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PLANT GROWTH-SUBSTANCES

PLANT
GROWTH-SUBSTANCES

THEIR CHEMISTRY AND
APPLICATIONS, WITH SPECIAL
REFERENCE TO SYNTHETICS

by

HUGH NICOL

Kek kezet mos (One hand washes the other)

— Hungarian proverb

LEONARD HILL LIMITED

17 STRATFORD PLACE, LONDON, W.l

ENGLAND

1940

THE AUTHOR

was born in 1898; was until recently Assistant
Bacteriologist at Rothamsted Experimental
Station, Harpenden, England; is Hon. Corres-
ponding Editor of Chronica Botanica, the
international plant science news magazine; and
has written Microbes by the Million, a layman's
introduction to the spirit of microbiological and
other science.

PRINTED IN GREAT BRITAIN BY
EBENEZER BAYLIS AND SON, LTD., THE
TRINITY PRESS, WORCESTER, AND LONDON

Dedicated
in fellowship and with sympathy
to those who in their respective continents

work their way through college
or must carry out their studies while the more
fortunate relax

PREFACE TO THE SECOND EDITION

This will be found as
Chapter III

\

CONTENTS

Chap, Page

List of Illustrations ----- xi

Notes ------- xii

I A chapter for the layman - - - - i

II Also for the layman : How to use the commer-
cial growth-substances - - - - lo

III The scope of this book - - - - 19
Further reading ----- 23

IV Introductory scientific work on effects of the

synthetic growth-substances

Introduction ; the oat-coleoptile test ; epinasty - 24
Growth-promotion and growth-regulation: a

distinction ---___ 25

Description of methods of application - - 27

Initiation of roots ----- 29

Practical methods of using the substances - 30

Uptake from soil - - - - - 33

References A- - - - - - 34

References B- - - - - -35

V Recent work with cuttings

Applications :

Ordinary woody cuttings - * - 37

Forest trees ------ 40

Sub-tropical species - - - - 40

Non-woody plants - - - - 42

Substances :

Solvents ------ 42

Additions or reinforcements to solutions 43

Other substances ----- 43

vii

53802

Chap. Page

VI The treatment of seeds _ - - - 46

References C------49

VII The use of synthetic substances in various

horticultural and other operations
Grafting; rooting of grafted cuttings - - 51
Inhibition and stimulation of budding and

flowering; forcing - - - - - 53
Possible non-biological applications - " 57

References D------57

VIII What is a root? 59

IX Growth-substances from natural sources

Agricultural researches having a possible
bearing on growth-substances in soil and
manure -------62

Vitamins -------68

References E------69

X Some constituents of urine. Occurrence of
growth-substances (other than the auxins
and vitamins), and bodies related thereto, in
urine ------- yi

XI Chemistry in relation to growth

Some notions on concentrations - - - 77

Inhibition and stimulation - - - 77

Plant growth substances in tissue culture 81

Chemical constitution and phytological activity 82

References F------89

XII Classification and nomenclature of growth sub-
stances -------91

What a hormone is - - - - 92

Group nomenclature for plant growth-
substances ----- 95

References G - - - - - 97

viii

Chap.
XIII

XIV

Envoi

Synthesis of growth-substances

Introduction ______

(Mainly about indole compounds)
Positioning and nomenclature
Early work on indole compounds
Relation to animal physiology
Recent indole-acetic acid syntheses
Microbiological preparation - -
Higher indole-acids - _ _ _
Other compounds - _ _ _

Naphthalene compounds - - _ _
Higher aryl compounds - _ _ _

An important hint - _ _ _ _

Note on the auxins - _ _ _ _

Vitamins ---__,._

References H ----- -

Identification of growth-substances, and some

substances related to them _ _ _

Phenyl derivatives - - _ _

Indole derivatives - - - - _

Indole-acetic acid - - _ _

The urorosein test - _ _ _

Methods of isolation - - - _

Various - - _ _ _

Creatine and creatinine - - - _

Oxalacetic acid - - - - _

Vitamins ---___

Went's pea test --____

References J - - -

XV Tabular index of substances

Author Index - _ _

Index of principal subjects

Index of important plants

ix

Page

98

100
103
104

105
107
108
109
III
112
112

113
114

116

120

121

123
123
126
128
128
128
129
129

136

137

143
146

148

AS

ILLUSTRATIONS

Facing
Figure i Page

Natural aerial roots, forty feet long, of a tropical vine
growing in the Missouri Botanical Garden, St. Louis,
U.S.A. 30

Figure 2

Tomato plants with i8-day-old roots on stems and
leaves. --------30

Figure 3

Collar of white roots on the upper part of the stem

of Kalanchoe daigremontiana - - - - 30

Figure 4

Cuttings of American holly, showing roots produced

five weeks after treatment with indole-acetic acid - 31

Figure 5

Cuttings of American holly, showing roots induced

by treatment with naphthyl-acetic acid - - - 31

Figure 6

Sketch indicating no, medium, and maximum
response of split stems in Went's pea test - - 131

FORMULA DIAGRAMS

Conjugations of phenol ------ 72

Analogues of indole ___--- 82

Auxin-a, indole-acetic acid, creatinine, and creatine - 88
Indole and some derivatives - - - - 101-103

The auxins -------- 113

Vitamins Bi and C « - - - - 115-116

NOTES

This book is based upon a series of articles which appeared
in The Manufacturing Chemist (Messrs. Leonard Hill
Limited, London) in June, July, and August, 1937 (Vol. 8,
pp. 175-180, 211-216, 254-257).

References to literature not cited in full in the text or in the
list at the end of the Chapter in which they are mentioned, are
distinguished by a capital letter indicating the list in which
they will be found. This has been done to avoid duplication,
while ensuring that each list bears broadly on one subject.

The quotations ascribed to Beaume are taken from the
English translation, by John Aikin, of the second edition of
Beaume's book on Chemistry. (Warrington, 1778).

xu

CHAPTER 1

A CHAPTER FOR THE LAYMAN

Vegetables are organised bodies, which extract from the
earth the juices proper to their nature. Vegetable substances
are much more compound than mineral. Their analysis is
consequently more difficult: certain principles, of too great
tenuity or volatility, escape us entirely. — Beaum^.

Thoughtful people have always been attracted by the
patterned development of a plant. Practitioners of several
branches of science allied to agriculture now find considerable
interest in the topic of growth substances.

The subject is not really new, as its beginnings can be
traced back to some observations of Charles Darwin, but it
has recently become popular with remarkable rapidity. This
onset of interest is possibly due less to an appreciation of the
importance of the subject of growth-regulation than to a
widening knowledge of the extraordinary results which can
be achieved by the use of some of the growth substances
lately prepared artificially. Botanists have long known that,
with certain plants at least, it is possible to induce roots to
grow from the middle of the stem of a plant, but that it could
not be done without some trouble. It is now easy to make
roots sprout on stems of almost any soft-wooded plant merely
by painting them with something out of a bottle. It is not
hard to understand why many laymen, as well as botanists,
have had their imagination quickened.

This initiation of rooting on stems and leaves is something
more than a laboratory trick, because the same substances
that produce roots on leaves will cause roots to appear on
cuttings more rapidly than they will appear in the ordinary
way after putting into compost. To the horticulturist, there-
fore, the recent artificial preparations of growth-regulating

1

Plant Qrowth'Suhstances

substances represent a real gain of immediate practical
importance. Moreover, the possibilities have not been fully-
explored, and it is confidently expected that the new growth-
substances will be useful in grafting and in further types of
propagative work in the nursery garden.

The substances are sometimes called growth-promoting
substances, but that name is not sound as a general rule. It
often happens that a plant that has been treated with one of
these substances produces a whiskery out-growth of roots
over most of its stem and a great part of its leaves, but becomes
dwarfed in comparison with an untreated plant. Dr. H. L.,
Pearse, writing in the January, 1937, issue of the Journal of
Pomology and Horticulture, has pointed out that the amount
of plant-capital is not altered by the application of an artificial
growth-substance such as phenyl-acetic acid, but that the
distribution of the plant's capital is thereby altered. In other
words, the more aerial root, the less stem and leaf, the sum
of matter belonging to the plant being apparently unchanged.
It is, therefore, better to call these substances growth-regulat-
ing substances, or, more simply, growth-substances. This
book is devoted to an exposition of the chemistry of plant
growth-substances; that is, substances, other than fertilizers
and nutrients, that can regulate the growth of plants.

The non-chemical reader who glances at the later portions
of this book and is affrighted by the array of formulae, and
the words, often of more than Johnsonian ponderosity and
length, with their uncouth-looking jumble of syllables
hyphenated with figures and Greek letters, may think that it
is something like English, but what does it mean ?

It means, in brief, the story of an attempt to adapt the
forces of nature whereby plants grow, not only in size, but in
the increase of their several parts — roots and leaves, stems and
fruit. It is the summary of an attempt to set our accumulated
knowledge to a new field of endeavour : the better control of
plant growth. Firstly, however, it has been necessary to under-

2

/

For the Layman

stand how plants grow, and to know what substances affect
their different kinds of growth.

The understanding of substances belongs to the science of
chemistry, which has built up for itself that queer-looking
but exact-meaning terminology which I have already men-
tioned. The search for answers to the questions that centre
around the "how" of plant growth belongs to the science of
phytology, or plant physiology, and, in what follows, I have
written practically nothing under that head, because I am not
a plant physiologist, and don't understand the subject suffi-
ciently to discuss it in detail. However, some hints as to how
plants grow can be gained by observing the effect of different
substances on plants. The preparation and study of such sub-
stances is the job of the chemist.

The substances that can be tested on any one kind of plant
fall into two groups: those which the plant can't produce
for itself, and those which it can.

Plants cannot produce for themselves the mineral foods
they need, and which they normally obtain from the soil ; these
are such things as phosphates, nitrates, sihca, lime, potassium
salts, and so on. They also require carbon and water, which
they obtain from the air and the soil, and a plant growing
from seed has a reserve of food in the seed to start life with.

Given these things, the plant grows. It gets bigger, but it
also becomes differentiated, that is, it acquires parts, such as
root, stem, and leaf. How does a seedHng "know" that it
must form a number of parts, all of them necessary, and of
several different types, and how does it arrange them into the
pattern which is recognizable by us, so that we can say "that
is a willow, and that is a tomato plant" ?

There is possibly a clue in the activities of some of the sub-
stances which the plant produces for itself. A large number of
plant-products are structure-materials and reserve foodstuffs,
together forming the framework of the plant and its kitchen
and pantry. Other substances seem to have a directive or
husbanding effect. These latter are the natural growth-
regulating substances. They are also called hormones, or
"chemical messengers".

3

Plant Qrowth'Suhstances

Professor Boysen Jensen, on pages 115-6 of his book on
growth-substances, has said: "Numerous experiments have
shown that without growth-substances, growth of the shoots
of higher plants cannot take place. . . . The increase in cell-
wall boundaries is only one manifestation of the fundamental
ability of an organism to build itself out of the materials of its
environment."

As Boysen Jensen reminds us, there are several modes or
kinds of growth. Growth in length or height may be due to
the lengthening of a fixed number of cells, without new cells
being formed: that is rather like a stretching, only, the force
comes from inside, not outside. Growth in length or height
may also take place by an increase in number of cells, so that
the growing-point is pushed out to make more room for the
older tissue. There is also a mode of growth which starts the
formation of new sorts of tissue, as when a leaf-bud begins to
form. This last kind of growth is due to cell-differentiation.

The effects of growth-substances that have been most
intimately studied are those that arise from the first mode,
namely, the increase in length of a fixed number of cells.
Young seedling oats have become a favourite material for
demonstrating the existence of this type of growth. This is
partly because it is relatively simple to see what is happening
when a certain part (the coleoptile, or first leaf-sheath) of a
young seedling oat-plant that has had its tip cut off, has been
treated with a trace of the substance that is being investigated.
The substance is applied to one side of the cut surface of the
stump (the tip, which contains the plant's own shoot-growth
hormone, is thrown away). The applied substance causes the
cells on the treated side to grow longer (provided, of course,
that it is active as a growth-substance). The result is that
the leaf-sheath bends to one side in order to relieve the strain
caused by the unequal growth. The bending is easily visible.

This is the essence of the oat-test. Oat seedlings are em-
ployed because they are cheap, easily grown and handled,
are responsive, and otherwise convenient.

The effect of light in causing bending of stems is known to

4

For the Layman

everybody who has grown plants inside a window-space. For
some years, plant physiologists sought to explain the effect
of the light, before they suspected the existence of growth-
substances. As soon as it became reasonably clear that there
was a connexion between light and the elaboration of some
growth-material by the plant, efforts were made to isolate in
a pure state the substance causing the cell-elongation.
Microscopic studies had shown that the observed bending
was an expression of greater growth in length of some of the
cells on one side of the stem.

The story of the isolation of highly-active growth-sub-
stances is an interesting one. The amount of growth-
substances in the tips of seedlings was too small to offer any
hope of obtaining much of it from that source. After it was
noticed that urine had a marked effect in making young
plant-cells become longer, efforts were made to obtain the
active substance (whatever it was) from urine, which could be
obtained easily in quantity. The effort was successful, and
enough of a substance, at first called auxin, was obtained
from urine, to enable its chemical structure to be ascertained.
It was found that there were in urine two rather similar
growth-substances ; the one first discovered is now called auxin-
a, the other is known as auxin-b.

In undertaking this chemical investigation of urine, the
researchers did not know whether the result of their labours
would after all be the finding of a substance identical with
or different from the growth-hormone of oat seedlings. Even
now it is not known with certainty — only with a very high
degree of probabiHty — that an auxin is in fact the natural
growth-substance of the seedling oat tip. The presence of
auxins in urine propounded the problem as to what they were
doing there. It was found that both auxins could be isolated
from some vegetable oils, and from malt, so their presence in
urine can be accounted for by their being included in our
vegetable foods.

Although the formulae of both auxins are known, they have
not yet been prepared by purely artificial methods in the

5

Plant Qrowth'Suhstances

laboratory. The synthesis of the auxins is a difficuh problem
in chemistry.

At this point I may make a digression for the benefit of
those whose ideas of chemistry are based on the reading of
popular novels involving a secret chemical formula, In
chemistry, a formula is not a recipe, but is an exposition of
the number and kinds of atoms comprising a substance, show-
ing also the way in which the atoms are arranged in relation
to one another. Before a natural substance can be imitated in
the laboratory, it is necessary to know the exact "configura-
tion" of the molecule of the substance; knowledge of the
formula, however, is far from being all that is necessary to
enable even an expert chemist to make the substance on the
spot. Those who are interested can find the formulae of the
auxins on page 113.^

In urine there is often a third growth-substance, the
existence of which was discovered in a curious way by Fritz
Kogl and others, of Utrecht. They found that it was not
always possible to extract auxin-a from the mixed urines of a
hospital. They therefore concentrated their attention on an
"especially appreciated source" — the urine of an eighteen-
year-old youth, which had been found by the oat test to be
four or five times as active as ordinary urines. An isolation
procedure applied to this urine led to the production of a
very small quantity of crystals of a substance quite different
from either of the auxins, though practically as eflfective in
causing cell-elongation in seedling oats. The nature of this
substance was a puzzle, but some hints were available, and
finally the "new" substance was definitely established to be
indole-acetic acid, which was already well known to chemists,
but was not then known to be a growth-regulating substance.
This shows that if you enter hospital, you never know what
purpose you are likely to serve !

Later, indole-acetic acid was found to be present in very

^The formulae of vitamins Bj and C can also be found on pages 115 and 116.
These substances are now made artificially, and are on sale.

6

For the Layman

minute amount in yeast, from which it was laboriously
isolated after more than a hundred and fifty separate treat-
ments had been made upon three hundredweights of yeast
in portions of a kilogram (about two pounds) at a time.

The story of these, and other, discoveries has been racily
told by Dr. Kogl and his collaborators in the"Zeitschrift fiir
physiologische Chemie," vol. 228, of 1934. Regarding the
role of chance in the sequence of isolation of the auxins and
of indole-acetic acid, I cannot do better than give the words
(translated, of course) of Dr. Kogl and Dr. Kostermans from
page 116:

"It was a lucky accident that from urine, which contained
both the growth-substances [i.e., auxin, and indole-acetic
acid], we isolated the "real" auxin first. If our attempts to
isolate the active substance had first of all led to the finding
of indole-acetic acid, probably no one would have taken the
trouble to separate the active substance [auxin] existing in
the residual liquors. Having acquired the knowledge that
yeast and other lowly plants form a substance capable of
producing cell-elongation, and identical with indole-acetic
acid, one would probably have been content with that, and
any variation in behaviour displayed by growth-substances
in cereals [oats, especially] would have been put down to
the existence of substances only incidentally present. The
existence of auxins a and b would, most likely, have re-
mained unsuspected for a considerable time."
Even now, it is not known why indole-acetic acid and the
auxins behave so similarly when applied to the decapitated
seedling oat and in some other tests of growth-promotion and
regulation. It is usually assumed that substances that display
similar physiological behaviour are at least related chemically,
but between the auxins and indole-acetic acid there is no
obvious similarity whatever. All three substances occur in
urine because they are derived from articles of diet, but the
origin of urinary indole-acetic acid is quite different from the
origins of the auxins.

An important practical consequence arose from the dis-

7

Plant Qrowth'Suhstances

covery that indole-acetic acid had growth-regulating pro-
perties. Whilst the auxins were new to science, indole-acetic
acid, and substances chemically similar to it, have been
known for a long time. A number of ways of artificially pre-
paring indole-acetic acid and its relatives were already known
before Kogl found the acid to be a growth-substance. It did
not take long to test the growth-regulating properties of it and
of other substances like it. It was found that a solution of
indole-acetic acid, for example, when painted on the stem of
a tomato or other soft-stemmed plant caused roots to grow
on the stem. From that it was but a step to the attempt to
quicken root-formation on cuttings. It was found that
indole-acetic acid did considerably hasten the appearance of
roots on cuttings put into compost. Hence, it and its relatives
are already of practical importance as an aid to horticul-
turists and gardeners. A number of the artificial growth-
substances, including indole-acetic acid itself, are now on sale
for horticultural purposes.

If you are a business man or woman, you will perhaps think
that here is a field to exploit, by selling manures to which
traces of the growth-substances have been added, while you
claim remarkable properties of growth-promotion for "Smith's
Auxonic Fertilizer". The idea has already occurred to a
number of people, but I think that to translate it into practice
without more knowledge of the modes of action of growth-
substances would be to start at the wrong end. In my view,
it is more important to investigate the old, well-tried, organic
manures of the farm, and to see whether they contain the
growth-substances, and in what proportions. If growth-
substances are present in organic manures, a knowledge of
their presence and distribution may help us to understand
more about soil fertility, and possibly about the healthfulness
of foods. Such research work on organic manures has hardly
yet begun, but a beginning has been made in America.

Some years ago, the foundation of an understanding of the
rotting-down of organic materials was laid at Rothamsted,
during an investigation of the decomposition of straw. One
practical result was the production of the so-called artificial

8

For the Layman

farmyard manure — a compost of straw. This artificial farm-
yard manure is not artificial in the sense that many purely
inorganic fertilizers are. It derives nothing from large live-
stock, but is nevertheless a biological product, because many
forms of living micro-organisms take part in the rotting.
Artificial farmyard manure ("Adco" manure) has been shown
to be the equal of farmyard manure as far as crop-producing
capacity is concerned. It looks, therefore, as though the
biological contribution to fertility is much the same, whether
it is supplied by a few large animals or by a multitude of small
forms of life.

It is too much to say that the auxins in plants, or growth-
substances in general, are the very stuff of life, but the con-
nexion between plant-growth and the natural growth-sub-
stances is so intimate that it is tempting to suggest that since
the discovery of the modes of action of growth-substances we
are a step nearer understanding the mechanism of life itself.

One paragraph more. Once Kogl had found the crystals
that turned out to be indole-acetic acid, it was easy, by means
of a few comparatively simple chemical tests, to say what the
substance was. This was because a great deal was already
known about the chemical relationships of indole-acetic acid.
The chemistry of indole-acetic acid and of its relatives had
been worked out over many years by researchers who were
mainly interested in pure, or theoretical — as distinct from
applied — chemistry; that is, many of these chemists were
interested in chemistry for its own sake. They suspected little
of the future importance of their work, but they were broaden-
ing knowledge, though, at the time their work was done,
a good deal of it must have seemed to have no practical value
except, possibly, as an exercise in chemistry. We benefit
to-day from the results of the work of these people who laid
the foundations of chemistry many years ago. A similar
remark would be true of the work of scientists in other fields
besides chemistry. That is the reason why I selected as the
keynote of this book the proverb that appears on the title-
page.

CHAPTER II

ALSO FOR THE LAYMAN

How to use the Commercial Growth- Substances
in Quickening the Rooting of Cuttings

Bees burnt to ashes, and a ly made with the ashes, trimly
decks a bald head, being washed with it. — [Unconfirmed
quotation from N. Culpeper's Herbal (1653); se non k vero,
i molto ben trovato.^

How to Buy

The best-known artificial growth-substance is that variously
called indole-acetic acid and indolyl-acetic acid, with or
without a hyphen and with or without the prefix 3- or p
(pronounced beta). It is still sometimes called heteroauxin.
The prefixes serve amongst chemists to distinguish difi^erent
relatives in a chemical family — all the members of which in
the present case consist of the same number of atoms of
carbon, hydrogen, oxygen, and nitrogen, but arranged in
certain systematically-different ways. Such relatives do not
necessarily have a similar action on plants. The prefix 3-
means here the same as p. Whenever the prefix is omitted, as it
often is in this book, a 3- acid is meant, for little is known
about the others, such as the 5- substances. You would be
quite safe in asking for simply indole-acetic acid, because only
the 3- acid — the one known to be eflPective in causing rooting
on cuttings — is available commercially.

