume measurements of the fruits may also be

determined by the water displacement method.

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 for comparison—5, 15, and

50 p.p.m. of para-chlorophenoxyacetic acid, or

10, 50, and 100 p.p.m. of gibberellic acid.

References

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

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

1951.

See also, "References," p. 62.

FRUIT THINNING

APPLE METHOD

Based on research by L. P. Batjer ; C. P. Harley, H. H. Moon, and L. 0. Regeimbal.

Description of method—Compounds are evaluated as a means of thinning fruit by applying

regulating chemicals to selected limbs of trees

1-3 wk. after full bloom.

By modifying the dosage, the method may be

useful in entirely removing undesirable fruits

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

Apparatus, Chemicals, and Other Materials

Analytical 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.)

Sprayers having a large tank divided into

several 50- or 100-gal. compartments facilitate the work

Hand-pressure sprayer (11-liter) to use if

individual limbs are to be sprayed

Hand tally registers, 2 or 3 per person, to

record data

10-20 g. of alpha-naphthaleneacetamide or

1-naphthyl N-methyl carbamate for standard treatment

Tween 20 or other wetting agent
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 65

Suggested plant material—Apple trees {Malus

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

variety in need of thinning.

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 5 or more trees. In

this way several treatments can be used on

each tree.

Procedure—Prepare aqueous sprays containing several concentrations of each chemical (10,

30, and 60 p.p.m. are suggested). (See p. 100.)

Apply sprays 1-3 wk. after full bloom and wet

thoroughly all parts of the tree or branch.

About 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 to have trees or

branches thinned an optimum amount for comparison.

Method of taking results—Determine the number of fruits thinned chemically after June

drop. 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 for comparison—50 p.p.m.

spray concentration of alpha-naphthaleneacetamide. 3 g./gal. of 1-naphthyl N-methyl carbamate.

References

Batjer, L. P. Fruit Thinning With Chemicals. U.S.

Dept. Agr., Agr. Inf. Bui. 289. 1965.

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 METHOD

Based on research by P. C. Marth and V. E.

Prince.

Description of method—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 ginkgo, mulberry, and

hickory, without injury to the trees.

Apparatus, Chemicals, and Other Materials

See p. 64, substituting 50-100 g. of isopropyl

N-(3-chlorophenyl) carbamate for standard

treatment. (''Apple method'')

Suggested plant material—Peach trees (Prunus

pérsica (L.) Batsch) bearing young fruits (30-

45 days after full bloom)—Elberta, Halehaven,

Hiley, or other varieties; or plum trees (Prunus

domestica L.) bearing young fruits (30-45 days

after full bloom)—Stanley, Italian, or German

varieties.

Preparation and selection of plant material—See

''Apple method,'' p. 64.

Procedure—Prepare aqueous sprays containing several concentrations of each chemical.

(See p. 100.) 125, 250, and 500 p.p.m. are 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 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 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, 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, degree of coloration of fruits, and effect

of treatment on time of maturation. Compare

theàa data to evaluate effectiveness of the chemical treatment.

'\

Suggestea^andard for comparison—Spray concentration of 200 p.p.m. of isopropyl N-(3-

chlorophenyl) carbamate.

Reference

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

Fruits of Peach. Science 117: 497-498. 1953.
66 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

GROWTH MODIFICATION

STERILE NUTRIENT METHOD

Based on research by L. G. Nickell.

Description of method—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 is

studied.

Apparatus, Chemicals, and Other Materials

Analytical 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 tapwater over a small amount of sandy soil.)

Table space in a room with constant light

intensity of 700 to 1,000 foot-candles for

12 hr. per day and with temperature controlled at 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 listed :

KNOs 0.002M Pyridoxine 800y/liter

Ca(N03)2 0.003M Nicotinamide 800y/liter

KH2PO4 O.OOIM B O.lp.p.m.

MgS04 O.OOIM Mn O.lp.p.m.

CaCl2 0.003M Zn 0.3 p.p.m.

KCl 0.002M Cu 0.1p.p.m.

MgCl2 O.OOIM Mo O.lp.p.m.

Sucrose 2% Fe 0.5 p.p.m.

Thiamine lOOy/liter

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 treatment

Solution of mercuric chloride (1:2,000 of

water) for sterilizing plants

O.T. Clear Aerosol or comparable detergent

Suggested plant material—Duckweed plants

(Lemna minor L.).

Preparation and selection of plant material—Select a vigorously growing supply of duckweed.

Sterilize this material by immersing rosettes

(3 or 4 fronds) in the mercuric chloride solution (1:2,000 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 them one by one at

5-minute intervals so a series of treated rosettes

results which have been exposed from 5-30

minutes to the mercuric chloride mixture. As

each rosette is removed, rinse it several times

in sterile water and finally place it in a flask

containing sterile nutrient medium. Allow all

the plants to grow in the flasks for a week to

determine which plants are aseptic. Uncontaminated cultures are used for further experimentation.

Procedure—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 it contains 300 p.p.m.

of the chemical. Remove contaminating organisms in this solution by passing it through the

sintered glass filter. Place 50 ml. of the sterile

nutrient medium aseptically into each of 5

flasks with a 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

sufficient volume of the sterile stock solution

containing the regulating chemical to make à

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, selected, duckweed plants in each flask aseptically.

Stopper the flasks with cotton and place them

under controlled environmental conditions,

22.5° C. and 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 wk.), remove the test

plants, blot them free of water, and for each

flask record the number of plants and the number of fronds on each. 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
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 67

and treated plants in terms of number of plants

and fronds, and the average weight per plant.

Suggested standard for comparison—Bacitracin,

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

References

Nickell, L. G. Effects of Antigrowth Substances in Normal and Atypical Plant Growth. ''Antimetabolites and

Cancer," A Symposium Presented on Dec. 28 and 29,

1953. American Association for Adv. of Science,

Washington, D. C. 312 pp. 1955.

Nickell, L. G. Some Simple Substituted Pyrimidines and

Their Effects on the Growth of Lemna minor. Phyton

18: 59-63. 1962.

Nickell, L. G., and Celmer, W. D. A Potent, Non-Toxic

Plant-Growth Inhibitor from an Antibiotic Fermentation. Nature 192: 1098-1099. 1961.

Nickell, L. G., and Celmer, W. D. Structure-Phytoactivity Relationships in the Penicillins. Nature 206: 302-

304. 1965.

Nickell, L. G., and Finlay, A. C. Growth Modifiers, Antibiotics, and Their Effect on Plant Growth. Jour. Agr.

and Food Chem. 2: 178-182. 1954.

Nickell, L. G., and Gordon, P. N. Effects of the Tetracycline Antibiotics, Their Degradation Products, Derivatives, and Some Synthetic Analogues on the

Growth of Certain Plant Systems. Antimicrobial

Agents Annual 1960: 588-594. 1961.

WATER CULTURE METHOD

Based on research by H. R. Offord.

Description of method—An aquatic plant, duckweed, is grown in water containing a regulating compound, and the growth-modifying effect of the chemical is observed.

Apparatus, Chemicals, and Other Materials

Analytical balance

Petri dishes

Tank in which to maintain a stock supply of

the test plant. Duckweed will grow vigorously through the year in dilute nutrient

solutions at a pH of 5-6 or in tapwater

over a small amount of sandy soil.

Table space in a room with constant light

intensity of 700-1,000 foot-candles for 12

hr. per day and temperature controlled at

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 sulfuric acid

Dilute solution of potassium hydroxide

Suggested plant material—Duckweed plants

(Lemna minor L.).

Preparation and selection of plant material—Select relatively large, uniformly green plants

that are growing vigorously.

Procedure—Prepare tapwater solutions (150

ml.) of each compound in a range of concentrations from 0.001-1,000 p.p.m. Place 50 ml.

of each concentration in each of 3 petri dishes.

Place the same number of duckweed plants in

each test solution, using 10 or more for each.

Include some dishes that contain plants in tapwater 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 duckweed. Maintain the

temperature of the solutions at 24°-27° C. and

the light intensity constant so 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.

There is an apparent increase in the number of

plants and in the total estimated area of the

fronds.

Suggested standard for comparison—Determine

by trial the minimum concentration of 2,4-

dichlorophenoxyacetic acid that induces marked

inhibitory effect. No standard suggested for

stimulation of growth.
68 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Reference

Offord, H. R. Rapid Estimation of the Phytocidal Action

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

FIELD METHOD

Based on research by J.

Marth and R. E. Wester.

R. Havis; P. C.

Description of method—Test chemicals are

sprayed on soil in which seeds have been planted

or on leaves and stems of plants growing under

field conditions, and the effect of the chemical

on growth and productivity of the plants is

measured.

Apparatus, Chemicals, and Other Materials

Analytical balance

Knapsack sprayer, to be operated at 30-lb.

pressure per sq. in.

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

growth regulator

5 g. of 2,4,5-trichlorophenoxyacetic acid or

2,4-dichlorophenoxyacetic acid for standard treatjnent

Solvent for preparing concentrated solutions

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

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—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.

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 4 or more 40-sq.-m. plots for each chemical

or concentration tested. Space the plants 46-60

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

the plants 15-20 cm. apart in rows 76-91 cm.

apart. Use 2 or more replications of rows 1.5-

3 m. long for each chemical or concentration

tested. It is sometimes advantageous to select

uniform plants 60 cm. or more apart. Mark

these and use 10 of them for each chemical or

concentration tested.

Procedure—For preemergence application, apply the test chemicals within a few days after

planting, using suggested rates of 1, 2, 4, and

16 lb./acre.

For postemergence treatment, apply the

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

stage of development after emergence. Since

the development of this early method, logarithmic sprayers have become available which automatically apply a range of dosage levels.

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 preemergence

experiments, plants near the center of each plot

should be measured.

Suggested standard for comparison—Aqueous

sprays at concentrations of 0.001-1,000 p.p.m.

of 2,4,5-trichlorophenoxyacetic acid or 2,4-dichlorophenoxyacetic acid if applied postemergence; or 0.5, 1, 2, and 7 kg. per 4.047 sq. m. if

applied preemergence.

References

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

Sugarcane. 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 Ecc.ns. Amer

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

GROWTH RETARDATION

VEGETATIVE SPROUT METHOD

Based on research by P. C. Marth and E. S.

Schultz; P. C. Marth.

Description of method—Pieces of carrots, small

onions, and potato tubers 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 to study the

growth and development of buds on any vegetative reproductive organ.

Apparatus, Chemicals, and Other Materials

Cutting spoon, 1 cm., such as one used for

making melon balls for salad
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 69

Analytical balance

Sphagnum, peat moss, or fine quartz sand

Petri dishes or other shallow containers about

10 cm. in diameter

Space in darkened room with temperature

controlled at 18°-24° C.

0.1-0.5 g. of each compound to be evaluated

0.5 g. of methyl ester of alpha-naphthaleneacetic acid; diethanolamine salt of 6-hydroxy-3-(2H)-pyrida2inone (MH-30) ; or

3-chloro-isopropyl-N-phenyl carbamate for

standard treatment.

Suggested plant material—Unsprouted vegetable storage organs such as carrots (Dauciis

carota L.), onions (Allium cepa L.), and potato

tubers (Solanuin tuberosum L.) that have experienced a rest period.

Preparation and selection of plant material—Carrot: Select carrots of about medium size for

uniformity. Sever petioles so about 1 cm. of

each petiole remains attached to each root. Remove and discard the lower part of the root

saving about 2.5 cm. of the upper part. Place

pieces in humid air at 4°-10° C. for 3-4 days

or until they become suberized.

Onion: Select small onions about 1 cm. in

diameter and of uniform shape. Remove 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.

29). Place the pieces in humid air at 4°-10° C.

for several days until they become suberized.

i

i|i

.vp

Procedure—Select 10 of the prepared carrot

pieces. Dip these momentarily in a tapwater

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 10 cm.

in diameter and without covers (fig. 30). Dip

comparable batches of carrot pieces in tapwater

and place them on sphagnum or sand in the

manner described to serve as controls. Keep the

moss or sand moist, and allow the plant material to grow in darkness at room temperature,

18°-24° C.

Apply the chemical to the onion sets and the

potato pieces, and culture them in the same

manner as for carrot pieces.

Method of taking results—After the controls

have developed a measurable amount of new

vegetative growth, measure the length of each

sprout and the fresh weight of the detached

sprouts. Compare these measurements with

similar ones for the treated material.

Suggested standard for comparison—1,000 p.p.m.

of methyl ester of alpha-naphthaleneacetic acid ;

1,000 p.p.m. of diethanolamine salt of 6-hydroxy-3-(2H)-pyridazinone (MH-30); 2,000

p.p.m. of 3-chloro-isopropyl-N-phenyl carbamate.

References

Heinze, P. H., Marth, P. C, and Craft, C. C. Further

tests with 3-Chloro-isopropyl-N-phenyl Carbamate as

a Sprout Inhibitor for Potato Tubers. Amer. Potato

Jour. 32: 357-361. 1965.

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.

PN-297 A

FIGURE 29.—Use of melon-ball cutting spoon to remove

eyes from potato tuber.

WHEAT SEEDLING METHOD

Based on research by H. F. Taylor and B. E.

A. Knight.

Description of method—The growth-retarding

activity of a compound is assessed by comparing the height of wheat seedlings grown in nutrient solution to which the compound has been

added with the height of seedlings grown in

nutrient solution alone. The seedlings are placed

on pieces of gauze made buoyant with polystyrene beads. After the first leaf has emerged

from the coleoptile, each gauze "raft" containing the seedlings is floated on the test solution.

After 13 days, measurements taken of plants
70 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

'''**''^^5**''

PN-297 B

FIGURE 30.—Left to right, Carrot roots, potato buds, and onion sets growing on moistened sphagnum moss contained

in petri dishes.

grown in the test solution are compared with

those of plants grown in solutions without the

growth-retarding chemical.

Apparatus, Chemicals, and Other Materials

Filter paper, 9 cm. in diameter

Petri dishes, 10 cm. in diameter

Table space in a room illuminated with red

light of low intensity and temperature controlled at 24° C.

Constant humidity boxes with glass covers

Forceps

Crystallizing dishes, 94 X 50 mm., unspouted

Plastic drinking cups, 10 cm. high

Razor blades

Circular pieces of coarse cotton gauze, 95

mm. in diameter

Washed, expanded polystyrene beads, 1-5

mm.

Growth room with controlled temperature,

humidity, and day length

Analytical balance

Transparent 50-mm. rule

Salts for nutrient and micronutrient solutions (See "Procedure")

100 mg. of each compound to be tested

1 g. of (2-chloroethyl) trimethylammonium

chloride (known as CCC) for standard

treatment

Distilled water

Suggested plant material—Germinated wheat

seeds (Triticum aestivum L.) Eclipse variety.

Preparation and selection of plant material—Soak

wheat seeds in distilled water for 2 hr. and

place them on wet filter paper in petri dishes.

Allow the seeds to germinate for 42 hr. in a

humidity box (relative humidity 80-85%) at

24° C. and in red light of low intensity. Make

a raft upon which the plants will be grown.

To do this, make 3 cuts around the plastic cup

at the position of the arrows to detach rings

a and b (fig. 31, A). Place the circular piece

of gauze (fig. 31, B, g) on rings a and b inverted in the position shown. Press the rings

together holding the gauze taut (fig. 31, C).

Make the raft buoyant by introducing expanded

polystyrene beads beneath the gauze. To do this,

proceed as shown diagrammatically in figure

31, D and E. Place the raft (fig. 31, D, r) over

a small perforated plastic cup (fig. 31, D, p)

containing dry polystyrene beads. Immerse the

raft slowly in water, allowing the cup to be removed and the beads to ñoat up into the raft

without trapping large air bubbles. Lift the raft

from the water and place it in a crystallizing

dish (fig. 31, E, d) containing 200 ml. of distilled water (fig. 31, E, w). Place 12 uniform

42-hr.-old wheat seedlings around the edge of

the gauze with the primary root passing

through the gauze to hold the seeds in place.

Wrap a black paper band (fig. 31, Í7, s) around

the dish to reduce the light intensity to which

the roots are exposed and then move the dish

to the growth room (23° C, relative humidity

80%, 500-600 foot-candles of light intensity at

bench level, day length 15 hr.). Place a filter

paper disk over each dish for the first 24 hr. to

shade the root tips until they grow through the

gauze. When 10-15 mm. of the first leaf has

grown through the coleoptile, remove the 2 least

uniform plants from each raft.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 71

Procedure—Prepare the following solutions :

Test solution: 100 ml. of solution at a concentration of 10"^ M of each compound to be tested.

Nutrient solution: Dissolve in 1 liter of distilled

water :

33 g. of KH2PO4

133g. of Ca(N03)2*4H20

17 g. of MgS04 • 7H2O

6.1 g. of Fe-EDTA

Micronutrient solution: Dissolve in 1 liter of

distilled water :

290 mg. of H3PO4

200 mg. of MnS04 • 4H2O

10 mg. of CuS04 • 5H2O

20 mg. of ZnS04 • 7H2O

10 mg. of Na2Mo04 • 2H2O

Dilute the 10"^ M test solutions mentioned

above with distilled water to give 200 ml. of

solution at each of the concentrations to be used,

usually 10"^ M-10'-^ M. Pour each of these solutions into a crystallizing dish and add a raft

bearing 10 wheat seedlings.

After 4 days, introduce the nutrients into

the culture medium. Replace the solution in

each dish with 200 ml. of fresh test solution,

which also incorporates 2 ml. of the nutrient

solution and 2 ml. of the micronutrient solution.

This change is readily achieved either by siphoning away the remaining liquid before pouring in the fresh solution, or simply by transferring the raft to a clean dish containing the fresh

solution. Conclude the test 9 days later, after the

plants have grown in the test solution for a

total of 13 days.

Method of taking results—Assess the growth of

each of the 10 plants in a dish by measuring

the distance between the ligules of the first and

second leaves (fig. 31, F, x). Compare the

''treatment mean'' obtained from these 10

measurements with the ''control mean'' obtained

from measurements taken from 10 plants that

had received distilled water in place of the test

solution. If the treatment mean is subtracted

from the control mean and the difference expressed as a percentage of the control mean, a

convenient percent retardation value is obtained. Where the base of the second leaf blade

has not emerged from the sheath of the first

leaf, negative values for x are obtained and

figures for the retardation will be greater than

100%.

An alternative method of assessment, which

avoids negative values and percentages greater

than 100, is as follows: Record the length y

(fig. 31, F). Obtain treatment means and control means as before and express the difference

between these means as a percentage of the control mean.

B

b

g

a

immmmasm -d

\— S

- w

FIGURE 31.— A, B, C, D, E, Drawings of equipment used to culture wheat seedlings; F, drawing showing methods of

taking measurements. (After diagram by H. F. Taylor.)
72 AGRICULTURE HANDBOOK 336^ U.S. DEPARTMENT OF AGRICULTURE

Suggested standard for comparison—Solutions of

(2-chloroethyl) trimethylammonium chloride

(CCC) at concentrations of 3 X lO""^, 10"^% and

3 X 10-6 M.

Reference

Method submitted by H. F. Taylor and B. E. A. Knight,

Plant Growth Substance and Systemic Fungicide Unit,

Agricultural Research Council, Wye College, Near

Ashford, Kent, England.

ROOT GROWTH

AGAR SLANT METHOD

Based on research by R. L. Jones, T. P. Metcalfe, and W. A. Sexton.

Description of method—Seeds of oat and rape

are germinated on agar slants containing a test

chemical, and the root growth is studied.

This method is also useful in studying the

root growth of a wide variety of other kinds of

germinating seeds.

Apparatus, Chemicals, and Other Materials

Analytical balance

Test tubes, 1 X 15 cm.

Test tube racks

Light-proof chamber at room temperature of

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-phenyl-carbamate

Agar

Suggested plant material—Seeds of a crop and

a weed plant, such as oat (Avena sativa L.) and

rape (Brassica naptts L.).

Preparation and selection of plant material—Sort

the seeds, eliminating any that are shriveled or

diseased.

Procedure—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 mixture into separate, labeled test tubes,

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

extends about 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 2 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 upright and store them in

racks in a dark chamber at room temperature

of 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 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 are 5-6 cm. long, 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 are 6-7

cm. long, 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% 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
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 73

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 % 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 for comparison—Use 2,4-dichlorophenoxyacetic acid at a concentration of

0.1 p.p.m. as a standard for rape, and isopropylN-phenylcarbamate at 0.1 p.p.m. as a standard

for oat. 2,4-dichlorophenoxyacetic acid and isopropyl-N-phenylcarbamate at 0.1 p.p.m. are

rated as very active in this test when applied to

rape and oat plants as described.

Reference

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 METHOD

Based on research by T. K. Pavlychenko.

Description of method—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.

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.

Apparatus, Chemicals, and Other Materials

Cone-shaped galvanized iron cylinders, 20

cm. diameter at top, 25 cm. at bottom, 61

cm. or more high and fitted with perforated wooden or metal removable bottoms

Analytical balance

Sandy loam soil free from undecomposed

organic material, uniformly mixed and

well packed into cone-shaped cylinders

Greenhouse maintained at about 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

Suggested plant material—Barley seeds (Hordeum vulgäre L.), well matured (stored for

8-9 months after harvest), or other grass

seeds.

Preparation and selection of plant material—Discard any diseased, broken, or malformed seeds.

Procedure—Dip a number of seeds in aqueous

mixtures of each plant regulator at each of 3

dosage levels: 0.002, 0.004, and 0.006% are

suggested. The chemicals are dispersed in

water with the aid of a small amount of

ethanol. The alcohol concentration in the final

mixture should not exceed 0.1%. Plant 3 seeds

in duplicate cylinders for each chemical and

each concentration level. Repeat to obtain

duplicate sets that can be examined after 3

wk., 6 wk., and at maturity. Three days after

emergence, remove all but the one most vigorous plant in each cylinder. Grow plants in a

cool (20°-21° C.) greenhouse for 2 wk.; then

increase the temperature to 24°-29° C. thereafter. Dip seeds in water only and grow them

in the same manner as 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 wk., 6 wk., and at maturity, tapping the metal cylinders and then lifting them

off over the tops of the plants. Separate roots

from tops and record the number of roots,

fresh and dry weight of leaves and stems, and

the number of tillers. Carefully remove the

roots from the soil by immersing the soil together with the roots in water. Gently wash the

soil away from the roots 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 for comparison—Seeds

soaked in 0.002, 0.004, and 0.006% alphanaphthaleneacetamide.

Reference

Unpublished method proposed by T. K. Pavlychenko,

Amchem Products, Inc., Ambler, Pa.
74 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

IMPREGNATED FILTER PAPER METHOD

Based on research by J. W. Brown; Vivian

K. Toole, W. K. Bailey, and E. H. Toole.

Description of method—Root growth of germinating seeds on filter paper treated with the

test chemicals is observed.

Apparatus, Chemicals, and Other Materials

Analytical balance

Pressure-sensitive tape with adhesive on

both sides, Scotch brand No. 400, 13 mm.

wide

Filter paper, Whatman No. 1, 8 X 23 or 8 X

46 cm., depending on seed size

Filter paper strips 1 cm. wide with 6-mm.

holes punched with their centers 1-3 cm.

apart. For large seeds such as bean, pea,

sunflower, and corn use holes 3 cm. apart;

for small seeds, 1 cm. apart

Paper drinking cups, 10 cm. tall

Metal trays 48 cm. wide X 56 cm. long X 15

cm. deep with false perforated bottoms 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 28° C.

10 mg. of each compound to be evaluated

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

for standard treatment.

Acetone

Suggested plant material—Wide variety of

seeds—morning-glory (Ipomoea spp.), sunflower (Helianthus annuus L.), pigweed

(Amaranthus 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 hirsutum L.), cucumber (Cucumis sativus L.), barley (Hordeum vulgäre L.),

corn (Zea mays L.), oat (Avena sativa L.),

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

L.) have been used.

Preparation and selection of plant material—Sort

the various kinds of seeds, eliminating any that

are shriveled or diseased.