Possibly slightly more powerful than indole-acetic acid
as far as root-production on cuttings is concerned, are two
other substances. One of them is 3-indole-butyric acid,
which is chemically very like indole-acetic acid, being just a
little more complex. When speaking of, or placing an order
for, this substance, you may again omit any prefix, and ask

10

Rooting of Cuttings

simply for indole-butyric acid. The y in this word is short,
like i in pin — not like y in why ; and the a of indole is always
short, as in doll. The other substance is derived from naph-
thalene, and is known as a-naphthyl-acetic acid (alpha-
naphthyl-acetic acid, the y this time being long, as in why).
There is also a beta-naphthyl-acetic acid on the market.
The beta acid is distinctly less useful for most horticultural
purposes, so if you decide to obtain a naphthyl-acetic acid for
ordinary gardening purposes, you should specify alpha-
naphthyl-acetic acid (or, alphd-naphthalene-acetic acid), not,
for the present, omitting the alpha part of the name.

All these substances are very expensive, as judged by
ordinary standards, and the indole compounds, at least, seem
likely to remain expensive for a long time ; no cheap method
of producing them is in sight. Indole, which may be regarded
as the parent of the indole-acetic (indolyl-acetic) group of
substances, is used in small quantities by perfumers, and
during the Great War it attained the price of ;^ioo ($500) per
pound. This seemed to me to be a record for a commercial
chemical. Indole, however, is not a material likely to be
bought by the general public even at its present reduced
price; yet, until a short while ago; indole-acetic acid was on
general sale at a price of about ;^220 ($1,100) per pound. It is
not very much below that price now, and indole-butyric acid
is even dearer.

You may think that at such prices the artificial growth-
substances are impossibly beyond our reach, and, indeed,
you will see that such prices make chemicals worth literally
more than their weight in gold. However, you do not need
more than minute quantities, and, since nobody expects to
sell indole-acetic acid by the pound, the usual commercial
unit of weight for retail sale is the gram : this is a metric unit
equal to about a thirtieth of an ounce.

Current prices for some of the better-known pure sub-
s .mces are given on next page from the comprehensive price-
lisi" of the British Drug Houses Limited, of Graham Street,
C'i 7 Road, London, N.I : ^.^^

LIEi^^ARYJ at

Plant Qrowth'Suhstances

3-indole-acetic acid ... 7s. gd. per one-gram tube

3-indole-butyric acid ... I OS. od. ,, ,,

a- or p-naphthyl-acetic

acid ... ... ... 6s. 8d. „ ,,

Since a usual strength for a solution of these substances is
5 parts, more or less, of the pure substance in 100,000 parts
of water, one gram will make 20,000 grams (20 litres) or
about sixteen quarts of usable solutions, the cost of a quart
thus being pennies only. Small users can buy still smaller
quantities: at least some of the substances are sold in tubes
containing one-tenth of a gram, and costing about a shilling
each.

The Choice of Substance

There is not much difference between the efficiency of
indole-acetic, indole-butyric, and a-naphthyl-acetic acids.
No one of them is "best" in all circumstances or for all plants,
and no definite figures in support of this or that substance
can be given. Dr. M. A. H. Tincker in his 1938 paper, of
which the reference is given on p. 36, reported that both
indole-acetic acid and a-naphthyl-acetic acid brought about
the formation of roots on about 55 per cent, of the species he
tried, and on about 40 per cent, of those species known to be
difficult to root in the ordinary way. These figures will give
you an idea of your chance of success in bringing about good
rooting of cuttings : with any one species the synthetic growth-
substances are about as likely as not to be effective. Even
when they bring about rooting of a given species, it may
happen that not every cutting of a batch will root. The
synthetic growth-substances will not work miracles. What
they do is to hasten root formation on some species (after
planting out into rooting compost) or to increase the length or
number of roots on other species.

Published work with indole-butyric acid has not taken
the form that would enable calculations to be made com-
parable with the above, but it seems to be about as effective as
the other two synthetics. There is, however, a growing im-

12

Rooting of Cuttings

prejsion that the roots produced on cuttings after a treatment
with indole-butyric acid are more desirable in type than those
produced by the naphthyl compound at least ; in other words,
indole-butyric acid scores on quality.

There is one artificial growth-substance which is almost
ridiculously cheap, costing only a few pennies an ounce.
This is p\ienyl-acetic acid. It is, however, much less potent
than the o'-.hers that have been mentioned. It often requires
to be used at about ten times the strength at which the
other substances are used, and even so it produces good root-
ing with relatively few species. In Dr. Tincker's experiments
only about 1 5 per cent, of species rooted after treatment with
phenyl-acetic acid solutions, and as those which rooted were
mostly species easy to root, the figure of 15 per cent, compared
with about 50 per cent, for the other substances probably
flatters phenyl-acetic acid. It has its special advantages,
nevertheless, and there is no reason why it should not be
tried. It has a delightful rose smell, and if you give it up as a
growth-substance you may like to use it to freshen pot-pourri
or for other fragrant purposes.

None of these substances, which are called acids for
reasons of theoretical chemistry, is corrosive in the ordinary
sense. They are all powders, practically bland. They dissolve
only to a minute extent in water, though phenyl-acetic acid is
soluble enough in water to give a rose odour to a clear (filtered)
solution. Compounds of the acids with potash and other bases
{e.g., soda, lime) are called salts; they have been used by
experimenters, because some of them have the advantage of
being appreciably soluble in water. These useful salts are not
widely available in the solid form.

How to Prepare

Only very low concentrations of the chosen substance are
necessary. As it is difficult to make a very dilute solution
straight away, the best way is to make, say, about a one per cent,
"stock" solution of the substance in water, by dissolving a
known weight of the substance in a hundred times its weight
of water. The dearer substances (that is, all of them except

13

Plant Qrowth'Suhstances

phenyl-acetic acid) are usually supplied in tubes contaidng
one gram (28-4 grams =one ounce avoirdupois). A measure
to contain a hundred grams (millilitres) of water can be bought
through a druggist, but three and a half ounces of wa:er will
be sufficient approximation to a hundred millilitres.

If the acid, and not a salt, has been obtained, it will be a
good plan to dissolve the acid first of all in about five or ten
times its weight of alcohol or methylated spirit, which is,
however, of no use for the salts. The volume of the solution
is then made up with water to the final volume desired for a
one per cent, solution.

The stock solution, when not required, should be stored in
a cool, dark place in a bottle of dark-coloured glass, because
light tends to decompose the substances.

From the stock solution a weaker solution can be made
freshly as needed, by adding one part of the stock solution to as
many parts of water as are desired. Dilutions of about one
part of the original substance in one thousand, ten thousand,
forty thousand, a hundred thousand, etc., can be made by
adding one part of the stock solution to 9, 100, 400, 1,000
parts of water respectively. These final dilutions will contain
respectively 100, 10, 2-5 and i part of substance per 100,000 of
water. No alcohol or other substance except water is required
to dilute the stock solution. Tap water is suitable. Any
unused weak solution should not be kept, as it will probably
become valueless on standing long.

A smaller measure (to contain 10 millilitres) will be found
a convenience both for measuring out the stock solution and
for dosing the plants.

To avoid the trouble of making up solutions from the solid,
you may also use commercial preparations. These consist of
solutions of a carefully-chosen and efficacious synthetic
growth-substance already made up into a stock solution. For
use, all you have to do is to follow the simple directions. The
size of the bottle you buy is not necessarily an indication of
what you are getting for your money, since what may seem
dear may be merely more concentrated.

To the small gardener an advantage of buying one of the

14

Rooting of Cuttings

reputable commercial preparations is that with each bottle is
given a list indicating accurately the special requirements of
cuttings of many named plants.

Stock I per cent, solutions of named synthetics can also be
bought. These are intended for the buyer who for experi-
mental or other reasons wishes to use a particular substance
and / or to have it at a known strength.

How to Use

In addition to solutions in water, pastes or salves consisting
of the substances mixed with lanolin (wool-fat) can also be
obtained or prepared. These are chiefly useful for local
applications, such as smearing on definite areas of stems or
wounds, and are of little practical value for inducing rooting
on cuttings, which is at present the main horticultural utility
of commercial growth-substances.

As the whole subject of utilization of growth-substances is
new, no definite rules for their use have yet emerged, and to
that extent every user must for a time be an experimenter.

The following indicates the general lines of procedure for
treatment of cuttings, starting with pure (solid) substances. It
may be varied to suit inclinations, and as experience is gained.

For best results, cuttings should bear some leaves.

Divide the batch of cuttings to be treated into three lots.
From the stock solution, if that is of indole-acetic acid,
prepare three solutions containing, say, about 5, 10 and 15
parts of acid per 100,000 of water. That is, to 10 millilitres of
the stock solution add 2,000 millilitres (roughly two quarts) of
water, to another 10 millilitres of the stock add 1,000 millilitres
(one quart), and to another 10 millilitres add 670 millilitres
of water (half a quart, and a bit). (Another way would be to
get your pharmacist to make up solutions for you, reminding
him that a gallon of water weighs 70,000 grains). Great
accuracy is in no case essential.

Plunge the bases of one-third of the cuttings into each
of the solutions in ordinary daylight at ordinary temperatures.
At the end of 12 or 16 hours withdraw one-third of the
cuttings from each vessel, and plant them out in potting

15

Plant Qrowth'Suhstances

compost, suitably labelled. At the end of 24 hours withdraw
half the remainder and plant them out, and withdraw the last
lot at from 36 to 72 hours after the beginning, and plant out.
You will have thus nine lots of cuttings, which have been
subjected to three concentrations for three periods of time.
Concentration of the growth-substance and length of exposure
thereto have often to be narrowly judged, but there is a
certain latitude — just as in exposing a photographic negative
— and at least one lot of your cuttings should have received
something like the proper treatment from the suggested
ranges of concentrations and times. It would add to the
interest if you planted out a few cuttings treated with water
only.

If phenyl-acetic acid is used, all the above-mentioned
concentrations might be increased about ten times. That is,
the amounts of water mentioned should be added to say 100
millilitres (3^ ounces) of stock solution, instead of to 10 milli-
litres.

After removal from the solutions the cuttings look just the
same as before. All that has happened is that transpiration
of water through the leaves has occurred, and some of the
solution has been sucked up. Visible roots will not be pro-
duced before at least a couple of weeks in compost. The time
will depend upon the season of the year, as well as on the kind
of plant.

Experience has shown that immersion for 12 or 24 hours in
a solution of about 5 to 10 parts of growth-substance per
100,000 of water is about right for several kinds of plants, and
this may serve as a rough guide if you are not willing to experi-
ment with several concentrations and times as I have sug-
gested. Immersion in a relatively strong solution for a short
time (say 10 parts per 100,000 overnight) seems to be usually
better than immersion for a longer time in a weaker solution.
On the other hand, an error in judgment of the concentration of
a "strong" solution may lead to the preparation of a solution
that is poisonous to the cuttings. Be prepared for disappoint-
ments ; for death of cuttings left too long in the solutions, or in
too strong a solution; and for no results, from insufficient

16

Rooting of Cuttings

treatments, and from treatment at an unsuitable time of year,
or of species which will not respond anyhow.

Some words by Richard Bradley, a famous eighteenth-
century gardener, can fittingly be quoted here:

"And again, the proportions of every Ingredient ought to
be as reasonably consider'd, and not to double our Quantities
over hastily, because a moderate Proportion has already begun
to shew its good Effects ; like some unthinking People, who
believe, that the more Physick they take, the more healthful
they shall be, because they have once been reliev'd from
Sickness, by a small dose of it : But a few Observations to an
ingenious Man, who loves a Garden, will soon bring him to
rights in these Matters."

An Excellent Compost

You may like to know of the two standardized composts
evolved at the John Innes Horticultural Institution, London,
S.W.17. One is for seed sowing and pricking off, the other for
potting. These unconventional composts have given superb
results, and though the sowing compost (I) does not im-
mediately concern my subject, I give it for the sake of com-
pleteness. The proportions are as recorded by W. J. C.
Lawrence and J. Newell, of the Institution, in Scientific
Horticulture, 1936, Vol. 4, 165-177.

I II

(potting)

Parts

Parts

Loam

••• 5

5

Moss-peat ...

• • • A.

4

Sand

2

I

Ballast

• • •

I

It is very important to observe the order of operations
exactly, as follows: I. The loam and sand are partially steril-
ized separately, and the unsterilized moss-peat, super-phos-
phate and chalk are added afterwards. One and a half ounces
of superphosphate (acid phosphate) and i ounce of pure chalk
are added per bushel of compost.

17

Plant Qrowth'Suhstances

II. The loam and ballast are sterilized together, and the
sand separately. The unsterilized moss-peat is added after-
wards, with the following per bushel of compost: il ounces
hoof and horn ; 1 2 ounces superphosphate ; I ounce sulphate
of potash ; i ounce pure chalk. The fertilizers must have been
finely ground, and all must be mixed thoroughly.

Further information about composts is given in the book
by the same authors (Lawrence and Newell) : Seed and
Potting Composts, with Special Reference to Soil Sterilization
(London: George Allen & Unwin, 1939. 3s. 6d.).

Note

American readers in particular should note that the treat-
ment of cuttings with indole-acetic acid, indole-butyric acid,
indole-propionic acid, and naphthalene-acetic acids (and
salts and esters of these) is covered by U.S. Patents respec-
tively numbered 2,129,598; 2,129,599; 2,129,601; and
2,129,600; these were granted in September 1938, and were
taken out by A. E. Hitchcock and P. W. Zimmerman on
behalf of the Boyce Thompson Research Foundation of
Yonkers, N.Y.

f

18

CHAPTER III

THE SCOPE OF THIS BOOK

It is interesting to look back upon some of the comments
made by reviewers of the first edition. The book was origin-
ally written for chemists, and after the second chapter it
assumed and still assumes the reader to have a knowledge of
chemistry at least equal to that of pass degree standard.
Chapter XIII, which one reviewer justly said could only give
the layman a headache, introduces the indole group of
compounds, but ignores most of the phenyl compounds, with
which the reader of that chapter can be expected to be suffi-
ciently familiar. Few chemists, except specialists in some
branches of organic chemistry, know much about the indole
(benz-pyrrole) nucleus, though they all know the phenyl ring.
Hence the chemical explanations start from the phenyl ring,
and no attempt is made to simplify organic chemistry further
by introducing explanations couched in terms the non-
chemist might possibly understand but of which he would
probably lose the thread long before he had an idea of the
meaning of the structural formula of benz-pyrrole.

Chemistry, like everything else, has to be learnt; but this
book is not the place to teach its elements.

Originally the book contained no plant physiology, and
some plant physiologists who reviewed the book apparently
concluded that it was therefore a quite unintelligible and
peculiarly useless piece of work. Another praised it (on the
whole) for what it was, but devoted a good share of his space
to regretting that the book did not set forth an explanation
of the action of all growth-promoting and growth-regulating
substances in every sphere. Such an explanation neither I

19

Plant Qrowth'Suhstances

nor anyone else can give, and it is not likely that the ideal will
be even approached for a long time.

The book now contains sketchy outlines of some physio-
logical aspects. These have been introduced not as a con-
cession to the over-weening specialists, but partly because one
paradox was not properly explained in the first edition (though
no reviewer noticed that), and partly because it is becoming
possible to account to some extent for actions which pre-
viously were brought about purely empirically.

The difference between just using the synthetic growth-
substances to produce a result (such as rooting of cuttings) and
understanding how growth occurs is like the difference
between pharmacy and physiology. A year or two ago there
was practically no knowledge about how the artificial growth-
substances produced their results: they were applied like a
drug from the outside, produced a certain or uncertain
effect, and tliat was all. There was an increasing under-
standing of the way normal growth was regulated inside
plants by their own natural hormones, but a discussion of these
belongs to books on plant physiology.

This book, then, is about drugs that produce, control, or
regulate growth in plants. Being foreign to the plant, such
applied substances are not hormones, though they are fre-
quently miscalled "plant hormones" or "phytohormones".
Both these latter terms connote, and should be restricted to
mean, the (natural) hormones of plants, which are the pro-
vince of physiology and are not discussed in this book,
except briefly on pages 53 and 93, and elsewhere incidentally
in relation to the synthetic substances.

Since the first edition was written there have been very few
changes of significance to the pure chemist, as far as the
phenyl- and indolyl-acetic group of substances is concerned.
No important new synthetic growth-substance of that type has
been prepared; no new method of preparation has been
published ; and no known substance, belonging to the group,
but previously unused, has received any extensive application.
The truth of this last can be realized from the fact that the
only fresh development worth mentioning from the chemical

20

^

The Scope of this Book

point of view has been the application of phenyl- butyric acid as
a reagent in physiological research. This had not been
so employed when the first edition was written.

The chief changes since the writing of the first edition have
been of horticultural value and interest, with one exception.
In 1937 the principal application of synthetic growth sub-
stances related to the treatment of cuttings; other develop-
ments were foreshadowed, but had not been extensively
tested. Since then the treatment of seeds with growth-
substances has occupied the attention of a number of workers,
the use of the synthetics in grafting has become more than a
suggestion, and the questions of bud-inhibition and retarda-
tion of flowering have assumed practical importance. These
developments are at least sketched in the present edition.

It is hoped, therefore, that the present book may be useful
to horticulturists as well as to chemists. The horticulturist
will probably get on reasonably well with the book if he skips
Chapters X, XIII, and XIV, and some other bits.

Chapter IV on applications of growth-substances to the
rooting of cuttings and the production of such freak results
as the formation of roots on stems has been maintained in
its original form. Some of the work mentioned in this chapter
already has not much more than a historical significance,
but it has been felt that, historical as they are, such results
provide a fairly easy introduction to the more recent practical
uses described in later chapters.

The first edition was written in November 1937 (which
accounts for some omissions), but could not be published until
July 1938. Now (May 1940) the book is being revised and
extended with a view of the second edition appearing in the
early autumn of 1940. It is not possible to mention every
application or to keep strictly up-to-date in a book, but
because it is desired to give here as good service as possible, a
hint may be offered to those who desire to keep fully informed
of developments soon after they are made.

It is possible, though relatively expensive, for an individual
to keep up-to-date if he has no access to libraries. Chemists
will not need to be reminded of such abstracting journals as

21 Bl.

Plant Qrowth'Suhstances

Chemical Abstracts and British Chemical Abstracts. The horti-
culturist, however, may not know of the monthly Experiment
Station Record (Washington, D.C., U.S.A.) or of the excellent
Horticultural Abstracts issued by the Imperial Bureau of
Horticulture, East Malhng, Kent, England. The latter
periodical is a quarterly, and costs 6s. 6d. (about $1.50) per
issue, or;fi 5s. od. ($6.00) per year. To subscribers to certain
other publications of the Bureau the price is reduced 20 per
cent. The issue for March 1939 contains about 20 abstracts
relating to natural and artificial growth-substances, and that
number, which may be expected to maintain itself quarterly
for a time, gives an idea of the rate at which knowledge of the
subject is expanding.

The exceptional development already mentioned as of
interest to chemists is the discovery that vitamin B^ (thiamin,
aneurin) is potent as a growth-regulating substance when
applied to plants. Vitamin C (ascorbic acid) acts somewhat
similarly. Thiamin, at least, may be a serious rival to sub-
stances of the indole-acetic acid type at present used in
horticulture.

In this book little space has been given to the chemistry
of the vitamins. Unlike the indole and naphthyl compounds
that act as regulators of plant growth, the vitamins have had
considerable publicity given to their chemistry. It has been
felt, therefore, that chemists will be satisfied with a few leading
references to the synthesis of vitamin B^, and that it is hardly
necessary to say much at this date about the synthesis of
ascorbic acid. Probably most chemists will be more interested
to learn of the new field for the vitamins — as drugs capable of
regulating plant growth when applied to plants from the
outside — than they will be to learn much about the artificial
syntheses of vitamins, or of their natural production in or by
plants and micro-organisms. It does seem, however, that
an understanding of the phytological functions of some vita-
mins, when outside the plant, may have great agricultural as
well as horticultural importance.

22

The Scope of this Book
FURTHER READING

The history and physiological aspects of hormones particu-
larly, and to a less extent, of synthetic growth-substances,
have been well reviewed in the following three books :
Boysen Jensen, P. (1938), Growth Hormones in Plants,

translated from the German, and revised, by G. S.

Avery, Jr. and P. R. Burkholder. McGraw Hill Co., New

York and London. 2nd edition.
Schlenker, G. (1937), Die Wuchsstoffe der Pflanzen. Ein

Querschnitt durch die Wuchshormonforschung. Leh-

manns Verlag, Munich.
Went, F. W., and Thimann, K. V. (1937), Phytohormones.

The Macmillan Co., New York.
A good short introduction for chemists to some physiological
matters such as curvature and the oat test (these are mentioned
in the next chapter) is the paper by Fritz Kogl, "On Plant
Growth Hormones", in Chemistry and Industry, Vol. 57, 15th
January, 1938, pages 49-54.

A comprehensive review of horticultural aspects of syn-
thetic growth-substances has been provided by H. L. Pearse
under the title Plant Hormones and their Practical Importance
in Horticulture: Imperial Bureau of Horticulture (East
Mailing, Kent, England) Technical Communication No. 12
(1939) ; 3s. 6d. post free, or from any bookseller.

23

CHAPTER IV

MODES OF APPLICATION

Introductory Scientific Work on Effects of the
Synthetic Growth- Substances

The rapidity with which the subject of synthetic growth-
regulating substances has developed can be judged from the
relative poverty of mentions of these substances in the first
edition of Boysen Jensen's book, which was published in
1936. This book is written mainly from the point of view of
the academic plant physiologist, rather than from that of the
horticulturist. To the chemist it will be of interest mainly on
account of the extent of the lists of references to work on the
physiological aspects of plant-growth stimulation; most of
these, however, are of the "pre-indole-acetic era", having
served to convey the ideas of botanists and plant physiologists
at a time when the chemistry of plant-growth stimulants was
non-existent.

Physiological Methods of Testing

The earliest methods of testing for the presence of growth-
promoting substances other than fertilizers were based upon
the amount of curvature occurring in a coleoptile (first leaf
sheath) of an Avena (oat) seedling after the growing tip had
been cut off and material containing the substance had been
applied to one side of the cut surface. This material was often
derived from another growing plant. The test depended upon
the stimulation of cell-elongation, and hence of growth on the
treated side, so that the cell-tissue became unequally devel-
oped, and bending was visible to the naked eye. The method
was suited to the detection of very small amounts of active

2+

Scientific Results

substance, as a relatively small number of cells were directly
affected. In many cases the active substance was that absorbed
from a plant-surface by a moist agar surface during a very
short time, a small block of the agar being then cut out and
applied to the cut surface of another (demonstration) plant,
which showed by a bending response that it re-absorbed
some of the substance present in the first plant. An Avena~
Einheit (AE) or Avena unit (A.U.) has been devised. Full
details of this technique and of its numerous refinements will
be found in Boysen Jensen's book.

It was soon realized that urine and the resources of bio-
logical and synthetic chemistry laboratories could provide
larger amounts of growth-promoting substances than those
yielded by a transverse section of a plant. The delicate
oat-coleoptile {Avena) test has remained in use for purely
physiological research, but for more practical purposes it has
been succeeded by grosser tests largely based either on root-
formation — often in highly unusual positions on the plant —
or else on the phenomenon known as epinasty} Epinasty is an
expression by a mature plant of a set of changes due to
unequal development of various parts, so that bending, and
sometimes twisting, develops. For example, if one side of a
stem or leaf receives more growth-promoting substance than
the other, the stem or leaf will bend or curl. The phenomenon
of epinasty is thus not different in principle from the simple
and measurable bending of a decapitated coleoptile of an oat
seedling, but visible epinastic changes in mature plants are
complicated by the presence of asymmetric structures of
various degrees of rigidity — such as the tapering "veins" in
the leaves. For this reason epinastic changes must be regarded
as gross qualitative manifestations.

Growth Promotion and Growth Regulation: a Distinction

The production of roots, or, to speak more accurately,
the initiation of root-formation, is that property of growth-

^ Epinasty and its opposite, hyponasty, are together known as nastic
movements or responses. The original papers should be consulted for fuller
details, including a discussion of the hyponastic effects, epinasty being taken
as the type of both for the purpose of this introductory explanation.

25

Plant Qrowth'Suhstances

regulating substances of the most practical importance at
the present moment. Before discussing the property of
root-initiation the author desires to draw the attention of
the reader to the fact that the adjective growth-promoting
has been twice used in explaining bending tests, but that
the term growth-regulating has been reverted to in speaking
of root-production. This is because at the cut surface of a
seedling coleoptile the initiation of cell-elongation is promo-
tion of growth. On the other hand, initiation of root-forma-
tion on a mature plant, while leading to the differentiation of
new tissue in some places, is often accompanied by diminished
growth in other parts, and the plant may even become dwarfed
(Hitchcock and Zimmerman, 1935 ; Pearse, 1936-7). Hence it
would be illogical to apply the term "growth-promoting"
indiscriminately in alluding to substances other than the
nutrients in fertilizers. There seems a need to distinguish,
as zoologists have done in their own sphere, between the
promotion of cell growth and the initiation of tissue dif-
ferentiation in plants.

The adjective "growth-promoting" is sometimes applied
by physiologists to those amino-acids, without which animal
growth (in size) cannot take place. It is doubtful whether the
adjective is valid in that connexion, which is mainly foreign
to the purpose of this book. Some of the work of R. W.
Jackson, and of Berg, and others, had its origin in a study of
the necessity of certain amino-acids in animal nutrition.

A method of actually observing the formation of roots
was reported by F. W. Went (1934), who found that the
number of roots formed on cuttings of sweet pea was approxi-
mately proportional to the concentration of rhizocaline
("the root-forming substance").

Hitchcock (1935), of the Boyce Thompson Institute at
Yonkers, New York State, caused initiation of roots, epinasty
of leaves, and bending and swelling of stems of intact tomato,
tobacco, African marigold, and other plants by making local
applications of synthetic indole-acetic and -propionic acids.
He made the interesting prophecy that "The fact that one

26

Scientific Results

homologue of 'heteroauxin' [indole-acetic acid] was found to
be active in causing certain formative responses indicates that
possibly other indole derivatives and perhaps other unrelated
chemicals might induce one or more of these same responses."
It will be seen how abundantly this remark has been justified.

Description of Methods

A description of methods may be quoted from a later paper

from the Boyce Thompson Institute (Zimmerman and

Wilcoxon, 1935):

"The compounds were used as distilled water solutions
or mixed with lanolin (U.S.P.).^ In a few cases where
cuttings were involved dilutions were made with Knop's
solution,^ The cuttings were placed in vials or flasks so
that the basal ends were immersed in the solutions. The
water solutions were introduced into the stems and petioles
[the stalks of the leaves] by means of glass tubes drawn to a
capillary at one end. The capacity of the tubes varied, but
held on an average approximately 0*3 c.c. of solution. The
capillary end of the tube was inserted into the stem or
petiole and left to drain into the plant. There was consider-
able variation in the length of time required for the tubes
to empty. Also the response of the plant varied with the
rate at which the substance drained from the tubes.

"Lanolin preparations were applied locally by rubbing
the mixture on stems or leaves with a glass rod. . . . The
usual concentration range was from o-oi per cent, to
2 per cent."
Another technique used in later work (Hitchcock and

Zimmerman, 1936) instead of injection was to dip a slit-up

portion of the stem of an otherwise intact plant into a vial

containing solution.

The following list of species of plants successfully used

in the experiments reported by Zimmerman and Wilcoxon

(1935) showed that the effects of certain growth-substances

^ The lanolin-smear method was introduced by Laibach (1935).
^ Physiologically balanced and isotonic with land-plant cells, Knop's
solution is a simple mixture of inorganic reagents in water.

27

Plant Qrowth'Suhstances

were not peculiar to a limited range of plants: African
marigold, tomato, buckwheat, sweet pea, Windsor (broad)
bean, sunflower, Jerusalem artichoke, dahlia, sensitive plant
(Mimosa pudtca), and goosefoot {Chenopodium album). A
number of other unrelated plants were used in later experi-
ments. Amongst them was the tropical vine Cissus stcyoides
illustrated in Fig. i.

Epinasty of leaves was induced by application of the
substances in lanolin or by injecting a solution by the capil-
lary-tube method. When the substance was applied in lanolin
to a narrow zone around the stem of an active plant, leaves
above and below the zone of application showed downward
bending. Epinasty of any single leaf was induced by applying
the lanolin paste lightly to a small region on the upper side of
the petiole. There was considerable similarity in the response
of plants to the cyclic compounds and to ethylene, propylene,
acetylene, and carbon monoxide.

Tomato plant cuttings 5 ins. long with their basal ends
immersed in Knop's solution containing the synthetic
substances showed declination of the uppermost leaves
within a few hours. The use of Knop's solution does not
appear to be essential, as Hitchcock (1935) and others have
reported a similar response to aqueous solutions of indole-
propionic acid. Zimmerman and Wilcoxon (1935) did not
determine the absolute lowest concentrations effective in
eliciting this rapid response, but they found that the following
low concentrations were effective in that way: Phenyl-acetic
acid 125, a-naphthalene-acetic acid 20, and indole-butyric
acid 4 parts per million of solution. On the other hand,
a-naphthyl-acetonitrile did not cause an evident response in
tomato plant cuttings until the second day after its applica-
tion in solution to the basal ends. Whether applied thus, or
injected, or in lanolin, a-naphthyl-acetonitrile evoked final
results with tomato similar to those obtained with a-naphthyl-
acetic acid.

Growth substances applied to one side of active shoots
caused negative or positive bending, according to the con-
centration of the chemical, and the plant species.

28

Scientific Results

Altogether eight compounds were found by Zimmerman
and Wilcoxon (1935) to cause unusual activity such as nastic
responses, root-initiation, etc., when applied to sizeable
plants; the list has been lengthened since.

Initiation of Roots

All the compounds mentioned in the preceding section, and
also anthracene-acetic, fluorene-acetic, and acenaphthene-
acetic acids, caused local initiation of roots (adventitious roots)
on aerial parts of growing plants of tomato, sunflower,
African marigold, Jerusalem artichoke, tobacco, dahlia, and
buckwheat, a-naphthyl-acetic and indole-butyric acids were
especially effective in initiating roots on stems and leaves.
The subjoined table, taken from the paper by Zimmerman
and Wilcoxon (Contrib. Boyce Thompson Institute, 1935, 7,
211), indicates the approximate effective concentration ranges
of five of the substances, expressed as percentages in lanolin.
Numerical results are, of course, affected by the mode of
application, the species of plant, and the type of effect sought.

Table — Effective Concentration Ranges of Five
Synthetic Growth- Substances for Tomato, ex-
pressed IN Percentages in Lanolin.

Causing Negative

Chemical Substances

Bending in Stem

Inducing Adven-

or Epinasty of

titious Roots.

Leaves.

a-naphthalene-acetic acid

o-oioo — 2-0

o-i — 2-0

Indole-butyric acid

o-oioo 2-0

o-i — 2.0

Indole-acetic acid

0-0003 — 2-0

0 -4 — 2 -o

Indole-propionic acid

0-0250 — 2-0

i-o — 2-0

Phenyl-acetic acid

0-0250 — 3-0

i-o — 3-0

Fluorene-acetic acid

00500 — 3-0

1-0—3-0

This remarkable initiation of roots is well shown by
Figs, z and 3, taken by permission from the paper by Zim-

29

B2

Plant Qrowth'Suhstances

merman, Hitchcock, and Wilcoxon (1936, p. 109). Though
this paper was mainly devoted to discussing the effectiveness
of esters of the cycHc acids, the mode of root-formation by the
acids, esters, and the one nitrile tested was similar. Fig. 2,
indeed, presents evidence of the root-initiating capacity of
both a-naphthyl-acetic acid (left) and its ethyl ester (right),
applied to a leaf and one side of the stem of each plant. Fig. 3
shows an especially beautiful demonstration, the plant on the
left being in this case untreated, that on the right having been
treated with a low concentration of methyl 3-indole-acetate
only ten days before the photograph was taken. The plant
shown in Fig. 3 belongs to an uncommon species — Kalanchoe
daigremontiana — which has the property (useful for teaching
purposes) of producing white roots in a dry atmosphere.
The initiation of roots on woody plants such as apple,
holly, and Cupressus is a problem which has not yet been
solved satisfactorily in all respects; there remains consider-
able scope for experimentation. The lanolin-smear method
appears to be less generally effective with the woody plants
than with such plants as tomato and Kalanchoe. F. W. Went
(1934) and Cooper (1935) suggested that the apical ends of
cuttings, and not the basal ends, should be treated with
preparations of the growth substances, in accordance with the
idea that "auxins" can move only downward in stems. Other
workers have shown that treatment of the basal ends of
cuttings with lanolin, water or dust preparations of synthetic
growth substances can induce root formation.

Practical Methods of Using the Substances

With respect to the practical use of solutions of growth
substances for inducing root-formation in woody cuttings,
Hitchcock and Zimmerman (1936) recommend leaving
the cut (basal) ends of cuttings for twenty-four hours or
several days in aqueous solutions containing relatively low
concentrations in preference to using higher concentrations
for periods shorter than twenty-four hours. Desirable con-
centrations of what they call the three principal substances
(indole-acetic, indole-butyric, and a-naphthyl-acetic acids)

30

Root-production extraordinary. This photo shows the natural aerial roots
of a tropical vine growing in the Cycad House of the Missouri Botanical
Garden, St. Louis, U.S.A. In this greenhouse these roots attain a length'of
40 feet, not branching at all unless they strike soil or moist material. The
roots here shown owe nothing to artificial growth-substances, and the
illustration is given as a matter of general interest.

According to John A. Moore (1933), from whose paper the photograph
is reproduced, the vine shown had been in the greenhouse only five years.
It was included by Zimmerman and Hitchcock (1935, H) in a series of plants
subjected to the action of root-forming substances, towards which it behaved
in no unusual way. The botanical name of this vine is Cissus sicyoides L. var.
jfacqiiini.

:/

Fig. 2. — Tomato plants, showing coloured roots grown in dry air of the
greenhouse, after treatment with lanolin preparations of the growth sub-
stances. There are roots on the long stem-like leaf-stalks bending towards
the camera, as well as on the true stems. The bending of the stems arose
from the treatment on one side. The photographs were taken i8 days after
treatment. Left: Leaf and one side of the stem above it, smeared with 0.50
per cent, a-naphthalene-acetic acid.
Right: Similar plant treated with i.o per cent, ethyl a-naphthalene-acetate.

Fig. 3. Kalanchoe daigremontiana, a tropical "greenhouse" species, here
kept under reasonably dry conditions. Left: Untreated. Right: Showing
the collar of white roots induced near the top of the stem ten days after
local treatment with a lanolin preparation containing 0.05 per cent, methyl
3-indole-acetate.

Fig. 4. — Rooting response of cuttings of American holly. The
bottom row had had their lower ends dipped for 54 hours in a
solution of 10 mg. of indole-acetic acid in 100 ml. of water.
The top row was placed in tap water for the same time. All
cuttings were then put into a compost of peat-moss and sand
for five weeks before the photograph was taken.

Fig. 5. — Two sets of cuttings similar to those of Fig. 4, except
that the lower row of cuttings were dipped for four days in a
solution of 2 mg. of a-naphthalene-acetic acid per 100 ml.
water, the controls being put into tap water for four days.

Scientific Results

would be from i to 4 parts of the substance in 100,000 parts of
water. A small quantity of alcohol may be used for prelimin-
ary solution of the substance ; the naphthyl-acetic acids are all
but insoluble in pure water. After treatment with the water
solution for two to four days the cuttings should be placed
in a potting compost. The novice should be warned not
to expect to see any extensive root-formation at the end of
a few days in the solution; the immediate purpose of the
treatment is to enable the cuttings to absorb the growth-
substance, which will then initiate root-formation while
the cutting is in the compost. The formation of roots,
however, usually takes place in appreciably less time than
when compost alone is used.

Figs. 4 and 5 show respectively the effects of solutions
of indole-acetic acid (10 parts per 100,000) and a-naphthyl-
acetic acid (2 parts per 100,000) on batches of ten cuttings
of American holly. These photographs are reproduced
by permission from the paper by Hitchcock and Zimmer-
man (1936, p. 66). Similar results were obtained with
Japanese holly and Taxus cuspidata (Chinese yew) — also
difficult plants on which to induce rooting with compost
alone. It will be noticed that the cuttings that were almost
leafless produced few roots, in agreement with the experience
of gardeners who have used compost only. Rooting responses
of a number of other species of plants are described in the
paper.

Japanese maple was one of the few species of woody
plants which responded consistently to treatment with
lanolin preparations, concentrations of from 25 to 100 mg.
of indole-propionic acid per gram of lanolin having to be
used. As these concentrations are very much higher than
those necessary for the solution method, it appears that
the lanolin smear method will be less generally applicable
than the solution method. Probably lanolin smears will
be restricted to treatment of wounds on the parent plant
(as after pruning) to hasten the formation of callus, or else
as a technique in suitable cases where it is desired to treat a
cutting before removal from its parent. Lanolin smears may

31

Plant Qrowth'Suhstances

also be useful in grafting, thus being a refinement on the use
of dung. The work of Pearse (1936-7) suggests that spraying
may be in some cases at least as effective as lanolin smears.

Cooper (1935), who applied a i : 2000 lanolin paste of
indole-acetic acid to a scraped surface at the tops of cuttings,
was able to induce formation of roots on leafless lemon
cuttings, which do not ordinarily form roots. He described
his experience as "very encouraging", and his 1936 paper gives
additional results with cuttings difficult to root.

Evenari and Konis (1938, D) whose work is mentioned at
length on p. 52, repeat (in agreement with other authors) that
there is no general method of applying indole-acetic acid to
suit all cases. They point out that a lanolin preparation
applied to leafed cuttings may vitiate root-formation, by
preventing the intake of water. A related suggestion has been
put forward by Jackson (1938), namely, that failures to secure
rooting with winter cuttings after treatment with synthetic
growth-substances (in solution) may be due to imperfect
absorption by the leafless cuttings.

Grace (1937, C) has also obtained excellent root-pro-
duction on woody cuttings, by dipping the bases of bunches
of 50 into a dust bearing o-i per cent, of growth substance by
weight, shaking off the excess dust, and planting immediately.
This technique should be very valuable, and appears to be
coming into general use.

The substances tried by Grace in his very promising study
included indolyl-acetic, -propionic, -butyric, and a-naphthyl-
acetic acids, and some salts and mixtures of these. A few
additional details of this procedure are given in the next
two chapters (pp. 37, 42, 46).

Pearse (1936-7) has examined the effects of occasional
and repeated spraying with solutions of phenyl-acetic and
indole-butyric acids, having investigated the action of
phenyl-acetic acid at greater length than any other worker.
Epinasty of leaves of young tomato plants was clearly visible
within thirty minutes of spraying with a o-i per cent, solution
of phenyl-acetic acid, which could also cause the production
of roots from the stem.

32

Scientific Results
He wrote:

"By the spray method of applying the growth-substances
to the aerial parts of plants, uniform entry of the substance
is secured so that no bending of the stem occurs. . . . The
experiments described have shown that with the treatment
given the growth-substances cause no increased growth of
the plant as a whole, but exert, on the other hand, a reducing
effect. With the appropriate concentration, this reduction is
minimal.

"... The treatments seem to have a very large effect
on the way in which plant capital is distributed. The
substances do in fact appear to control the amount of
plant capital that shall go to the making up of each particu-
lar organ, without altering the total amount of plant
material."

Templeman (1938) obtained decreases, but no increases, in
dry-matter production of plants grown in pots of sand, by
spraying the foliage or watering the sand with indole-acetic or
a-naphthyl-acetic acids, ascorbic acid (vitamin C), or skatole.

Uptake from Soil

Hitchcock and Zimmerman (1935) reported that the addi-
tion of synthetic growth substances to soil induced all the
responses previously observed by them after applying the
materials to the aerial parts of plants. There were differences
in the extent and rapidity of response to substances applied to
soil, these differences depending not only on the species of
plants, but also upon the type of growth, magnitude, and
environment of the plant. As before, only a few results can
be quoted here. The plants were grown in 4-inch pots holding
the equivalent of about 450 gm. of air-dry soil. The solutions
were made by extensive dilution with water of an alcohol
solution of each compound. Phenyl-acetic, phenyl-propionic,
indole-acetic, -butyric, and -propionic, and naphthalene-
acetic [? a] acids were tried.

Epinasty (downward bending) of some or all leaves of

33

Plant Qrowth'Suhstances

tomato plants from 4 to 10 or more inches high occurred after
treatment of the undisturbed soil with any of the six acids
except phenyl-propionic. Pronounced responses were often
displayed within two hours of the application of the solution :
plants about 5 inches high required only about 3 mg. of the
substance in order to show the effect, but larger and older plants
required 10 to 20 mg. On the whole, the indole compounds
were more effective than the phenyl ; and toxic effects were
observed after application of more than 20 mg. of the indole
compounds [to the youngest plants?] or about 50 mg. of
the phenyl compounds.

The responses of tobacco plants were generally similar to
those exhibited by tomatoes, except that no noticeable
response was evoked in tobacco in less than three to six hours.

Root growth from the stems of tomatoes was essentially the
same after application of the active substances to the soil as
has already been described for local applications. However,
"Roots induced by phenyl-propionic acid (applied to soil)
appeared sooner and in larger numbers during the first eight
days than did roots induced by other compounds. Phenyl-
propionic acid was the only compound to induce many roots
to appear from parts of the stem that did not exhibit noticeable
swelHng or other type of proliferation. Naphthalene-acetic
acid was the most toxic to root systems in soil. ... In addition
to root growth from the stem of the tomato, roots also appeared
from the petiole [leaf-stalk] and along the entire midrib of the
middle leaves. This type of response was induced by indole-
propionic acid (27 mg. per pot). . . . Indole-butyric acid was
especially effective in causing roots to be initiated in stems of
tobacco plants 10 to 12 inches in height." Optimum initiation
of aerial roots in tobacco resulted from the addition of 27 mg.
of indole-butyric acid to the soil.