Procedure—Dissolve 10 mg. of the test chemical in 5 ml. of 100% acetone; then dilute to 1

liter with tapwater, 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 1 cm. past the edge of the large

paper. Place the filter paper strip with the

evenly spaced holes on top of the tape. Make

the length of paper strip such that it will leave

fifteen 6-mm. disks of adhesive exposed. Place

15 seeds of the first species to be tested on the

exposed disks, one seed to a disk. Roll the filter

paper with the seeds inside and hold the roll in

place with the exposed pressure-sensitive 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 the 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 the

control rolls in the metal trays filled with water

to a depth of 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 28° C.

Large seeds such as peanuts require a relatively large quantity of water to germinate

and they are sensitive to submergence in a

liquid. For these the following procedure is

suggested. Line plastic boxes about 20 X 25 X

10 cm. with paper toweling or filter paper,

using 2 thicknesses of the paper (25 X 100

cm.) folded to form corrugations 2 cm. high

(fig. 32). Wet the paper with 260 ml. of the

test solution and place the seeds (100 per box)

between the grooves of the paper with the radicle end down. Place covers on the boxes. Temperatures, solutions, and method of taking results are the same as those of the method

described more fully here.

Method of taking results—After 72-96 hr. (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

(acetone-water solution) minus

average treated root length

^ X 100 = percent

Average control root length inhibition

(acetone-water solution)
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 75

FIGURE 32.—Equipment used to germinate large seeds.

BN-28986

If 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, rerun the test.

Suggested standard for comparison—1 p.p.m. of

2,4-dichlorophenoxyacetic acid in tapwater containing a concentration of 0.5% acetone.

References

Brown, J. W. Two Tests for Herbicide Screening. Northeastern Weed Control Conference Proc. (Sup.) 7:

71-74. 1953.

Toole, V. K., Bailey, W. K., and Toole, E. H. Factors

Influencing Germination of Peanut Seeds. Plant

Physiol. 39: 822-832. 1964.

NUTRIENT MIST METHOD

Based on research by B. T. P. Barker; W.

Carter; L. J. Klotz; M. C. Vyvyan and G. F.

Trowell.

Description of method—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 to study growth

responses of many different kinds of plants.

Apparatus, Chemicals, and Other Materials

Mist chamber*

Analytical 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

Suggested plant material—Young trees—^maple

(Acer spp.), pine (PmMs spp.), oak (Querem

spp.), apple (Malus sylvestris Mill., also called

Pyrus malus L.), peach (Prunus pérsica [L.]

Batsch), and Citrus spp.

Preparation and selection of plant material—Select young trees at a uniform stage of development and growing at approximately the same

rate.

Procedure—Support trees (2-6 of each species) so 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

* Construction of various types of mist chambers is

described in articles listed at the end of this method.
76 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

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 overall 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

grown in the same chamber. The size of the

chamber may be varied from approximately

1-liter capacity to room size.

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 for comparison—No standard treatment suggested.

References

Barker, B. T. P. Studies of Root Development. Lon^

Ashton Res. Sta. Ann. Rpt. 1921: 9-20.

Carter, W. A Method of Growing Plants in Water Vapor

to Facilitate Examination of Roots. Phytopatholoe:v

32: 623-625. 1942.

Klotz, L. J. A Simplified Method of Growing Plants

with Roots in Nutrient Vapors. Phytopathologv 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. 1953.

ROOT INDUCTION

ROOTING COFACTOR METHOD

Based on research by C. E. Hess; variation

by S. Challenger, H. J. Lacey, and B. H. Howard.

Description of method—A bioassay designed to

detect natually ocurring substances that stimulate root initiation in the presence of indole-3-

acetic acid. Extracts of easy-to-root plants,

such as the juvenile form of English ivy

(Hederá helix, L.) and Chrysanthemum, contain 4 chromatographically separable substances that stimulate root initiation of mung

bean cuttings in the presence of indole-3-acetic

acid. An increase in the number of roots per

cutting over the controls (cuttings with indole3-acetic acid alone) indicates the presence of

a rooting cofactor. A decrease in the number of

roots is an indication of the presence of an

inhibitor.

Apparatus, Chemicals, and Other Materials

Vermiculite

Porcelain pan, 43 X 30 X 6 cm.

Growth chamber maintained at 27° C, 60%

relative humidity, and with about 3,000-

foot-candle light intensity from a combination of fluorescent and incandescent

lamps; 16-hr. photoperiod. A greenhouse

can be used but the results wi/ll be variable

because of variations in light intensity.

Shell vials, 19 X 65 mm.

Razor blade

Water aspirator

Waring Blendor

Separatory funnel

Chromatographie equipment (ascending or

descending)

Chromatographie paper, Whatman No. 3

MM

Lyophilizer

pH meter

Ether

Chloroform

About 500 mg. of indole-3-acetic acid and an

equal amount of catechol for standard

treatment

Methanol

Ethanol

Isopropanol

Distilled water

Sodium hypochlorite

Suggested plant material—Nine- to 10-day-old

seedlings of mung bean (Phaseolus aureus

Roxb.—large oriental variety).

Preparation and selection of plant material—

Treat 95-100 ml. of dry mung bean seeds for

3 minutes in a 0.33% solution of sodium hypochlorite. Rinse the seeds and then soak them

in running tapwater for 18-24 hr. Place the

seeds on moist vermiculite in a porcelain pan

and cover them with a layer of moist vermiculite about 1.5 cm. thick. Germinate the seeds in

a growth chamber.

Make cuttings from selected, uniform seedlings 9-10 days old. Do this when the primary

leaf is fully expanded and the trifoliolate leaf

bud has not expanded. Prepare the cuttings by
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 77

cutting off the seedling root system and removing any cotyledons that have not abscised. The

cutting consists of 3 cm. of hypocotyl, plus the

epicotyl, the primary leaves, and the unexpanded trifoliolate leaf bud. Select cuttings of

uniform size and shape to avoid wide variations between individual cuttings in the same

vial.

Procedure—Place 5 or 10 cuttings in each of

17 shell vials, together with 4 ml. of a 5 X 10"^

M solution of indole-3-acetic acid per vial. The

incubation solution will be taken up by the cuttings within 18-24 hr. Whenever required, replace with distilled water the amount of solution taken up during the 6- to 7-day period

allowed for root growth.

To obtain substances to use in this rooting

test, extract lyophilized tissue or fresh tissue.

If lyophilized tissue is used, extract 50-200

mg. of ground, lyophilized tissue 3 times with

25- to 100-ml. portions of absolute methanol.

Combine the extracts and concentrate them

under reduced pressure, using a water aspirator to provide the vacuum. Streak the extracts

across a strip of Chromatographie paper 5 cm.

wide. Develop the chromatogram (descending)

with isopropanol-water (8:2 v/v) after a 3-hr.

period for equilibration. Allow the solvent to

descend 30 cm. below the origin.

If fresh tissue is used, boil it in ethanol or

methanol for at least 1 minute, and then grind

it in a Waring Blendor. Filter and extract the

residue 2 more times with fresh portions of

ethanol or methanol. Combine the extracts and

evaporate to dryness. Dissolve the extract in

80% ethanol and streak the solution on the

Chromatographie paper. Chromatograph as decribed above.

After drying the chromatogram, divide it

into fifteen 2-cm. segments. Fold each piece

(2X5 cm.) and place each separately in a

shell vial. For controls, cut from the same

chromatogram 2 comparable pieces of paper,

i.e., one 2 X 5-cm. piece from above the origin,

and one piece of this size from below the solvent front. When each segment is in a corresponding vial, add 4 ml. of a 5 X 10"^ M solution of indole-3-acetic acid to each vial. Place

the cuttings in the vials as described above

and return them to the growth chamber.

In addition to separation by paper chromatography, preliminary fractionation is possible.

Evaporate the extract. Dissolve the extract by

shaking it, together with chloroform and

water, in a separatory funnel. The chloroform

layer will contain Cofactor 4. Acidify the remaining aqueous layer to a pH of 3.0 and partition it with ether. Cofactor 3 will go into the

ether layer, and Cofactors 1 and 2 will remain

in the aqueous layer.

Method of taking results—Count the number of

roots on each cutting 6-7 days after the cuttings are made. Compare the average number

of roots per cutting for each treated segment

with the average number of roots per cutting

for the control segments. Prepare a histogram

to show peaks of promotion and inhibition.

The approximate Rf values of the 4 rooting

cofactors using the isopropanol-water solvent

system are:

Rf value

Cofactor 1 0.1

Cofactor 2 0.3

Cofactor 3 0.6

Cofactor 4 0.8

Suggested standard for comparison—Indole-3-acetic acid 5 X 10"^ M; indole-3-acetic acid 5 X

10« M together with catechol 3 X 10"^ M; and

catechol 3 X 10"^ M. the combination of indole3-acetic acid and catechol is normally synergistic.

Variation used by Challenger, Lacey, and Howard

—To prepare plant extracts for use in testing

for root cofactors, cut 3-cm. segments from

stems of the plants to be tested. Place these,

basal end downward, in moist sand for a period

sufficient to allow callus to develop or root

primordia to become apparent. Remove the cuttings, wash the sand from the basal ends, and

then immerse the cuttings for 24 hr. in 96%

methanol at 3° C. Collect the extract and repeat the extraction for another 24 hr. Combine

the 2 extracts and evaporate the alcohol under

vacuum at 25° C. Centrifuge the aqueous residue. Extract the water-soluble materials with

ether, first at pH 8 and then at pH 4. Apply

replicate aliquots of the acid fraction to strips

of Whatman No. 1 filter paper and develop,

using descending chromatograms and isopropanol-water-ammonia (10:1:1 v/v) as a solvent system. Dry the strips in darkness and

divide each strip into 10 segments. Determine

the amount of activity in each segment by

means of the rooting cofactor bioassay.

References

Challenger, S., Lacey, H. J., and Howard, B. H. The

Demonstration of Root Promoting Substances in Apple

and Plum Rootstocks. Ann. Report of the East Mailing

Res. Sta. 1964: 124-128. 1965.

Hess, C. E. A Physiological Analysis of Root Initiation

m Easy and Difficult-to-Root Cuttings. XVI Internatl.

Hort. Cong. Proc. 4: 375-381. 1964.

Hess, C. E. Naturally Occurring Substances Which

Stimulate Root Initiation. Régulateurs Naturels de la

Croissance Végétale, Cent. Nati, de la Rech. Sei.,

Paris, 123: 517-527. 1964.
78 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

ROOT PRIMORDIA METHOD

Based on research by Tetsuo Takematsu.

Description of method—The number of root

primordia induced on young radish stems

(hypocotyls) depends on the kind and concentration of growth substance applied. Roots and

hypocotyls of intact radish seedlings are placed

for a specified length of time in a solution of

the test compound and then transferred to

sand. The number of induced root primordia

that form on hypocotyls of treated plants is

compared with the number of primordia that

form on hypocotyls of the control plants. The

difference represents the root-inducing capacity of the compound.

Apparatus, Chemicals, and Other Materials

Petri dishes, large

Sand

Analytical balance

Glass cylinder (jar or vial), 3.5 cm. deep and

3 cm. in diameter

Transparent glass plate

Black paper

Dissecting needle

Tweezers

Magnifying glass or microscope of low magnitude

10 mg. of each compound to be evaluated

10 mg. of indole-3-acetic acid or alphanaphthaleneacetic acid, and 10 mg. of 2,4-

dichlorophenoxyacetic acid for standard

treatments

Suggested plant material—Hypocotyls of young

radish plants (Raphanus sativus L.)—Riso

Daikon variety, or other varieties with a high

degree of sensitivity to root-inducing compounds.

Preparation and selection of plant material—

Plant radish seeds 0.5 cm. deep in the large

petri dishes two-thirds filled with sand and

maintain the moisture content of the sand at a

level of 20% water by weight. Place'the dishes

in diffused light at a temperature of about 24°

C. Three to 4 days after germination, add sufficient water to the dishes so seedlings can be

pulled easily from the sand without damaging

the roots or hypocotyls. Select seedlings of uniform size and shape with well-formed cotyledons, with hypocotyls approximately 3-4 cm.

long, and with taproots of approximately the

sanie length. Keep the selected plants temporarily in petri dishes containing water to prevent wilting. It is not necessary to wash the

sand from the roots.

Procedure—Prepare a series of dilutions of

the growth substances to be tested by dissolving each regulating compound in a small

amount of alcohol (not to exceed 0.1% of the

water on a volume basis). Then, while stirring,

add the alcoholic solution to the required

amount of water at concentrations from 0.01-

500 p.p.m. Place each concentration in a separate glass cylinder to two-thirds of the cylinder's depth. Soak roots and hypocotyls of each

group of 15-20 radish plants in respective test

solutions contained in the glass cylinders. The

duration of the treatment varies according to

the growth substances to be tested and their

concentrations. For example, use a 12- to 24-hr.

period for 5-p.p.m. solution of indole-3-acetic

acid, 5-p.p.m. solution of alpha-naphthaleneacetic acid, and 0.5- to 1-p.p.m. solution of 2,4-

dichlorophenoxyacetic acid. For comparison,

place one lot of plants into a cylinder containing distilled water for a corresponding period.

Transplant the treated and untreated seedlings from cylinders to the large petri dishes

containing sand maintained at a moisture level

of 25% and keep them in a room at 70-80%

humidity and about 20°-24° C.

Method of taking results—Seventy-two hours after the treated plants and the controls are

placed in the sand, remove each plant from the

petri dishes and wash the sand from the roots.

Discard wilted plants. Place the plants on a

transparent glass plate backed with black

paper. Strip off the cortex of the hypocotyls

with a dissecting needle and tweezers, exposing

the central cylinder (stele). Examine the stele

of each hypocotyl with a magnifying glass or

microscope and record the number of root

primordia. The number of primordia on the

treated hypocotyls compared with the number

on the hypocotyls kept in distilled water indicates the effectiveness of the growth-regulating

substance as a root-inducing compound. Measure 10-20 plants to obtain the average root

primordia formation for each lot of seedlings.

Suggested standard for comparison—5 p.p.m.

of indole-3-acetic or alpha-naphthaleneacetic

acid solutions and 0.5-1 p.p.m. solution of 2,4-

dichlorophenoxyacetic acid for 12-24 hr.

Reference

Takematsu, Tetsuo. New Methods for the Qualitative

and Quantitative Assays of Growth Substances; Research on the Raphanus Test and Its Application.

Utsonomiya Univ., Col. of Agr. Spec. Bui. 12, 193 pp.

1961.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 79

STEM-CUTTING METHOD

Based on research by J. W. Mitchell and P.

C. Marth; H. B. Tukey; J. S. Wells.

Description of method—Cuttings are soaked,

dusted, or dipped in mixtures containing chemicals, and the effect on root initiation and

growth is determined.

Compounds that affect top growth can also

be applied separately or simultaneously with

root-inducing substances.

Apparatus, Chemicals, and Other Materials

Knife with sharp, thin blade 10-15 cm. long

Analytical 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 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, 8 cm. in diameter

Composted soil

100-200 mg. of each compound to be evaluated

500 mg. of 3-indolebutyric acid or alphanaphthaleneacetamide for standard treatment

Solvent such as 95% ethanol

0.5 kg. of dust carrier such as talc, fuller's

earth, or finely ground clay

Suggested plant material—Pieces of stems of

woody plants—privet (Ligustrum spp.), yew

{Taxus spp.), osmanthus (Osmanthus spp.),

and holly (Ilex spp.) ; or of herbaceous plants

—chrysanthemum (Chrysanthemum supp.),

geranium (Geranium spp.), lantana (Lantana

spp.), and carnation (Dianthus caryophyllus

L.).

Preparation and selection of plant material—Cuttings from 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 10-

15 cm. long or pieces including 3-6 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 10-20 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.

Procedure—Three methods of treating cuttings have proved suitable for this test:

Soak 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 3 cm. (fig. 33, A). 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 3-6 hr. in

diffused light, remove the cuttings and insert

the basal ends of the stems to a depth of 5-8

cm. in the rooting medium contained within the

propagating chamber (fig. 33, B). Pack the

moist rooting medium firmly around each stem

and water thoroughly. Keep the medium moistened throughout the test.

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 regulator-dust 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 3 cm. of the basal part in the dust mixture so that a thin coating of dust adheres to it.

If the dust fails to adhere, moisten the end of

the cutting before applying the dust (fig. 33,

C). Tap off any loose dust and set the treated

portion of the cutting in the rooting medium

(fig. 33, B). Do not rub the dust from the cuttings when inserting them in the medium;

place in a trench wide enough so the dust remains on the stem.
80 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

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

3 cm. in one dilution of a compound, and insert

the treated portion of these cuttings individ-

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FIGURE 33.—Applying regulating chemicals to determine their effect On initiation of roots by stem cuttings: A, Soaking cuttings in solution of test compound ; B, inserting cuttings in rooting medium ; C, dusting cuttings with a mixture of test compound; D, examining cuttings to determine whether roots have formed.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 81

ually in the rooting medium. Repeat, using

other bundles of cuttings for each concentration of each test chemical.

Method of taking results—Examine several of

the cuttings at intervals to determine whether

roots have formed (fig. 33, D),

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 for comparison—Soak

method, using 3 concentrations of 3-indolebutyric acid or alpha-naphthaleneacetamide (25,

50, and 100 p.p.m.) for 2-4 hr.

References

Mitchell, J. W., and Marth, P. C. Growth Regulators.

129 pp. Univ. of Chicago Press, Chicago, 111. 1947.

Tukey, H. B., editor. Plant Regulators in Agriculture.

269 pp. John Wiley and Sons, Inc., New York. 1954.

Wells, J. S. Plant Propagation Practices. 344 pp. Macmillan Co., New York. 1955.

SEED FORMATION

LANOLIN PASTE METHOD

Based on research hy S. L. Emsweller and

N. W. Stuart; R. E. Wester and P. C. Marth.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Clay pots, 15-25 cm.

Composted soil

Space in greenhouse maintained at about

21^-29° C. by day and 18°-21° C. by night

Small vials, 25-50 ml., or other containers

Analytical balance

250-500 mg. of each compound to be evaluated

1 g. each of alpha-naphthaleneacetamide,

para-chlorophenoxyacetic acid, and 3-indolebutyric acid for standard treatment

Lanolin

Tv^een 20

Suggested plant material—Species of Lilium

such as L. longiflorum Thunb, (Creole, Croft,

or Ace clones) ; bush lima beans (Phaseolus

lunatus L.), Fordhook, Early Market, or Peerless varieties; or other plants difficult to hybridize.

Preparation and selection of plant material—

Grov^ the plants, knov^n to be difficult to cross,

under usual greenhouse conditions.

Procedure—Prepare a range of concentrations

of the chemical to be tested in lanolin, as described on p. 7.

Lilies: Immediately before hand-pollinating

the flowers, separate one ''petaF' (i.e., one

member of the outer v^horl 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 to use 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 seeds. Determine the viability of the

seeds.

Suggested standard for comparison—For lima

beans, make a lanolin mixture containing sufficient 3-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

3-indolebutyric mixture to one part of the phenoxyacetic acid mixture.
82 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

References

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.

DETECTION OF EXOGENOUS GROWTH REGULATORS IN PLANTS

GRAFT METHOD

Based on research by T. J. Muzik and J. W.

Whitworth.

Description of method—The presence of a

growth-regulating substance or a metabolite of

it in a plant previously treated with the chemiical is determined qualitatively by grafting a

scion from an untreated plant of the same kind

to the plant being tested. Malformation of

leaves that develop on the scion, typical of

those induced by the compound involved, indicates that there is an effective, translocatable

amount of the compound or a metabolite of it

in the test plant.

Apparatus, Chemicals, and Other Materials

Beaker, 1 liter

Razor blade or scalpel

Composted soil

Clay pots

Analytical balance

0.5 g. of compound to be evaluated

0.5 g. of 2,4-dichlorophenoxyacetic acid for

standard treatment

Suggested plant material—Young, vigorously

growing tomato plants (Lycopersicon esculentum Mill.). Other kinds of plants that can be

readily grafted and are sensitive to the grovrthregulating chemical involved can also be used.

Preparation and selection of plant material—

Grow tomato plants in composted soil maintaining conditions that afford vigorous growth

throughout this period. Select 24 plants of uniform size about 6-8 wk. old and set aside 8-10

others as a source of scions.

Procedure—While the fourth leaf from the

base on each plant of the first group of 24 is

still attached, dip each of these leaves into a

water solution containing 1,000 p.p.m. of the

compound to be evaluated. Excise each of the

treated leaves after 8 days. Collect 4 buds or

4 short branches from the untreated plants.

Graft one of these on each of the 4 treated

plants at the second internode above the excised leaf. Collect additional scions from the

untreated plants 2 wk. later and graft them to

4 more of the treated plants. Repeat the grafting procedure at 2-wk. intervals for 60-90

days.

Method of taking results—Allow the scions on

all grafts to grow and produce several leaves.

Examine these and record the extent to which

the new leaves are malformed. Presence of

malformed leaves indicates that sufficient

growth regulator or a derivative of it was

translocated from the treated plants to the scion

to influence grov^h. Determine the length of

time after treatment during which positive

results are obtained.

Suggested standard for comparison—2,4-dichlorophenoxyacetic acid at concentration of 1,000

p.p.m.

Reference

Muzik, T. J., and Whitworth, J. W. Growth-Regulating

Chemicals Persist in Plants: Qualitative Bioassay.

Science 140: 1212-1213. 1963.

DETECTION OF EXOGENOUS PLANT GROWTH REGULATORS IN

ANIMALS

BEAN STEM METHOD

Based on research by J. W. Mitchell, R. E.

Hodgson, and C. F. Gaetjens.

Description of method—Regulating chemicals

are extracted from animal tissues, fluids, secretions, or excrement. The extract is applied to

stems of young bean plants, and elongation in-
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 83

dicates 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.

Apparatus, Chemicals, and Other Materials

Millimeter rule

Pipette, 0.1 ml. capacity graduated in 0.01

ml.

Fluorescent lights that will supply 800 footcandles of light intensity at leaf surface

Wiley Mill or suitable means of grinding

samples to 60 mesh

Glass containers for holding samples

Analytical balance

1-2 g. of sodium or ammonium salt of 2,4-

dichlorophenoxyacetic acid for standard

treatment

Centrifuge

Composted soil

Clay pots, 8 cm.

Suggested plant material—Young bean plants

(Phaseolus vulgaris L.)—Black Valentine or

Pinto variety.

Preparation and selection of plant material—

Germinate bean seeds in composted soil contained in 8-cm. clay pots, using several seeds

per pot. Allow the plants to grow in a wellilluminated greenhouse until they are about 9

cm. tall. If the temperature is maintained near

27° C. during germination and adequate sunlight is available, the plants will produce

sturdy stems and reach the required height in

about 5 or 6 days. 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 be, whenever possible, 13

mm. but may in some cases be 12 or 14 mm.

Select a sufficient number of plants so 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 the plants can

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.

Procedure—Prepare 4 aqueous solutions of

sodium 2,4-dichlorophenoxyacetate, the first

containing 60 p.p.m. of the salt (6 mg. of 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 ingested the chemical under test, and also serum

from animals that have been fed the test compound. Measure five 3-ml. portions of the control serum. Prepare standard growth-regulator-serum mixtures by adding none of the

aqueous salt solutions to the first portion of

control serum, 1 ml. of the 60 p.p.m. solution to

the second portion of serum, 1 ml. of the 30

p.p.m. solution to the third portion of serum,

continuing until each concentration of salt has

been added to a corresponding portion of control serum. Measure several 3-ml. portions of

serum from the animals that were fed the

growth regulator.

Evaporate separately all the serum samples

on a steam bath. Grind the residues quantitatively through a 60-mesh screen and 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 5-10 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, with 12 hr. of light, followed by 12 hr.

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 that received

serum from untreated animals with that of

plants that received serum from 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 animals. Using this standard curve, express the

amount of elongation obtained with serum
84 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

from the treated animals 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 for comparison—Sodium or

animonium salt of 2,4-dichlorophenoxyacetic

acid used as described above.

Reference

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.

DETECTION OF REGULATORS IN WATER

ROOT GROWTH METHOD

Based on research by D. Ready and V. 0.

Grant.

Description of method—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.

Apparatus, Chemicals, and Other Materials

Petri dishes, 15 cm. in diameter

Filter paper

Analytical balance

Darkened constant-temperature room maintained at about 28° C.

1 g. of 2,4-dichlorophenoxyacetic acid for

standard treatment

Distilled water

Suggested plant material-

{CucuTYiis sativus L.).

-Cucumber seeds

Preparation and selection of plant material—Use

seeds having a high percentage of germination ;

sort them, and discard those that are defective.

Procedure—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 28° C. Prepare 5 dishes,

using distilled water instead of the test solution, and maintain these in the same manner

for controls.

Method of taking results—After 96 hr., 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 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.

Reference

Ready, D., and Grant, V. O. A Rapid Sensitive Method

for Determination of Low Concentrations of 2,4-Dichlorophenoxyacetic Acid in Aqueous Solution. Bot.

Gaz. 99: 39-44. 1947.

PENETRATION OF SOIL BY REGULATORS

SEED METHOD

Based on research by P. J. Linder.

Description of method—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 method 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. This method also

can be adapted to the study of persistence of

regulating chemicals in soil as affected by fac-
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 85

tors such as temperature, moisture, duration of

exposure, and microbial content of the soil.

Apparatus, Chemicals, and Other Materials

Straight glass tubes, 3 cm. in diameter X 14

cm. long

Cork stoppers to plug one end of the glass

tubes

Fertile soil passed through a screen with 0.6-

cm. openings

Cork stoppers with a maximum diameter

slightly less than the inside diameter of the

glass tubes

Dowel, 0.6 cm. in diameter, 30 cm. long

Analytical 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, about 6 cm. diameter

Dark chamber maintained at about 27° C.

with a high relative humidity

About 5 g. of each compound to be tested

Suggested plant material—Seeds of mustard

(Brassica spp.) or other small-seeded plants

known to be sensitive to the compounds to be

tested.

Preparation and selection of plant material—

Sort the seeds, eliminating any that are

shrunken or defective.

Procedure—Tightly stopper one end of the

glass tube. 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. to

compress 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. m. on the floor. If necessary, 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 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 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-

100%. 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 sufficient 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 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 growth of

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 that have been treated with a test chemical.

Suggested standard for comparison—No standard treatment suggested.

Reference

Linder, P. J. Movement and Persistence of Herbicides

Following Their Application to the Soil Surface.

Northeast. States Weed Control Conf. Proc. 6: 7-11.

1952.
86 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

RESIDUE OF REGULATORS IN SOIL

SUCCESSIVE CROP METHOD

Based on research by A. J. Loustalot and R.

Ferrer; W. W. Robbins, A. S. Crafts, and R. N.

Raynor.

Description of method—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.

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 environment on the

persistence of the chemical in soil.

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

Greenhouse space

About 2-3 g. of each compound to be evaluated

Suggested plant material—Use plants known to

be highly sensitive to the compound to be tested.

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.

Preparation and selection of plant material—Sort

the seeds, eliminate any that are defective.

Procedure—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 0.5 kg. of the chemical applied to 4,047 sq. m.

of clay loam soil and mixed to a depth of 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-

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 depth of 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 10-15

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 in which

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 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 succès-
MP:THODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 87

sive 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 for comparison—No standard treatment suggested.

References

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. Ed. 1, 503 pp. McGraw, Hill Book Co., New

York. 1942.

SOME PHYSICAL METHODS

INTRODUCTION

Physical methods, such as gas chromatography and measurement of radioactivity, are

used widely for detection and identification of

exogenous regulating substances in plants.

Some chemical methods are also useful in this

way. We are, however, not attempting to describe these physical and chemical methods comprehensively here. On the other hand, because

of their importance in the study of growth regulators, radioactive isotopes must be at least

briefly dealt with.

The use of radioactive tracer methods is based

on the generally valid assumption that the active isotope behaves biologically in the tagged

molecule exactly as does the inactive counterpart in a similar untagged molecule. An important characteristic of radioactive tracer

methods is their extreme sensitivity when used

to detect and measure tagged compounds. For

instance, satisfactory quantitative measurement

of C^^ can be obtained on 2 X lO^^^ g. of this

substance.

Isotopes emitting alpha particles and beta and

gamma rays have proved very useful in detecting tagged growth-regulating substances in entire plants and to some extent in thin sections of

plant parts by means of radioautographs. As a

general rule, adequate images are obtained on

film when as little as 4 X lO'^^ g. of C^^ per

cm.- is exposed to the film for about one month.

In using radioisotopes, it must be kept in

mind that degradation of at least some regulating substances, such as naphthaleneacetic acid,

may occur on the surface of the plant even before the regulator is absorbed. Furthermore,

absorbed regulating substances are sometimes

altered within the plant in many ways. Because

of these changes which result in degradation

products or metabolites, it is desirable, whenever possible, to identify the molecule with

which the tag is associated once the compound

enters the plant.

Comprehensive information concerning isotope techniques and other physical and chemical

methods of studying regulating substances can

be found in references listed at the end of this

section. Since radioactivity is used so widely

to study regulating substances, some representative isotope techniques are described here.

Some radioactive materials are dangerous.

Experiments with radioactive substances

should not be undertaken without a thorough knowledge of the hazards involved.

RADIOAUTOGRAPHS OF ENTIRE PLANTS

RADIOAUTOGRAPH METHOD

Based on research by Shogo Yamaguchi and

A. S. Crafts; variation by Edgar Inselberg.

Description of method—Compounds labeled

with beta ray emitters are applied to leaves or

to the culture medium where they are absorbed

by the roots. The plant is later freeze-killed and

freeze-dried under vacuum. X-ray film is then

exposed to the plant for the required period. The

image obtained on the film is registered with

the dried plant and the exact position of the

tagged compounds recorded.

Apparatus, Chemicals, and Other Materials

Test tubes, 10 X 75 mm.

Micropipette, 10-lambda

Lanolin stiffened with granular starch or

water
88 AGRICULTURE HANDBOOK 336^ U.S. DEPARTMENT OF AGRICULTURE

Small wire loop

Facial tissues

Dry ice

Aluminum foil

Masking tape

Hardware cloth tray large enough to support

plant involved

Cardboard box, 15-20 cm. deep, large enough

to contain the hardware cloth tray

Vacuum tank

Freezing compartment maintained at approximately — 15° C.

Vacuum pump

Vacuum hose

Vapor trap with freezing mixture of dry ice

and alcohol

Humidity chamber

Glossy white paper

Rubber cement

Blotters

Sheets of plywood

Plant press

X-ray film, 25 X 30 or 36 X 43 cm.

Sponge rubber, 0.6 cm. thick

Elastic web belts

Light-tight box or light-proof wrapping material

Dark room

X-ray film development supplies and equipment

Treatment solution containing approximately

0.05-1.0 microcurie of the labeled substance per 10 microliters of 50% ethyl alcohol and 0.1% Tween 20 in water

Suggested plant material—Any kind of plant

can be used and a radioautograph can be made

of any part of a plant, provided the part is of

suitable size.

Preparation and selection of plant material—Select plants to be treated that are growing vigorously since rapid growth is associated with

active transport. For leaf treatments, select

leaves that are three-fourths or fully expanded.

Procedure—With a wire loop apply lanolinstarch paste to make a ring of the lanolin mixture, about 6 mm. in inside diameter, near the

base of the leaf. (See also, ''Application of a

measured amount of regulating chemical to a

known area of leaf surface," p. 98.) With the

micropipette apply a measured amount of the

labeled compound in solution covering completely the area within the lanolin ring. For full

development of the distribution pattern, allow

2 days for a fast-growing plant and 4-10 days

for others.

After this period, carefully wipe off the lanolin paste with tissue, making certain that the

tagged compound does not contaminate any

parts of the plant. To guard further against

contamination of other areas, cover the treated

area on the leaf with masking tape or remove

the entire treated area. (See ''Separation of leaf

areas treated with a regulating chemical from

the remaining untreated portion of the leaf,"

p. 123.) Place the plant on a hardware cloth

tray.

Place the hardware cloth tray into the vacuum tank which is then placed in a freezer compartment at — 10° or — 15° C. Connect the tank

with a vacuum line, and evacuate it. Protect the

vacuum pump from moisture by placing a vapor

trap with a dry ice-alcohol mixture in the line

leading from the tank to the pump.

Allow 4-10 days' drying time with pressures

of 0.02-2.0 mm. of mercury. After the plant is

dried, place it in a humidity chamber until it

becomes flexible. Mount the plant with rubber

cement that has not thickened on glossy white

paper and press overnight between blotters in

a press board. Place the mounted plant against

an X-ray film. Sandwich the film and plant between sheets of sponge rubber and between 2

pieces of plywood and wrap the entire assembly

with aluminum foil. If many plants are involved, repeat the procedure, forming a stack of

sandwiches. Fasten an elastic belt around the

stack and store it in a light-tight box for 2 wk.

At the end of the storage period, develop the

film using standard methods but reduce the

time of development by one-half to three-fourths

to obtain relatively light backgrounds.

Method of taking results—Place the radioautograph against glossy white paper and record

the position of the darkened image that shows

distribution of the labeled compound. Determine the exact location of the labeled compound

by comparing the darkened image with the

pressed and dried plant material.

Suggested standard for comparison—No standard treatment suggested.

Variation used by Inselberg—Another method

which might be of interest when high-energy

beta, low-energy gamma, or X-ray radiation is

involved provides for using the film without removing it from its original container, thus eliminating the need for light-proof equipment.

References

Crafts, A. S., and Yamaguchi, Shogo. Gross Autoradiography of Solute Translocation and Distribution in

Plants. Med. and Biol. Illus. 10: 103-109. 1960.

Inselberg, Edgar. Simplified Procedures for Radioassay

and Autoradiography of Plant Materials. Agron. Jour.

51: 301-306. 1959.

Yamaguchi, Shogo, and Crafts, A. S. Autoradiographic

Method for Studying Absorption and Translocation of

Herbicides Using C^-^-Labeled Compounds. Hilgardia

28: 161-191. 1958.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 89

TRANSLOCATION OF RADIOACTIVELY TAGGED COMPOUNDS

GROUND TISSUE METHOD

Based on research by J. W. Mitchell and P. J.

Linder.

Description of method—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.

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. 34)

Brass rings (inside diameter 25 mm., outside

diameter 31 mm., 3 mm. high)

Cellophane tape, 4 cm. wide

Analytical balance

0.5-1.0 mg. of 2,4-dichloro-5-iodophenoxyacetic acid tagged with r-^\ one of its salts,

or 2,4-dichlorophenoxyacetic acid tagged

with C^"^ for standard treatment

Lanolin

Tween 20

FIGURE 34.—Diagram of remote-control applicator used

to apply small volumes of solutions containing radioactive regulating chemicals to leaves and stems of

plants.

Suggested plant material—Any kind of terrestrial plant.

Preparation and selection of plant material—The

following method is designed for use in studying translocation from leaves of plants such as

bean (Phaseolus vulgaris h.), tomato (Lycopersicon esculentum Mill.), 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.

Procedure—Make a remote control applicator

as follows: Support an aluminum rod (fig. 34,

a) approximately 65 cm. long and 5 mm. in

diameter with two 13-mm. aluminum angles

(6). Thread one end of the rod and screw it

through a plate into a metal box (c) so it rests

against a movable plate (d). Turn the rod to

press the plate against the piece of soft rubber

tubing (6), which is held in place by clamps.

Close the upper end of the tubing with a glass

plug. Insert a 0.1-ml. pipette (/) 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 0.02 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 tip of the pipette to spread

the liquid evenly over the plant surface.

Prepare an aqueous mixture which contains

0.1% of Tween 20 (or other suitable cosolvent)

and 10 fxg, of the radioactive plant regulator

per 0.01 ml. This can be easily accomplished,

using the proper precautions, by weighing the

required number of mg. of the radioactive substance into a vial and adding the required

amount of distilled water containing the cosolvent. With 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. (See p. 98.) With the

remote control applicator, place 0.01 ml. of the
90 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

mixture within the lanolin ring. If the entire

upper surface of the leaf is to be covered with

the mixture, use the method described on p. 12.

Replicate treatment with any one tagged 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

with fluorescent tubes so 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 depends 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 difficult 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, difficult to grind into

particles of relatively small size. Care must be

exercised in the utilization of this method so

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 for comparison—2,4-Dichloro-5-iodophenoxyacetic acid tagged with

V-^\ one of its salts, or 2,4-dichlorophenoxyacetic acid tagged with C^^.

Reference

Mitchell, J. W., and Linder, P. J. Some Methods Used

in Tracing Radioactive Growth-Regulatinp: Substances

in Plants. Bot. Gaz. 112: 126-129. 1950.

VOLATILITY ' OF PLANT REGULATORS

DISPOSABLE CONTAINER METHOD

Based on research by P. C. Marth and J. W.

Mitchell.

Description of method—This method 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

•' Carelessness in the use or disposal of highly volatile

regulating chemicals can result in contamination of an

entire laboratory or greenhouse, thus causing loss of

valuable plants and space.

compounds or from surfaces of other materials

to which these substances have been applied.

Apparatus, Chemicals, and Other Materials

Clay or paper pots, 8 cm., or clean discarded

tin cans, about 450-g. capacity

Analytical balance

Greenhouse space maintained at 24°-29° C.

for growing test plants

Table or bench space in darkened room maintained at 21°-32° C. for conducting tests

Several 1- and 5-ml. pipettes

Vials, 20-30 ml. with tightly fitted stoppers

or screwcaps
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 91

Filter paper No. 1 or other absorbent paper

disks, 7-9 cm. in diameter

Cellophane bags, 10 cm. square and 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

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 esculentum Moench), cucumber {Cucumis sativus L.),

and tomato (Lycopersicon esculentum Mill.).

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

to produce rapidly growing seedlings for the

tests. Select bean plants that have primary

leaves measuring 3 cm. across, and with trifoliolate leaves still folded in the terminal

buds (about 5-7 days old) ; or tomato plants

6.4-7.6 cm. tall (about 25 days old). Thin the

plants, leaving a single plant, all of uniform

size, in each pot.

Procedure—Prepare an alcohol or acetone solution of the pure chemical by placing a weighed

amount of the chemical 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 disk with 1 ml. of the alcohol 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, about 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 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 at room temperature; then remove,

and allow them to remain in a greenhouse for

later observation. Dispose of the filter paper

containing the chemical, bags, etc., so as to

avoid contamination of the 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 second, 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 any remaining contaminated materials.

Suggested standard for comparison—Impregnate

a filter paper disk with 0.2 mg. of butyl ester

of 2,4-dichlorophenoxyacetic acid and expose

the plants exposed to the vapors for 48 hr. at

27^-32° C.

Reference

Marth, P. C, and Mitchell, J. V^. Comparative Volatility

of Various Forms of 2,4-D. Bot. Gaz. 110: 632-636.

1949.

GERMINATING SEED METHOD

Based on research by W. P. Anderson.

Description of method—Relative volatility of

regulating compounds is determined by exposing germinating seeds to vapors of these chemicals in closed containers.

Apparatus, Chemicals, and Other Materials

Petri dishes, about 15 cm. in diameter X 2

cm. deep and 5 cm. in diameter X 2 cm.

deep

Filter paper, Whatman No. 1, to fit the petri

dishes

Forceps

Wide-mouth moisture- and air-tight containers, 0.5-1 liter
92 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Constant-temperature box or room controlled

at 21°-24° C.

Analytical 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

Suggested plant material—Cucumber (Cucumis

sativus L.), buckwheat (Fagopyrum esculentum Moench), corn {Zea mays L.), or other

seeds.

Preparation and selection of plant material—

Germinate seeds in the large petri dish containing moistened filter paper at a temperature of about 21°-24° C. When the seed coat

has cracked and the tip of the radicle 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 hr., respectively, after the seeds are placed in moist

petri dishes.

Procedure—Impregnate a disk of filter paper

(5 cm. in 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

m the constant-temperature box along with

others for the same test. The period of exposure may vary from a few to about 48 hr., 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 the

dishes, 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 decrease in length of the root hair

zone, the relative number of hairs, increase or

decrease of branching along the taproot, the

relative lengths of the taproot and branch

roots; abnormalities occurring in the growth

of the hypocotyl.

Suggested standard 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.

Reference

Unpublished method submitted by W. P. Anderson,

Amchem Products, Inc., Ambler, Pa.

ACTIVATED CHARCOAL METHOD

Based on research by P. J. Linder.

Description of method—Measured amounts of

the radioactive test chemical are applied to

filter paper pads suspended in a closed chamber

containing activated charcoal. Volatility of the

compound is determined by subsequently measuring the radiation emanating from the test

chemical adsorbed on the activated charcoal.

Apparatus, Chemicals, and Other Materials

Thin copper wire

Fruit jar (approximately 1 liter) or similar

glass chamber that can be tightly closed

Sealing w^ax

Filter paper

Brass ring to fit the radioactivity chamber to

be used

Cellophane tape, 3 cm. wide

Activated charcoal

Aluminum foil

Conventional equipment for detection of

radioactivity

0.25 g. of compound (radioactively tagged)

to be evaluated

250 micrograms of C^Magged methyl 2,4-

dichlorophenoxyacetate for standard treatment

Alcohol

Procedure—Attach 4 wires to the inner surface of the fruit jar lid, using sealing wax

(fig. 35, a). Cut uniform disks of filter paper

(c) 1-2 cm. in diameter. Pierce each of these

near the edge with the end of one wire (&),

bending the wire so that each disk will remain

suspended separately within the jar. Dissolve

0.5 mg. of each tagged chemical to be tested

per 0.05 ml. of solvent (alcohol). Place 0.05 ml.

of the alcohol solution of the regulator on each

paper disk. Prepare a brass ring (/) covered
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 93

with cellophane tape. (See ''Ground tissue

method/' p. 89.) Cover the surface of the tape

with activated charcoal. Place the brass ring

in the bottom of the jar and support a piece

of aluminum foil (d) above the brass ring to

protect the charcoal from any radioactivity

that might accidentally fall from the impregnated paper disks. Close the chamber (e) tightly and allow it to stand for a period from 30

minutes to overnight.

Method of taking results—Remove the brass

ring and record any radioactivity present using

the conventional equipment for detection of

radioactivity.

The possibility exists that the growth regulator being tested may be contaminated with a

volatile substance, the regulator itself being

nonvolatile. It is, therefore, suggested that the

radioactive compound adsorbed to the activated

charcoal be dissolved and chromatographed

along with an aliquot of the original sample.

Compare Rf's of chromatographed samples

by means of radioautographs to determine

whether one or more compounds are involved.

Suggested standard for comparison—C^^ carboxyl-tagged methyl ester of 2,4-dichlorophenoxyacetic acid.

Reference

Method submitted by P. J. Linder, Agricultural Research Service, U.S. Department of Agriculture.

FIGURE 35.—Closed system used to measure volatility of

radioactively tagged regulating substances.

REFERENCES TO OTHER PHYSICAL METHODS

The publications listed below are recommended for those wishing to gain a more thorough understanding of how^ radioisotopes can

aid in the study of growth-regulating substances. For extraction of these, see p. 118.

Aronoff, Sam. Techniques of Radiobiochemistry. 145 pp.

Iowa State College Press, Ames. 1956.

Biddulph, Orlin. Translocation of Radioactive Nutrients

in Plants. Conf. Use of Isotopes in Plant and Animal

Research, U.S. Atomic Energy Comm., TID-5098

(April 1953) ; Agr. Expt. Sta., Kans. State Col., Rpt.

4: 48-58. 1953.

Boyd, G. A. Autoradiography in Biology and Medicine.

399 pp. Academic Press, Inc., New York. 1955.

Calvin, M., Heidelberger, C, Reid, J. C, and others.

Isotopic Carbon. 376 pp. John Wiley & Sons, New

York. 1949.

Comar, C. L. Radioisotopes in Biology and Agriculture.

481 pp. McGraw-Hill Book Co., New York. 1955.

Hevesy, Georg von. Radioactive Indicators. 556 pp.

Interscience Publishers, Inc., New York. 1948.

Overman, R. T., and Clark, H. M. Radioisotope Techniques. 476 pp. McGraw-Hill Book Co., New York.

1960.

Schweitzer, G. K., and Whitney, I. B. Radioactive Tracer

Techniques. 241 pp. D. van Nostrand Co., Inc., New

York. 1949.

U.S. National Bureau of Standards. Safe Handling of

Radioactive Isotopes. Nati. Bur. Standards Handb. 42,

30 pp. 1949.

U.S. National Bureau of Standards. Control and Removal of Radioactive Contamination in Laboratories.

Nati. Bur. Standards Handb. 48, 24 pp. 1951.

Wolf, George. Isotopes in Biology. 173 pp. Academic

Press, Inc., New York. 1964.

Yagoda, H. J. Radioactive Measurements with Nuclear

Emulsions. 356 pp. John Wiley and Sons, New York.

1949.
94 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

CHROMATOGRAPHY

Chromatography is an essential part of many

methods involved in the study of hormones and

regulating chemicals. All of the major types of

chromatography, including paper, column, gas,

and thin-layer techniques, have played an important role in our gaining an understanding of

hormones and regulating substances. For example, with chromatography it is possible to

determine the purity of a regulator before it is

applied to plants and to purify and identify

hormones obtained from plants. With chromatography it is possible to determine whether an

externally applied substance is absorbed and

translocated as such, or whether some of the

applied substance is degraded, how it is degraded, and, finally, what happens to the products of degradation. Therefore, investigators

in the field of hormones and regulating substances must have a thorough knowledge of

the methods and techniques involved in chromatography and must be skilled in the application of these methods wherever they are

needed.

Although chromatography is mentioned in

many of the methods described in this book, it

is not possible, nor necessary, to give Chromatographie procedures in detail here. Excellent theoretical considerations and descriptions

of Chromatographie techniques can be found

in the following and other texts. (See also p.

118 for hormone extraction methods.)

References

Block, R. J. A Manual of Paper Chromatography and

Paper Electrophoresis. 2nd ed., rev. and enlarged. 710

pp. Academic Press, Inc., New York. 1958.

Block, R. J., Le Strange, Raymond, and Zweig, Gunter.

Paper Chromatography, A Laboratory Manual. 195

pp. Academic Press, Inc., New York. 1952.

Bobbitt, J. M. Thin-Layer Chromatography. 208 pp.

Reinhold Pub. Corp., New York. 1963.

Burchfield, H. P., and Storrs, Eleanor E. Biochemical

Applications of Gas Chromatography. 680 pp. Academic Press, Inc., New York. 1962.

Dal Nogare, Stephen. Gas-Liquid Chromatography,

Theory and Practice. 450 pp. Interscience Publishers,

Inc., New York. 1962.

Hais, I. M., ed. Paper Chromatography; A Comprehensive Treatise. Ed. 3, 9ij5 pp. Publishing House of the

Czechoslovak Academy of Science, Prague. 1963.

Heftmann, Erich, ed. Chromatography. 753 pp. Reinhold Pub. Corp., New York. 1961.

Keulemans, A. I. M. Gas Chromatography. Ed. 2, 234 pp.

Reinhold Pub. Corp., New York. 1959.

Linskens, H. E. Papierchromatographie in der Botanik.

408 pp. Springer-Verlag, New York. 1959.

Littlewood, A. B. Gas Chromatography; Principles,

Techniques and Applications. 514 pp. Academic Press,

Inc., New York. 1962.

Pollard, F. H., and McOmie, J. F. W. Chromatographie

Methods of Inorganic Analysis. [With special reference to paper chromatography.] 192 pp. Butterworths

Scientific Publications, London. 1953.