REFERENCES A

The lists appended to this chapter refer to but a few papers
published before 1935, as most of those already have only a
historical interest for chemists. Papers mainly of pure plant-

3+

Scientific Results

physiological interest have also been omitted. For the earlier

references consult Boysen Jensen's book.

Cooper, W. C. (1935), Plant Physiol., 10, 789; (1936), ibid.y

11, 779-
Hitchcock, A. E. (1935), Indole-3-n-propionic acid as a

growth hormone and the quantitative measurement of

plant response. Contrib. Boyce Thompson Inst., 7, 87;

see also ibid., p. 349.

Hitchcock, A. E., and Zimmerman, P. W. (1935). Absorption
and movement of synthetic growth substances from soil
as indicated by the responses of aerial parts. Contrib.
Boyce Thompson Inst., 7, 447.

Hitchcock, A. E., and Zimmerman, P. W. (1936). Effects
of growth substances on the rooting responses of cut-
tings. Contrib. Boyce Thompson Inst., 8, 63.

Jackson, T. H. (1938). Nature, 14, 835.

Laibach, F. (1935). Uber die Auslosung von Kallus- und
Wurzelbildung durch (3-Indolylessigsaure. Ber. Deut.
bot. GeselL, 53, 359; see also ibid., p. 469.

Moore, J. A. (1933). Missouri Bot. Garden Bulletin, 21, 139.

Pearse, H. L. (1936-7). Journ. Pomol. Hort., 14, 365.

Templeman, W. G. (1938). Emp. Journ. Exp. Agric, 7, 76.

Went, F. W. (1934). Versl. K. Akad. Wetensch., Amsterdam,
37, 445.

Zimmerman, P. W., Hitchcock, A. E., and Wilcoxon, Frank
(1936). Several esters as plant hormones. Contrib. Boyce
Thompson Inst., 8, 105.

Zimmerman, P. W., and Wilcoxon, Frank (1935). Several
chemical growth-substances which cause initiation of
roots and other responses in plants. Contrib. Boyce
Thompson Inst., 7, 209.

REFERENCES B

Afanasiev, M. (1939). Journ. Forestry, 37, 37.
Amlong, H. U. (1938). Ber. Deut. bot. Gesell, 56, 239.
Curtis, O. F. (19 1 8). Stimulations of root growth in cuttings

by treatment with chemical compounds. Cornell Agric.

Expt. Sta. Mem., No. 14, 69-138.

35

Plant Qroivth'Suhstances

Deuber, C. G. (1923). Amer. Journ. Bot., 13, 276.

Doak, B. W. (1939). New Zealand Journ. Sci. Tech. ,20, 269A.

Gillett, S., and Jackson, T. H. (1937). East African Agric.

Journ., 3, 229.
Hubert, B., and Rappaport, J. (1939). Invloed van para-

phenyleendiamine op de beworteling van stekken.

Natuurwet. Tijdschr., 21, 56. (Flemish, with EngHsh

summary).
Johnston, S. (1939). Mich. Agric. Exp. Sta. Quart. Bull, 21,

255-
Komisarov, D. A. (1938). Compt. rend. {Doklady) Acad. Set.

U.R.S.S., 18, 63.

Laurie, A. (1928). Amer. Florist, 70, No. 2074; after Exp.

Sta. Record 1928, 58, 838.
Lek, H. A. A. van der, and Krijthe, Eltien (1937). Meded.

Landbouwhoogesch. Wageningen, 41, Verh. 2. (Dutch,

with long English summary).
Myers, M. C, Bowden, R. A., and Hardisty, F. E. (1938).

Science, 88, 167. /
Nowosad, F. S. (1939). Sci. Agric, 19, 494.
Pearse, H. L., and Garner, R. J. (1937). Journ. Pomol. Hort.,

14, 365-
Pollacci, G., and Oddo, P. (1920). Gazz. Chim. Ital, 50, 54;

(1932), Atti R. 1st. Botan. Pavia, Ser. IV, 3, 69.
Snow, A. G., Jr. (1938). Amer. Journ. Forestry, 582.
Tincker, M. A. H. (1936 a). Journ. Roy. Hort. Sac, 61, 380;

(1936 b), ibid., 510; (1938), ibid., 63, 210.
Traub, H. P. (1938). Proc. Amer. Soc. Hort. Sci., 35(?), 438.
Traub, H. P., and Marshall, L. C. (1937)- Ibid., 34, 291.
Webster, M. E., and Robertson, I. M. (1937). Nature, 139, 71.

The actual or possible effect of a number of factors that may
affect the production of roots on cuttings treated with syn-
thetic growth-substances has been well reviewed by B.
Hubert, J. Rappaport, and A. Beke {Meded. Landbouw-
hoogesch. OpzoekSta. Gent, 1939, 7, 1-103), who have com-
pared most of the methods of application. They appear to
prefer the dusting technique, with charcoal as the dust.

36

CHAPTER V

RECENT WORK WITH CUTTINGS
[References B on pp. 35-36)

Applications

Ordinary woody cuttings

Effects of natural and synthetic growth-substances apphed
to cuttings of a large number of species of shrubs and plants of
horticultural importance have been described by Tincker
(1936, 1938). Amongst the illustrations given in Tincker's
second paper is one showing roots induced on cuttings of
Viburnum carlesii, perhaps the most deliciously fragrant-
flowered of all shrubs. It is hoped that use of the new syn-
thetic growth-substances, by cheapening the cost of plants,
will enable this species to be more widely grown.

Tincker's first paper (1936 a) is a review which may be
commended to those interested in the history of the use
of growth-substances on plants. His second paper (1936 h)
describes his own experiments; he used synthetic phenyl-
acetic, indole-acetic, and a-naphthalene-acetic acids, and
an extract of urine (the active substance in which was mainly
"heteroauxin" — indole-acetic acid), in the form of lanolin
pastes of various strengths. He found tomato and Pelar-
gonium zonule useful as test plants for the smear preparations.
With some woody and "recalcitrant" species the lanolin-paste
method of application was ineffective.

However, aqueous solutions of indole- and a-naphtha-
lene-acetic acids were strikingly effective in inducing rooting
to occur in cuttings of woody plants such as holly. Tincker's
control cuttings, and those that had been treated with the
active solutions, were planted in sand, not compost; other-

37

Plant Qroivth'Suhstances

wise his technique was similar to that used by the Boyce
Thompson Institute workers.

As has already been suggested on pages 12-17, the artificial
growth-substances should be regarded only as an aid to
propagation, for they will not infallibly compel cuttings to
form roots. Doak (1939) has recommended that cuttings
intended to be treated with synthetic growth-substances
should be taken at the time of year and condition of wood
most likely to lead to success without any treatment. Two
commercial preparations were found by Johnston (1939) to
be of little value in the rooting of hardwood blueberry, which
is known to be difficult to propagate in the ordinary way.

Pearse and Garner (1937) have applied the solution-
technique to accelerate root-production of soft-wood cuttings
of plum and pear rootstocks; immersion of the bases for
twelve hours in a solution containing 30 to 40 parts of
a-naphthyl-acetic acid per million of water markedly improved
rooting. With fig and blackcurrant cuttings, accelerated
rooting was also obtained.

Van der Lek and Krijthe (1937) studied the effects of indole-
acetic acid solutions on the rooting of plum, quince, and other
cuttings. They used the East Mailing types A and B (plum)
and A, B, and C (quince). The various types of one species
responded differently to a given concentration of sub-
stance or time of treatment. These workers are the only
ones who have measured the actual uptake of solution (and,
consequently, of growth-substance) by cuttings, and it appears
from their account that the same dose can produce different
results, depending upon the species and type of plant, and
upon concentration of solution and time of administration.
It was confirmed that, in general, a relatively high concentra-
tion over a short period of immersion is best. This paper
offers several points of novelty, and is of considerable practical
interest.

As an example of the variability encountered in treatment,
the following fairly full abstract may serve (Komisarov (1938) :
"Application of growth-substances to increase the rooting
capacity of woody species and shrubs") :

38

Recent Work with Cuttings

Results with indole-acetic acid (about 0005 per cent,
solution for about 36 hours) : 50 to 100 per cent, rooting with
16 out of 24 trials on 18 species. No root-formation with
Pinus sylvestris and Thuja occidentalis 6-7 years old, but 92 per
cent, of Thuja cuttings rooted if taken from trees 20 years old.
In general, the treatment gave (i) greater percentage of root-
ing; (2) greater root system, especially pronounced with Acer
dasicarpum and Ribes rubrum — most cuttings apparently
leafless at the start; (3) earlier rooting — mostly 2-6 days' gain,
but up to 16 days with Thuja occidentalis of age not stated.
Most cuttings were made in summer. Populus alba showed no
significant difference in the behaviour of winter and summer
cuttings, but otherwise the data were insufficient for forming
an opinion as to the advantage of taking winter or summer
cuttings. Treatment was 12-36 hours for summer cuttings;
20-48 hours for winter cuttings.

Phenyl-acetic and phenyl-propionic acids behaved similarly
to indole-acetic acid, but had a less pronounced effect.
Summer and winter cuttings of Populus alba and Ribes rubrum
responded readily to the phenyl compounds, but other species
did not. (This author is almost the only one who has tested
the rooting effect of phenyl-propionic acid.)

a-naphthyl-acetic acid acted similarly to indole-acetic acid
on Populus alba, Quercus pedunculatus, and Ribes aurea (the
last, however, not having been treated with indole-acetic
acid), but had no effect on Larix sibirica: on which indole-
acetic acid caused roots to form on 26 per cent, of not-woody
cuttings and on 6 per cent, of woody.

No satisfactory theory was advanced to account for the
difference in responses. Komisarov refers to his 1935 work
with Salix caprea and S. cinerea, wherein he obtained 40-70
per cent, better rooting after treatment with urine, and maize
flour "auxins".

A modified treatment of cuttings has been announced by
Amlong (1938). He placed vine cuttings iyitis vinifera) in a
warm bath for 24 hours, before transferring them to aerated
tap water, in which they were kept five weeks. The maximum
number of roots thus produced without any artificial growth-

39

Plant Qrowth'Suhstances

substance was obtained when the first bath was at 25° C.
Addition to the bath of small amounts of one-thousandth or
one-hundredth normal^ solutions of indole-acetic acid mark-
edly increased the number of roots formed under the same
conditions.

Forest Trees

Some work with woody cuttings has already been cited.

Only two other papers will be mentioned here. They are
given as illustrations of the type of work that has been done,
of the variability of the results obtained, and of the fact that
generalization is not possible. See also p. 39.

Snow (1938) reported that (i) dormant cuttings of trembling
and large-toothed aspen (Populus tremuloides Michaux and P.
grandidenta Michaux) can be rooted to the extent of at least
65 per cent, (of cuttings treated) by dipping them into a
solution of 10 milligrams of indole-butyric acid per litre (one
part in 100,000) for about 27 hours; (2) maximum rooting is
obtained when dormant cuttings of these species are taken just
before the buds are beginning to burst in the spring; (3)
abundant callus is not indicative of potential root-formation ;
(4) new hybrids of white poplar treated similarly behaved
variously, some rooting to the extent of 70 per cent., others
not at all.

Afanasiev (1939) tried the effect of indole-butyric acid on
the rooting of greenwood cuttings of deciduous trees, using
two species each of Betula, Acer, and Populus. No rooting was
obtained with hard maple and aspen, but in four other species
30-60 per cent, of the plants rooted in about 7- 11 weeks after
treatment with a 2 in 100,000 or weaker solution for 6 to 24
hours.

Sub-tropical Species

Since most of the work on the applications of synthetic
growth-substances has been done in temperate regions, many
workers in warmer regions will be interested in this section.

^For explanation of the term normal, see p. 55.

40

Recent Work with Cuttings

Traub (1938) (of Orlando, Florida) has reported tests of
over a hundred chemicals on the rooting of sub-tropical
plants. Most of the reagents he tried are not usually regarded
as growth-substances, and many of those he used have not
been tested by other workers. A number of substances that
gave some improvement of rooting (over controls) of Passi-
flora quadrangularis or Bignonia venusta are listed in his paper ;
most of them were inferior to indole-butyric acid in 002
per cent, aqueous solution, even in qualitative tests. Salicyl-
idene acetamide (quarter-saturated solution), and tetra-
furfuryl alcohol and sodium naphthyl-4-sulphonate (both in
o-oi per cent, solution) were found to have an approximately
equal efficacy with indole-butyric acid within the scope of
these rough tests. Amongst substances of the more customary
class, phenyl-acetic acid, diphenyl-acetic acid, indole-3-«-
propionic acid, and ascorbic acid, were tried; they were less
useful than the indole-acetic, indole-butyric, and a-naphthyl-
acetic acids, but it is not clear whether they were found by
Traub to have any value at all.

Many plants (listed in the paper) were considered from the
standpoint of their suitability as test objects, and the out-
standing ones for sub -tropical use were found to be Passiflora
species, especially P. quadrangularis and P. ligularis. Cuttings
of the former can be used in the upright or inverted positions.
Inversion has the advantage that the controls root late and
sparely. Cuttings were made with the major portion of one
internode and one node with leaf attached.

Though reported in 1938, this work was begun in 1933, that
is, prior to the identification of heteroauxin as indole-acetic
acid. In another paper Traub and Marshall (1937) briefly
reported on the rooting of papaya cuttings {Carica papaya) ;
they have worked with three Carica species.

Gillett and Jackson (1937) and other workers have obtained
encouraging results with rooting of cuttings of Cojfea arahica
treated with 10 parts of indole-acetic acid per 100,000 of
water, and with appropriate dilutions of a commercial
preparation.

41

Plant Qroivth'Suhstances

Non- Woody Plants

Demonstration experiments with some non-woody plants
have already been mentioned; these were performed to test
the efficacy of the then new synthetic growth-substances
rather than for purposes of propagation. Recently Nowosad
(1939) has described some preliminary tests with indole- and
a-naphthyl-acetic acids in the rooting of cuttings of some
forage plants, and has concluded that the synthetics may be of
value to the plant breeder. It may be recalled that the
planting of alfalfa (lucerne) on the field scale from cuttings
was practised experimentally in England in the eighteenth
century.

The optimal treatments for alfalfa and clover were : (i )
50 parts per million in talc, applied to the fresh scar of
cuttings; (2) dipping basal ends 12 hours in 5-50 p. p.m.
solution; (3) feeding 10 p. p.m. dissolved in nutrient culture
solution.

No satisfactory rooting of timothy {Phleum pratense) was
obtained. It is pointed out that the degree of success with
plants reacting positively is influenced by such factors as
temperature, humidity, and light, in the greenhouse ; degree of
maturity of cuttings; pH and chemical composition of the
medium in which the cuttings are planted — as well as by the
kind of growth-substance chosen.

See also the work with kudzu {Pueraria hirsuta) men-
tioned on p. 45.

Substances
Solvents

As already mentioned, the potassium and sodium salts of
indole-acetic acid and related acids are sufficiently soluble
in water. Such salts, particularly the potassium salt, have been
used and recommended by a few workers. Most of the alkali
salts are not generally available.

The usual practice with the indole- and naphthyl-acetic
acids as growth-substances is to rub them up with a little
ethyl alcohol, in which they dissolve fairly readily, and
to dilute that alcoholic solution with water to the desired

42

Recent Work with Cuttings

volume. The presence of a small percentage of ethyl alcohol
in the treatment solution is not harmful.

Other solvents have been tried by Tincker (1938).

Vapours

Vapours of "synthetics" do not appear to have been tried
on cuttings, but see p. 54.

Additions or Reinforcements to Solutions

It seems that only one report has been made (Amlong and
Naundorf (1938, C) ) of the use of mixed solutions, containing,
say, both indole- and naphthyl-acetic acids, and the effects of
mixture do not appear to have been very marked, as compared
with the effects of either component alone. Hubert et al. (1939 ;
bottom of list B) found that a mixture of indole-acetic acid
with ascorbic acid gave better results than the single sub-
stances. (See p. 68 and 80 for some work with ascorbic acid
(vitamin C) alone applied to cuttings),

Evenari and Konis (1938, D) tried potassium perman-
ganate alone and in conjunction with indole-acetic acid in
solution, with various results, but the salt alone was not as
effective in inducing rooting as was the acid alone. They
found that sugar (sucrose ; cane sugar) added to solutions of
indole-acetic acid improved the rooting response, and they
suggest that the addition of sugar to solutions of growth-
substance may have considerable value.

Other Substances

The use of permanganate and of sugar as aids to root-
formation is not new. A weak water solution of potassium or
sodium permanganate is used by practical gardeners as a
rooting medium for cuttings of some woody and semi-woody
plants. Its rationale is obscure. Some suppose the per-
manganate to act by suppressing microbial growth, such as
would occur if the cuttings were left long in unchanged
plain water. It may conceivably produce growth-substances
by oxidation of tissue-material of the cuttings, and it supplies
potassium, which some writers overlook.

43

Plant Qrowth'Suhstances

The anti-microbial action of potassium permanganate
received no support from the work of Curtis (1918), who has
contributed the most extensive report of the action of the
substance on the rooting of cuttings. His discussion of the
role of permanganate may be commended to those interested.

Curtis also found that cane sugar in simple solution notably-
improved rooting of some woody cuttings. A favourable
effect of glucose on the rooting of cut etiolated pea shoots has
been reported by Amlong (1938, C). Cane sugar, given alone,
was found by Evenari and Konis (1938, D) to stimulate callus
formation in Bignonia and Myrtus.

Went (1934, A) used potassium permanganate (0.05 per
cent, solution in water) to give "an effective disinfection" to
etiolated pea seedhngs from which the roots had been cut off
in the course of preparation of the seedlings for a test of
"rhizocaline, the root-forming substance". It is clear that
Went did not impute to potassium permanganate any positive
root-forming activity on such pea seedlings, and he wrote
that the salt "decreases the number of roots on the controls".
Webster and Robertson (1937) have reported, in a note, that
application of potassium permanganate and related salts to
some (presumably intact) plants (particularly the cactus
Opuntia leucotricha growing in sand) produced a marked
growth response (? of the aerial parts), which the authors
thought was not solely a manurial effect. No further details
have been found.

Laurie (1928) found the following substances to hasten
the appearance of roots on cuttings of the plants named,
though they did not increase the ultimate percentage of
rooting :

2 per cent, cane sugar in water: lilac, carnation, heliotrope,
climbing Euonymus.

o-i per cent, potassium permanganate in water: chrysan-
themum and the above-mentioned four and some
others.

Vinegar (three teaspoonfuls to a gallon (U.S.) of water) :
lilac, Euonymus, chrysanthemum.

44

Recent Work with Cuttings

Potassium permanganate (one ounce to 8 gallons (U.S.) of
water) was found superior to any "synthetic" tried, for root-
production on kudzu cuttings. (Myers et al., 1938).

The action of some of the minor elements, such as boron,
zinc, and manganese, has been compared to that of vitamins.
This is not the place for a discussion of the roles of simple in-
organic combinations of elements necessary for plant growth,
but one organic compound has a special interest, as it has
been claimed to be a succedaneum for inorganic iron. It has,
apparently, no special effect in root-induction.

Iron is usually regarded as essential for plant growth, and
it is supposed to have a special part in the formation of
chlorophyll, although chlorophyll contains no iron. Pollacci
and Oddo (1920) claimed that solution-cultures deprived of
iron but supplied with other necessary elements and with
magnesium 2-pyrrole-carboxylate, permitted normal growth
of several species of plants. The pyrrole ring brings to mind
the indole compounds and nicotinic acid ; like inorganic iron,
these may be growth-catalysts.

The work of Pollacci and Oddo was challenged by Deuber
(1926) who found none but a toxic effect to be exerted by the
pyrrole-carboxylate, even in minimal doses. That the
substance had a stimulating effect was reaffirmed by Pollacci
and Oddo (1932) for higher plants, and again by Pollacci for
algae {Ber. Deut. hot. GeselL, 1935, 53, 540).

Para-phenylenediamine has been successfully used by
Hubert and Rappaport (1939) to stimulate root-formation on
cuttings of several species of shrubs. It was used at a concen-
tration of loo-iooo mg. per litre, at which strengths its action
was reported to be very similar to that of the usual concen-
trations of indole-acetic acid.

Honey has recently been found by R. W. Oliver {Sci. Agric,
1939, 19, 586) to exert a considerable stimulating action on
the rooting of cuttings of Thuja and chrysanthemum.

45

CHAPTER VI

THE TREATMENT OF SEEDS

In 1937 Grace suggested that if young lettuce, tomato, and
other seedlings growing in sand or soil are treated with a solu-
tion containing indole-acetic acid or other synthetic growth-
substance, a real stimulation is obtained with a dose of active
substance at the rate of 50 to 250 mg. per acre (120 to 600 mg.
per hectare). This is, so far, the ultimate in agricultural micro-
therapy, and, should active substances be present in organic
manures in only corresponding traces, their detection will
provide a problem for the analyst. A pictorial height-curve
of nasturtiums treated with such minute doses has been
provided by Grace in two publications (1937, 1938 a).

Grace (1937) has also proposed two dusting techniques
which could be used not only for cuttings but also for seeds.

An inert dust such as talc, or a standard mercurial dust
disinfectant such as is used for cereals, had a small proportion
of indole-acetic, indole-butyric, or a-naphthyl acetic acid
mixed with it. Grace claimed that treatment with such dusts
is safer than treatment with solutions. A toxic excess of
growth-substance can be easily avoided in the solution treat-
ment of such relatively massive material as cuttings. In the
case of seeds, which are not only small but may be supposed
while germinating to have little need of auxin-like material, it
is very easy to give a toxic overdose of artificial growth-sub-
stance— as, indeed, the work of Grace has shown. Some toxic
effects of solutions applied to seedlings are discussed in
Chapter XI.

The dust technique appears to be peculiarly suitable for the
treatment of seed with artificial growth-substances. Grace
treated seeds of wheat, barley, and other plants, with a dust

4-6

The Treatment of Seeds

containing about 2-5 parts of growth-substance per million of
dust, and grew them on in sand. Stimulation of the early
stages of growth of the aerial parts was shown, and there was
also a stimulated growth of the normal (underground) roots of
wheat and barley in presence of concentrations of this order of
magnitude.

Grace (1938 b) has shown that at least two synthetic growth-
substances are compatible with, and stable in, formalin solu-
tion. A formalin treatment is widely used in Canada and
elsewhere for the repression of that fungal condition in cereals
known as smut. Smutted seeds treated by formalin have re-
duced germination (some of the seeds being dead) and the
survivors have a weakened growth. According to Grace, such
damage to seed oats and wheat can be considerably reduced, or
entirely offset, by adding minute amounts of a-naphthyl or
3-indole acetic acid to the formalin solution before it is used
on the seed. The strengths recommended were o-oi-i part
per million of solution. Such a mixture of formalin and
growth-substance can be stored at least ten weeks. It is
thought that the effect on the seed of the mixture of formalin
and growth-substance should be attributed to stimulation of
damaged embryos, rather than to prevention of damage at the
time of treatment. It might seem that the artificial growth-
substance replaces some natural hormone inactivated by the
treatment, but there is evidence that the benefit cannot be so
simply explained.

A demonstration of "the undoubted physiological activity"
on winter wheat of mercurial dusts containing about 2-10
parts per million of the indole- and a-naphthyl-acetic acid
was made by McRostie and others (1938). The dusts were
applied to the seed, the comparison being between treatment
with mercurial fungicide alone and fungicide plus growth-
substances. Small but significant increases of yield of both
grain and straw were reported, but the experiment is regarded
as preliminary, and further work must be done to define the
effects of variety, season, and other variables. This was the
first field experiment with growth-substances applied to an
agricultural crop.

47

Plant Qrowth'Suhstances

It may be noted that even should indole-acetic acid regain
its one-time price of about los. ($2-50) a gram, or about ;f200
($1,000) a pound^, treatment of agricultural seeds on the field
scale with a dust or formalin solution containing the recom-
mended doses of synthetic growth-substance cannot add
appreciably to the cost of dressing. Mercurial dusts, costing
about 2S. a pound, are already applied free by many seed-
merchants, and the added cost of material, if five parts of
indole-acetic acid were incorporated, would not exceed a
farthing (half a cent.) per pound of dust. It is, in fact,
practicable to give away a very expensive synthetic chemi-
cal!

Work on the treatment of seeds with synthetic growth-
substances has been done by Amlong and others (1937, 1938,
1939), who investigated mainly the effects of water-soluble
potassium salts of indole-acetic, indole-butyric, and a-naph-
thyl acetic acids, in 10-^ and lo-^ equimolecular solutions to
which had been added traces of magnesium nitrate and
manganese chloride. 20 gm. of seed of sugar-beet (2 strains),
lucerne, maize, and spring wheat were steeped 24 hours in
60 ml. of solution and sown by hand in the field. The results
with maize (ears), wheat (grain) were not significant, but both
strengths gave remarkable increases (24-62 per cent.) of yield
of leaves and root of beet, and of lucerne hay. The authors
conclude that the treatments are economically practical for
these two crops. Seed treatment was also given to a number of
herbs and vegetables grown in pots, and it was concluded that
each species has its optimal dose-requirements.

Sprays of a commercial growth-substance preparation
applied from four weeks after sowing gave especially remark-
able results with sugar beet (about 60 per cent, increase in root
and 80 to over 100 per cent, increase in tops). It was suggested
that spraying has a specially beneficial influence upon the tops,
and that the effect of seed-treatment is principally upon the

^The cost is now slightly reduced ; the prices of indole-butyric acid and the
naphthyl-acetic acids are similar. There is reason to think that a-naphthyl-
acetic acid should now cost shillings (a few dollars), rather than many
pounds (hundreds of dollars) per pound. A cheapening of the other sub-
stances can scarcely be looked for.

48

The Treatment of Seeds

leaves. The spray treatment had little effect upon lucerne,
however, and depth of rooting may be a factor.

In their 1938 paper Amlong et al. reported on the com-
bined effect of two synthetics in mixed solution applied to
seeds, and are probably the first workers to do so. The effect
of mixture does not seem to be very marked.

The first investigation of the effect of a synthetic growth-
substance on seeds was done by Cholodny (1936) in connec-
tion with the Russian work on vernalization {syn. yaroviza-
tion), that is, the effect of applying physical treatments, such
as cooling, to seed, for the purpose of hastening growth.
Cholodny and others suspected a hormonal effect, and his so-
called "hormonization" was an outcome, from which the
whole of the other work on application of growth-substances
to seed has probably derived.

The term "hormonization" is a vague one, because it can
connote treatment with at least three classes of substance:
(i) hormones proper to the species tested; (2) hormones
belonging to another species of plant; (3) animal hormones. It
has been used wrongfully to mean (4) treatment of seeds with
synthetic growth-substances, and was in fact used by Chol-
odny in both senses (2) and (4) in his 1936 communication,
wherein he described the effect of maize embryos on seedling
growth of oats. (See also p. 39).

The treatment of seeds with urine containing animal
hormones has been revived by Tovarnitzky and Statkovskaia
(1938), who used urine as a source of sexual hormone; cf.
Eyster and Ellis (1924).

REFERENCES C

Amlong, H.U., and Naundorf, G. (1937), Forschungsdienst, 4,

417; (1938), ibid., 5, 292-303; (1939) ibid., 7, 465-482.

The last is well illustrated.
Cholodny, N. (1936), Nature, 138, 586.
Eyster, W. H., and Ellis, M. M. (1924), Growth of maize

seedlings as affected by glucokinin and insulin. Journ,

Gen. Physiol., 6, 653.

49

Plant Qrowth'Suhstances

Grace, N. H. (1937)- Canadian Journ. Res., Sect. C, 15, 538;

(1938 fl), Nature, 141, 35; (19386), Canadian Journ.

Res., Sec. C, 16, 313-329. An account of the last paper

is given by Hugh Nicol, Manuf. Chem., 1938, 9, 388.
McRostie, G. P., Hopkins, J. W., and Grace, N. H. (1938).

Canadian Journ. Res., Sect. C, 16, 510.
Tovarnitzky, V. I., and Statkovskaia, E. I. (1938), Khim.

Sotsial. Zemled. (Chemisation of Socialistic Agriculture),

Part 3, 37-45.

50

CHAPTER VII

The Use of Synthetic Growth- Substances in Graftings
in Producing Bud-Inhibition, and Other Operations

When a tree is grafted into another at the time of a certain
conjunction of sun and moon, and is fumigated with certain
substances whilst a formula is uttered, that tree will produce a
thing that will be found exceedingly useful. . , . — Moses
Maimonides, The Guide of the Perplexed (trans. M. Fried-
lander) as quoted by S. Tolkowsky, Hesperides (London: John
Bale, Sons & Curnow, 1938).

Grafting ; Rooting of Grafted Woody Cuttings
In view of the earliness of the demonstration {e.g., Laibach
(1935, A) ) that a synthetic substance such as indole-acetic
acid applied in a lanolin paste may hasten or improve the
formation of wound-callus, it is rather surprising that so few
investigations on the application of the artificial growth-
substances to practical grafting have been published. So late
as the spring of 1939, a discussion of the possibility of grafting
olives on to an Abyssinian stock plant was made, in which
there was nothing but surmise concerning the mutual effects of
the natural hormones of stock and scion and of an artificial
growth-substance, if one were used (Tallarico (1939) ). It is
not, of course, to be expected that in the present state of plant-
physiological knowledge any sound predictions regards such
mutual effects could be made for any plant; still less could
they be made for this almost unknown stock. The reason for
mentioning this discussion is that it quoted no previous work,
in spite of the fact that its author came from Rome, where the
library facilities are excellent. One would suppose a priori that
the lanolin smear method would be peculiarly suitable for
grafting operations, though this suitability has been recently
denied.

51

Plant Qrowth'Suhstances

A number of trials of indolyl-acetic acid as grafting re-
agent and some other new applications are recorded in the
papers of Evenari (Schwarz) and Konis (1938). As these were
published in a journal that is somewhat difficultly accessible,
it may be useful to give here a rather full abstract, though it is
not possible to mention every question touched upon in the
papers.

These workers described, in their first paper, experiments
with figs, olive, Rosaceae (apple, cherry, pear), and vine;
and in their second paper, with several less important species
{Hibiscus, Ligustrum, Myrtus, and some ornamental conifers).
They used leafless (Part I) and leafed (Part II) cuttings, and
applied the growth-substance in three modes: solution,
lanolin paste and as actual crystals. They appear to be the
first investigators to report the placing of crystalline growth-
substance directly in contact with plant tissues.

These workers report with figs a number of findings
regarding the relations between rooting and the possession of
terminal buds by cuttings, untreated, and treated apically and
basally. Indole-acetic acid applied to olive cuttings led to
precocious shedding of leaves ; in olive and Rosaceae, marked
formation of callus was induced, but little root-formation. An
apple grafting was improved by treatment, but in a pear and a
cherry grafting the union of stock and scion was hampered by
treatment.

On all varieties of Vitis vinifera tested, treatment improved
rooting all round, though one mode of application was some-
times more effective than another, depending on the variety.
In all varieties the union of stock and scion (so-called "English
graft") was markedly improved by treatment, and in 1938
vines were grafted on a large scale at Ein-Harod with an indole-
acetic acid treatment. The mode of treatment with growth-
substances that was adopted in these two cases is not stated.

Traub (1938, B) has investigated the transport of natural
hormones over the graft union of citron on sweet orange (the
former being known to root readily, the latter with difficulty),
and concluded that Went's factor for callus formation was
transmitted, but not the factor for rooting. The cuttings

52

Synthetic Qrowth'Suhstances in Qrafting

formed no roots after being planted in a rooting medium. No
artificial growth-substance was used, and it is not excluded
that a synthetic substance might be useful if it were desired
to root grafted cuttings.

Perhaps the most cogent paper that has appeared so far
on the application of artificial growth-substances to grafting is
that of Lefevre (1939)- He worked with vines {Vttis vinifera) :
scions of Pinot d'Ay on a strain of Berlandieri. He used four
methods of application : two modes of spraying, immersion of
the whole graft in a solution for 48 hours, and dipping the cut
ends, only, for 48 hours. This last procedure seemed to be the
best, both as regards production of roots and callus-formation
of the union. He tentatively recommends a solution of 10
parts of growth-substance per 100,000 of water, and he thinks
that for use on grafts indole-acetic acid may be superior to
indole-butyric acid.

The review by Chouard (1939) should be consulted.

Inhibition and Stimulation of Budding and Flowering. Forcing
This is a development likely to have considerable import-
ance. There is reason to suppose that much work of a mainly
practical tendency is being done to extend and perfect the
control of dormancy, and also that the results are being
to some extent regarded as trade secrets, so that but little
is being published.

The existence, in terminal buds, of a hormone (auxin) or
hormones, affects the growth of lateral shoots, as has been
shown by Thimann and Skoog (1933, F) and others. From
this knowledge it is not a long step to consideration of an
endeavour to control bud-development and flowering by the
application of artificial growth-substances. If such control
were achieved, it would be an important gain in several
fields of horticulture: for example, for retarding flowering,
and thus loss of blossom and of fruit, in fruit-growing dis-
tricts liable to late frosts; and to the plant exhibitor, who
could more accurately time the optimal development of his
show material.

Though it does not directly concern the subject of syn-

53

Plant Qrowth'Suhstances

thetic growth-substances, Chailakhian's hormonal theory
(1937) of flowering may be mentioned. This worker pos-
tulated the existence of a factor for flowering, which he
named florigen. The mobility, and therefore the existence,
of such a hormone was demonstrated in several ways; for
example, grafts of Perilla on Perilla at different stages of
flower-ripeness were made, and the grafts behaved as if the
factor passed through the unions {cf. the grafting experiment
of Traub mentioned on p. 52).^ Practical applications of
hormone theory in horticultural practice are explained in the
1937 paper, which has some 300 references. By appropriate
techniques of vernalization [see below) transplanting, grafting,
and by taking advantage of photoperiodism, the concentration
of the flowering hormone can be controlled, and flowering
may thus be either forced or retarded.

The vapour of methyl a-naphthalene-acetate has been
found by Guthrie (1939) to inhibit the growth of buds of
potato tubers. The substance is sufficiently volatile at a warm
room temperature (25° C.) to produce its effects. The sprout-
ing of whole tubers can be retarded by merely storing them at
that temperature in presence of paper impregnated with the
ester, e.g. in a bag. Epinasty of tomato leaves is readily
induced if a little of the substance is placed in a bell jar with
the plant.

Zimmerman et al. (1939) discuss "Responses of plants to
growth substances applied as solutions and as vapours."
There are similarities between the effects of unsaturated
hydrocarbon gases (ethylene, etc. ; used for forcing) and those
produced by vapours of a number of "synthetics".

Gustafson (1936) seems to have been the pioneer in show-
ing that known chemicals could cause the ovary of a flower
to develop into a fruit. By using indole-butyric acid he
obtained almost normal, but seedless, fruits from unfertilized
tomato flowers. A good set of (parthenocarpic) fruit was
secured by Gardner and Marth (1937-8) with holly and

^In another paper Chailakhian and Zhdanova (1938) concluded that the
natural growth-hormones affect growth only, and do not supply the stimulus
for the formation of buds and flowers.

5+

Synthetic Qrowth'Suhstances in Qrafting

strawberry by the use of synthetic growth-substances, the
results with holly being thought to be of practical value to the
florist. See also Nixon and Gardner (1939).

In a preliminary report, Bennett and Skoog (1938) give the
results of applying yeast extracts and solutions of vitamin Bj,
indole-acetic acid, and other substances to the cut tips of dis-
budded fruit trees kept in a warm greenhouse. The point
about the greenhouse being warm is that cold is normally
essential for the breaking of dormancy of fruit trees, just as
subjection to a low temperature is an essential feature of the
process of vernalization, which is used to "break dormancy"
(induce earlier growth) in such seeds as those of winter wheat.

In these experiments of Bennett and Skoog the effect of the
applied substances was not uniformly well marked. Vitamin
Bj, while not notably influencing the number of buds pro-
duced, appeared to induce strong growth of such buds as did
develop. The best effect was obtained by "injection" of
extracts of autolyzed yeasts (brewers' and bakers'), especially
in young Bartlett pear trees.

The following is a translation of the summary of a paper by
Amlong and Naundorf (1938), after Horticultural Abstracts:^

"Coating the dormant buds of lilac (variety, Charles X) once
a day for seven days in succession with a hundredth- or
thousandth-normal solution of indole-acetic acid, or with a
thousandth-normal solution of a-naphthyl-acetic acid, or with
certain other stimulating mixtures, results in a considerably
earlier bloom. Covering the terminal buds with growth-
substance pastes has a similar effect. The forcing effect of a
hot-water bath can be increased by spraying the treated plant
with indole-acetic acid solution once a day for a week."^

^B.C.A. quotes what is apparently the same paper but gives elder as the
experimental plant.

*A "normal solution", sometimes written N solution, is a chemical term
here denoting the molecular weight, in grams, of a growth-substance theoret-
ically dissolved in a litre of water. The molecular weight of indole-acetic
acid is about 170 ; that of the naphthyl acids and the other indole acids which
are used as growth-substances is rather more. There is, however, some per-
missible latitude in the strength of solutions, and for horticultural purposes
no harm will be done if the molecular weight of all these substances is taken
as 200. A hundredth-normal solution of indole-acetic acid would be pro-
hibitively expensive, as it would contain (nearly) two grams in about a

55

Plant Qrowth'Suhstances

The mainly physiological work of Stoughton and Plant
(1938) on sea-kale {Crambe maritima) should be consulted by
those interested.

Warne (1937) has used sprays of indole-acetic and of
a-naphthyl-acetic acids in 0-05 per cent, water solution to
induce curling of chrysanthemum petals.

In a study which principally aimed at elucidating some
physiological problems, Ferman (1938) suggested that it is
not necessary to assume the existence of a hormone newly
postulated by Went (1938) under the name caulocaline,
and supposed to be a factor in the elongation of stems and
lateral buds. Ferman described a number of experiments
with solutions and pastes of indole-acetic acid applied to
seedlings and cuttings, giving varying results. An aqueous
solution of one part in 100,000 applied to the cut surface of a
decapitated seedling of Lupinus albus inhibited the develop-
ment of axillary buds, but more dilute solutions of that
substance rather promoted their development.

For a report of trials with phenyl-acetic and indole-acetic
acids (mostly as sprays) to attempt to bring about the breaking
of dormancy of strawberries during the season September-
April, see Roodenburg and Tiddens (1938).

Dihydrofolliculin has been shown to have an action similar
to that of hypothetical flowering hormones (florigen, etc.),
which may be chemically related to that animal derivative
(Chouard (1938) ). Butenandt has also suggested that one
sexual hormone may be common to the animal and plant
kingdoms.

The effect of some synthetic growth-substances on the
production of new roots on transplanted trees (young red
oaks) has been studied by Tilford (1939). Indole-butyric
acid was more effective than indole-acetic acid; the cut ends
were wrapped in sphagnum moss soaked in the solution, or
the roots were soaked in a solution.

quart of water ; the thousandth-normal solution is still costly. This applica-
tion would seem to be one for which a-naphthyl-acetic acid is well adapted,
and for which it could be extensively applied if its price comes down to a
reasonable figure (see p. 48).

56

Synthetic Qrowth'Suhstances in Qrafting

Possible Non- Biological Applications

Robbins and Jackson (1937) have recorded an effect of
indole-acetic acid on elongation of dead stem-wall material
(cotton thread and hemp cord). These textiles stretched more,
under the influence of a weight, when treated with lanolin
containing 0-2 per cent, of indole-acetic acid, than when
treated with lanolin alone. Stewart (1938) attempted to
repeat this work and also extended its scope. He found no
conclusive evidence of an effect of indole-acetic acid on the
extensibility of living or dead stem- and root- cell-walls.
Artificial silk (called by him "regenerated cellulose"), however,
was found by Stewart to have an increased extensibility in
dilute solutions of certain organic acids, such as acetic and
oxalic, which he says are "known not to be growth-sub-
stances",^ as well as in 0*2 per cent, aqueous indole-acetic acid.

'At the Chelsea Flower Show of 1939 an unknown interlocutor told the
author that several years ago he had obtained excellent rooting of Bougain-
villea cuttings in India by treating them with dilute formic acid ; untreated
cuttings failed almost wholly to root. It is not known to the author how this
substance was introduced into use for that purpose ; it may represent an echo
of some work by H. E. and E. F. Armstrong {Annals Bot. (191 1), 25, 507;
Proc. Roy. Soc. Lond. B, (1910), 82, 588), who examined some effects of
dilute acids on leaves, but did not report root-formation, or apply the
substances to cuttings.

REFERENCES D

Amlong, H. v., and Naundorf, G. (1938). Gartenbauwiss.,

12, 116 {homHort.Abs.)
Bennett, J. P., and Skoog, Folke (1938). Plant Physiol., 13,

219.
Chailakhian, M. H. (1937). Bull. Acad. Set. U.R.S.S., 198

(after Hort. Abs. 1939, 9).
Chailakhian, M. H., and Zhdanova, L. R. (1938). Compt.

rend. Acad. Sci. U.R.S.S., 19, 303 {irom Hort. Abs.).
Chouard, P. (1938). Chron. Bot., 4, 13.
Chouard, P. (1939). Les "Hormones" de croissance et leur

emploi pratique, specialement dans le bouturage. Rev.

bot. appl. Agric. trop., 19, 332-350.

57

Plant Qrowih'Suhstances

Evenari, M. (W. Schwarz), and Konis, E. (1938). The effect

of heteroauxin on root-formation and on grafting: I.

Palestine Journ. Bot., Ser. J, 1, 13-26; II (with D.

Zirkin), ibid., 125-130.
Ferman, J. H. G. (1938). The role of auxin in the correlative

inhibition of the development of lateral buds and shoots.