Randerath, Kurt. Thin-Layer Chromatography. 250 pp.

Academic Press, Inc., New York. 1963. [Translated bv

D. D. Libman.]

Stahl, Egon, ed. Thin-Layer Chromatography, A Laboratory Handbook. 553 pp. Academic Press, Inc., New

York. 1965.

SUPPLEMENTARY TECHNIQUES

Some techniques developed by scientists are

of general use in studying responses of plants

to regulating chemicals. A number of these are

described on the following pages. Some of the

less complex ones may be of particular interest

to those who are undertaking work with regulating chemicals for the first time. Other more

complicated techniques are described with the

hope that they will be useful to the more advanced research worker. It is not possible to

determine the originators of all of the techniques listed; therefore, credit for some of

them is not given.

TREATING PLANTS

Absorption Cell for Introducing Compounds Into

Limited Portions of Roots

Construct an absorption cell for applying

radioactively tagged chemicals to limited regions of individual roots by using a glass tube

10 mm. in diameter with a 3-mm. sidearm.

Draw the end of the larger tube out to make

it smaller (about the diameter of the sidearm),

and bend it around so the narrow end faces

the sidearm as shown in fig. 36. Connect this

narrow end of the tube with the sidearm using

pure gum amber rubber tubing 3 mm. in diameter. Burn a hole through both walls of the

rubber tubing with a hot nichrome wire of
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 95

FIGURE 36.—Absorption cell for introducing compounds

into limited portions of roots. (Photo—courtesy of

Plant Physiology.)

somewhat smaller diameter than the roots.

The size of the hole should be slightly smaller

than the root so the root fits snugly. Connect

the holes by a slit. Bend the rubber tubing

slightly to open the slit and insert a rootlet of

an intact plant. Seal the slit in the tubing and

the junction between the tubing and the root

with lanolin that has been warmed to make it

almost liquid. Twist a piece of nichrome wire

to make a support for the plant as illustrated.

Place the solution of the chemical to be absorbed by the root in the cell and immerse the

apparatus in tapwater or a nutrient solution

as illustrated. Tap the apparatus gently to

dislodge air bubbles trapped in the rubber

tubing. Insert glass tubing in the main tube

of the cell and bubble air through this tube to

circulate and aerate the solution in the cell.

Keep the level of the solution in the cell 1.3

cm. below the level of external nutrient solution so that, if leaks develop, they can be detected by the rise in the level of the solution

within the cell. Aerate the external nutrient

solution. (See "Plastic culture vessel ("plasticon") for small plants," p. 107.) Change the

nutrient solution frequently to keep to a minimum contamination due to exudation from the

roots. Maintain the whole apparatus at 25° C.

It has been suggested that, with some modification, this method might be useful to indicate

root uptake of regulators by determining the

growth response of the plant after it absorbs

the chemical. Absorption of the compound

might also be detected by bioassay and chromatography of tissue extracts.

This technique was successfully used to introduce P32, Ii»i, Rb86, and S»«. It was not successful in introducing Ca*^. This method is described for workers who may wish to attempt

its adaptation for use with radioactively

tagged growth regulators.

Reference

Wiebe, H. H., and Kramer, P. J. Translocation of Radioactive Isotopes from Various Regions of Roots of Barley Seedlings. Plant Physiol. 29: 342-348. 1954.

Absorption Through a Leaf Flap

A flap absorption technique that has been

used successfully is as follows: Place the leaf

of an intact plant under water. Using a razor

blade, cut parallel to and on both sides of the

midrib of a leaf, such as that of a bean plant,

for a distance of 2-3 cm. (fig. 37, a). Connect

FIGURE 37.—Leaf-flap absorption technique.
96 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

the 2 cuts at the ends nearest the petiole attachment while the leaf is still immersed. Remove the leaf from the water and dip the flap

(b) immediately into the test solution contained in a small vial (c) to prevent air from

entering the xylem vessels. Allow the test solution to be absorbed for 1-24 hr. Remove the

flap and immerse it in a vial containing water

to prevent drying. Some absorbed test solutions will be translocated through the leaf and

into the plant. One principal lateral vein of a

bean leaflet has also been used for making the

flap in a method similar to the one described

above.

Reference

Biddulph, O. Diurnal Migration of Injected Radiophosphorus from Bean Leaves. Amer. Jour. Bot. 28: 348-

352. 1941.

Agar Block Preparation and Use

Agar blocks are useful for applying exogenous substances and hormones to plants and

plant segments used in bioassays and translocation experiments. When a moist agar block

containing a test compound evenly dispersed

throughout the agar is placed on a freshly cut

plant surface, the compound in the agar block

readily diffuses from the agar into the aqueous

film between the agar and the plant surface.

In most instances the compound then diffuses

into the plant cells. Conversely, some endogenous substances, as well as exogenous substances that have been introduced into plants,

will diffuse in a similar manner from freshly

cut surfaces into an agar block. Thus, under

suitable conditions, agar blocks represent a

means of supplying substances to and collecting some substances from plants.

To make agar blocks, prepare 1% agar by

mixing together and bringing to a boil 0.1 g.

of the agar (ionagar) and 10 ml. of water.

Pipette the hot agar mixture into a glass tube

(15-30 cm. long, 5 mm. inside diameter). Hold

a finger over the lower end of the tube to keep

the agar in place. After the tube is full, place

another finger over the upper end of the tube

and hold the entire tube under a stream of

cold running water. When the agar has solidified (1-2 minutes), tap the tube endwise

gently on a hard surface to loosen the agar

from the inside wall. Force the agar column

from the tube with a solid glass rod. Repeat

this process until sufficient molded agar has

been obtained. Slice each column of agar into

segments of appropriate length (2 mm.) with

the common cutter and place them in a petri

dish lined with moist filter paper. Store the

segments at 5° C. until used. (See p. 101.)

A somewhat more precise method has been

suggested by McCready. Prepare a 2% agar

solution and draw it into a number of lengths

of precision glass tubing (3.0 mm. diameter).

After the agar has solidified, insert a plug of

nonabsorbent cotton into one end of each tube.

Extrude the columns of agar from the tubes

by pushing the cotton. Cut the columns into

blocks of appropriate length. Use the common

cutter with the desired number of separator

plates and a corresponding number of razor

blades, the width of each plate adjusted to

give the desired length of agar block. After

cutting the columns, use a comb with teeth

properly spaced to displace alternate blocks

from between the blades. Move the comb

slightly and remove the remaining blocks so

that 2 rows of blocks are conveniently spaced

for handling with forceps. If the agar contains

a growth substance, use a separate set of cutter blades and a different comb and forceps

when preparing and handling receiver blocks

of plain agar.

A variation in the preparation of agar

blocks and the method of supporting the blocks

and segments was used by McCready. Prepare a 2% agar solution and draw this solution into a number of lengths of precision glass

tubing (3.0 mm. diameter). Allow the agar to

solidify. Insert a plug of nonabsorbent cotton

into one end of each tube. Push the cotton

through the tube extruding the column of agar

gel. Cut the gel column into blocks of the desired length using the common cutter with the

required number of blades spaced 2-3 mm.

apart. Use a comb with teeth properly spaced

to move alternate blocks from the cutter onto

a glass plate. Move the cutter slightly and

expel the remaining blocks so that 2 rows of

blocks are conveniently spaced for picking up

with forceps. If the agar contains a growthregulating substance, do not use the same cutter, combs, and forceps as those used with receiver blocks of plain agar. Place the donor

block on the apical end of the plant segment

and the receiver block at the basal end of the

segment. Lay each segment, with its blocks retained in position by surface tension, horizontally across a 3-mm. gap between 2 glass

strips cemented to a glass plate. Prepare as

many replications of this treatment as required. Repeat the procedure, this time applying the donor block to the basal end and the

receiver block to the apical end of the segment,

arranging the segments across the 3-mm. gap

between additional plates as described above.

Use the replications equal to the number in
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 97

the first treatment. Place the plates with the

segments and agar blocks in a moist atmosphere in a dark chamber at a controlled temperature of 25° C.

It is sometimes desirable to make blocks

that will accommodate more than one plant

segment. Leopold, Lam, and de la Fuente have

suggested methods for doing this. To make

equipment for this purpose, drill a hole 20 mm.

in diameter in a brass or aluminum plate 2 mm.

thick. Place this mold on a microscope slide

that is cooled from below with ice. Pipette into

the mold about 1 ml. of hot 1.5% agar solution.

After the agar has solidified, remove the metal

plate leaving the agar block on the slide. Place

10-20 segments of the desired plant material

vertically on this agar block. Prepare a similar

agar block that contains 10"'' M C^^-tagged

indole-3-acetic acid or other radioactive compound to be tested. Carefully slide this block

onto the standing plant segments so that the

block is in contact with the upper end of each

segment. Place the microscope slide and segments in a petri dish or plastic box lined with

moistened filter paper and cover the dish or

box. After the desired time interval, measure

the amount of compound in the lower agar

block using the method described in the "Agar

block polar transport method," p. 8.)

It is sometimes convenient to substitute a

disk of moist filter paper for the receiver block

or to dry the agar receiver block and then

measure directly the radioactivity in it, correcting for self-absorption. When direct scintillation counting of the receiver block is desired, make blocks of an aqueous paste of

Cab-o-Sil. To avoid the quenching effect of the

agar in the scintillation solution, extract the

blocks with ethanol and determine the radioactivity in the extract.

Diffusible endogenous hormones can also be

collected from plant segments or plant parts

by placing their cut surfaces against agar

disks.

References

de la Fuente, R. K., and Leopold, A. C. Kinetics of Polar

Auxin Transport. Plant Physiol. 41: 1481-1484. 1966.

Leopold, A. C, and Lam, S. L. The Auxin Transport

Gradient. Physiol. Plant. 15: 631-638. 1962.

McCready, C. C. Movement of Growth Regulators in

Plants. I. Polar Transport of 2,4-Dichlorophenoxyacetic Acid in Segments From the Petioles of Phaseolus vulgaris. The New Phytol. 62: 3-18. 1963.

Zaerr, J. B. Submitted the principal polar transport

method described. (See "Agar block polar transport

method," p. 8.)

Application of Aqueous Mixtures Quantitatively

to Stems

Apply two narrow lanolin bands about 1-2

cm. apart and extending around the stem of the

selected plant. Allow 0.01 ml. of the aqueous

mixture containing the regulating chemical to

now from a 0.1-ml. pipette so 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. 38). Revolve

the plant and spread the mixture over the

PM-SIO

FIGURE 38.—Applying an aqueous mixture of a growth regulator quantitatively to a stem.
98 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

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.

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 a

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. 39, A). 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, and 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. 39, B). 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%.

Application of Regulators as a Part of a

Coating on Seeds

When it is desirable to apply regulating

chemicals to seeds without the use of water,

which generally initiates germination, the

compounds can be mixed with various kinds

of nonaqueous carriers and the mixture applied to the seeds as a coating. A typical seedcoating procedure, which can be varied readily,

is as follows : Place the seeds in a rotating pan

such as that used by pill and candy manuPN-306, A and B

FIGURE 39.—A, Marking a known area of a leaf with

lanolin; B, applying a measured volume of solution

containing a known amount of growth regulator to

this area.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 99

facturers.* Wet the seeds with methyl cellulose, an adhesive material, and then dust the

seeds with a mixture of 65% feldspar and 35 Çf

fly-ash (ash obtained from the burning of powdered coal) containing the required amount of

growth regulator. Repeat the wetting and dusting procedure until the desired thickness of

coating has been obtained. Use a coarse feldspar which passes through a 40-mesh screen to

build up most of the coating. For a technique

of coating individual particles of this coarse

feldspar with the regulating chemical, see

"Easy method of impregnating dust carriers

with regulating chemicals," p. 100.

Application of Growth Substances With

Capillary Tubes

Small, pointed glass capillaries that hold a

required volume of growth regulator solution

are useful for introducing minute quantities

of the solution into a speciñc part of intact

8 Stokes Tablet Coating Machine, Model 29, F. J.

Stokes Machine Company, Philadelphia, Pa.

plants. Draw out glass capillaries about 1-2

mm. outside diameter. Draw out the end of

each tube to a point. Make each tube so it can

hold more than the volume required. Fill an

opsonic pipette (graduations about 3 cm. to

each 0.01 ml.) with distilled water. Hold the

opsonic pipette horizontally and place the

capillary to be calibrated against the orifice

of the opsonic pipette. In this way, draw the

water into the capillary tube. When the meniscus in the opsonic pipette reaches the required

volume, remove the capillary tube, mark the

meniscus in the tube with a glass knife or file,

and then break the capillary tube at this mark.

Touch the capillary tube to filter paper to

empty it. Tubes made and calibrated in this

way will take up the appropriate volume of

growth substance solution. The length of the

tubes with equal capacity may vary depending upon the bore of the capillaries.

After allowing the tubes to draw up a known

volume of the growth regulator solution, stick

them to adhesive tape (sticky side out) on a

microscope slide (fig. 40). To apply the regulators, remove a tube from the slide and stick

the pointed end of the filled capillary into the

appropriate part of the plant.

BN-289S6

FIGURE 40.—Capillary tubes for introducing known amounts of growth-regulating substances into intact plants.

(Illustration—after photo by H. W. B. Barlow.)
100 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Direct insertion into a young internode on

apple or plum shoots caused damage to that

internode, even though there were responses

to gibberellins above and below the point injected. Insertion into the petiole was preferable,

particularly when the lamina was removed, as

it did not induce injury response to the stems.

If one capillary empties at a much slower

rate than the majority of the others, remove

this one and reinsert it after breaking a small

piece off the tip.

This method is particularly valuable when

only very small quantities of material are

available or when application of measured dosages to particular sites is required. When used

in the field, the capillaries remain attached to

the plant reasonably firmly, even on -windy

days.

Reference

Submitted by H. W. B. Barlow, East Mailing Research

Station, East Mailing, Maidstone, Kent, England.

A somewhat similar technique can also be

used. Supply the solution of growth-regulating

substance to a pedicel or stem of an intact

plant, such as tomato or cucumber, through a

glass tube, 4-6 mm. in inside diameter tapering to not more than 0.5 mm. in outside diameter. Fill the tapered end of the tube with the

growth-regulator solution by capillarity. Plug

the large end of the tube with cotton. Insert

the tapered end of the tube directly into the

pedicel or stem. Support the tube by threads

tied around the tube and around a stationary

support.

Reference

Homan, D. N. Auxin Transport in the Physiology of

Fruit Development. Plant Physiol. 39: 982-986. 1964.

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 it 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 alcohol solution with the dust

(fig. 41). Stir the slurry thoroughly and allow

the alcohol to evaporate at room temperature.

Mix the impregnated dust thoroughly before

applying it to plants.

Useful concentrations of regulating chemicals in dust carriers often range from 500 to

5,000 parts of the chemical in 1 million parts

of carrier. Eflfectiveness of some regulators apPN-304

FIGURE 41.—Impregnating dust carriers with regulating

chemicals.

plied in talc dust has been slightly enhanced

experimentally by the addition of small

amounts of a hygroscopic agent, such as glycerine, to the alcoholic mixture. During evaporation of the alcohol, the hygroscopic agent is

incorporated into the dust preparation.

Convenient Method of Dispersing Relatively Insoluble

Organic Compounds in Water

Many growth-regulating substances in the

form of acids or esters are not readily soluble

in water. In dispersing these substances in

water, the final mixture may be a suspension

of relatively large groups of molecules of a substance. These particles, however, may be so

small that the final mixture is clear and looks,

temporarily at least, like a true solution. Upon

standing, however, such a suspension of minute

particles may aggregate, with the result that

the chemical falls out of mixture. The size of

the dispersed molecular aggregates, with the

growth regulator initially in the mixture and

subsequently maintained in the mixture, governs to some extent the amount of the compound absorbed by the plant. The amount of

response obtained, therefore, indirectly depends to some extent on the way a relatively

insoluble growth regulator is dispersed in a

water mixture.

To obtain and maintain even dispersion as

long as possible, it is often desirable to use an

adjuvant, sometimes termed a "cosolvent."
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 101

Such cosolvent should be relatively nonvolatile,

should be a solvent for the growth regulator,

should have strong surfactant properties, and

should be nontoxic to plants at effective concentrations.

Many commercially available surfactants can

be used as cosolvents in experimenting with

relatively insoluble growth-regulating chemicals. Tween 20, a sorbitol derivative obtainable

from the Atlas Powder Company, Wilmington,

Del., is used here to illustrate how a cosolvent

aids in dispersing a regulating chemical in

water and in keeping a regulator finely divided

even after the water in the mixture evaporates

from the leaf or stem surface.

Measure volumetrically a sufficient amount

of the surfactant to make a final concentration

of 0.1%. Add the weighed amount of regulating chemical to the surfactant, warm the mixture (not above 50° C), and stir until the

chemical is dissolved in the surfactant. With

rapid stirring, introduce the surfactant-growth

regulator mixture into a volume of warm water

sufficient to give the desired final concentration. If relatively small amounts of chemical

and surfactant are involved, use a microbeaker made by cutting off the lower end of a

small shell vial. Weigh the required amount of

surfactant into the microbeaker and dissolve

the required amount of test compound in the

surfactant by gently warming and stirring the

mixture with a micro glass rod. Place the entire microbeaker containing the mixture into

the required amount of water while rapidly

stirring the water. Care should be taken that

the surfactant mixture is poured into the water

and not the water into the surfactant mixture.

It is impractical to describe here how the

many other equally effective surfactants can be

used. No discrimination is intended against

these other surfactants which include, for example, Tween 80 (Atlas Powder Co.), Carbowax 1500 (Union Carbide Chemicals Co., New

York, N. Y.), Triton X 100 (Rohm & Haas

Co., Bristol, Pa.), and dimethyl sulfoxide,

known as DMSO, (Crown Zellerbach Corp.,

Camas, Wash.).

For further information regarding surfactants see :

Schwartz, A. M., and Perry, J. W. Surface Active

Agents—Their Chemistry and Technology, v. 1. 579

pp. Interscience Publishers, Inc., New York, and Interscience Publishers, Ltd., London. 1949.

Schwartz, A. M., Perry, J. W., and Berch, Julian. Surface Active Agents and Detergents, v. 2, 839 pp. Interscience Publishers, Inc., New York, and Interscience

Publishers, Ltd., London. 1958.

Segment Cutters

Segments of plant parts, such as coleoptiles

and stems, are used to detect and measure plant

growth-promoting and growth-inhibiting substances. Various instruments have been devised

so uniform segments of required lengths can

easily be made from these and other plant

parts. Diagrams of 3 easily constructed cutters

are shown in fig. 42.

A - BARLOW CUTTER B-WIGHTMAN CUTTER C-COMMON CUTTER

FIGURE 42.—Segment cutters. (A, Diagram—courtesy of Journal of Experimental Botany; B, diagrammed from

photo by F. Wightman.)
102 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

Barlow cutter (fig. 42, A): Make the separator

bar (x) wide enough to hold the razor blades

the required distance apart. Construct several

of these interchangeable separator bars of required widths, if segments of different lengths

are needed. Make separator bar (z) wide

enough to hold the razor blade nearest the

guide plate 3 mm. from the guide plate. Base

plate (y) can be made of either wood or metal,

the other parts of aluminum or brass. To make

coleoptile segments with this cutter sever the

coleoptiles from the seeds with a scalpel. Place

the coleoptiles in rows on wet microscope

slides, keeping them in the chronological order

in which they were cut. Arrange the coleoptiles

at right angles to, and with their tips coincident with, one edge of the slide. Place the slide

against the zero line of an aluminum grader

scribed at 5-mm. intervals. Place coleoptiles

with bases falling between any 2 adjacent lines

in one length grade, maintaining chronological

order. Align the tips with the slide as before.

Using a separator bar that holds the blades 10

mm. apart, invert the slide containing coleoptiles and place it on the blades of the cutter.

Keep the tips against the guide plate and then

exert a gentle downward pressure. Remove the

unwanted 3-mm. tips thus cut from the coleoptiles by passing a needle between the guide

plate and the nearby blade; also remove the

unwanted basal ends cut from the coleoptile.

Remove the cut segments by inserting the

needle between the blades and under the coleoptile segments. While pressing the segments

gently against the glass surface, lift off and

invert the slide. Retain only the segments that

are completely filled with leaf tissues. (See

''Wheat coleoptile straight growth method,''

p. 36.)

Wightman cutter (fig. 42, B): Construct the cutter of aluminum or brass so the distance (a)

is 3 mm. The distance (b) can be changed to

make any desired length of segment by varying

the thickness of the separator plates in the

part of the instrument holding the coleoptile

and also the width of the plates separating the

razor blades. In making segments, discard the

3-mm. tips and the basal ends of the coleoptiles.

Remove the primary leaves by threading the

measured segments onto fine glass capillaries,

2 segments per capillary, and then allow the

segments to float on distilled water in a petri

dish until required to start the experiment. Do

not allow this pretreatment period to exceed

3 hr. (See ''Wheat coleoptile straight growth

method,'' p. 36.)

Common cutter (fig. 42, C): Construct aluminum or brass razor blade holder as illustrated.

Adjust the blades to the desired distance apart

(s) by varying the width or number of

rator plates used.

sepaDevice That Delivers Measured Amounts of

Regulating Chemical

The microinjector shown in fig. 43 delivers

measured volumes of lanolin paste containing

a regulating chemical (fig. 43, upper). 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. 43, lower) 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

"A microdevice for rapid application of toxicants to individual insects." publication No.

ET-306 of the former Bureau of Entomology

and Plant Quarantine, U.S. Department of

Agriculture, 1953.

Direct Measurement of Volatility^

Prepare an alcohol solution that contains

10,000 p.p.m. of the growth-regulating chemical to be tested. (This technique is not suitable

for measuring the volatility of hygroscopic

compounds.) Weigh a sheet of tinfoil 3X6 cm.

(Aluminum foil is not satisfactory for this

test.) Dip the tinfoil into the alcohol solution

and allow the excess to drain off for a few

moments. Immediately attach the tinfoil to the

beam of a balance so that the foil swings freely. Record the combined weight of the tinfoil,

alcohol, and growth regulator at frequent intervals within the next few minutes to determine when the relatively rapid rate of weight

7 Carelessness in the use or disposal of highly volatile

regulating chemicals can result in contamination of an

entire laboratoiy or greenhouse, thus causing loss of valuable plants and space.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 103

FIGURE 43.—Device that delivers measured amounts of regulating chemicals.

PN-303. A and B
104 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

loss due to evaporation of alcohol changes to a

slower rate of weight loss, which in most instances represents the evaporation of the

growth regulator. Weigh the remaining growth

regulator and the tinfoil at intervals to determine the rate of evaporation of the growth

regulator. Pieces of thin glass about 25 square

cm. in area may be used in place of the tin foil.

For other methods of measuring volatility of

plant regulators, see "Volatility of plant regulators," p. 90.

Reference

Anderson, W. P., Linder, P. J., and Mitchell, J. W.

Evaporation of Some Plant Growth Regulators and Its

Possible Effect on Their Activity. Science 116: 502-

503. 1952.

Fractionated Lanolin as a Carrier for

Growth-Regulating Compounds

Lanolin is an excellent carrier for growthregulating substances, provided the compound

is first dissolved in a suitable cosolvent and then

mixed with the lanolin. Lanolin, however, remains relatively solid or viscous at ordinary

room temperatures, even though there may be

heat from a nearby lamp being used for illumination when treating small areas on plants with

such a mixture. By fractionating lanolin, a fat

with slightly more fluidity than that of lanolin

can be obtained. This fraction usually becomes

very fluid when applied under a lamp, thus affording excellent contact between the fractionated lanolin mixture and the surface of the

plant. Use the following procedure to make this

lanolin fraction.