Thesis, Univ. Utrecht.
Gardner, F. E., and Marth, P. C. (1937). Bot. Gaz., 99, 184.
Gustafson, F. G. (1936). Proc. Nat. Acad. Sci., 22, 628.
Guthrie, J. D. (1939). Inhibition of the growth of buds of

potato tubers with the vapour of the methyl ester of

naphthalene-acetic acid. Contrib. Boyce Thompson Inst.,

10, 325-
Lef^vre, J. (1939). Compt. rend. Acad. Agric. France, 25, 629.
Nixon, R. W., and Gardner, F. E. (1939). Bot. Gaz., 100, 868.
Robbins, W. J., and Jackson, J. R. (1937). Amer. Journ. Bot.,

24, 83.
Roodenburg, J. W. M., and Tiddens, B. A. (1938). Chron.

Bot., 4, 18.
Stewart, W. S. (1938). Amer. Journ. Bot., 25, 325.
Stoughton, R. H., and Plant, W. (1938). Nature, 142, 293.
Tallarico, G. (1939). L'innesto dell'olivastro di Etiopica

[Chrysophila etiopica). Proc. VIII Cong. Trop. Agric.

(Tripoli, 1939); also (illus.) in L'ltalia Agricola, 76,

295-304-
Tilford, P. E. (1939). Proc. 14th Nat. Shade Tree Conf. (St.

Louis, 1938), p. 51 (from Hor^ Abs.).

Warne, L. G. G. (1937). Nature, 140, 1065.

Went, F. W. (1938). The dual effect of auxin on root-forma-
tion. Amer. Journ. Bot., 26, 24.

Zimmerman, P. W., et al. (1939). Contrib. Boyce Thompson
Inst., 10, 363.

58

CHAPTER VIII

WHAT IS A ROOT?

In popular language the idea "root" implies fundamentality,
but, although the rooted plants normally bear their roots as
their lowermost parts, there are many plants that have no
roots at all and manage quite well without them. The algae —
whether microscopic {e.g. diatoms), or big (the seaweeds) —
have no true roots, the whole plant living bathed in a nutrient
solution.

There are numerous parasitic plants which utilize other
plants' roots. Some, like the sandalwood tree of Southern
India, grow apparently independently, but have no true
roots of their own and are root-parasites deriving nourishment
from the roots of plants of other species growing near.

Another kind of rootless parasite grows on the ordinary
aerial stems of its host without necessarily having any contact
with the ground ; parasitic plants of this type rely indirectly on
the roots of their hosts — indirectly, because a portion of the
host's stem intervenes between parasite and host-roots.

A root may be defined as an absorptive organ having a
characteristic structure and possessing a growing-point pro-
tected by a [root-] cap.

Roots need not be underground; witness the pendant
aerial roots in Fig. i and the supporting or buttress-roots of
the banyan tree and the stilt-plant {Pandanus sp.). The latter
possesses large aerial roots upon which the cap is often
distinctly visible to the naked eye.^

A root cannot, therefore, be defined as a fundamental part of

^Excellent pictures of the plant Pandanus sanderae, including a close-up
view of the aerial root-cap, are given in the article by John A. Moore, in the
Missouri Botanical Garden Bulletin, 1933, 21, No. 10.

59

Plant Qrowth'Suhstances

a plant, nor can it be defined in terms of its position in the
plant. Even thorns may grow underground.

It is now fairly well known that true roots can be induced to
form in unusual positions: the property of being able to
induce the formation of aerial roots, and to cause cuttings to
form roots, is possessed by artificial growth-substances. This
has given rise to no small amount of confusion, regarding the
effects of the substances on existing roots.

Many people have mistakenly assumed that an application
of a synthetic growth-substance to the normal underground
roots of common plants must be beneficial. To explain why
this assumption is wrong would take me beyond the province
of this book into the sphere of plant physiology, but it may be
hinted that the induction of new roots on rootless parts of
plants — cuttings or stems — is one thing, and the elongation or
growth of normally formed roots is another. While a root
cannot be defined by an appeal to its position, the effects of a
chemical stimulant may and do differ considerably, according
to the position and original function of the part of the plant to
which the substance is applied.^

Went (1938 D) has shown that it is possible to separate two
phases in the action of indole-acetic acid on the formation of
roots on cuttings. The first phase is tentatively identified
with a redistribution of the hormone rhizocaline within the
stem. This phase can be induced by a number of substances
not active in root-formation proper. The second phase can be
induced only by indole-acetic acid and similar substances;
this phase may be an activation of the accumulated rhizocaline.

There is at present no warrant for supposing that the
addition of a synthetic growth-substance to a manure, or its
presence in a manure, must inevitably improve the growth of
ordinary underground roots. It is tempting to suggest that
part of the special value of organic manures lies in their
content of, or ability to form, substances like indole-acetic
acid. But, until more is known about the presence in organic

^The formation of shoots on or near the tips of roots of Pogonia ophioglos-
sides has been recorded by Margery C. Carlson (Bot. Gaz., 1938, lOO, 215),
but the effect of synthetic growth-substances on this process does not seem
to have been examined.

60

What is a Root?

manures of such substances, and the part they play (if present)
in organic manures, invocation of growth-substances in such
manures can be little more than insecurely-based sales talk.
Vitamin B^ may be responsible for some of the growth-promot-
ing effect of organic manures. (See p. 68).

Many workers have found that synthetic grov/th-substances,
and even auxin, are actually toxic under certain conditions to
normal roots growing in solution-cultures. Some workers,
such as Grace, have produced methods of application that
are claimed to minimize the toxic effect, or even to convert it
into a real stimulation of normal roots in sand or soil.

Some of the reported toxic effects are discussed in Chapter
XI. It is not possible to sum them up, and the reader is
therefore left to form his own conclusions about the paradox.
It may resolve itself as knowledge grows.

"

CHAPTER IX

GROWTH-SUBSTANCES FROM NATURAL

SOURCES

Agricultural Researches Having a Possible Bearing on
Growth- Substances in Soil and Manure

I have experienced, that the black water taken from a
Dunghill, will make a Cabbage, or any of that Race, prosper
extremely. — R. Bradley, New Improvements of Planting and
Gardening, 171 8.

The action of mixed micro-organisms on proteins and trypto-
phan (3-indole-a-amino-propionic acid; indole-alanine) pro-
duces indole-acetic acid and other related growth-sub-
stances, as well as substances capable of acting as nutrient
materials for plants. In nature and in agricultural practice,
such substances reach the plant directly from the excretions of
animals or via manure-heaps, in which they are mixed with
decomposition-products of straw and other vegetable mater-
ials. Natural decay of leaves and roots, and the operation of
composting, may also be supposed to yield growth-substances
and related bodies. Rhizopin (indole-acetic acid) has, for
example, been recognized as a product of a fungus, and
fungi play an important part in the decay of plant-residues.
Recent work by plant physiologists and by botanists has
tended to ignore the not inconsiderable mass of work done by
botanists on the assimilation of amino-acids such as tyrosine,
and by chemists on the existence of organic nitrogenous
compounds in soil. The early work on amino-acids was
almost all directed to proving their value as nutrients (sources
of nitrogen) when the amino-acid was used as sole source of
nitrogen. It therefore has only a comparative interest.

62

Natural Sources

On the other hand, the work of the school of Schreiner and
Shorey seems to bear with some directness on the question of
the relation of protein-decomposition products and soil
fertility. The studies of Schreiner and Shorey on nitrogenous
compounds were more concerned with bodies related to the
purine group than those of the indole group and phenylic
amino-acids. After their preparation of creatinine from soil,
they broke new ground in two respects (Schreiner et al.,
1 911): by trying the effect of creatinine on wheat-plants in
water-cultures both with and without nitrate, and by demon-
strating the presence of creatinine in stale extract of farmyard
manure and in fresh cowpea vines.

Creatinine, and its relative creatine, which was also tested
on wheat-plants, may have acted simply as nutrients. Creat-
inine is the anhydride of creatine, which is methyl-guanid-
ino-acetic acid.

H

N CO

HN:C

\
H3C-N CH,

creatinine

The experiments of Schreiner et al. were not fine enough
for it to be possible to say that the increases of plant yield
observed with creatinine and creatine in presence of small
amounts of nitrate showed that creatinine, or creatine, is a
growth-substance. The importance of the work of Schreiner
and Shorey in the present connexion is rather that it focuses
attention on the organic constituents of soils. As Schreiner
and Brown wrote, in 1912, "the purely mineral-requirement
theory of soil fertility has proven itself inadequate to cope with
the accumulated facts."

A re-reading of the paper by Schreiner and Lathrop (191 2)
suggests in the light of present-day knowledge that decomposi-
tion products of protein (not necessarily of nucleo-proteins
only) may be associated with the phenomena of "sickness" of

63

Plant Qrowth'Suhstances

highly-manured greenhouse soils, such as is treated by means
of partial sterilization: growth-substances of the indole and
other groups can be toxic in too great a concentration.

In a letter to Nature under the title "Role of Heteroauxones
in Legume Nodule formation, Beneficial Host Effects of
Nodules, and Soil Fertility", Link (1937) has suggested that
"the beneficial effects of suitable concentrations of (3-indole-
acetic acid and other auxones may account in part for the
characteristically beneficial effects of: (i) nodules for some
host plants; (2) green manuring with nodule-bearing plants;
(3) fertilizing with manures rich in dung and urine, or with
compost; (4) humus soil; and (5) mycorrhizal fungi for some
host plants."

This claim has been anticipated to some extent by other
workers^, and notably at some length in relation to green
manures by Schreiner, Reed, and Skinner in 1907. Whereas
unchanged tyrosine was found by Schreiner, Reed and
Skinner (1907) to be toxic to young wheat seedlings, a
tyrosine solution, after having become discoloured on stand-
ing, produced excellent growth of young wheat seedlings.
The colour of this old tyrosine solution resembled that of
manure extract, and the wheat plants grown in it resembled
plants grown in manure extract. The darkening was possibly
due to the oxidation of tyrosine to homogentisic acid (2:5-
dihydroxyphenyl-acetic acid) which gave rise to further
oxidation compounds of dark colour. The effect on plants
of homogentisic acid has not otherwise been investigated.
The chemical relationships of this substance have received
attention owing to the occurrence of homogentisic acid in the
urine of people subject to the rare condition known as "alcap-
tonuria" (see p. 74).

^ In 1935 the author wrote: "One of the most remarkable effects of the
leguminous crop, whether in mixture or in rotation, is its apparent ability to
supplement animal manures. In peninsular Indian practice (the best-studied
case) it would seem that legumes grown in mixture fill the place of animal
manures. It appears unlikely that this ability is due solely to the nutrient
nitrogen compounds supplied by the legumes, and it is probable that
leguminous plants everywhere make a definitely biological contribution to
the fertility of soil." — Hugh Nicol, "Mixed cropping in primitive agricul-
ture," Empire Journ. Exper. Agric, 1935, 3> i8q.

6+

Natural Sources

Another ring compound investigated by Schreiner is
picoline carboxylic acid (2-methyl-4-carboxy-pyridine). This
was originally isolated from a virgin Hawaiian soil by Shorey
(1907) and from other soils, and appears to have afforded the
first instance of isolation and identification of a definite
crystalline organic compound from soil. It was isolated from
the virgin Hawaiian soil to the extent of 200 to 300 parts per
miUion of soil — a not negligible amount. It was isolated from
a Maryland soil by Schreiner and Shorey (1909) by a pro-
cedure fully described, though no quantitative details were
given. The Maryland soil was "exceedingly" infertile, and
Schreiner and Shorey were inclined to ascribe the infertility
in part to the presence of the pyridine compound. On testing
its eflFect on wheat seedlings, these authors found that in
distilled water at a concentration of about one part per million
"this soil constituent ... is a stimulant, an effect which is,
however, characteristic of small doses of poisons when
applied to plants." Uvitonic acid (2-methyl-4: 6-dicarboxy-
pyridine) showed no clear evidence of stimulation. It seems
possible that a mono-carboxy-pyridine or similar compound
may yet be found to have a real "growth-promoting" effect.
The Maryland soil did not give notable yield-responses to
application of either farmyard manure or artificial fertilizers.
Whether this failure to respond was due to sufficiency of ring-
compounds, or to excess of a toxic substance such as dihy-
droxy-stearic acid, must remain for the present an open
question.

Thornton and Smith (19 14-15) found that growth of
the chlorophyll-bearing unicellular animal Euglcena was
greatly stimulated by small amounts of phenyl-alanine

H O

and tyrosine (hydroxy-phenyl-alanine; \, hydroxy-phenyl-

acetic acid +NH3), also by hay infusion ; fresh hay infusion, or
an infusion that has been long subject to decomposition by
microbes, was appreciably less effective towards Euglcena
than an infusion subjected for a short time to the action of
microbes. Tryptophan was not used in these experiments.
Bottomley (191 5-1 7) suggested that the quantity of

65

Plant Qrowth'Suhstances

auximone (growth-substance) present in stable manure
increased with the progressive decomposition of the manure,
though here again a very well-rotted manure contained
less auximone than did fresh manure.

Marked effects of dilute extracts and fractions of farmyard
manure upon the extent of production of roots (in their
normal positions in water-cultures) have been reported in-
dependently by Breazeale (1927) and by Niklewski (1931,

1935)-

Breazeale used several dialyzed fractions of manure on wheat

seedlings and concluded that the effect of the manure extract

seemed to depend largely on the presence of black organic

matter, and not upon the amount of plant-food ingredients

it contained.^ Minute quantities of peat-extract had a very

striking effect on the growth of citrus seedlings deprived of

organic matter but well supplied with nitrogen, phosphorus,

and potash. Niklewski believed that the favourable effect of

very dilute extracts of manure on the root-development of

plants was due to colloids, but his earlier work was not

chemically critical, as he apparently performed no dialysis

on his manure extracts.

In a recent publication, Niklewski and Wojciechowski
(1937) have brought forward a notable dossier of evidence
that minute amounts of manure and peat extracts produce
marked effects on the amount of growth of several kinds of
plants grown in water, sand, and soil pot, cultures. Plants
supplied with the organic extracts and complete mineral
nutrients grew much more sturdily than did those supplied
with complete minerals only. The extracts were not dialyzed.
In the discussion there is, however, but little reference to
colloids.

In a few preliminary field experiments, Niklewski and
Wojciechowski attempted to see whether yield effects of
normal doses (25 and 50 dz. per hectare) of compost applied as
a late top-dressing to cereals had a relation to the water-
soluble humus substances in the compost. Increases in yield

^ Hitchcock and Zimmerman (1935, A) found that three indole acids
could pass through a membrane impermeable to the dye Fast Green.

66

Natural Sources

resulted, but it is not clear that these increases were signifi-
cantly different from those produced by an equivalent
amount of N-P-K.

The experiments of Hartley and Greenwood (1933) which
showed marked increases in crops after application of the
small dressings of i and 2 tons of farmyard manure, were for
some time believed to show a special value of farmyard manure,
since the equivalent amounts of nitrogen in the form of artifi-
cials failed to produce a similar effect. It was later shown,
however (Hartley, 1937) that increases similar to those given
by farmyard manure in these small dressings could be
obtained by the use of equivalent amounts of readily-avail-
able phosphate.

The remarkably long-lasting yield responses to farmyard
manure on one of the Hoosfield (Rothamsted) classical barley
plots, still await a satisfactory explanation. Plot 7-1 annually
received farmyard manure at the rate of 14 tons per acre from
1852 until 1 871, but has been unmanured since. The yields of
both straw and grain on this plot continue to be appreciably
higher than those on the unmanured plots or on those
receiving certain artificials without nitrogen. The average
yield (since 1871) is about the same as that of the plot annu-
ally receiving 206 lbs. of sulphate of ammonia only, but is
less than that of the plots receiving 275 lbs. of nitrate of
soda.

In this connexion, the rather neglected work of von
Liebenberg (19 16) may be recalled. For a rotation manured
once with a normal dose of farmyard manure and artificials,
Liebenberg showed that doses of 20 and 50 kg. of farmyard
manure per hectare, applied annually each autumn there-
after, produced appreciable, though small increases, in crops.
Considerable tabular details of yields and composition were
given. He wrote of the 50 kg. dose that nutrients as well as
organic matter were active, but in the 20 kg. dose "diirfte
wohl die organische Substanz allein oder in der Hauptsache
die ertragsteigernde Wirkung verursachen."

By Mr. N. W. Barritt the author has been shown quince

67

Plant Qrowth'Suhstances

prunings, such as are usually discarded as valueless, which had
developed well-marked roots after being "skewered" during
March and April, 1937, into a compost heap formed largely of.
grass and turves.

Root-induction and cell-elongation are distinct. Zimmer-
man, Hitchcock, and Wilcoxon (1936) recorded that "so far,
no growth-substances have been found to accelerate elonga-
tion in root-tissue. This peculiar response of roots raises the
question whether there might exist in nature an entirely
different type of hormone effecting elongation of the cells."
Thornton and Nicol (1936) noted that elongation of one type
of cell — the root-hairs of leguminous plants — was induced by
secretions of legume nodule bacteria. Some properties of
these secretions have been examined, but their chemistry is
still obscure. Bottomley (1915-17) found that legume nodules
were a potent source of auximones.

Vitamins

J. Bonner and Greene (1938) (with D. Bonner) have pub-
lished figures of the vitamin B^ content of three samples of
cattle manure; they found o -08-0 -13 mg. of vitamin Bj per
kilo, of dung. A laboratory culture of Azotobacter, the free
living nitrogen-fixing bacterium frequently present in fertile
soils, contained over a thousand times as much. Since vitamin
Bj (thiamin, aneurin) seems to have a potently favourable
effect on the growth of normal plant-roots, these findings may
be important. The authors do not quote the pioneering work
of Mockeridge (1920), but they say that the "long-disparaged
'auximones' of Bottomley" (1914, 1915-17) seem to rest upon
a sounder basis than has been hitherto admitted.

V. G. Lilly and L. H. Leonian {Science, 1939, 89, 292)
have reported a demonstration of vitamin Bj in soil.

Vitamin C was first used to accelerate root-production on
cuttings [Salix, willow) by Davies, Atkins, and Hudson
(1936), who found it to act similarly to indole-acetic acid,
while less toxic. See also p. 43.

68

Natural Sources

REFERENCES E

Bonner, J., and Greene, J. (1938), Vitamin B^ and the

growth of green plants. Bot. Gaz., 100, 226.
Bottomley, W. B. (1914) Annals Got., 2S, 531 ; Proc. Roy. Soc,

Lond., 88 B, 237; (1915-17), Proc. /?ojy. Soc. Lond.,89B,

102 and 481. See also Mockeridge (1920).
Breazeale, J. F. (1927), Vitamin-like substances in plant

nutrition. Arizona Agric. Expt. Sta.y Tech. Bull. No. 16.
Davies, W., Atkins, G. A., and Hudson, P. C. B. (1936).

Nature, 137, 618.
Hartley, K. T. (1937), An explanation of the effect of farm-
yard manure in Northern Nigeria. Empire Journ.

Exper. Agric, 5, 254.
Hartley, K. T., and Greenwood, M. (1933), The effect of

small applications of farmyard manure on the yields of

cereals in Nigeria, ibid., 1, 113.
von Liebenberg, A. R. (1916), Mitt, landw. Lehrk., Hochschule

far Bodenk. (Wien), 3, 225.
Link, G. K. K. (1937), Nature, 140, 507.
Mockeridge, Florence A. (1920), Biochem. Journ., 14, 432-
Niklewski, B. (193 1), Dostoiadczalnictwo Rolnicze, 7, Part HI,

3 (Polish, with 45 figs, and short French summary);

also Annales Agron. (193 1), 337.
Niklewski, B., Kahlowna, M., and Dyd6wna, M. (1935),

Uber die chemotropische Reizung der Wurzel. Rocz.

Nauk Roln. (Poznan), 34, 457 (Polish, with German

summary).
Niklewski, B., and Wojciechowski, J. (1937), Bodenk. u.

Pflanzenerndhr., 4 (49), 294.
Schreiner, O., and Lathrop, E. C. (1912), The chemistry of

steam-heated soils. U.S. Dept. Agric, Bur. Soils,

Bull. No. 89.
Schreiner, O., Reed, H. S., and Skinner, J. J. (1907), ibid.,

No. 47.
Schreiner, O., and Shorey, E. C. (1909), ibid., No. 53.
Schreiner, O., Shorey, E. C, Sullivan, M. X., and Skinner,

69

\

Plant Qrowth'Suhstances

J. J. (191 1 ), A beneficial organic constituent of soils:

creatinine. Ibid.y No. 83.
Shorey, E. C. (1907), Hawaii Agric. Expt. Sta., Rept. for

1906, p. 37.
Thornton, H. G., and Nicol, Hugh (1936), Nature, 137, 494.
Thornton, H. G., and Smith, G. (191 4-1 5), Proc. Roy. Soc.y

Lond.,88 B, 151.

70

CHAPTER X

SOME CONSTITUENTS OF URINE

Occurrence of Growth- Substances {other than the auxins
and vitamins) in Urine

Urine, in a heat below that of boiling water, yields a clear
liquor, of a very disagreeable smell, and which is found to
contain nothing saline. — Beaum6.

A LARGE number of substances able to affect growth and
differentiation in plants are to be found in urine. Some of
them (auxin-a, auxin-b) probably pass unchanged through the
digestive tract, and, if they do, are integral plant-products.
Others are fission-products of plant or animal protein, the
latter being broken down either endogenously (in the body
processes of the excreting animal) or exogenously (in the
digestive tract, which is outside the body proper; or, less
commonly perhaps, as a result of eating decomposed food,
e.g., high game). Still another class is formed by those animal
hormones which are capable of excretion in urine ; of these the
sex hormones are the chief.

In discussing urinary excretion, it should be borne in mind
that to speak of urine as if it were one material, is often
convenient, but does not always imply accuracy. The kind
and amount of substances naturally present in urines, or
excreted in urine after administration of a substance not a
foodstuff, vary widely with the species, sex, age, and idiosyn-
crasy of the animal. Thus, phosphates are abundant in
human urine, but are present in only small amounts in the
urine of horned cattle ; and hippuric and ornithuric acids, by
their very names, remind us of special occurrences. The
following outline should be read with such reservations, and

71

Plant Qroivth'Suhstances

with the further one that it is not intended as an introduction
to urine chemistry in the wider sense.

Normally, benzene compounds possessing short side-
chains are excreted as phenols. Free phenols sometimes exist
in traces in urine, but phenol and cresols are usually found in
urine as compounds of sulphuric acid and glucuronic acid.
Such compounds are said by biochemists to be "conjugated".
The combination (conjugation) prevents the phenols from
attacking the walls of the urinary passage. It will be seen
later that othier irritant substances also have their caustic
group "protected" or "detoxicated" by the formation of
nearly neutral organic compounds.

V

A

V

OK,HS0.

phenol

potassium

sulphate

phenol-

glucuronic

acid

/\°-7

CH

V

OH-C-H

O H-C-OH

OH-C- H

C-H

I
COOH

72

Constituents of Urine

These phenolic compounds are not growth-substances.
They represent the end-products of normal metabolism of
such substances as tyrosine.

Phenylalanine, a constituent of almost all proteins, is
oxidized in the body to phenyl-acetic acid, a growth-substance
of considerable potency, and to phenyl-propionic acid.
Phenyl-acetic and phenyl-propionic acids can also arise from
the metabolism of tyrosine. When fed to animals, phenyl-
acetic acid is said to form phenaceturic acid (a bland com-
pound of phenyl-acetic acid and glycine) but human beings
seem to be peculiar in employing glutamine as the detoxicat-
ing agent, forming phenacetoyl-glutamine.

It is interesting to note that glutamine is used to conjugate
phenyl-acetic acid only, and not its derivatives. Some of the
urinary phenyl-acetic acid in human urine is combined with
glucuronic acid.

Phenaceturic acid (phenylacetoylglycine), being a com-
pound of glycine with phenyl-acetic acid, is analogous to the
better-known hippuric acid (benzoyl-glycine) a compound of
glycine with benzoic acid. In fact the best natural source for
the isolation of phenaceturic acid, as for hippuric acid, is
horse urine, and both acids may be isolated from one sample
of urine. It appears that the urine of herbivores is richer
than that of carnivores in phenaceturic acid. The degraded
sweet odour of horse urine is probably referable to its phenyl-
acetic acid.

There seems to be a tendency for aromatic compounds
having a fatty acid side-chain to be excreted in urine (in
combination with glycine) as either benzoic or phenyl-acetic
acid. The substances which, when fed, yielded benzoic acid,
were those having an odd number of carbon atoms in the
chain (phenyl-propionic, phenyl-valeric acids); while those
acids having an even number of carbon atoms (phenyl-
butyric, phenyl-caproic) appeared as phenyl-acetic acid. It
will be noted that the formation of indole-acetic acid from
tryptophan (indole-a-amino-propionic acid) upsets the beauti-
ful regularity thus suggested.

The value of phenaceturic acid as a growth-regulating

73

Plant Qrowth'Suhstances

substance has not been tested, but it is easily hydrolyzed, and
probably has the same value as its equivalent of phenyl-acetic
acid. A few laboratory experiments have suggested that
glycine, when used as sole source of nitrogen in the growing of
plants, is fairly readily assimilable by plants, but such
experiments bear little relation to practice. Some may be
tempted to think that since phenaceturic acid comprises both
a growth-substance and a source of nitrogen, it might be an
especially valuable substance for plants, but it should be re-
membered that the amount of phenyl-acetic acid useful to the
plant is probably much less than a molecular equivalent of the
nitrogen required for growth. The growth-substance/nitro-
gen ratio in phenaceturic acid is i, when expressed in mole-
cules, but this is probably much too low for physiological
balance; if the plant is to take up from the phenaceturic
acid all the nitrogen it requires for growth, it will be left
with a possibly toxic excess of phenyl-acetic acid. The point
might be interesting to examine experimentally, but its
nutritional imphcations do not seem likely to be useful.
Phenaceturic acid, however, may find horticultural appHca-
tions as a source of phenyl-acetic acid in cases where growth-
regulation and not simple nutrition is the main object.

Hydroxy-phenyl-acetic acid in the form of ^-hydroxy-
phenaceturic acid has been reported in urine after administra-
tion of hydroxy-phenyl-ethylamine, but its importance under
natural conditions is not known. It is almost certainly a
growth substance. (See p. 65.)

Phenyl-propionic and p-^-hydroxy-phenyl-propionic acids
also have been reported in urine. 2 : 5-dihydroxy-phenyl-
acetic acid (homogentisic acid) occurs naturally in urine — in
the free state, apparently, not conjugated — in persons subject
to the rare condition known as "alcaptonuria". This is an
inborn error of metabolism, owing to which the sufferers are
unable to oxidize tyrosine and phenyl-alanine beyond the
stage of the 1:2:5 compound. Alcaptonurics do not appear to
suffer in health, but their urine blackens on exposure to air,
owing to the rapid oxidation of the dihydroxy-benzene

radical.

74

Constituents of Urine

Chemists may wonder how tyrosine (p-/>-hydroxy-phenyl-a-
amino-propionic acid) and phenyl-alanine (^-phenyl-a-
amino-propionic acid) acquire the 2:5 hydroxy groups.
It may be stated that proof of the physiological origin of
2 : 5-dihydroxy-phenyl-acetic acid from both tyrosine and
phenyl-alanine rests upon the increased excretion of homo-
gentisic acid after ingestion of either of the amino-compounds
by an alcaptonuric.

Alcaptonuria is too rare a condition to have an agricultural
importance, but consideration of it suggests that the black
matter of manure- and compost-heaps may be in part a
complex of oxidation products of dihydroxyphenols. For
further information about the acid, see the section "Nachweis
and Bestimmung der Homogentisinsaure im Harn", by W.
Weise, in Abderhalden's Handbuch (193 1), Abt. 4, Teil

5 (i). 551-560.

Coupling of a ring-acid with an ahphatic amino-acid
(conjugation) seems to be of a fairly general occurrence. The
methyl-pyridine compounds in 2-methylpyridine and pico-
linic acid, after administration by mouth, appears in the urine
of dogs, rabbits, and frogs, as pyridinuric acid, a compound of
picolinic acid and glycine, analogous to phenaceturic and
hippuric acids.

Picolinic acid administered to birds becomes combined with
the peculiar avian detoxicating amino-acid, ornithine, and is
excreted as pyridinornithuric acid. Ornithine is normally
found in combination with benzoic acid as ornithuric acid,
but acetyl-ornithine has been reported as a plant constituent
by Manske.

Indoxyl (hydroxy-indole) and skatoxyl are other phenols
found in urine, of carnivora particularly. They are usually
conjugated in the form of potassium sulphates; the indoxyl
potassium salt is known as urine-indican. Their effect on
plants is not known. Indole-propionic acid is stated to undergo
hydroxylation before excretion.

3-indole-acetic acid is the substance revealed by the old
"urorosein" test (see Chapter XIV). Its properties and a brief
account of its isolation from urine are given elsewhere.

75

Plant Qrowth'Suhstances

Creatine and creatinine, already mentioned on p. 50, are
important and constant constituents of urine. They can
almost certainly supply nitrogen to plants, at least after they
have undergone bacterial decomposition. It is not known
whether they can act as accessory growth-substances.

For much of the information given in this section, the
author has relied upon the textbooks of biochemistry of
H. H. Mitchell and T. S. Hamilton ("The Biochemistry of
the Amino Acids": Chemical Catalog Co., New York, 1929,
and of B. Harrow and C. P. Sherwin ("A Textbook of
Biochemistry": W. B. Saunders Co., Philadelphia, 1935),
and particularly in the latter the section on detoxication by
A. M. Ambrose and C. P. Sherwin. See also the sections:
"Nachweis und Bestimmung von Abkommlingen des Trypto-
phans im Ham", by W. Weise, in Abderhalden's Handbuch,
193 1, Abt. 4, Teil. 5(1), 765-784, and "Von Tryptophan
ableitbare biochemisch wichtige Verbindungen," by A.
Ellinger in the same Handbuch, Abt. i, Teil 7, 779-806,
especially for E. and H. Salkowski's method of preparation
from decomposed protein material.

To these and to similar sources the reader is referred for
references and more details.

76

^*^-,
>

CHAPTER XI

CHEMISTRY IN RELATION TO GROWTH

Some Notions on Concentrations

Inhibition and Stimulation

The grovyth-substances can act in extremely high dilutions.
By the Avena test, auxin-a has been shown to be active at a
concentration of i part in 110,000,000 of water; though
ethylene in some circumstances is from 100 to 600 times more
potent for equivalent molecular weights (Crocker, Hitchcock
and Zimmerman (1935), as corrected by Hitchcock and
Zimmerman (1935, A) ).

Acute disturbances not unlike the effects of poisoning can
be produced by the synthetic growth-substances in quite small
doses ; they are especially pronounced with young seedlings,
grown in solution-cultures, and the question has on that
account been asked whether it is not misuse of a term to call
the synthetics growth-promoting substances (Leonian and
Lilly, 1937). It is true that the substances do not always
promote growth, even in large plants (Hitchcock and Zim-
merman, 1935, A; Pearse, 1936-7A, ) though they may init-
iate or control it. The doubt evoked by the toxic phenomena
shown by seedlings is probably based on a misunderstanding
of results of an unrealized overdosage.

This contention has received support from the work of
Macht and Grumbein (1937) who have considered the
question of toxicity of indole-acetic and indole-butyric
acids and a naphthalene-acetic acid, from the point of view
of the so-called diphasic biological effect familiar to toxi-
cologists and pharmacologists. Their work is discussed in
more detail below. In the present connexion it will suffice to
quote a few words from their paper:

77

Plant Qrowth'Suhstances

"In the experiments reported by other writers (with the

exception of Amlong [1936]) only the toxic phase of the drug

action was recorded because of the concentrated solutions

employed and the longer periods in which such plants were

exposed to their action."

A fair size for the diameter of a plant cell is 0*02 mm.
Supposing the cell to be a cube of 0-02 mm. side, a seedling
of total volume of o-8 ml. will have 100,000,000 cells. If all
the indole-acetic acid contained in i-o ml. of a solution of
o-i gm. of the acid per litre is uniformly absorbed by that
seedling, each cell will contain 3 -5 thousand million molecules
of active substance. The number is roughly one-fiftieth of
the number of molecules in a volume of air at N.T.P. equal
to the volume of a cell.

Caution in dosage is still more necessary in working with
excised root-tips having an initial total volume of the order of
1 cubic millimetre. As the excised tips are usually surrounded
with many thousand times their weight of solution, the weight
of active substance in the solution will have to be infinitesimal
if harm is not to result.

Kogl et al. (1934) have shown that auxin-a can exert an
inhibition of root-elongation in young oat seedlings. The
efi"ect was perceptible when tap water bathing the roots con-
tained 0-0 1 mg. of auxin per litre. Indole-acetic acid was
found to exert a similar, but less powerful, inhibition.

Marmer (1937) has made observations on the inhibition of
primary root-elongation in wheat seedlings, testing the three
homologous acids, indole-acetic, -propionic, and -butyric,
with the refinement of working at two hydrogen-ion concen-
trations in phosphate-buffered solutions. At pVL 4-6 the con-
centrations causing a 50 per cent, reduction in root-growth
were respectively 0-012, 0-250, and 0-055 mg. per litre, while
at pW 7-5, the same results required respectively I7'58,
4'57, and 0-56 mg. per litre.

The magnitudes of such figures must depend very much
upon the conditions of experiment.

The apparent paradox that synthetic growth-substances,
capable of promoting growth of apical cells of seedlings, and

78

I

Chemistry and Qrowth

of causing initiation of adventitious root growth, inhibit
elongation of true roots, has been studied by Amlong (1936;
his paper has not been seen by the author) and by Macht
and Grumbein (1937). The latter workers studied a new
variable — the time of exposure of roots (of Lupinus albus ; white
lupin) to low concentrations of indole-acetic and indole-
butyric acids, and an unspecified naphthalene-acetic acid.
Concentrations as low as one part of acid in 5,000,000,000
of water or salt solution (/)H about 5) were tried. Instead of a
toxic effect, a small stimulation of root-growth was observed
24 hours after transferring the plants from growth-substance
solutions in which their roots had been dipped for 15-20
minutes ; an exposure twice as long usually produced a small
inhibition of root-growth. No estimate of error is given,
however, and it is not clear whether the plants not receiving
growth-substance were subjected to a control operation of
transference.

Hare and Kersten (1937) have appealed to a final absence
of toxicity, towards plant roots, of dilute aqueous solutions of
indole-propionic acid irradiated with ultra-violet light, as
evidence of the disappearance of the substance. (They give
reasons for thinking that methyl anthranilate was formed).

Unfortunately, none of the published values for minimal
doses of solutions affecting root-elongation is comparable with
the values for minimal doses able to produce cell-elongation
by the oat coleoptile test ; this is true even when oats were used
for determination of both types of activity. The minimum
dose producing bending of the coleoptile in the usual Avena
coleoptile test is determined from results of tests on single
plants, each plant being supplied with a minute dose of sub-
stance, of which it absorbs the whole or the greater part. On
the other hand, tests of the toxicity of growth-substances
towards roots have been made with batches of plants having
their roots bathed in relatively massive amounts of solution,
and the figures for the limiting doses that inhibit root-
elongation to a prescribed degree have not been furnished
as a dose absorbed per plant, as must be done for compara-
bility with the coleoptile test. In reported results of the oat

79

Plant Qrotvth'Suhstances

coleoptile test, limiting doses have been expressed as milli-
grams per plant (or what is in practice the same thing, a
prescribed degree of bending of so many plants per milli-
gram or gram of growth-substance), while the limiting values
for root-inhibition have been recorded as milligrams per litre
of solution, with either the number of plants or the volume
of solution left unspecified.

Grace (1937, C) has made an attempt to solve the paradox
of growth-inhibition caused by the synthetic growth-sub-
stances. He concluded that inhibition of growth was usually
due to overdosage. His colleagues Newton and Jack found
that as little as i part of a-naphthyl-acetic acid in 200,000,000
parts of water depressed the growth of wheat roots in length,
though not in weight. Grace's dust treatment of seed (see
page 46) apparently gets round the paradox by slowly releas-
ing the active agent in amounts so small as to be stimulatory.

An excellent review of the subject has been presented by
Borgstrom (1939) who has performed numerous experiments
in an attempt to resolve the paradox. He used genetically
uniform material [Allium) to determine the limits of con-
centration of various substances for the retardation or promo-
tion of growth. Inhibition at relatively high concentrations
was succeeded by a slight promotion at very dilute ones.^
Like most other workers in this field, Borgstrom worked with
roots attached to the plant, but he has carefully reviewed the
possible interactions between applied synthetics and the
plant's own hormones.

Borgstrom has also reviewed phytological eflFects of the
vitamins ascorbic acid (C) and thiamin (Bj). The effects of
these substances on roots is not restricted to a narrow range of
concentrations. He suggests, therefore, that they may have a
practical application, as they are "more convenient and far
safer to use as actually growth-promoting chemicals for roots".

'On page 79 the author has pointed out that there is no comparability
between results of tests on roots and shoots, just because there is no common
unit. Borgstrom has made a slip in thinking that the author brought this
contrast forward as an explanation of the paradox discussed in this chapter.
The author had no other intention than to make a plea to future workers to
use consistent, or at least comparable, modes of stating their results.

80

Chemistry and Qrowth

These two vitamins, according to Borgstrom, really acceler-
ate growth of roots, and therefore are growth-promoting, in
the sense used in this book.

Further evidence of a favourable action of vitamin B^ has
been provided by Bonner and Greene (1938, E) whose con-
clusions are interesting (see p. 68).

It may be suggested here that the glycerides of acids active
as growth-regulators might be safe vehicles of application.
They resemble the true fats in being non-volatile and in
being practically insoluble in water, but can hydrolyze slowly.
Methods of preparation of some lower phenyl glycerides are
given by Darzens (1937).

Plant-Growth Substances i?i Tissue Culture

Some very interesting work has been briefly reported by
LaRue (1935). He cultivated minute pieces of the hypocotyl
(first shoot) of dandelion, wild lettuce, and ox-eye daisy. A
mineral culture solution was used, one lot receiving indole-
acetic acid in the very high dilution of one part in 20,000,000.
Successful growth took place only in the culture receiving the
indole-acetic acid. The resulting plants developed numerous
leaves and extensive root systems, and were believed by
LaRue to be the first successful tissue cultures in which com-
plete plants had been grown. See also the paper of Gautheret
(1937), who found that indole-acetic acid in appropriate con-
centration was favourable to the growth of cambial tissue of
willow.

Kaufmann (1938) removed the cotyledons of seeds, let the
seeds swell for two days in water, then placed them in the
dark in media containing potassium salts, sugar (sucrose),^
and indole-acetic acid. A 24-hour treatment with concentra-
tions of one-millionth and one-hundred thousand millionth
per cent, of growth-substance induced maximal growth of
root.

Indole-acetic acid added to nutrient agar in which excised

^This obligatory use of sugar in cultivation of excised roots, for which
it replaces the normal photosynthetized sugar of whole plants, should not be
confused with the use of sugar mentioned on pages 43 and 44. For other work
on excised roots see p. 115.

81

Plant Qroivth'Substances

coleoptiles were grown retarded their growth in length
(Avery and LaRue (1938) ).

Duhamet (1939) has shown that indole-acetic acid inhibits
or accelerates the growth of isolated (excised) roots according
to dose, and has brought forward the fact that the substance
acts not only on cellular growth, but also on the proliferation
of root meristem. This he claims as new.

The effectiveness of salts, of esters, and of a nitrile as
growth-substances shows that the favourable action of the
synthetic growth-substances in proper concentration is not
due to acid irritation, as has been pretended by a Continental
school of plant physiologists. Quantitatively at least, the
action of the natural and synthetic plant-growth substances
accords with that of vitamins and hormones in animals. Very
small quantities of either of the latter suffice to exert an optimal
action upon the animal organism. The quantitative similarity
is strengthened if the animal doses are calculated per gram
instead of per kilogram of body-weight as usual. The animal
is to some extent protected against overdosage of vitamins by
the necessity of ingesting them through the gut. The plant
itself has no obvious protection against excess of auximones
offered to it in water solution, but it is possible that the
colloids of manure and of soil are the plant's defence in this
respect.

Chemical Constitution and Phytological Activity

The following compounds (three of which are heterocyclic)

have been shown to possess varying degrees of phytological

activity, when provided with an acetic-acid side-chain.

(R= — CH2.COOH). The names under the formulae are

^R

vV

N

indolyl-
(various)

/v

coumaryl-
Thimann

A

A

VV-" VV W

CH

indenyl-
Thimann

"thio-naphthenyl-
Shackell

82

Chemistry and Qrowth

those of workers who have tested the action of the derivatives
on plants, and are not necessarily those of the synthetizers.
Thimann's coumaryl-acetic acid was called the i-acid by
him, as he excepted the oxygen atom from numbering. It is
substituted in the same position as is the better-known
coumarilic acid, called coumaryl-2-carboxylic acid by
Heilbron (1934).^

Shackell (1937), who reported the preparation of ^thio-
naphthene-3 -acetic acid (by whom, is not clear, but see also
the paper by Crook and Davies (1937) ) found that compound
inactive in the Avena (oat) test of coleoptile bending, but
active in fairly high dilution by Went's etiolated pea method
(see p. 129).

Thimann's results are most succinctly given in the form of
a table, which also brings out the typical difference in activities
revealed by different methods of assay, thus suggesting how
much caution should be brought to attempts to correlate
chemical constitution with an unspecified "activity".

Table. — Approximate Activity expressed as Percentages
OF that of Indole-3-acetic Acid. (Thimann (1935) )

By Went's

By

By

By

By

(1934, J)

inhibition

root-

Avena

Avena

etiolated

of bud-

formation

coleoptile

straight

pea

formation

on etiol-

curvature.

growth.

method

on etiol-

ated pea

ip. 129).

ated pea
stems.

cuttings

Went (ig24

A).

Indene-3-

acetic acid ...

I

7

20

14

definite'

Coumaryl-i (2)

-acetic acid...

0

about 0-3

4

4

definite*

^ The opportunity has been taken of correcting some mistakes v^^hich
were made in Part III of the article in the Manufacturing Chemist (August
19:^7) before the author had access to the original of Thimann's 1935 paper.

* True naphthenes are C2H2n. Thionaphthene is analogous to indene
and indole, but the name thio-indene is not, however, sanctioned by custom,

* Dependent on mode of application, etc.

83

Plant Qrowth'Suhstances

Four other furane derivatives tested by Thimann were
found to be completely inactive by the two Avena tests.