Warm carefully on a steam bath 10 g. of anhydrous lanolin (U.S.P. Adeps lanae) dissolved

in acetone (6 parts of acetone by weight to 1

part of lanolin) until the temperature of the

mixture reaches about 50° C. The steam bath

is preferred because of the flammability of acetone, which boils at 56° C. However, proximity

to flame or electrical contacts that might spark

is to be avoided since acetone flashes at —15°

C, far below its boiling point. Stir the mixture

carefully until the lanolin is dissolved. Allow

the solution to cool to room temperature

(25° C.) overnight. Cover the beaker with foil

to retard evaporation during this period. Recover the solution from the precipitate by any

convenient method such as filtration, décantation, or centrifugation followed by décantation.

Warm the solution on a steam bath to evaporate

the acetone and recover a fraction that is slightly more fluid than the lanolin itself. Lower precipitation temperatures will produce a more

fluid fraction but with reduced yield.

Investigators may also wish to determine the

possible usefulness of another fraction of lanolin known as Viscolan, available from the American Cholesterol Products, Inc., Amerchol Park

Edison, N. J. 08817.

Reference

Method submitted by E. A. Wilder, S. C. Johnson and

bon, Inc., Racine, Wis.

Injecting a Regulating Substance or Hormone Into a

Single, Living Cell

Intracellular responses to regulators and hormones can be observed and photographically recorded during and following injection of the

accelerator or inhibitor into an individual cell.

In addition to plant material to be studied, the

followmg equipment is needed: capillary hard

glass tubing (such as Pyrex or Kimax), a pipette puller, micromanipulators, high-powered

microscope, injection equipment, water-immersion objective with which a magnification of

400 X can be obtained, still camera for photomicrographs, and equipment for cinematography if the response of living cells is to be

studied in terms of protoplasmic movements

and changes as the responses progress.

Prepare sections of living plant tissue to be

studied. (See "Freehand sections for studying

cell responses to regulators," p. 120.) Mount

these sections in tapwater on a microscope slide

without a cover slip. Use two hypodermic

needles to hold the plant section against the

surface of the sHde, both needles being held by

a single micromanipulator. With the beveled

side of the 2 needles facing downward, press the

edges of the section against the glass while leaving the cells to be studied uninjured.

Make a micropipette with a tip that is barely

visible when magnified 400X. A pipette of this

diameter generally has an opening that is 0.5

micron or less in diameter. Such an opening is

not visible at this magnification. Fill the micropipette by capillary action by immersing the tip

for several minutes in the solution to be injected. Join the micropipette to a microinjection

apparatus mounted on another micromanipulator.

Position the micropipette and section under

35 X magnification. Figure 44 shows tip of

micropipette at cell wall (a) of fiber cell (a-&).

Switch to the water-immersion objective

(400X) and inject the cell by inserting the

micropipette through the cell wall and for a

very short distance into the cytoplasm. Transfer sections containing successfully injected

cells to tapwater contained in a slide chamber.

(See ''Slide chamber for the study of living
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 105

BN-28953

FIGURE 44.—Micropipette touching surface of a fiber cell

(a) before puncture of cell wall and injection of

growth-regulating substance; (6), wall on opposite

side of fiber cell.

cells," p. 123.) Observe the cells through the

cover slip and record the responses photographically.

A droplet of castor oil can be injected into

some kinds of cells without apparent injury to

the protoplasm. Dissolve the required amount

of the test substance in the oil and inject it as

a single droplet into the cell to be studied.

Various pipette pullers are commercially

available, including the following: Livingston

Micropipette Puller made by Otto K. Hebel, 80

Swarthmore Avenue, Rutledge, Pa.; one made

by E. Leitz, Inc., 468 Park Avenue South, New

York, N. Y. A Livingston Micropipette Puller

was used in developing the method described

above.

Reference

Method submitted by J. F. Worley, Agricultural Research Service, U.S. Department of Agriculture.

bored in their branches or trunks. Effects of

these substances on growth, fruit set, fruit

drop, longevity, and on systemic disease control

can be studied. With a specially made tool (fig.

45, a), the female part of a plastic quick connector (fig. 45, b) is driven into a hole previously

made with a hand drill. The male part of the

quick connector, with rubber tubing and plastic

bottle attached to one end, is fitted into the

female part already in the tree (fig. 46, p. 107).

Small quantities of test solution can be forced

into the bottle through a small hole with a large

hypodermic syringe and allowed to flow by

gravity into the tree. With large trees, 2 or

more injection sites may be used with containers with a capacity of 1 liter or more.

Reference

Method submitted by H. L. Keil, Agricultural Research

Service, U.S. Department of Agriculture.

Injecting Substances Into Trees

Growth regulators, antibiotics, or other substances can be injected into trees through holes

Introduction of Chemicals Directly Into Xylem

Elements in Stems of Herbaceous Plants

Small amounts of radioactively tagged compounds have been introduced directly into

xylem elements to study transport of the substances. The method described here applies to

young bean plants, but the technique should also

be useful for studying translocation in other

herbaceous plants. Carry out a preliminary

microscopic examination of the stem anatomy

of the test plant chosen to become acquainted

with the location of xylem elements leading to a

primary leaf. To do this, cut freehand cross

sections through the first internode of several of

the plants at a level midway between the first

and second nodes. Observe tracheary elements

that lead to one primary leaf and note their

position in relation to the outside contour of the

stem.

Select a plant comparable to those examined

microscopically and determine the exact location on the first internode to which a droplet

should be applied so the droplet will be directly

outside of the tracheary elements leading to the

primary leaf. At this point on the stem, apply

a 0.005-ml. droplet of water containing the

plant regulator to be tested. Make a tool to sever

some of the tracheary elements leading to the

leaf by forming a wedge-shaped chisel approximately 0.8 mm. wide on the end of a length of

fine steel wire. This provides a sharp cutting

edge. Immerse the chisel in the droplet and,

far as the pith (fig. 47), thus severing, in the

with slight pressure, insert it into the stem as

case of young plants, the primary tracheary elements connected with the leaf. When the instrument is withdrawn, the droplet is sucked into
106 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-2S942

FIGURE 45.—Special tool (a) used to insert quick connector (6) into tree.

the plant through the opening into the stem. The

rapidity of droplet intake may vary from a few

seconds to a minute or more. The investigator

may wish to classify the plants on the basis of

time required to absorb the droplet and thus include the rate of absorption along with other

factors involved. The absorbed liquid is moved

upward from the place where the xylem elements are severed into the primary leaf. Repeat

the procedure to obtain the required number of

treated plants. After the desired interval following treatment, determine the distribution of

the radioactivity by means of radioautographs.

(See "Radioautograph method," p. 87.)

Reference

Linder, P. J., and Mitchell, J. W. Rapid Transport of

Alpha-Methoxyphenylacetic Acid Introduced Directly

Into the Water Stream of Bean Plants. Bot. Gaz. 121 :

139-142. 1960.

not injure plants. It makes close contact with

the plant surface, and regulators, when mixed

with lanolin, diffuse readily Jrom 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 semisolid. Apply the mixture to

stems or leaves of broadleaf plants with a toothpick or other disposable applicator.

In the case of grass leaves, use a glass rod 2

mm. in diameter to spread the lanolin mixture

in a thin, even layer completely covering a portion of the upper surface of the leaf for a distance of 1 cm. To do this, draw the rod from a

position near the base of the leaf toward the

tip. With practice, uniform amounts of compound can be applied rapidly by this means.

Lanolin as a Carrier for Regulating Chemicals

Lanolin is widely used as a carrier for regulating chemicals (fig. 48). Applied in small

amounts, this semisolid, fatty substance does

Movement of Regulating Substances Through

Plant Membranes or Tissues

A simple apparatus has been used for studying movement of substances through plant
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 107

BN-28941

FIGURE 46.—Injecting a regulator into tree with plastic

bottle, rubber tubing, and quick connector.

membranes or layers of plant tissue (fig. 49).

Fill a screwcap vial (approximately 9 mm. in

inside diameter at the open end) to about fourfifths of its capacity with a solution of the compound to be tested. Cut off the closed end of the

cap, thus making a threaded collar. This collar

must fit the threaded area on the screwcap vial.

It must not be wider than the threaded area.

Place the plant membrane over the open end of

the vial and screw the collar completely on the

threaded area of the screwcap vial to fasten the

membrane tightly across the opening. Insert the

vial through a hole in a disk of relatively thick

sheet rubber, a support for the screwcap vial.

Make the diameter of the hole in the rubber

slightly smaller than the outside diameter of the

vial. Place a measured amount of distilled water

in a shell vial of greater inside diameter than

that of the screwcap vial. Invert the screwcap

vial with its membrane attached and adjust its

position in the rubber support so that the membrane is below the surface of the water in the

shell vial. Allow the substance being tested to

diffuse through the membrane for the required

length of time (2, 4, or 8 hr.). Remove the

screwcap vial, stopper the shell vial, and subsequently determine the concentration of the substance that has diffused through the membrane

by appropriate bioassay methods, colorimetric

methods, or the use of radioactively tagged compounds.

Reference

Szabo, S. S., and Buchholtz, K. P. Penetration of Living

and Non-Living Surfaces by 2,4-D as Influenced by

Ionic Derivatives. Weeds 9: 177-184. 1961.

Plastic Culture Vessel ("Plasticon") for

Small Plants

Plastic culture vessels (fig. 50, p. 110) are

useful for supporting small plants with their

roots suspended in nutrient solutions or liquids

containing chemicals to be tested. Construct a

BN-28940

FIGURE 47.—-Severing tracheary elements in stem of

young bean plant with tip of metal chisel immersed in

droplet containing plant regulator.
108 AGRICULTURE HANDBOOK 336. U.S. DEPARTMENT OF AGRICULTURE

PN-SOO

FIGURE 48.—Applying lanolin containing a regulating chemical to stem.
METHODS OF STUDYING PLANT H0RM0NF:S AND GROWTH-REGULATING SUBSTANCES 109

////////77

^^;iii:i:;;^^

/

m

¿Z/////7/////.

Screwcap vial

Level of solution

Ti^ Rubber support

and air seal

Shell vial

Screwcap

Level of wate

Membrane

FIGURE 49.—Apparatus for studying movement of regulating substances through membranes or tissues.

(Photo—courtesy of Weeds.)

plastic vessel with an upper and lower part,

using Plexiglas (methyl methacrylate synthetic

plastic) (fig. 51). Close the lower part with

Plexiglas, forming a cylindrical reservoir about

8 cm. in diameter. For the upper part, make a

disk slightly greater in diameter than the cylinder of the reservoir and seal a hollow Plexiglas

cylinder of smaller diameter in the center of

this disk as shown. Stretch a netting of nylon

across the lower end of this small cylinder and

seal the netting as shown. Use dissolved plastic

to seal the various pieces together. Place the required number of seeds on the netting and adjust the volume of liquid as illustrated. Construct a cylinder of opaque paper to fit over the

reservoir or apply aluminum paint to partly

exclude light and prevent growth of algae.

To aerate the liquid in the plasticon, construct

a manifold using ï-tubes connected with plastic

tubing. Attach pieces of plastic tubing to ''bubblers/' such as are used to aerate an aquarium,

and insert the tubing through a hole drilled in

the top of the plasticon. Connect the plastic tubing with the outlets of the manifold. Immerse

the bubblers in the liquid with the plasticon.

Connect one end of the manifold to an aquarium

pump and leave the other end of the manifold

open but with a piece of plastic tubing and pinch

clamp attached. Force compressed air through

the bubblers with the pump. Adjust the rate of

airflow by means of screw clamps on each manifold outlet and the pinch clamp at the end of the

manifold.

Reference

Hagborg, W. A. F. The Effect of Antibiotics on Infection of Wheat by Xanthomonas translucens. Canad.

Jour. Microbiol. 2: 80-86. 1956.

Simulated Spray-Droplets

Droplets of diflierent sizes and various patterns of distribution can be obtained to simulate

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 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. 52).

Technique for Studying Absorption and Translocation

of Regulating Substances by Aquatic Plants

The following technique has been used to

separate different parts of submersed water

plants so absorption and translocation of a

growth-regulating substance can be studied.

Place the stem (7.5 cm. long) of a polyethylene funnel, with a capacity of 750 ml. of liquid,

through a hole in a No. 12 rubber stopper (fig.

53). Thread a water plant, such as sago pondweed (Potamogetón pectinatus L.), through the

stem of the funnel, allowing the roots to protrude from the stem. Lightly block the hole in

the funnel stem with one finger and fill the stem

of the funnel with melted eicosane (fig. 53, e),

When the surface of the eicosane has solidified,

immerse the plant and funnel with the eicosane

plug in cold water to solidify the entire plug.

Place 250 ml. of the solution containing the

growth-regulating substance being tested in a

wide-mouth bottle that can be tightly closed

with the rubber stopper. Submerse the roots of

the plant in this solution and fit the stopper

snugly into place. Pour 500 ml. of water into the

funnel, thus submersing the upper part of the

plant. Use a 5-7 mm. glass tube leading from

inside the bottle through the stopper to a level

above the funnel as a vent to accommodate expansion or contraction of the liquid in the closed
no AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-28939

FIGURE 50.—Plastic culture vessels ("plasticons") for small plants. (Photo—courtesy of Canadian Journal of Microbiology.)

BN-289S8

FIGURE 51.—Parts of a plasticon. (Photo—courtesy of Canadian Journal of Microbiology.)
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 111

PN-S09

FIGURE 52.—Simulating spray-droplets on a leaf.

part of the system. If absorption and translocation from the top of the plant to the root is to be

measured, place a solution of the growth regulator in the funnel. Fill the bottle with water.

After an appropriate treatment period, remove

the stopper and funnel, pour the liquid from the

funnel, and separate the roots from the top by

severing the stem within the eicosane plug. Blot

the plants dry after washing them in running

water and arrange them on blotting paper.

Freeze the plants with solid carbon dioxide and

allow them to dry. Prepare radioautographs to

show absorption and translocation based on the

distribution of the isotope. Assay aliquots of the

liquid in the top and bottom of the apparatus

at the end of the experiment to be sure that

leaks did not develop. Do this by evaporating

aliquots of the liquid in planchets and then

measuring any radioactivity present with a conventional gas-flow counter.

Reference

Frank, P. A., and Hodgson, R. H. A Technique for

Studying Absorption and Translocation in Submersed

Plants. Weeds 12: 80-82. 1964.

FIGURE 53.—Diagram of apparatus and aquatic plant

arranged to study absorption and translocation of

regulating substances. (Diagram—courtesy of

Weeds.)

Use of Dwarf Corn to Detect and Identify

Gibberellins and Gibberellin-Like Substances

Dwarfism in some plants can be partly or

wholly overcome by the addition of gibberellins

or gibberellin-like substances. Some dwarf mutants, therefore, are useful in detecting and

classifying regulating substances that induce

elongation and growth of these plants. Six

single-gene mutants of maize {Zea mays L.) are

especially useful for studying gibberellins and

gibberellin-like substances. These are: Anther

ear-1. Dwarf-(5232), Dwarf-1, Dwarf-(8201),

Dwarf-(4963), and Dominant-dwarf. The first

4 of these mutants are relatively sensitive to

gibberellic acid, the fifth responds slightly to

this compound, and the sixth does not respond

to the ac'd. Since the response of these mutants

to growth-accelerating substances varies, all 6
112 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

mutants can be used as a composite bioassay

technique in studying gibberellins and plant

hormones. Thus, a wide spectrum of response

can be obtained by considering the response

pattern of all 6 mutants as a unit characteristic

of the growth-accelerating activity of the compound.

A few (about 25-50) seeds of the mutant

dwarf corn alíeles in this experiment can be obtained for propagation from E. P. Patterson,

Department of Agronomy, College of Agriculture, University of Illinois, Urbana 61803. The

recipient of these seeds will need to increase the

seed stock of each dwarf if he wishes to have

experimental amounts.

One way of applying the test compound is to

place a measured amount of solution containing

the chemical and an appropriate wetting agent

to the upper surface of the first leaf of the

germinating plant. Repeat the treatment at 2- to

3-day intervals using new leaves as they develop during the following weeks. Compare

variations in growth rate due to treatment.

References

Phinney, B. O. Growth Response of Single-Gene Dwarf

Mutants in Maize to Gibberellic Acid. Nati. Acad. of

Sei. Pi-oc. 42: 185-189. 1956.

Brian, P. W., and Hemming, H. G. The Effect of Gibberellic Acid on Shoot Growth of Pea Seedlings.

Physiol. Plant. 8: 669-681. 1955.

GROWING PLANTS

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 suitable for tests with regulators. Plant

the seeds in composted soil ; keep the soil uniformly moist and at a temperature of not less

than about 26° C. until the seedlings appear

above the surface of the soil ; then reduce the

night temperature to about 21°-24° C. About

5-8 days is 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. 54,

PN-301. A and B

FIGURE 54.—A, Applying regulating chemical to sensitive plant; B, growth response to chemical.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 113

A) will often induce a marked growth response

within a few hours or during the following

overnight period (fig. 54, B).

References to Methods of Maintaining Constant

Relative Humidity

Plant scientists working with regulating substances are sometimes concerned with maintaining known, constant relative humidities.

Solutions of inorganic salts, sulfuric acid, bases,

or carbohydrates have been commonly used for

this purpose. The following references contain

complete, reliable data concerning methods of

maintaining constant relative humidities.

Carbohydrates

(1) Grover, D. W., and Nicol, J. M. The Vapor Pressure

of Glycerin Solutions at 20°. Soc. Chem. Indus. Jour.

59: 175-177. 1940.

Methods of preparing solutions having relative humidity values from 98 to 27 percent at

20° C. by varying the percentage by weight of

glycerol are described. Refractive indices for

these solutions are given so that by using a

refractometer the exact amount of glycerol can

be determined. Relative humidity is substantially independent of temperature. These and

other data have been redrawn. Sharpf, R. F.

A Compact System for Humiditv Control.

Plant Dis. Rptr. 48: 66-67. 1964.

(2) Shaw, L. Intercellular Humidity in Relation to Fireblight Susceptibility in Apple and Pear. New York

(Cornell) Agr. Exp. Sta. Mem. 181. 40 pp. 1935.

Values for relative humidities between 93.4

and 99.9 at 25° C. are listed for 31 sucrose solutions ranging in molality from 0.1 to 3.0.

Inorganic Salts

(3) Carr, D. S., and Harris, B. L. Solution for Maintaining Constant Relative Humiditv. Jour. Indus.

Engin. Chem. 41: 2014-2015. 1949.

Relative humidities ranging from 27.4 to 85

which are maintained by 12 different saturated

salt solutions at temperatures from ambient to

90° C. are given.

(4) Winston, P. W., and Bates, D. H. Saturated Solutions for the Control of Humidity in Biological Research. Ecology 41(1): 232-237. 1960.

This is a very extensive listing of the relative

humidity values of saturated salt solutions at

temperatures from 5° to 50° C. at 5° intervals

with their properties and methods of preparation. Drying agents that maintain relative humidities of less than 1 to 5.6 percent at 25° C.

are also listed. Excellent bibliography.

Sulfuric Acid

(5) Greenewalt, C. H. Partial Pressure of Water out

of Aqueous Solutions of Sulfuric Acid. Jour. Indus.

Engin. Chem. 17: 522-523. 1925.

The water vapor pressure of concentrated

H2SO4 solutions (62 to 96%) are plotted as a

function of the reciproca-, of the absolute temperature.

(6) Wilson, R. E. Humidity Control by Means of Sulfuric Acid Solutions, With Critical Compilation of

Vapor Pressure Data. Jour. Indus. Engin. Chem. 13:

326-331. 1921.

Solutions maintaining relative humidities

from 0 to 100 percent at 25° C. and their densities are plotted as a function of the percentage

H2SO4. A method is given for calculating the

relative vapor pressure at any temperature that,

however, changes very little over a 5-10° C.

range.

Bases

(7) Buxton, P. A., and Mellanby, K. The Measurement

and Control of Humidity. Bui. Ent. Res. 25: 171-175.

1934.

The grams of KOH used per 100 ml. of water

and the specific gravities are presented for solutions maintaining from 10 to 100 percent relative humidity at 30° C.

(8) Stokes, R. H., and Robinson, R. A. Standard Solutions for Humidity Control at 25° C. Jour. Indus.

Engin. Chem. 41: 2013. 1949.

The molality of NaOH solutions which will

maintain relative humidities from 5 to 95 percent is given at 5-percent intervals.

Reference

References submitted by R. D. Durbin, Agricultural

Research Service, U.S. Department of Agriculture.

Microisolation Device

The isolator illustrated (fig. 55) is of the

^'biscuit cutter'' type and is mounted on the

microscope objective so that the cutter remains

in the field of view continuously during the

isolation of small plant parts, cells, pollen, and

spores.

The isolator consists of the following parts:

a holder (fig. 56, e) fitted snugly over a 16-mm.

microscope objective (d) and held against the

base of the objective by a small thumbscrew.

Make the holder from a small piece of aluminum

or brass approximately 7 mm. thick; fashion a

cutting device (b) from a 4-cm. portion of 18-

gauge nichrome wire bent as illustrated. Flatten one end of the wire with a hammer and bend

the flattened portion into a cylindrical cutter

(a) having a diameter of 1.5 mm. Secure the

cutter in the holder with a thumbscrew to permit adjustment of its vertical and radial positions. Fix a handle (c) permanently to the

holder to facilitate removal for flaming.

Use the isolator as follows: fill convenient

containers to a depth of at least 3 mm. with
114 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-28937

FIGURE 55.-

tached.

-Microscope with microisolation device at-

(Photo—courtesy of Phytopathology.)

4% clear agar. When agar of less than 4% is

used, moisture may be exuded by the pressure

of the cutter and the particle being isolated may

float out of position. Spread the particles to

be isolated individually over the surface of the

agar by any of the methods commonly used for

spores. Before making isolations, fasten the

isolator against the base of the objective with

the thumbscrew. Then adjust the cutter in the

field of view until it appears slightly above the

focal plane. Remove the isolator from the objective and sear the agar surface for a wellplaced cell, pollen grain, spore, or whatever

particle is involved. If an aseptic transfer is to

be made, rack the objective away from the specimen, flame the isolator, and replace it on the

objective exactly as before so the cutter automatically lines up in the center of the field of

view. Focus the objective until the specimen

comes into view through the center of the

slightly out-of-focus cutter, then adjust the objective down until the cutter penetrates the

agar to at least twice its depth. On raising the

objective, a minute agar plug is removed. Part

of this plug with the specimen being isolated

on its surface protrudes above the top of the

cutter. Remove the protruding agar and specimen for study or transplant. To perform further isolations readily, remove the isolator from

the objective, flame the cutter, replace the isolator, and repeat the above procedure.

With this device, the cutter can be observed

as it penetrates the agar, the isolator can be

removed easily and quickly for flaming, and it

can be replaced in exactly the same position

without removing the objective from the microscope.

Reference

Leach, C. M. A Simple Device for Single Spore Isolation. Phytopathology 45: 405-407. 1955.

Suggestions Concerning the Production of

Greenhouse Plants for Experiments With

Regulating Chemicals

When experimenting with regulating chemicals, use test plants that are healthy, are of

uniform shape and size, and have reached a

stage of development most suited to the experiment at hand. To produce test plants efficiently

and rapidly, use fresh, well-filled seeds with

FIGURE 56.—Diagram of isolator. (Illustrationdiagram by C. M. Leach.)

-after
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 115

unbroken seed coats, together with the most

suitable environmental conditions.

It is, of course, not practical here to describe

how all kinds of experimental plants can best

be grown. The following shows, therefore, one

method of efficiently producing a test plant that

has been widely used in experimentation with

regulating chemicals, namely, the common bean

(Phaseolus vulgaris L.).

Obtain seeds of a readily available type of

bean such as Black Valentine, Pinto, Bountiful,

or red kidney types. Use a fertile composted

soil that will not pack tightly when placed in a

clay pot and watered.

Use small clay pots about 7.5 cm. in diameter.

Fill these to within about 2.5 cm. of the rim with

the soil. Place from 2-4 bean seeds in each pot,

spaced as illustrated in fig. 57. Add enough soil

to cover the seeds to a depth of about 1 cm. Use

a flat surface to smooth the soil over the seeds

but do not pack it. Leave a space of about 1 cm.

from the soil surface to the rim of the pot

(fig.. 57).

Fill this space with water and allow the water

to seep into the soil. Place the filled pots at a

temperature of about 27°-32° C. Do not allow

the surface of the soil to become even slightly

dry.

Young plants will appear above the surface

of the soil within 3-4 days. This stage of development is termed the "staple" stage (fig. 58).

The amount of light received by plants in the

staple stage is critical. To obtain short, sturdy

plants, reduce the temperature at this stage to

21°-24° C. and place the plants where they will

receive direct sunlight, or expose them to artificial light from fluorescent tubes equal to an

intensity of 3,000 foot-candles for 12 hr. each

day. To obtain etiolated plants, expose the plants

as they break through the soil to a lower light

intensity and maintain the temperature at

24°-27° C.

BN-28949

FIGURE 57.—Bean seeds before they are covered with

soil.

BN-28946

FIGURE 58.—Bean plants at the "staple" stage of development.
116 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OP AGRICULTURE

Bean plants grown at the relatively low

temperature and high light intensity will develop primary leaves that are approximately 3.5

cm. across within 5-6 days after the seeds are

planted (fig. 59). As soon as these leaves are

relatively smooth (6-7 days), they are capable

of absorbing and translocating regulating chemicals to other parts of the plants. Stems of these

plants grow rapidly and respond readily to regulating chemicals that induce cell elongation or

cell proliferation, or both.

After the plants have reached a desirable

size, sever the stems (just below the surface of

the soil) of all but the most suitable plant in

each pot. Test plants of uniform size and responsiveness thus can be obtained within 1 wk.

or lesf

Storage of Seeds Used as a Plant Source for the

Study of Hormones and Regulators

To carry out experiments with plant hormones and regulating substances, it is necessary to have seeds of the required kinds that

will germinate uniformly and produce vigorous

plants. It is also necessary to have a continuous

source of a given kind of seed to produce plants

that will respond consistently during the experimental period involved. To check biologically

any changes in growth-regulating activity that

occur in stored hormones and regulating compounds, it is sometimes necessary to use the

same lot of seeds over an extended period of

time. Conditions most suitable for seed storage

are, therefore, essential.

First, obtain the appropriate kind of freshly

grown seeds in an amount sufficient to last for

a number of years. If the seeds are not uniform

in appearance, sort them to eliminate cracked,

small, wrinkled, and discolored ones. Make sure,

through appropriate tests, that seeds in each lot

have a high degree of viability, germinate rapidly, and are relatively free from disease.

To maintain seeds over a long period so they

will produce uniform seedlings capable of vigorous growth, store the seeds at a low temperature

and with an appropriate moisture content. If

seeds are maintained under the conditions listed

below, they should remain essentially unchanged for at least 10 years.

Dry the following kinds of seeds to the designated moisture content:

6% moisture

Cucumber

Cocklebur

Radish

Squash

Sunñower

Tobacco

Tomato

8% moisture

Bean

Cereals (including corn)

Pea

Soybean

Do not allow the temperature of the stored

seeds to rise above 2° C. If it is more convenient

BN-28952

FIGURE 59.—Bean plants 5-6 days after seeds are planted.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 117

to store the seeds in a deep freeze (— 10° to

— 18° C), moisture content of the seeds may

be approximately 1% higher than given above.

To dry the seeds to the desired moisture content, determine the amount of moisture that

they contain when they are received. To do this,

place 10-20 g. of seeds in a tared dish and

weigh accurately. Dry large seeds, such as bean

and corn, in a forced-draft oven at 100°-103° C.

for 48 hr. and small seeds for 24 hr. Determine

the percentage of moisture lost on the basis of

the original fresh weight of the sample.

Spread the entire lot of seeds in a thin layer

on a cloth or paper in a warm, dry place with

free movement of air, using a heated room or a

protected area that receives sunlight. Do not

allow the temperature of the seeds to exceed

45° C. After several days, determine the progress of drying by weighing the entire seed lot.

From the original weight and from the loss of

weight, calculate the moisture content at the

time of the weighing as follows:

When the seeds have dried to the desired

moisture content, mix the seeds thoroughly and

again determine the moisture content with a

final test.

In order to facilitate removal of small

amounts of seeds from storage for a series of

tests without raising the temperature and without changing the moisture content of the remaining seeds, divide the entire seed lot into

small samples, each sample adequate for use

over a short period. Place each sample in a

separate cellophane or polyethylene sack with a

label describing the seed lot. Place the sacks

into Mason jars and seal them. Store the jars

at the appropriate temperature. When seeds are

needed, remove a sack quickly and reseal the

jar to avoid increasing the temperature or

moisture of the remaining seeds.

Reference

Information assembled by E. H. Toole, Agricultural Research Service, U.S. Department of Agriculture.

If _ desired moisture %

""" ÏÔO

and X = weight of water to be lost from lot to reach desired moisture content,

(original weight of water in lot)-x'

(original w^eight of lot)-.i*

«(original weight of lot)-ax = (original w^eight of w^ater in lot)-i'

{l-a)x = (original weight of water in lot)-a(original w^eight of lot)

_ (original weight of water in lot)-a(original w^eight of lot)

^ — ' —— — ij1-a

Exa7npie:

original w^eight of seed lot 10,000 g., moisture 16%

1,600 g. water in original lot

If 6% moisture is desired, water to be lost {x) =

1,000

1,600-0.06(10,000)

1-0.06

- = -^-^^^^^'^^^

10,000-1,063.82 = 8,936.18 g. = weight of lot dried to 6% moisture

OBSERVING AND MEASURING PLANTS

Aphid Honeydew Technique for Study of

Sieve Tube Content

When aphids are allowed to feed on the stems

of rooted willow cuttings, the leaves of which

have been supplied with C^^02, honeydew from

the insects contains radioactive sugar. The fact

that aphids feed within sieve tubes offers a

means of detecting and, under certain conditions, measuring the tagged sugar in these

tubes.

The honeydew method is described here on

the assumption that some radioactively tagged

growth-regulating substances, when carried in

the sieve tubes, might be ingested by the aphids

and then excreted along with honeydew, thus

offering a means of determining whether the

radioactivity reached a known position in the

stem by moving through the sieve tubes. This

method is not suitable, however, for studying

the metabolism of transported regulating sub-
118 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

stances since the tagged molecules must pass

through an insect which might in itself metabolize the compound.

Use the aphid Tuberolachnus salignus

(Gmelin) (Homoptera, Aphididae). Cut segments about 15 cm. long from branches of 2-

to 4-yr.-old willow trees, Salix viminalis L. Immerse the lower 4-5 cm. of each cutting in

water and, when the new roots are 3-4 cm. long,

pot the cuttings in a mixture of peat moss and

sand. Supply the plants with natural light or

with artificial light from a suitable source that

gives an intensity of at least 500-1,000 footcandles at the leaf surfaces.

Apply a C"-tagged regulator to the leaves.

(See "Bean stem curvature method (absorption

and translocation)," p. 12, ñg. 7, for application.) Collect the honeydew from the aphids

feeding on stems of the cuttings near leaves to

which the regulator has been applied (G. P.

Hill, The Sources of Sugars in Sieve-Tube Sap.

Ann. Bot. (n.s.) 27: 79-87. 1963). Place the

honeydew directly on a smooth nickel planchet.

Flood the planchet with distilled water so tho

honeydew is dissolved and spread evenly over

the entire surface of the planchet. Evaporate

the water at room temperature and then bring

the planchets to constant weight in an oven at

22° C. Finally, dry the planchets for several

days in a desiccator. Count the samples with a

gas-flow counter and calculate the results as

counts/minute/mg. of sample. Self-absorption

would, of course, be involved in the use of this

method. The amount of self-absorption could be

estimated by mixing known amounts of the

radioactively tagged regulator with weighed

amounts of honeydew collected from aphids

feeding on cuttings to which the regulating

substance has not been applied.

Reference

Based on research by A. J. Peel and P. E. Weatherley.

Studies in Sieve-Tube Exudation Througli Aphid

Mouth-Parts: The Effects of Light and Girdling. Ann.

Bot. (n.s.) 26: 633-646. 1962.

Aphid Stylet Technique for Studying Regulating

Substances in Sieve Tubes

Aphid mouth parts have been used to study

the content of sieve tubes and of single sieve

elements. This technique is described here with

the thought that it might also be useful for

measuring radioactively tagged regulating substances and metabolites of them translocated in

sieve tubes.

Anesthetize aphids, Tuberolachnus salignus

(Gmelin) (Homoptera, Aphididae), with a

gentle stream of CO2 while they are feeding on

stems of willow cuttings, the leaves of which

have been treated with a radioactive growth

regulator. (See "Bean stem curvature method

(absorption and translocation)," p. 12, fig. 7,

for application.) Using a binocular microscope

and a fine scalpel made from a sliver of a razor

blade, sever the proboscis of one of the insects

as close to the head as possible. Remove the

labial sheath surrounding the stylet with a

fine brush. Repeat the procedure, severing as

many embedded stylets as are needed to obtain

a suflScient amount of exúdate (fig. 60). Collect

the sieve tube exúdate using a glass capillary

(0.5-0.1 mm. inside diameter) held with a

manipulator against the surface of the exuded

droplet. Measure the length of the column of

liquid in the capillary with a vernier microscope

and calculate its volume. Exudates collected in

this way can be subjected to various Chromatographie techniques to separate out the tagged

growth regulator or metabolites of it. Measure

the tagged regulating substance present by conventional counting methods or by radioautography.

Reference

Based on research by P. E. Weatherley, A. J. Peel, and

G. P. Hill. The Physiology of the Sieve Tube. Preliminary Experiments Using Aphid Mouth Parts. Jour.

Expt. Bot. 10: 1-16. 1959.

Extraction of Hormones and Growth-Regulating

Substances From Plants

Endogenous hormones can be obtained by allowing these substances to diffuse from freshly

cut plant surfaces into agar. A method useful

for this purpose is described on page 96.

BN-28964

FIGURE 60.—Phloem exúdate (a) that collected on detached aphid stylet (6) embedded in a stem. (Photo—

courtesy of M. H. Zimmermann and Science.)
METHOUtí OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 119

Methods of chemically extracting hormones

and growth-regulating substances from plants

are well known and widely used in physiological

studies. Since some of these methods were recently described by Mitchell and Smale (cited

below), a detailed description of them is not

given here.

The amount of extractable hormone in a plant

or plant part may vary, depending upon environmental conditions and factors controlling

hormone synthesis, utilization, and degradation.

Solvent used, pH, and also some physical factors

can influence the amount of hormones obtainable from a plant part. For example, the amount

of extractable hormone in some kinds of stems

has been increased by subjecting the plant part

to a centrifugal force (Kawase, Monselise). It

is not known whether this increase is due to

increased hormone synthesis, to an unbinding

effect of centrifugation that accelerates outward

movement of the hormone, or to accelerated

basipetal transport of hormones that makes them

readily extractable. Regardless of the mechanism involved, the investigator should bear in

mind that quantitative extraction of plant hormones, using any of the present methods, depends on several factors and that the results

may not be truly quantitative.

References

Goldschmidt, E. E., and S. P. Monselise. Private communication. Unpublished data. 1966.

Kawase, Makoto. Centrifugation, Rhizocaline and Rooting in Salix alba L. Physiol. Plant. 17: 855-865. 1964.

Mitchell, J. W., and Smale, B. C. Bioassay—Plants.

Analytical Methods for Pesticides and Food Additives.

V. 1, pp. 176-191. Academic Press, Inc., New York.

1963.

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. 61, right).

PN-S06

FIGURE 61.-^Right, Malformed leaf from a plant treated with a small amount of growth-regulating substance, compared with (left) a comparable leaf from an untreated plant.
120 AGRICULTURE HANDBOOK 336, U.S. DP]PARTA1ENT OF AGRICULTURE

Interferometer Method of Detecting Minute

Growth Movements

Minute plant movements associated with

growth response induced with hormones and

regulators can be detected by means of a sensitive interferometer method. Support a mici'oscope slide (fig. 62, b) by placing one end in

a groove in a wooden block. Glue 2 sharp glass

pegs to one end of a shorter slide. Rest one slide

against the other with the shorter one supported

by the pegs. Using cocoa butter, fasten one end

of a fine glass rod to the shorter slide, the other

end to the leaf or other part of a plant where

movement is to be detected. Heat sodium

chloride in a porcelain dish or spoon (c) over

a flame so yellow light made by the sodium

chloride reflects from the surfaces of the slides.

Focus a horizontal telescope (a) equipped with

cross hairs on the image of the flame reflected

from the glass. Observe the interference rings

that are apparent when the inner surfaces of

the slides are the appropriate distance apart.

Without producing air currents, allow small

spray droplets of the test solution (with a wetting agent added, if needed) to strike the leaf

or other portion of the plant used. Movement

of the interference rings in one direction indicates movement of the plant, the distances involved being in the range of wavelengths of

the light used.

FIGURE 62.—Diagram of apparatus used to detect minute

growth movements.

Freehand Sections for Studying Cell Responses to

Regulators

In studying cellular responses to hormones

and regulating substances, it is sometimes desirable, or even essential, that living cells be

used so that the actual size, shape, and behavior

of the cell parts can be observed. Freehand sections can be made of succulent parts of plants

that contain uninjured cells suitable for detailed studies of mitochondria and larger cell

parts and for use in still and cinema photography in recording the responses to hormones

and regulators.

To make freehand sections of succulent tissue, use the sharpest single-edge razor blades

possible and discard each blade at the first

evidence that it has become dull. In the case of

new blades, without touching the sharp edge,

remove excess oil or grease with tissue. Dip the

blade several times in acetone to remove any

remaining oil. Hold the segment to be sectioned

under a fluorescent lamp with magnifying glass

attached.

The plane of the blade should pass through

the cells parallel with the long axis of the plant

segment, i.e., parallel with the long axis of the

cells (ñg, 63). If the plane of the blade is not

parallel with the long axis of the segment, the

number of cells injured is greatly increased,

probably due to development of greater stress

in the tissue. Make the sections about 35-40

microns thick. After each slice, make sure the

blade is held in a position that maintains the

plane of the cut surface parallel with the long

axis of the segment.

Move the blade through the tissue so the cutting edge forms an angle 15° off perpendicular

with the long axis of the segment. Slide the

blade through the tissue at a uniform rate.

Avoid a sawing motion. Keep the blade and

the tissue wet during the entire procedure and

immediately transfer the tissue section to tapwater that has been boiled to remove any

chlorine and then aerated to replace the dissolved air driven off during the boiling. This

tapwater should be at room temperature. In

making the transfer, float the section off the

razor blade by immersing the blade in the

water.

To observe the tissue microscopically, transfer each section from the water in which it has

been stored temporarily by floating it into a

droplet of the water in the well of a special slide
METHODS OF STUDYING PLANT HORMGNi^S AND (ÎROWTH-RKGULATING SUBSTANCES 121

BN-28it48

FIGURE 63.—Making freehand sections foi- studying cell

responses to regulators.

chamber. (See "Slide chamber for the study of

living cells," p. 123.)

In the microscopic study of the living cells,

it is generally necessary to ignore the first row

below the cut surface since these are usually

injured in the process of making the section.

The second row below the cut surface may contain injured cells, but all or most of the cells in

the third row are generally uninjured.

Reference

Submitted by Marjorie D. Montgillion, Agricultural Research Service, U.S. Department of Agriculture.

Measurement of Root Response

Place a disk of blotting paper impregnated

with a water mixture of the regulating chemical and another impregnated with tapwater or

distilled water in separate petri dishes. Ordinary blotting papers often contain chemicals

that inhibit root growth. Blotting paper especially prepared for seed tests can be obtained

from the Standard Paper Manufacturing Co.,

P.O. Box 1554, Richmond, Va. 23212.

Place several cucumber seeds of uniform size

on the surface of each paper. Store the closed

dishes at room temperature in darkness for 24

hr. ; then measure growth of the roots repeatedly during the following 2 or 3 days with the

dishes closed (fig. 64). Compare growth of the

roots on the chemically treated papers with that

of roots on the papers impregnated with water.

Radioautographs of Sectioned Plant Material

Although problems still remain to be solved,

radioautographs of sectioned plant material

have been obtained that show clearly the position of the tagged compounds within various

tissues. Attempts to use radioautographs to

locate soluble tagged compounds within the

cells, however, have not been very successful.

The following procedures are described in general with the hope that they will be useful in

locating tagged regulators in tissues at least,

and perhaps, with some modification, certain

of these will be useful for intracellular studies.

Conventional methods cannot always be used

to fix, embed, and section the tissues to prepare them for radioautography because many

growth-regulating substances are soluble in the

alcohol and in some of the other liquids used in

these procedures.

One method for tissue radioautographs involves cutting the sections while the tissue is

frozen and then exposing the sections to an

appropriate type of film at a temperature below

freezing. A cryostat is the most suitable apparatus for this purpose since, with it, sections

can be made easily at very low temperatures.

Wrap aluminum foil around a pencil to make

a cylinder about 1 cm. long. Fill the metallic

cylinder with liquid gelatin (10%) and place

a small piece of the plant tissue in the gelatin.

Then freeze the gelatin and tissue in the cryostat at - 16° to —20° C. Place a drop of water

on the microtome specimen holder and freeze

the gelatin block in place for sectioning. While

operating the cryostat, cut the desired number

of sections 10-25 microns thick.

Within the cryostat, using a red safe light

(Wratten Series No. 1), place the sections flat

against the emulsion of a Kodak Type A lantern slide. X-ray film cut into a suitable size

may be substituted for the lantern slide. Place

a freshly and carefully cleaned microscope

slide or one freshly coated with silicon above

the sections and clamp the 2 slides together

with binder clips. Place this assembly in a

light-tight container and expose the emulsion
122 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

PN-S02

FIGURE 64.—Measuring root responses.

to the radioactivity within the cryostat or

transfer without thawing to a freezer compartment. Prepare several slides this way so they

can be developed one at a time at various intervals to determine the correct exposure. In

making this test, the exposure time must be

doubled in order to increase the density of the

image significantly. In developing the emulsion,

use methods suggested by the manufacturer

of the particular emulsion. For example, if the

Type A lantern slide is used, develop in D-19

developer for 4 minutes, stop in a bath of

water for 15 seconds, fix in 20% hypo for 20

minutes, and then wash thoroughly in water.

The sections of plant tissue that survive the

development and fixation are in place, and thus

the position of radioactivity in relation to different tissues and cells can be determined.

Reference

Procedure compiled by J. F. Worley, Agricultural Research Service, U.S. Department of Agriculture.

Two other types of sensitive emulsions have

been used, namely liquid emulsion and stripping film. To use these emulsions, the tissue

must be fixed and then embedded or dried. It

is emphasized that in the process of fixing,

embedding, or drying, soluble substances may

be removed from the tissue. In addition, the

position of the substance in question or the

position of the cell part with which it is associated may change during various steps in the

procedure. Some references concerning use of

these emulsions are:

Belanger, L. F., and Leblond, C. P. A Method for Locating Radioactive Elements in Tissues by Covering Histological Sections With a Photographic Emulsion. Endocrinology 39: 8-13. 1946.

Fitzgerald, P. J., Simmel, Eva, Weinstein, Jerry, and

Martin, Cynthia. Radioautography : Theory, Technic,

and Applications. Lab. Invest. 2: 181-222. 1953.

Kamen, M. D. Radioactive Tracers in Biology, An Introduction to Tracer Methodology. Academic Press, New

York. Ed. 2, 429 pp. 1951.

Pele, S. R. Autoradiograph Technique. Nature 160:

749-750. 1947.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 123

Separation of Leaf Areas Treated With a Regulating

Chemical From the Remaining Untreated Portions of

the 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 untreated portion which

remains can be assayed for radioactivity. The

following simplified method is useful since the

equipment required is inexpensive and, if accidentally contaminated, 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 so 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. 65). Treated portions of several leaves can be removed without contaminating untreated portions by using only 1 vial and

1 piece of abrasive material and then discarding

the equipment.

Slide Chamber for the Study of Living Cells

Place a No. 1 square cover glass on top of a

No. 2 square cover glass and align their edges

accurately. Use parafiin melted with a hot

needle to seal the edges (fig. 66, y, if) of 2

pairs of aligned cover glasses (x, x') to opposite

ends of a microscope slide. Immerse the sections of plant tissue in tapwater placed in the

center of the well formed by the square cover

glasses. Cover the sections with rectangular

cover glass (a) supported as a bridge about

0.26-0.32 mm. above the glass slide by the

square cover glasses. Use a slide 25 mm. wide

PN-307

FIGURE 65.—RemovinR the treated area of a leaf.
124 AGRICULTURE HANDBOOK 3:56, I .S. DEPARTMENT OF AGRK^ULTURE

FIGURE 66.—Diagram of slide chamber for studying living cells.

washer. Do this carefully so air bubbles are not

trapped within the chamber. Apply the metal

washer and threaded ring. Tighten the threaded

ring to press the coverslips gently against the

plastic w^asher. Construct inlet and outlet tubes

with 25-gauge hypodermic needles and insert

these through the plastic washer as illustrated

(c). Connect the chamber to an electrolytic

pump (d) or to a constant-flow mechanical

pump if relatively large amounts of liquid are

used. Observe or record photographically cellular responses by placing the assembly under a

microscope.

(narrow dimension), square cover glasses 25

mm. wide, and a rectangular cover glass 22

mm. wide.

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 stems is therefore one means

of evaluating the regulating properties of the

chemical.

Apply the chemical in a suitable carrier (lanolin or aqueous mixture), for example, to bean

plants that have developed primary leaves that

are partially expanded (3.5-5 cm. across) and

when the trifoliolate leaves are still folded in

the terminal bud (ñg. 67, left). Measure the

length of the terminal bud from second node to

tip of bud at the time of treatment. Measure

from the same node (second) to the tip of the

terminal bud about 1 wk. after the treatment

(ñg. 67, right), and compare growth with comparable measurements of untreated plants.

Observation Chamber for Continuous Application of

Hormones or Growth Regulators to Living Cells

Obtain an observation chamber ^^ (fig. 68, a).

Prepare sections of living tissues to be studied

as described on p. 120. Place a glass plate (6),

made by cutting a microscope slide to the required size, on the coverslip that serves as the

lower side of the chamber. Place into position

the soft plastic washer that separates the two

glass sides that form the top and bottom of the

chamber. Fill the chamber with water or the

test solution, immerse the freshly cut sections

in the liquid, and position them directly over

the glass plate. Close the chamber by dropping

the second coverslip into position on the plastic

'^ Obtainable from the Baltimore Instrument Co., Inc.,

716 West Redwood Street, Baltimore, Md. 21201.

Motion Detector for Recording Plant Growth

This sensitive instrument (fig. 69) can be

made to detect, measure, and record growth and

minute movements of plants continuously, including even slight growth responses to hormones and regulating chemicals.

The basic system consists of an angle transducer connected to the plant with a coiinterbalanced variable-length rod. Output from the

transducer is adjustable by means of a voltage

divider so that the voltage matches the desired

sensitivity of a recorder.

Obtain an angle transducer with a d.c. voltage output in excess of the recorder. Connect

the transducer to a voltage divider and then

connect the divider to a recorder such as a

0-10 mv data logger. Attach a part of an aluminum, rifle-cleaning rod to the transducer shaft

so that the rod is counterbalanced and free to

move vertically (fig. 70). Adjust the length of

the rod by adding sections when needed.

Construct a spring clip that can be attached

to the stem or leaf of the plant to be studied and

fasten this clip to the counterbalanced rod. Adjust the counterbalance so that the tip has a

tracking weight of less than 0.25 g. Connect the

transducer to the voltage divider and recorder.

Adjust the voltage divider and rod length so

that the sensitivity of the instrument (as fine as

5 X 10^^ in.) meets your needs.

To make an arrangement with which both

vertical and horizontal movements of plants can

be measured, mount the sensor portion of a

second transducer piggyback fashion and at a

90° angle in relation to the first one (fig. 70).

Arrange the first transducer to measure the

vertical movement and the second to measure

the horizontal movement in a plane perpendicular to the connecting rod.

To prevent the recorder from moving off

scale, place the transducer system on a motor-
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 125

PN-S08, A and B

FIGURE 67.—Measuring terminal growth.

ized jack and control the height of the jack platform by a feedback system for the output of

the transducer. Arrange the system (fig. 71) so

that when the output of the transducer reaches

a preset upper limit, the motor that operates

the jack is energized, thus raising the platform

upon which the transducer system rests. The

transducer output then decreases until it

reaches a lower preset mark and the motor is

stopped. Adjust the upper and lower limits of

recorder movement by variable potentiometers.

Reference

Method developed hy H. H. Kleuter, R. J. Downs, W. A.

Bailey, and D. T. Krizek, Agricultural Research Service, U.S. Department of Agriculture.
126 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

BN-2894B

FIGURE 68.—Observation chamber for continuous application of hormones or growth regulators to living cells: (a)

Plate of observation chamber; (6) glass plate; (c) hypodermic needle that serves as outlet; (d) electrolytic pump.

BN-28944

FIGURE 69.—Complete motion detector with counterbalanced rod attached to plant.
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 127

BN-28943

FIGURE 70.—Method of counterbalancing rod that conveys plant movement to transducer. Sensor portion of

a transducer (a) bearing the counterbalanced variable-length rod is mounted piggyback at a right angle

to the intact transducer (6).

FIGURE 71.—Schematic drawing showing method of wiring apparatus when arranged to prevent the recordeifrom moving off scale: a, 110 volt a.c. current supply;

ft, transducer; c, voltage divider; d, recorder; e, relay;

/, motor connected with jack; g, high-low limit control

switch; h, feedback lines.
128 AGRICULTURE HANDBOOK 336, U.S. DEPARTMENT OF AGRICULTURE

SUPPLEMENTARY INFORMATION

MEASURES, EQUIVALENTS, AND ABBREVIATIONS

WEIGHTS

1 kilogram (kg.) — 1,000 grams (g.) — 2.2 pounds

(lb.)

1 gram (g.) = 1,000 milligrams (mg.) = .035 ounce

avoirdupois (oz.)

1 milligram (mg.) = 1,000 micrograms (7 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 appi'oximately 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 gram in 1 liter = 1,000 parts per million (p.p.m.) oi0.1%

1 gallon (gal.) = 4 quarts = 3.785 liters

1 quart (qt.) = 2 pints = 0.946 liter

1 pint (pt.) = 16 fluid ounces = 0.473 liter

1 fluid ounce (oz.) = 29.6 milliliters or cubic centimeters

1 gallon (gal.) = 16 cups = 128 fluid ounces

1 quart (qt.) = 4 cups = 32 fluid ounces = 64 tablespoons

1 pint (pt.) = 2 cups = 16 fluid ounces = 32 tablespoons

1 cup m 8 fluid ounces = 16 tablespoons = 48 teaspoons

1 fluid ounce (oz.) — 2 tablespoons = 6 teaspoons

1 tablespoon (tbsp.) = 3 teaspoons (tsp.)

LENGTHS

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.) = 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.) = 0.039 inch = 1,000 microns

1 micron (M.) = 1,000 millimicrons = 0.0000394 inch

1 millimici'on (mM.) = 10 angsti'om units

1 square kilometer (sq. km.) = 1,000,000 square meters

= 0.386 square mile

square meter (sq. m.) 10,000 square centimeters =

10.764 squai-e feet

square centimeter (sq. cm.)

= 0.155 square inch

square millimeter (sq.. mm.)

100 square millimeters

0.002 square inch

AREAS

640 acres = 2.590 square

4046.873 square me25.293

1 square mile (sq. mi.)

kilometers

1 acre (A.) = 160 square rods

ters = 43,560 square feet

1 square rod (sq. rd.) = 30.25 square yards

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

TEMPERATURE CONVERSIONS

Short-Cut Method of Converting From Centigrade to

Fahrenheit Degrees

Double 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 ninefifths and add 32.

(°C. X 9/5) + 32 = °F.

9 Reprinted by permission from Chemical and Engineering News, Nov. 21, 1949.

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 STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 129

METHODS OF MAKING NUTRIENT SOLUTIONS

If plants are to be grown only for 2-3 wk. in

nutrient solution, a suitable nutrient can be prepared by adding only macronutrients to tapwater. If the plants are to be grown for a longer

period, however, or if distilled water is used,

micronutrients must generally be added.

Nutrient Solution Made With Tapwater

Prepare a i/^-molar stock solution of each of

the following macronutrients in separate containers using tapwater :

KH2PO4 (68.07 g. per liter)

MgSO 4 • 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 tapwater :

156 ml. of y2-mo\8iY solution of KH2PO4

581 ml. of y2-molar solution of MgS04

•7H2O

306 ml. of i/^-molar solution of CaCk

• 6H2O

375 ml. of i/^-molar solution of NaNOs

Nutrient Solution Made With Distilled Water

Prepare a i/4-molar stock solution of each of

the following macronutrients in separate containers using distilled 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)

In preparing the macronutrient solution, add

the macronutrient from the stock solutions in

the following amounts to 50 liters of distilled

water :

156 ml. of i/2-molar solution of KH2PO4

581 ml. of 1/2-îï^olar solution of MgS04

• 7H2O

306 ml. of 1/2-i^olar solution of CaCb

• 6H2O

375 ml. of l^-molar solution of NaNOs

Prepare micronutrient stock solutions :

A. Add the following micronutrients to 2

liters of distilled water :

572 g. of H3BO4

3.62 g. of MnCl2

0.44 g. of ZnS04 • 7H2O

0.16 g. of CuS04 • 5H2O

0.18 g. of H2M0O4

Add 1 cc. of the micronutrient stock

solution per liter of prepared macronutrient solution.

B. Add 1 ml. of 0.5% ferric citrate per

liter of macronutrient solution.

POTASSIUM PHOSPHATE BUFFER MIXTURES

Mixtures of potassium monohydrogen phosphate and potassium dihydrogen phosphate

required to give various pH values

Volume of 0.1 Volume of 0.1 Volume of 0.1 Volume of 0.1

molar K2ÍIP()4 molar KH2PO4 pH of mixture molar K2HPO4 molar KH2PO4 pH of mixture

solution 1 solution 2 solution 1 solution 2

Ml. ML Ml. ML

10.00 8.302 4.00 6.00 6.643

9.90 0.10 8.171 3.00 7.00 6.468

9.75 0.25 8.038 2.00 8.00 6.239

9.50 0.50 7.863 1.00 9.00 5.910

9.00 1.00 7.648 0.50 9.50 5.600

S.OO 2.00 7.347 0.25 9.75 5.303

7,00 3.00 7.146 0.10 9.90 4.976

6.00 4.00 6.976 — 10.00 4.529

5.00 5.00 6.813

1 0.1 M — K2HPO4 = 17.4 g. dissolved in water and diluted with water to 1 liter.

2 0.1 M — KH2PO4 = 13.6 g. dissolved in water and diluted with water to 1 liter.

DESCRIPTIVE INDEX

Page

Abbreviations 128

Abscission 1

Apple method 64

Apple petiole method 1

Attached petiole method 2

Coleus petiole method 4

Expiant method 5

Lanolin paste method 7

Peach and prune method 65

Abscission, fruit 64

Apple method 64

Apple petiole method 1

Peach and prune method 65

Abscission, leaf

Apple petiole method 1

Attached petiole method 2

Coleus petiole method 4

Expiant method 5

Absorption

Cell for introducing regulators into roots 94

Into leaves 95

Of growth regulators from agar block 96

Of regulators by aquatic plants 109

Radioautograph method 87

Through a leaf flap 95

Absorption and translocation 8

Agar block polar transport method 8

Bean root method 10

Bean stem curvature method 11

Absorption cell for introducing compounds into

limited portions of roots 94

Absorption, radioactively tagged compounds

Radioautograph method - 87

Absorption through a leaf nap 95

Acceleration, giowtli (Sec "(îrowth uccek^ration")

Activated charcoal met hod 92

Addicott, F. T.

Expiant method, with 2 variations 5

Aerosol method 60

Agar block polar transport method 8

Agar block preparation and use 96

Agar slant method 72

Air, contamination of, with regulators

Activated charcoal method _ 92

Disposable container method 90

(îei ininating S(MM1 nuMh()(L ÍH

Alpha-methoxyphenylacetic acid

Bean method 49

Root exudation method 52

Alpha-naphthaleneacetamide

Apple method 64

Cone container method 73

Stem-cutting method 79

Alpha-naphthaleneacetic acid

Apple petiole method 1

Coleus petiole method 4

Lanolin paste method 7

Radish cotyledon method 31

Root primordia method 78

Alpha-naphthaleneacetic acid, methyl ester of

Vegetative sprout method 68

Ammonium 2,4-dichlorophenoxyacetate

Bean stem curvature method 11

Amo-1618

Lanolin paste method 56

Page

Anderson, W. P.

Direct measurement of volatility 102

Germinating seed method 91

Angle and elongation of branches 13

Lanolin method 13

Animals, detection of exogenous plant growth

regulators in 82

Bean stem method 82

Aphid honeydew technique for study of sieve tube

content --- 117

Aphid stylet technique for studying regulating

substances in sieve tubes 118

Aphid technique for studying translocation of

regulators 118

Apple

Apple method 64

Apple petiole method 1

Lanolin method „ 13

Lanolin paste method 7

Nutrient mist method 75

Apple method 64

Apple petiole method 1

Application

Aqueous mixtures to stems 97

To roots with plastic culture vessel 107

Application of a measured amount of regulating

chemical to a known area of leaf surface 98

Application of aqueous mixtures quantitatively to

stems ^ ; •>7

Application of growth substances with capillary

tubes 99

Application of hormones and growth regulators to

living cells 124

Application of plant regulators to seeds 98

Application of radioactively tagged growth

substances

Ground tissue method 89

Application of regulators as a part of a coating on

seeds 98

Application of regulators—measured amounts 102

Application of regulators to leaves - 98

Application of regulators to localized areas

Application of a measured amount of regulating

chemical to a known area of leaf surface i)!^

Apj)lication of aqueous mixtures quantitatively to

stems - •>7

Lanolin as a carrier for regulating chemicals 106

Application of regulators to stems 97

Application of regulators using lanolin ring 98

Applicator, remote control

Ground tissue method 89

Aquatic plants, absorption and translocation of

regulators by 109

Attached petiole method 2

Avena colee)ptile method (Cell elongation) 2:i

Bacitracin

Sterile nutrient method 66

Bailey, W. A.

Motion detector for recording plant growth 124

Bailey, W. K.

Impregnated filter paper method 74

Barker, B. T. P.

Nutrient mist method 75

Barley

Barley endosperm method 48

131
132 AGRICULTURE HANDBOOK 38G, U.S. DEPARTMENT OF AGRICULTURE

Page

Barley—Continued

Barley leaf method 43

Cocklebur leaf disk method 44

Cone container method 73

Impregnated filter paper method 74

Barley endosperm method 48

Barley leaf method _ 43

Barlow, H. W. B.

Application of growth substances with

capillary tubes 99

Lanolin method 13

Segment cutters 101

Wheat coleoptile straight growth method, with 3

variations 36

Batjer, L. P.

Apple method 64

Bean

Agar block polar transport method 8

Attached petiole method 2

Bean hypocotyl method 25

Bean leaf method 55

Bean method, with 1 variation 49

Bean root method 10

Bean second internode method 26

Bean stem curvature method (absorption and

translocation) 11

Bean stem curvature method (effect of one

compound on translocation of another) 47

Bean stem method 82

Disposable container method 90

Ground tissue method 89

Lanolin paste method 56

Leaf disk enlargement method 42

Microculture method 15

Ovary culture method 58

Root exudation method 52

Submerged cultivation method 21

Bean hypocotyl method 25

Bean leaf method 55

Bean method 49

Bean root method 10

Bean second internode method 26

Bean stem curvature method (absorption and

translocation) 11

Bean stem curvature method (effect of one compound

on translocation of another) 47

Bean stem method 82

Bender, Robert

Attached petiole method 2

Berthold, R. V.

Oat first internode method 29

Beta-naphthoxyacetic acid

Aerosol method 60

Tomato method _ 62

Biddulph, O.

Absorption through a leaf flap 95

Biennial or perennial plant method 54

Biological methods 1

Blotting paper for germinating seeds, source of 121

Bonner, James

Oat coleoptile section method, with 2 variations,. 23

Bottomley, W.

Tobacco pith method (micro) 22

Branches, angle and elongation of 13

Lanolin method 13

Brown, J. W.

Bean hypocotyl method 25

Bean leaf method ')ö

Impregnated filter paper method 74

Buchholtz, K. P.

Movement of regulating substances through

membranes or tissues 106

Bud inhibition

Vegetative sprout method 68

Page

Buckwheat

Disposable container method 90

Germinating seed method 91

Impregnated filter paper method '74

Buffer mixtures, potassium phosphate ] 29

Capillary tubes, use in ai)plying re^uhitinii substances 99

Caplin, S. M.

Root culture method 16

Carbamates

Agar slant method 79

Peach and prune method [[ 65

Vegetative sprout method [ 68

Carnation

Stem-cutting method _ 79

Cams, H R.

Expiant method 5

Carrier, fractionated lanolin 104

Carriers

Dust.___ 100

Lanolin ]06

Carrot

Microculture method 15

Root culture method IQ

Vegetative sprout method ^ _____ 6S

Carter, W.

Nutrient mist method 75

Catechol

Rooting cofactor method 76

Cell

Continuous ti-eatincnt of, willi lionnones and

^Towt li i-e,ii;ulat ors 124

Freehand sections of „ 120

For measuring absorption of growth regulators

into roots 94

^ Injection of, with regulating substances, 104

Cell division, abnormalities

Endosperm method 13

Cell division or multiplication 13

Endosperm method 13

Microculture method 15

Root culture method 16

Single isolated cell method 18

Soybean callus tissue method 19

Stem tissue culture method 20

Submerged cultivation method 21

Tobacco pith method (micro) 22

Cell elongation 23

Bean hypocotyl method 25

Bean second internode method 26

Oat coleoptile section method, with 2 variations,, 28

Oat first internode method 29

Pea stem section method 30

Radish cotyledon method 31

Split pea stem method 32

Tomato hvjxx'otyl nu^tliod 34

Wheat coleoptile straight growth method,""with

3 variations 36

Young wheat coleoptile method 40

Cell enlargement 41

Citrus petal method 41

^ Leaf disk enlargement method 42

Cell expansion

Cell enlargement 41

Cell multiplication

Cell division or multiplication 13

Cells—study of 123

Centigrade, conversion to Fahrenheit degrees 128

Centrifugation, effect on extractable hormones 118

Century plant

Submerged cultivation method 21

Challenger, S.

Rooting cofactor method 76

Chamber, for continuous appHcation of hormones

and growth regulators to living cells 124
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 133

Page

Chandra, G. R.

Barley endosperm method. 48

(2-Chloroethyl) trimethylammonium chloride (CCC)

Wheat seedling method 69

3-Chloro-isopropyl-N-phenyl carbamate

Vegetative sprout method 68

Chlorophyll retention 43

Barley leaf method 43

Cocklebur leaf disk method 44

Dock leaf disk method 45

Wheat leaf senescence method 46

Christiansen, G. S.

Pea stem section method 30

Chromatography 94

Chrysanthemum

Stem-cutting method 79

Cis-cinnamic acid

Oat first internode method 29

Citrus

Citrus petal method 41

Expiant method 5

Nutrient mist method 75

Citrus petal method 41

Coating seeds 98

Cocklebur

Cocklebur leaf disk method 44

Cocklebur leaf disk method 44

Colchicine

Endosperm method 13

Coleus

Coleus petiole method 4

Coleus petiole method 4

Cone container method 73

Contamination—prevention of 123

Contamination of air with re^uhitors (See 'Air,

contamination of, with regulators") i)2

Continuous application of hormones and growth

regulators to living cells 124

Convenient method of dispersing relatively insoluble organic compounds in water 100

Conventional method of converting from Centigrade

to Fahrenheit degrees 128

Conventional method of converting from Fahrenheit

to Centigrade degrees 128

Corn

Dwarf, use in detecting and identifying gibberellins 111

Germinating seed method 91

Impregnated filter paper method 74

Cosolvent—for dispersing insoluble compounds in

water 100

Cotton

Attached petiole method 2

Expiant method 5

Impregnated filter paper method 74

Tomato hypocotyl method 34

Crabgrass

Impregnated filter paper method 74

Crafts, A. S.

Bean stem curvature method (absorption and

translocation) 11

Bean stem curvature method (effect of one compound on translocation of another) 47

Radioautograph method 87

Successive crop method 86

Crosby, D. G.

Oat first internode method 29

Cruzado, H. J.

Underground growth method 57

Cucumber

Agar block polar transport method 8

Disposable container method 90

Germinating seed method 91

Impregnated filter paper method 74

Lanolin paste method 56

Ovary culture method 58

Root growth method 84

Page

Culture vessel for small plants 107

Cutters for making segments 101

Cuttings (See "Root induction") . . 70

Cytokinins

Barley leaf method 43

Cocklebur leaf disk method 44

Leaf disk enlargement method 42

Soybean callus tissue method 19

Wheat leaf senescence method 46

Day, B. E.

Bean stem curvature method 11

Defoliation (See "Abscission") 1

De La Fuente, R. K.

Agar })lock preparation and use 9t)

Detection and identification of gibberellins with

dwarf corn 111

Detection of exogenous growth regulators in plants_ 82

Graft method 82

Detection of exogenous plant growth regulators in

animals 82

Bean stem method 82

Detection of regulators in water 84

Root growth method 84

Device that delivers measured amounts of a regulating

chemical 102

2,4-Dichlorophenoxyacetic acid

Agar slant method 72

Bean hypocotyl method 25

Bean leaf method 55

Bean stem curvature method 11

Bean stem method 82

Field method 68

Graft method 82

Ground tissue method 89

Impregnated filter paper method 74

Lanolin paste method 56

Radioautograph method 87

Radish cotyledon method - 31

Root growth method 84

Root primordia method 78

Seed method 84

Submerged cultivation method 21

Successive crop method 86

Underground growth method 57

Water culture method 67

Wheat coleoptile straight growth method 36

2,4-Dichlorophenoxyacetic acid, butyl ester

Disposable container method 90

2,4-Dichlorophenoxyacetic acid, isopropyl ester

Germinating seed method 91

2,4-Dichlorophenoxyacetic acid, methyl ester

Activated charcoal method 92

Diffusible hormones, collection from plant segments

or parts 96

Diffusion of hormones and regulators from multiple

segments into agar 96

2,4-Dinitrophenol

Tomato hypocotyl method 34

Direct measurement of volatility 102

Dispersion of compounds in water 100

Disposable container method 90

Dock leaf disk method, with 1 variation 45

Dormancy, prolongation of

Vegetative sprout method 68

Dowding, L.

Wheat coleoptile straight growth method, with 'A

variations 30

Downs, R. J.

Motion detector for recording plant growth 124

Droplet, simulated spray 109

Duckweed

Sterile nutrient method 66

Water culture method 67
134 AGRICULTURE HANDBOOK 336^ U.S. DEPARTMENT OF AGRICULTURE

Page

Dugger, W. M. Jr.

Bean stem curvature method (absorption and

translocation) 11

Bean stem curvature method (effect of one comcompound on translocation of another) 47

Durbin, R. D.

References to methods of maintaining constant

relative humidity 113

Dust carriers, for regulating chemicals 100

Dwarf corn, detection of hormones with 111

Easy method of impregnating dust carriers with

regulating chemicals 100

Edgerton, L. J.

Apple petiole method 1

Effect of one compound on the absorption and

translocation of another 47

Bean stem curvature method 47

Elongation

Lanolin method 13

Emsweller, S. L.

Lanolin paste method 81

Endive

Microculture method 15

Endosperm

Barley endosperm method 48

Endosperm method 13

Endosperm method 13

Endothal acid monohydrate

Attached petiole method 2

Enzyme production 48

Barley endosperm method 48

Equivalents 128

Exogenous growth regulators, detection of

Bean stem method 82

Graft method i__ 82

Expansion, cell

Cell enlargement 41

Expiant method, with 2 variations 5

Extraction of hormones and growth-regulating

substances from plants 118

Exudation of regulators from roots 49

Bean method 49

Root exudation method 52

Fahrenheit, conversion to Centigrade degrees 128

Ferrer, R.

Successive crop method S6

Field method 68

Fletcher, R. A.

Dock leaf disk method 45

Flowering 54

Biennial or perennial plant method 54

Form or growth modification 55

Bean leaf method 55

Lanolin paste method 56

Underground growth method 57

Formative effects of regulating chemicals 119

Fractionated lanolin as a carrier for growth-regulating compounds 104

Frank, P. A.

Technique for studying absorption and translocation of regulating substances by aquatic

plants 109

Fransson, Par 23

Oat coleoptile section method, with 2 variations 23

Freehand sections for studying cell responses to

regulators 120

Freeman, Greta D.

Bean method 49

Fruit abscission

Apple method 1

Apple petiole method 1

Peach and prune method 65

Page

Fruit growth 58

Grape method 62

Ovary culture method 58

Fruit set 60

Aerosol method 60

Grape method 62

Tomato method 62

Fruit size 64

Grape method 64

Fruit thinning 64

Apple method 64

Peach and prune method 65

Gaetjens, C. F.

Bean stem method 82

Gauch, H. G.

Bean stem curvature method (absorption and

translocation) H

Bean stem curvature method (effect of one compound on translocation of another) 47

Geranium

Stem-cutting method 79

Germinating seed method 91

Gibberellic acid A3

Bean second internode method 26

Biennial or perennial plant method 54

Dock leaf disk method 45

Grape method (fruit set) 62

Grape method (fruit size) 64

Leaf disk enlargement method 42

Oat first internode method. 29

Submerged cultivation method 21

Tomato hypocotyl method 34

Use of dwarf corn to detect and identify gibberellins

and gibberellin-like substances 111

Wheat coleoptile straight growth method 36

Young wheat coleoptile method 40

Gibberellin-like substances, detection of with dwarf

corn (See ''Gibberellic acid A3 and Gibberellins"). _ HI

Gibberellins

Barley endosperm method 48

Bean second internode method 26

Detection and identification of with dwarf corn__ 111

Dock leaf disk method 45

Oat first internode method 29

Submerged cultivation method 21

Tomato hypocotyl method ^4

Use of dwarf corn to detect and identify gibberellins and gibberellin-like substances 111

Ginkgo

Peach and prune method 65

Goldacre, P. L.

Tobacco pith method (micro) 22

Goldschmidt, E. E.

Citrus petal method 41

Extraction of hormones and growth-regulating

substances from plants 118

Goodhue, L. D.

Aerosol method 60

Gortner, W. A.

Split pea stem method 32

Graft method 82

Grant, V. O.

Root growth method 84

Grape

Grape method (fruit set) 62

Grape method (fruit size) 64

Grape method (fruit set) 62

Grape method (fruit size) 64

Ground tissue method 89

Growing plants 112

Growth acceleration

Bean second internode method 26

Cell division * 13
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 135

Page

Growth acceleration—Continued

Cell elongation 23

Cell enlargement 41

Measurement of root response 121

Terminal growth measurements 124

Underground growth method 57

Growth, measurement of 124

Growth modification 66

Field method ^ 68

Form or growth modification 55

Sterile nutrient method 66

Water culture method 67

Growth movement

Interferometer method of detecting 120

Motion detector method of measuring 124

Growth retardation 68

Vegetative sprout method 68

Wheat seedling method 69

Hagborg, W. A. F.

Plastic culture vessel C'plasticon") for small plants 107

Hamner, C. L.

Aerosol method 60

Hancock, C. R.

Wheiit coleoptile straight growth method 3()

Harley, C. P.

Apple method 64

Havis, J. R.

Field method 68

Hemphill, D. D.

Tomato method 62

Hess, CE.

Rooting cofactor method 76

Hickory

Peach and prune method 65

Highly sensitive test plants 112

Hildebrandt, A. C.

Microculture method 15

Single isolated cell method 18

Stem tissue culture method 20

Hill, G. P.

Aphid honeydew technique for study of sieve tube

content 117

Aphid stylet technique for studying regulating substances in sieve tubes 118

Hodgson, R. E.

Bean stem method 82

llodgscm, R. II.

Technique for studying absorption and translocation of regulating substances by aquatic

plants 109

Hoffman, M. B.

Apple petiole method 1

Holly

Stem-cutting method 79

Submerged cultivation method 21

Homan, D. N.

Application of growth substances with capillary

tubes 99

Honeydew technique for sieve tube transport of

regulators 117

Hormones, extraction of 118

Howard, B. H.

Rooting cofactor method 76

Howlett, F. S.

Tomato method 62

Humidity, methods of maintaining 113

Hunter, J. K.

Expiant method 5

6-Hydroxy-3-(2H)-pyridazinone (MH-30)

Underground growth method 57

Vegetative sprout method 68

Impregnated filter paper method 74

Page

rndole-;^-acetic acid (auxin)

Agar block polar transport method 8

Citrus petal method 41

Coleus petiole method 4

Expiant method 5

Lanolin paste method 56

Oat coleoptile section method 23

Oat first internode method 29

Pea stem section method 30

Root primordia method 78

Rooting cofactor method 76

Split pea stem method 30

Submerged cultivation method 21

Young wheat coleoptile method 40

3-Indolebutyric acid

Lanolin method 13

Lanolin paste method 81

Stem-cutting method 79

Underground growth method 57

índole compounds

Agar block polar transport method 8

CitrUs petal method 41

Coleus petiole method 4

Expiant method 5

Lanolin method. 13

Lanolin paste method (absorption and translocation) 56

Lanolin paste method ( seed formation) 81

Oat coleoptile section method 23

Oat first internode method 29

Pea stem section method 30

Root primordia method 78

Rooting cofactor method 76

Split pea stem method 32

Stem-cutting method 79

Submerged cultivation method 21

Underground growth method 57

Young wheat coleoptile method 40

Inhibitors (See "Retardants")

Injecting a regulating substance or hormone into a

single, living cell 104

Injecting substances into trees 105

Injection into living cells 104

Inselberg, Edgar

Radioautograph method 87

Insoluble compounds, dispersion of, in water 100

Interferometer method of detecting minute growth

movements 120

Introduction of chemicals directly into xylem

elements in stems of herbaceous plants _ _ 105

Introduction of regulators into roots 94

Introduction of regulators into xylem elements 105

Isolation of tissue 113

Isonicotinic hydrazide

Sterile nutrient method 66

Isopropyl N-(3-chlorophenyl) carbamate

Germinating seed method 91

Peach and prune method 65

Isopropyl N-phenylcarbamate

Agar slant method 72

Jackson, W. T.

Endosperm method 13

Jones, L. E.

Microculture method 15

Jones, R. L.

Agar slant method 72

Tomato hypocotyl method 34

Kefford, N. P.

Tobacco pith method (micro) 22

Keil, H. L.

Injecting substances into trees 105

Kende, H.

Barley leaf method 43
136 AGRICULTURE HANDBOOK 336^ U.S. DEPARTMENT OF AGRICULTURE

Page

Kent, Martha

Split pea stem method 32

Kinetin

Barley leaf method 43

Cocklebur leaf disk method 44

Leaf disk enlargement method 42

Soybean callus tissue method 19

Tobacco pith method (micro) 22

Wheat leaf senescence method 46

Young wheat coleoptile method __ 40

Kleuter, H. H.

Motion detector for recording plant growth, 124

Klotz, L. J.

Nutrient mist method 75

Knight, B. E. A.

Wheat seedling method 69

Kramer, P. J.

Technique for introducing regulators into roots___. 94

Krizek, D. T.

Motion detector for recording plant growth 124

Lacey, H. J.

Rooting cofactor method 76

Wheat coleoptile straight growth method, with

3 variations 36

Lam, S. L.

Agar block preparation and use 96

Lanolin as a carrier for regulating chemicals 106

Lanolin, fractionated 104

Lanolin method 13

Lanolin mixtures of regulators, measured amounts of _ 102

Lanolin paste method ( abscission) 7

Lanolin paste method (form or growth modification) 56

Lanolin paste method ( Procedure) 7

Lanolin paste method ( seed formation) 81

Lanolin ring, for localized treatment with regulators. 98

Lanolin-Tween 20 mixture as a carrier for growth

substances 8

Lantana

Stem-cutting method 79

Leach, CM.

Microisolation device 113

Leaf

Abscission of

Apple petiole method 1

Attached petiole method 2

Coleus petiole method 4

Expiant method 5

Absorption of regulators by 95

Application of regulators to 98

Simulated spray treatment of 109

Leaf disk enlargement method 42

Leaves

Malformed by regulators 119

Removal of treated areas of 123

Leopold, A. C.

Agar block preparation and use 96

Lettuce

Microculture method __ 15

Lily

Endosperm method 13

Linder, P. J.

Activated charcoal method 92

Bean method 49

Direct measurement of volatility 102

Ground tissue method 89

Introduction of chemicals directly into xylem

elements in stems of herbaceous plants 105

Root exudation method 52

Seed method 84

Liquid culture

Submerged cultivation method 21

Livingston, G. A.

Expiant method 5

Page

Localized areas (See "Application of regulators to

localized areas")

Loustalot, A. J.

Successive crop method __ S6

Luckwill, L. C.

Coleus petiole method 4

Dock leaf disk method 45

Tomato method _ 62

Lynch, R. S.

Explant method 5

Lyon, L. J.

Expiant method 5

McCalla, D. R.

Cocklebur leaf disk method _ __ 44

McCready, C. C.

Agar block preparation and use _ _ 96

McCune, S. B.

Grape method (fruit set) 62

Grape method (fruit size) 64

McMeans, J. L.

Expiant method __ __ 5

McRae, D. H.

Oat coleoptile section method, with 2 variations. _ 23

Maeda, Shizuko

Device that delivers measured amounts of regulating chemical 102

Maleic hydrazide

Underground growth method 57

Vegetative sprout method 68

Malformation (See "Formative effects of reguhiting

chemicals") Hi)

Maple

Nutrient mist method 75

Marigold

Microculture method 15

Single isolated cell method __ 18

Marsh, P. B.

Attached petiole method __ _ _ 2

Marth, P. C.

Aerosol method 60

Disposable container method 90

Field method 68

Lanolin paste method (abscission) 7

Lanolin paste method (form or growth modification) 56

Lanolin paste method (seed formation) 81

Peach and prune method 65

Stem-cutting method 79

Vegetative sprout method 68

Meader, E. M.

Aerosol method 60

Measurement of growth and growth responses 124

Measurement of root response 121

Measurements, terminal growth 124

Measures, equivalents, and abbreviations 128

Membranes, movements of regulators through _ _ 106

Metcalfe, T. P.

Agar slant method 72

Methods of making nutrient solutions 129

MH-30—6-hydroxy-3-(2H)-pyridazinone

Underground growth method 57

Vegetative sprout method 68

Microculture method 15

Microinjector for measured amounts of regulators. _ 102

Microisolation device 113

Micropipette puller 105

Micropump

Observation cham])er for continuous application

of hormones or growth regulators to living cells. _ 124

Miller, C. O.

Leaf disk enlargement method 42

Soybean callus tissue method 19

Mitchell, J. W.

Attached petiole method 22

Bean method 49
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 137

Page

Mitchell, J. W.—Continued

Bean second internode method 26

Bean stem curvature method (absorption and

translocation) 11

Bean stem curvature method (effect of one compound on translocation of another) 47

Bean stem method 82

Direct measurement of volatility 102

Disposable container method 90

Ground tissue method 89

Introduction of chemicals directly into xylem

elements in stems of herbaceous plants 105

Lanolin paste method ( abscission) 7

Lanolin paste method (form or growth modification) 56

Root exudation method 52

Stem-cutting method 79

Mobilization

Effect of one compound on translocation of another 47

Monselise, S. P.

Citrus petal method --_ 41

Extraction of hormones and growth-regulating

substances from plants 118

Montgillion, M. D.

Freehand sections for studying cell responses to

regulators 120

Moon, H. H.

Apple method 64

Morning-glory

Impregnated filter paper method 74

Motion detector^for recording plant growth 124

Movement of plants, measurement of 124

Movement of regulating substances through plant

membranes or tissues 106

Muir, W. H.

Single isolated cell method 18

Mulberry

Peach and prune method 65

Multiple segments, extraction of hormones from and

polar transport through 96

Mung bean

Rooting cofactor method 76

Murneek, A. E.

Tomato method 62

Mustard

Impregnated filter paper method 74

Seed method 84

Muzik, T. J.

Bean root method 10

Graft method 82

Underground growth method 57

Naphthalene acetamide

Apple petiole method 1

Naphthalene compounds

Apple method 64

Apple petiole method 1

Coleus petiole method 4

Cone container method 73

Lanolin paste method 7

Radish cotyledon method 31

Root primordia method 78

Stem-cutting method 79

Vegetative sprout method 68

Naphthyl maleimide

Attached petiole method 2

Nickell, L.G.

Sterile nutrient method oo

Submerged cultivation method 21

Nitsch, Colette

Oat first internode method 29

Nitsch, J. P.

Oat first internode method, with 1 variation zy

Ovary culture method 58

Nutrient mist method 75

Nutrient solutions, method of making ^ 1^9

Page

Oak

Nutrient mist method ___ 75

Oat

Agar slant method 72

Impregnated filter paper method 74

Oat coleoptile section method, with 2 variations. _ 23

Oat first internode method 29

Split pea stem method 32

Wheat coleoptile straight growth method 36

Oat coleoptile section method, with 2 variations 23

Oat first internode method, with 1 variation 29

Observation chamber for continuous application of

hormones or growth regulators to living cells. 124

Observing and measuring plants 117

Offord, H. R.

Water culture method 67

Onion

Vegetative sprout method 68

Osborne, Daphne J.

Cocklebur leaf disk method 44

Dock leaf disk method 45

Osmanthus

Stem-cutting method 79

Ovary culture method 58

Para-chlorophenoxyacetic acid

Grape method (fruit set) 62

Grape method (fruit size) 64

Lanolin paste method 81

Tomato method 62

3-(Para-chlorophenyl)-l,l-dimethylurea

Bean root method 10

Pavlychenko, T. K.

Cone container method 73

Pea

Impregnated filter paper method 74

Pea stem section method 30

Split pea stem method 32

Tomato hypocotyl method 34

Pea stem section method 30

Peach

Nutrient mist method 75

Peach and prune method 65

Peach and prune method 65

Peel, A. J.

Aphid honeydew technique for study of sieve tube

content 117

Aphid stylet technique for studying regulating i

substances in sieve tubes 118

Pegg, G. F.

Tomato hypocotyl method ^._ _ _ _ 34

Wheat coleoptile straight growth method, with

3 variations 36

Penetration of membranes and tissues 106

Penetration of soil by regulators 84

Seed method 84

Penicillin G

Sterile nutrient method 66

Petiole (See 'Abscission")

Phenoxy compounds

Activated charcoal method 92

Agar slant method 72

Bean hypocotyl method 25

Bean leaf method _ 55

Bean stem curvature method H

Bean stem method 82

Disposable container method 90

Field method 68

Germinating seed method 91

Graft method 82

Grape method (fruit set) ^2

Grape method (fruit size) 64

Ground tissue method 89

Impregnated filter paper method - - - 74

Lanolin paste method (form or growth modification) 56
138 AGRICULTURE HANDBOOK ;^SG, U.S. DEPARTMENT OF AGRICULTURE

Page

Phenoxy compounds—Continued

Lanolin paste method (seed formation) 81

Radioautograph method 87

Radish cotyledon method 31

Root growth method 84

Root primordia method 78

Seed method 84

Submerged cultivation methods 21

Successive crop method 86

Tomato method 62

Underground growth method 57

Water culture method 67

Wheat coleoptile straight growth method 36

Phenyl mercuric acetate

Attached petiole method -- 2

Phillips, I. D. J.

Tomato hypocotyl method 34

Phinney, B. O.

Use of dwarf corn to detect and identify gibberellins

and gibberellin-like substances 111

Phytokhiins (See "Kinetin")

Pigweed

Impregnated filter paper method 74

Pine

Nutrient mist method -_ 75

Pipette puller 105

Plant production

For testing regulators -- 114

Storage of seed for -- 116

Plants

For testing activity of regulating substances 112

For testing regulators -- 114

Growth regulators in

Graft method _ 82

Residual regulators in

Graft method _ 82

Plastic culture vessel ("plasticon") for small plants. _ 107

'Tlasticon," culture vessel 107

Polar transport

Agar block polar transport method 8

Pollitt, G. P.

Endosperm method 13

Pondweed, sago 109

Potassium phosphate buffer mixtures 129

Potato

Microculture method 15

Vegetative sprout method 68

Preparation of agar blocks 96

Preston, A. P.

Lanolin method 13

Preston, W. H., Jr.

Bean method 49

Lanolin paste method ( abscission) 7

Lanolin paste method (form or growth modification ) 56

Prince, V. E.

Peach and prune method 65

Privet

Stem-cutting method 79

Production of test plants 114

Prune

Peach and prune method 65

Radioactive growth substances, application of

Ground tissue method 89

Radioactive growth substances, extraction of

Extraction of hormones and growth-regulating

substances from plants 118

Radioautograph method, with 1 variation 87

Radioautographs

Of plant sections 121

Of sectioned plant material 121

Radioautographs of entire plants 78

Radioautograph method 87

Page

Radish

Cocklebur leaf disk method 44

Radish cotyledon method 31

Root primordia method 78

Radish cotyledon method 31

Rape

Agar slant method 72

Raynor, R. N.

Successive crop method __ _ 86

Ready, D.

Root growth method §4

Reeve, W.

Bean method 49

References to methods of maintaining constant

relative humidity 113

References to other physical methods 93

Regeimbal, L. 0.

Apple method 64

Regulating substances, extraction of, from plant

tissues 118

Relative humidity, how to maintain constant levels of _ 113

Removal of treated areas of leaves 123

Residue, detection of phenoxy compounds in plants

Graft method 82

Residue of regulators in soil S6

Successive crop method S6

Retardants

Lanolin paste method (form or growth modification) 56

Vegetative sprout method 68

Wheat seedling method 69

Rice

Impregnated filter paper method 74

Riker, A. J.

Microculture method 15

Single isolated cell method 18

Stem tissue culture method 20

Roan, C. C.

Device that delivers measured amounts of a

regulating chemical 102

Robbins, W. W.

Successive crop method S6

Roberts, R. H.

Tomato method 62

Robinson, Melba B.

Root exudation method 52

Root

Application of regulators with plastic culture vessel. 107

Cell for introducing regulators into roots 94

Measurement of response 121

Root culture method, with 1 variation 16

Root exudation method 52

Root growth 72

Agar slant method 72

Cone container method 73

Impregnated filter paper method 74

Nutrient mist method 75

Root growth method 84

Underground growth method 57

Root growth method 84

Root induction 76

Root primordia method 78

Rooting cofactor method 76

Stem cutting method 79

Root primordia method 78

Rooting (See ''Root induction") 76

Rooting cofactor method, with 1 variation 76

Rose

Submerged cultivation method 21

Rye

Impregnated filter paper method 74

Sato, H.

Endosperm method 13

Schneider, C. L.

Split pea stem method 32
METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES 139

Page

Schomer, H. A.

Aerosol method 60

Schultz, E. S.

Vegetative sprout method 68

Second internode method 26

Section cutters

Segment cutters 101

Sections

Freehand, for cell studies 120

Radioautographs of sectioned material 121

Slide chamber for study of living cells 123

Slide chamber for treating living cells 124

Seed

Application of regulators to 98

Seed formation 81

Lanolin paste method (seed formation) 81

Seed method 84

Seed storage 116

Segment cutters 101

Segments, extraction of hormones from and polar

transport through 8

Agar block preparation and use 96

Senescence

Barley leaf method 43

Cocklebur leaf disk method 44

Dock leaf disk method 45

Wheat leaf senescence method 46

Sensitive test plants 112

Separation of leaf areas treated with a regulating

chemical from the remaining untreated portions

of the leaf 123

Sexton, W. A.

Agar slant method 72

Short-cut method of converting from Centigrade

to Fahrenheit degrees 128

Sieve tubes—^honeydew technique for study of 117

Sieve tubes—^regulators in 118

Simulated spray-droplets 109

Single isolated cell method 18

Sirois, J. C.

Oat coleoptile section method, with 2 variations-_ 23

Slide chamber for study of living cells 123

Smith, O. E.

Expiant method 5

Soil, penetration of, by regulators

Seed method 84

Soil, residue of regulators in

Successive crop method 86

Solution of growth regulators in water

Convenient method of dispersing relatively insoluble organic compounds in water l^^^^

Some physical methods 87

Soybean

Soybean callus tissue method 19

Soybean callus tissue method 1^

Spencer, Roy, Jr.

Oat first internode method for auxins and gibberellins 29

Split pea stem method, with 1 variation 32

Spray, aerosol ^^

Spray-droplets, simulated -' 1 J|j|

Spray, preparation of 100

Sprout inhibition

Vegetative sprout method 68

Stem

Introduction of regulators into -- 105

Quantitative application of aqueous mixtures to- _ 97

Stem-cutting method -- 79

Stem tissue culture method 20

Sterile nutrient method ^^

Storage of seeds used as a plant source for the study

of hormones and regulators 116

Strawberry

O^^ary culture method 58

Page

Struckmeyer, B. E.

Stem tissue culture method 20

Tomato method 62

Stuart, N. W.

Lanolin paste method (seed formation) 81

Submerged cultivation method 21

Successive crop method 86

Sugarcane

Submerged cultivation method 21

Suggestions concerning the production of greenhouse

plants for experiments with regulating chemicals. _ 114

Sunflower

Impregnated filter paper method 74

Lanolin paste method (form or growth modification) 56

Microculture method 15

Supplementary information 128

Supplementary techniques 94

Surfactant

Convenient method of dispersing relatively insoluble organic compounds in water 100

Szabo, S. S.

Movement of regulating substances through membranes or tissues 106

Takematsu, Tetsuo

Radish cotyledon method 31

Root primordia method 78

Taylor, H. F.

Wheat seedling method 69

Technique for studying absorption and translocation

of regulating substances by aquatic plants 109

Techniques 94

Temperature conversions 128

Terminal growth measurements 124

Thimann, K. V.

Pea stem section method 30

Split pea stem method 32

Throne, J. A.

Bean hypocotyl method 25

Tissue culture

Endosperm method 13

Microculture method 15

Ovary culture method 58

Root culture method 16

Single isolated cell method 18

Soybean callus tissue method -___ 19

Stem tissue culture method 20

Submerged cultivation method 21

Tobacco pith method (micro) 22

Tissue, isolation of 113

Tissues, freehand sections for study of 120

Tissues, movement of regulators through 106

Tobacco

Microculture method 15

Ovary culture method 58

Single isolated cell method 18

Tobacco pith method (micro) 22

Tobacco pith method (micro) 22

Tomato

Aerosol method 60

Agar block polar transport method 8

Disposable container method 90

Graft method 82

Microculture method 15

Ovary culture method 58

Root culture method 16

Tomato hypocotyl method, with 1 variation 34

Tomato method 62

Tomato hypocotyl method, with 1 variation 34

Tomato method, with 1 variation 62

Toole, E. H.

Impregnated filter paper method 74

Seed storage 116
140 AGRICULTURE HANDBOOK 336, U.S

Page

Toole, E. H.—Continued

Storage of seeds used as a plant source for the

study of hormones and regulators 116

Toole, Vivian K.

Impregnated filter paper method 74

Translücation (See 'Absorption and translocation")

Agar block polar transport method 8

Beari root method 10

Bean stem curvature method 47

Effect of one compound on translocation of another 47

Ground tissue method 89

Of regulators by aquatic plants 109

Of regulators in sieve tubes 118

Radioautograph method 87

Translocation of radioactively tagged compounds 89

Ground tissue method 89

Radioautograph method 87

Translocation—radioautographs of sectioned plant

material 121

Translocation, regulators in xylem elements 105

Treated areas—removal of 123

Treating plants 94

Treatment (See "Application")

Trees, injection of 105

2,4,5-Trichlorophenoxyacetic acid

Field method 68

Trowell, G. F.

Nutrient mist method 75

Tukey, H. B.

Stem-cutting method -- 79

Tween 20-lanolin mixture as a carrier for growth

substances

Lanolin paste method ( Procedure) 7

Underground growth method, with 1 variation 57

Use of dwarf corn to detect and identify gibberellins

and gibberellin-like substances 111

Van der Weij, H. G.

Agar block polar transport method 8

Van Overbeek, J.

Wheat coleoptile straight growth method, with

3 variations 36

Varner, J. E.

Barley endosperm method 48

Vegetative sprout method -_ 68

Verner, Leif

Lanolin method 13

Volatility, direct measurement of . _ 102

Volatility of plant regulators -. 90

Activated charcoal method 92

Disposable container method - - 90

Germinating seed method 91

Vyvyan, M. C.

Nutrient mist method 75

Water, contamination of, with regulators

Root growth method 84

Water culture method 67

Water, detection of regulators in

Root growth method 84

Weatherley P.E.

Aphid honeydew technique for study of sieve tube

content - . _ 117

Aphid stylet technique for studying regulating

substances in sieve tubes 118

Weaver, R. J.

Grape method (fruit set) 62

Grape method (fruit size) - 64

Weintraub, R. L.

Bean hypocotyl method 25

Bean leaf method - 55

DEPARTMEVr'vOF AGRICULTURE

Wells, J. S.M"^

Stem-cutting method 79

Went, F. W.

Agar block polar transport method 8

Split pea stem method _ 32

Wester, R. E.

Field method 68

Lanolin paste method 81

Wheat

Cocklebur leaf disk method 44

Impregnated filter paper method 74

Wheat coleoptile straight growth method, with

3 variations 36

Wheat leaf senescence method 46

Wheat seedling method 69

Young wheat coleoptile method 40

Wheat coleoptile straight growth method, with

3 variations 36

Wheat leaf senescence method 46

Wheat seedling methods _ _ 69

White, P. R.

Root culture method, with 1 variation 1()

Stem tissue culture method 20

Whitworth, J. W.

Graft method 82

Underground growth method 57

Whyte, Pamela

Dock leaf disk method __ 45

Wiebe, H. H.

Technique for introducing regulators into roots 94

Wightman, F.

Segment cutters 101

Wheat coleoptile straight growth method, with

3 variations 36

Wilder, E. A.

Fractionated lanolin as a carrier for compounds 104

Williams, W. O.

Grape method (fruit set) 62

Grape method (fruit size) 64

Willow

Stem tissue culture method 20

Wittwer, S. H.

Tomato method 62

Worley, J. F.

Injecting a regulating substance or hormone into

a single, living cell 104

Radioautographs of sectioned plant material 121

Wright, S. T. C.

Wheat leaf senescence method 46

Young wheat coleoptile method 40

Wu, J. H.

Microculture methods_ . 15

Xylem elements, introduction of regulators into 105

Yamaguchi, Shogo

Radioautograph method

Yeatman, J. N.

Bean hypocotyl method _

Yew

Stem-cutting method

York, Greta D.

Bean second internode method

Young wheat coleoptile method

Zaerr, J. B.

Agar block polar transport method.

Agar block preparation and use

Zwar, J. A.

Tobacco pith method (micro)

87

25

79

27

40

8

96

22

-jîrU.S. Government Printing Office: 19C8 O—263-310