For synthesis, and effect on plants, of coumaryl-2(3 ?)—
acetic acid, see V. Titoff, H. Miiller, and T. Reichstein,
Helv. Chem. Acta, 1937, 20, 883 (from B.C. A).

A number of discrepant results have been reported about
the "activity" or "inactivity" of substances, in relation to
their effects on growth. Substances judged as "inactive" or
only slightly active, by workers using one test, have been found
highly active by later workers using a different test-plant and
technique, and possibly evoking a different type of physiolo-
gical effect on cell-growth, proliferation or differentiation.

For this reason, amongst others, it has not seemed worth
while to list and classify the numerous substances of which
the physiological activity has been tested on plants.

A comparison of three plant tests (oat-coleoptile, etiolated
pea stem, and "cylinder") has been made by Haagen-Smit
and Went (1935), using a large number of synthetic sub-
stances, mostly of the indolyl and phenyl types. No naphtha-
lene or higher aryl compound was tested in this series. AllO'
cinnamic acid, ethyl wo-propyl indole- acetate, and 5-methyl-
indole-acetic acid were reported to be equally as eflFective as
3-indole-acetic acid in the (Went's) pea test. Examined by
the other two tests, no substance tried was more than a fifth
as effective as indole-acetic acid, and only indole-3 -pyruvic
acid, i-methyl-indole-3-acetic acid, 5-methyl-indole-acetic
acid, and a//o-cinnamic acid reached that degree of effective-
ness. Though so eflFective in the pea and cylinder tests, allo-
cinnamic acid and i-methyl-indole-acetic acid were only
weakly effective in the Avena coleoptile test, in which they
produced an "abnormal" type of bending. Ordinary cinnamic
acid was inactive in all three tests.

These authors wrote: "That steric conditions play an
important part in the activity of these substances was con-
firmed by the activity of m-o-methoxy-cinnamic acid, whilst
the trans compound was inactive. In the meantime, the sub-
stitution with the methoxy groups has greatly diminished the
activity." Cw-o-methoxy-cinnamic acid, however, was

84

Chemistry and Qrowth

inactive in the Avena coleoptile test. The effects of other
substitutions were briefly discussed.

The differing activities of the artificial growth-substances
has been referred to their different behaviour towards what
plant physiologists call "polar transport" in the plant.

An inhibitory influence of the terminal bud of plants upon
the lateral ones has been known for a long time : it is the basis
for some operations of pruning. It has been shown by Thi-
mann and Skoog {1933) and others that the effect is due to
nothing else but the production of auxin by the terminal
bud : auxin is able to prevent lateral buds from developing.

Van Overbeek (1938) has attempted to explain how this
inhibition might work. Went (1938) found that phenyl-
butyric acid has no activity in the Avena test, yet is able to
inhibit bud development to a considerable degree. Van Over-
beek made use of this property of phenyl-butyric acid in order
to be able to distinguish between the effects of hormone (auxin)
produced by the plant, and the effects of an artificial growth-
substance (phenyl-butyric acid) applied externally. Whereas
other workers have recorded amounts of auxin or the activities
of natural or synthetic growth substances in Avena units and
thus in terms of auxin itself, Van Overbeek employed the
novel unit of "gammas of indole-acetic acid equivalent per
1,000 gm. of water contained in the plant". This is a step
towards standardization in terms of a known and widely-
accessible pure substance.

The reaction of dormant leafless cuttings of willow to
lanolin pastes or water solutions of indole-butyric acid has
been studied by Pearse (1938). The formation of roots was
greatly stimulated. When the portion of the base that had
been treated was removed, the effect of the treatment was
eliminated. A second treatment applied to a truncated
cutting shortly after the end of the first treatment (by a 24-
hour immersion in growth-substance solution) induced root-
formation. Pearse thought therefore that the indole-butyric
acid itself was the active agent that promoted root formation,
and was itself used up or chemically changed in the process.
He also thought that it may be more effective to apply

85

D1

Plant Qrowth'Suhstances

growth-substances to the apical end of dormant woody
cuttings. {Cf. Cooper (1935, A)).

No general correlation between chemical structure and
phytological activity can yet be postulated. A substance
affecting growth in one species of plant may be inactive
towards another species, and, even within the simple series of
analogues given at the head of this section no attempt to
correlate activity towards plants with the 3-substituted
position can logically be made.

D. Bonner (1938) has suggested that "compounds posses-
sing the essential molecular structure probably all have the
same activity in the ^H-dependent and stoichiometric grovvth
reaction, and that the differences in their observed activities
are due to differences in activities in secondary processes."
The latter may perhaps refer to Went's biphasic phenomenon
(p. 60).

By correcting for difference in /)K, so that only equimolecu-
lar concentrations of the free acid were compared, he found
that m-cinnamic acid had the same activity in the pea test as
has 3-indole-acetic acid, which, on this mode of calculation, is
superior to phenyl-acetic acid. He claimed to find a correlation
between the dissociation curves and the activities at different
internal pH of m-cinnamic and phenyl-acetic acids.

For preliminary work on the effect of ^H on the number of
roots of pea formed in indole-acetic acid solution (an "opti-
mum" curve was established), see Hubert (1938).

A short review of the chemistry and actions of growth-
substances, from the standpoint of plant physiology (hormon-
ology) rather than from that of the efficacy of external applica-
tions, has been given by Thimann (1938).

A more detailed discussion from the chemical point of view
has been given by Koepfli, Thimann and Went (1937-8).

Tincker (1936 a, A) has reported that tyo-indolinone-3-
acetic acid (synthetized by Professor F. M. Rowe of Leeds)
was inactive towards plants. Indole has been reported as
inactive towards plants, but according to Glover (1937), the
activity of skatole in the oat-coleoptile test appears to have
been of the same order as that of indole-acetic acid. . His

86

Chemistry and Qrowth

suggestion that skatole may act in the plant as a growth-
promoting hormone fails to convince, for lack of evidence that
skatole is elaborated by the plant.

The report by Snow (1937) that benzoyl oxide and peroxide,
applied in lanolin, produced perceptible bending in dark-
grown oat coleoptiles, further complicates the issue. The
activity of these substances was, however, only about one
four-hundredth of that of indole-acetic acid.

^/-indolyl-lactic acid was the only one of several indole
compounds prepared by Bauguess and Berg (1934) that was
inactive as a growth-substance (Bauguess, 1935). With one
exception it appears to be the only substance capable of
optical activity or resolution (other than /-tyrosine and /-try-
ptophan) of which the growth-regulating activity has been
tested, hence nothing can be said at present about the relative
growth-regulating power of most optical isomers.

The exceptional case which has been investigated is that of
a- (Pi indolyl) -propionic acid, synthetized by Kogl (1938)
by a method not stated. This substance is optically active (its
racemic form is probably the same as Ellinger's indole-
methyl-aceticacid,p. 102 and 104). Kogl found that the Avena
activities of the racemic (+), and ( — ) acids were, in lo*
A.U. per gram, respectively 23, 48, and 1-6. Thus the (+)
acid is about 30 times as active in the Avena test as the
opposite enantiomorph is. See page 23 for reference.

Compounds related to tyrosine as well as those related to
tryptophan have physiological activity. Extracts of thyroid
gland (containing thyroxin, an iodo-derivative of tyrosine)
were among the first of the animal hormones shown to affect
growth in plants (Nicol, 1937). Hammett (1936) writing
from a zoological standpoint, has suggested that attention
given to the iodine in thyroxin has overshadowed the import-
ance of its tyrosine residue.

As a matter of interest, and without comment (except to
point out that creatinine and creatine have not been shown to
be growth substances in the modern sense of an accessory
rather than a nutrient), the formulae of auxin-a, indole-3-
acetic acid, creatinine, and creatine may be given side by side :

87

Plant Qrowth'Suhstances

H,C

f^5(?)HC-HC

ch-chC£7h

\

CH

C-CHOH-CHj-('CH0H),-C00H

au.Tin-a

HN-

indole-3-acetic acid

CH,-COOH

HN-

hUC-N

HN:C

creatinine

--0H

•CO

•CH.

HC

\

COOH
C

N=

■CH

^CH

\

/

CH

nicotinic acid;
pyridine-S-carboxylic acid

H,N-C-N-CH2-COOH
^ II I
HN CH.

creatine

88

Chemistry and Qrowth
REFERENCES F

Book:
Heilbron, I. M. (1934), Dictionary of Organic Compounds.
London.

Periodicals :
Amlong, H. U. (1936)^^/1^6. zviss. Bot., 83, 773. (As quoted

by Macht and Grumbein (1937) ).
Avery, G. S., Jr., and LaRue, C. D. (1938). Bot. Gaz., 100,

186.
Bauguess, L. C. (1935), Amer. jfourn. Bot., 22, 910.
Bauguess, L. C, and Berg, C. P. (1934), Jowm. Biol. Chetn.,

104, 675.
Bonner, D. (1938). Bot. Gaz., 100, 200.
Borgstrom, G. (1939), Botaniska Notiser, 207.
Crocker, W., Hitchcock, A. E., and Zimmerman, P. W.

(1935), Similarities in the effects of ethylene and the

plant auxins. Contrib. Boyce Thompson Inst., 7, 231.
Crook, Eric M., and Davies, William (1937), Journ. Chetn.

Soc, 1697.
Darzens, G. (1937), Compt. rend., 205, 682.
Duhamet, L. (1939). Compt. rend., 208, 1838.
Gautheret, R. (1937), Compt. rend., 205, 572.
Glover, J. (1936), Nature, 137, 320.
Haagen-Smit, A. J,, and Went, F. W. (1935), Versl. K. Akad.

Wet., Amsterdam. 38, 852.
Hammett, F. S. (1936), Protoplasma, 27, 52.
Hare, Dorothy, and Kersten, H. (1937), Plant Physiol., 12,

509-
Hubert, B. (1938). Biol. Jaarhoek, 5, 321 (in English).

Kaufman, E. (1938). Naturwiss., 26, 773.

Koepfli, H. W., Thimann, K. W., and Went, F. W. (1937-8).

Journ. Biol. Chem., 122, 763-780.
K6gl, F., Haagen-Smit, A. J., and Erxleben, Hanni (1934),

Ztschr. physiol. Chem., 228, 104 and 121.
LaRue, C. D. (1935), Amer. Journ. Bot., 22, 914.
Leonian, L. H., and Lilly, V. G. (1937), Amer. Journ. Bot.,

89

Plant Qroivth'Suhstances

24, 135; see also Solacolu, T., and Constantinesco, D.,
Compt. rend., (1936), 203, 437.

Macht, David I., and Grumbein, Mary L. (1937), Amer,
Journ. Bot., 24, 457.

Marmer, Dina R. (1937), Amer. Journ. Bot., 24, 139.

Nicol, Hugh (1937), Animal hormones and plant growth.
Chetn. and Ind., 56, 23.

Overbeek, J. Van (1938), Bot. Gaz., 100, 133.

Pearse, H. L. (1938). Annals Bot. N.S., 2, 227.

Shackell, Ethel M. (1937), Austral. Journ. Exper. Biol., 15, 33.

Snow, R. (1937), Nature, 139, 27.

Thimann, K. W. (1935), Verslag. K. Akad. Wet., Amster-
dam, 38, 896,

Thimann, K. W. (1938). Plant Physiol, 13, 437.

Thimann, K. W., and Skoog, Folke (1933). Proc. Nat. Acad.
Sci., 19, 714.

"Went, F. W. {1938). Specific factors other than auxin affect-
ing growth and root development. Plant Physiol., 13, 55.

90

CHAPTER XII

I

CLASSIFICATION AND NOMENCLATURE OF
PLANT GROWTH-SUBSTANCES

Becher and Stahl, pursuing further and further the combina-
tions of the primitive principles, and of those which they
term secondary, establish diiferent orders of compound
bodies, to which they give improper denominations. The
signification of many of these terms is even contrary to the
ideas usually affixed to them, and is liable to occasion
obscurity. — Beaum6.

From at least a historical point of view, the three following
growth-substances seem to form a class : auxin-a (auxentriolic
acid); auxin-b (auxenolonic acid); and "heteroauxin" (3-
indole-acetic acid). To these may be added the synthetic
substances, homologous with or akin to indole-acetic acid,
and principally interesting the readers of this book.

In addition to these, however, there is a bewildering variety
of more or less well-characterized substances derived from
animal glands, for at least some of which claims have been
made that they are capable of affecting plant growth (see, for
example, the paper by Havas and Caldwell, 1935). Amongst
these may be mentioned the male and female hormones, the
chemistry of which has reached a fairly advanced stage,
owing largely to the work of Marrian and of Butenandt. For
the chemistry and names of most of these substances, the
books by Harrow and Sherwin (1934) and Bredereck (1936)
should be consulted.

Another, but ill-defined, class of substances is that known
chiefly for its effects on microbial growth. Amongst these
may be mentioned Williams's (1933) "pantothenic" acid, so
named on account of its wide occurrence in living material,

91

Plant Qrowth'Suhstances

and Allison and Hoover's (1933) "co-enzyme R", which can
be obtained from cane sugar; also reported to be widely
distributed.

One more class of substances needs to be mentioned as
having growth-regulating properties towards plants, and that,
strangely enough, is formed by the vitamins, especially
vitamins C (ascorbic acid) and Bj (thiamin).

This outline classification has been made partly for the
sake of completeness, and partly to render intelligible the
paragraph on nomenclature which follows: it is uneasy to
discuss the action of any group of substances on plants unless
we are sure which group we are discussing. No doubt the
borderlines between the above-mentioned groups will shift
as well as become clearer with increasing knowledge. As
things are, it would be a pity to lump together in one com-
prehensive group such substances as : auxenolonic and ascor-
bic acids; the phenyl-, indolyl-, and naphthyl-acetic acids
and esters and nitriles thereof; together with adrenalin and
impure theelin: under some such name as "phytohormone",
merely because they can all affect plant growth in some way.
Cf. Eyster and Ellis (1924, C).

What a Hormone is^

The term "hormone" is inapplicable to designate a class of
substances which affect the plant's growth only after having
been applied from without. The first hormone was discovered
by Bayliss in 1902. It was a substance (not yet properly
identified) secreted by a part of the intestine, and acting as a
sort of release for digestive processes lower down. Pre-
viously, it was thought that body processes were controlled
by telegraphic impulses along nerves, but Bayliss showed that
some processes were controlled, not by intangible messages,
but by actual substances. The word "hormone" was adopted
to signify a chemical messenger. Whereas a painful sensation,

* Part of this section, as well as the introductory paragraphs of Chapter I,
have been reproduced from the article "What is a plant-hormone?" (Fertil-
iser, Feeding Stuffs, and Far?n Supplies Journal of 28th July, 1937, 23, 407)
by kind permission of the Editor of the Fertiliser.

92

Nomenclature

for example, is like an unpleasant telegram, a hormone can be
likened to a key, a bundle of manuscripts, or a sample of
merchandise — that is, something passing between two parties
and essential to the carrying out of a transaction.

Note, however, that Bayliss's hormone, and the many
others discovered since, all pass between two parts of the same
body. A medicine or a poison administered from without may
produce a very marked result on the body, but it is not there-
fore a hormone. Vitamins are not hormones, because they
have to be supplied to the human or animal body from a
plant source — that is, from outside.

Phenyl-acetic acid or its more powerful relatives, such as
indolyl-acetic acid, produce remarkable effects on plants,
particularly by way of initiating adventitious root-growth.
They are, however, more like vitamins than they are like
animal hormones, and it would be fair to call them vitamins-
for-plants. In any case, it would be misleading to continue
to call them hormones, unless it can be proved that they are
formed within the plant and act as natural chemical messen-
gers within it.

There can be no objection to an extension of the meaning
of the word "hormone" to connote substances so acting in
plants. There is a small class of growth-substances, which do
occur in plants and do regulate growth within them. Vitamin
C is produced in the plant and regulates some growth-
processes within it (von Hausen, 1936). On the basis of von
Hansen's work, vitamin C is a type of true plant-hormone,
in which class come also the two auxins, the plant female
hormone of Butenandt, and possibly a male hormone. On
the other hand, such products as those of decomposition and
of putrefaction, whether derived from processes taking place
in the animal gut or in the compost heap, or derived from
synthetic processes, are supplied externally to the plant and
cannot be called hormones : they are auximones, or vitamins-
for-plants. Unless it can be proved that the "synthetic"
growth-substances occur naturally in plants, and regulate
growth within them, it would to-day be as absurd to give the
name "hormone" to these exterior substances which affect

93 oa

Plant Qrowth'Suhstances

plant growth as it would be to confuse vitamins with hormones
in speaking of animal physiology.

Link et al. (1937, H) have proposed a complicated nomen-
clature for all growth substances.

Their "autoauxone" is apparently an unnecessary synonym
of "hormone" {sensu stricto sive plantar um sive animalium) . A
hormone is excellently characterized by the phrase of Link et
al. : "produced by the individual whose development it
affects." "Auxone" is much the same as Bottomley's "auxi-
mone", and not obviously preferable thereto. The additional
term "heteroauxone" (or possibly "heterauximone") brings to
the nomenclature a little more definition than does "auxim-
one" alone, but only if "auximone" is used in the most
general sense to mean any accessory substance (not an
ordinary nutrient) affecting growth and cell-differentiation.
It appears that historical and linguistic propriety would be
satisfied if the term "hormone" were reserved in its usual
sense, and "auximone" (or "phytamin") were used for
substances affecting growth of an individual not producing
them.

Link et al. have performed a useful service in pointing
out the need for distinguishing between different modes of
tissue growth, but the necessary distinction can be made with-
out resort to ponderous words to designate the incitants of
such growth-modes.

An unexceptionable definition of a hormone is that given
by Went and Thimann in their book (mentioned on p. 23).
It is:

"A hormone is a substance which, being produced in any
one part of the organism, is transferred to another part and
there influences a specific physiological process."

This covers both animal and plant hormones. If it is
desired to distinguish hormones of plants from those of
animals, the former may be called phytohormones. This term
is widely known, though comparatively seldom used in its
proper sense ; it is often used to connote a synthetic growth-
substance, i.e.y a drug applied from outside. The acceptance of

9+

'Nomenclature

phytohormone may ease the way for the word phytamin. The
latter term has been tentatively accepted by Loehwing (1937)
and has been substantively used in its French form phytatnine
(f.) by some Italian and French authors.

The definition of a hormone given by Went and Thimann
excludes drugs, and is sound from a physiological point of
view. It is to be hoped that other physiologists working with
phytohormones or interested in the effect of synthetic drugs
will call upon their physiological training so as to discriminate
between what is a hormone and what is not.

The physiologists present at the First Phytohormone
Conference, held in Paris in October 1937 under the chair-
manship of Professor Boysen Jensen, apparently viewed the
matter in a similar light. For their preliminary classification,
see Chronica Botanica (1938), 4, 48.

Lefevre (1939) has recorded the concentrations of 3-indolyl-
acetic acid in fresh leaves and roots of various normal and
pathological plants. If the natural occurrence of this substance
in plants is accepted, it may be a true hormone.

Group Nomenclature for Plant Growth- substances

In 1934 the author suggested the name "phytamin" as a
generic term for the accessory food factors of plants (vitamins-
for-plants), for which numerous names and adjectives have
been proposed (auxins, plant hormones, growth-regulating
and growth-promoting substances, etc.). The name phytamin
was intended to cover the sense of Bottomley's (191 5-1 7, C)
"auximone", of which Bottomley wrote: "This term may
usefully serve as a general descriptive name for these organic
plant growth-promoting substances until our knowledge of
their true nature and composition is sufficiently extended to
warrant the application of a more satisfactory name." The
author proposed the term "phytamin" in substitution of
"auximone", because Bottomley's work was generally dis-
credited as a result of the exaggerated claims made for his
commercial preparation, "bacterized peat".

Bottomley may have been led too far in his inferences, but
it can now be suggested that his laboratory demonstrations

95

Plant Qrowth'Suhstances

of the growth-promoting value of extracts from rotted organic
matter were sound.

In view of this, it seems desirable to restore the term
"auximone", which is historically valid. It should be remem-
bered that growth-regulation and growth-promoting are
not synonymous. If "auximone" is thought not to be logical
(Greek auximos : promoting growth) there would seem to be
no alternative to the generic use of either "phytamin" or
growth-regulating substance: the latter may, however, be
shortened to growth-substance.

The term growth-substance, or growth-regulating sub-
stance in the current sense as applied to plants, was intro-
duced by R. Snow about 1928.^ It is admittedly not free from
objection, being vague (Avery (1937) ) but, if used in con-
junction with the adjective "synthetic", expressed or under-
stood, it at least avoids confusion with hormonal substances.
These also are, of course, growth-substances or -factors, but,
being hormones, such can be definitely named as a class. We
thus have the division of all plant-growth substances into :

(i) Hormones (phytohormones) :

(a) auxins (auxin-a, auxin-b).

(b) vitamins (B^, C, . . .)

(c) rhizocaline, florigen, . . .

(2) Synthetic growth-substances (drugs).

This classification is not ideal, for it suffers from the classifi-
catory disadvantage that vitamin C is a true hormone in the
plant, but when applied as a drug it enters division (2),
producing as a drug a possibly different effect from that which
it brings about hormonally.

Division (2) might be divided in terms of Went's biphasic
actions, but his name sub-auxin for e.^., phenyl-butyricacid,
does not seem logical, as that substance is not an auxin or
chemically auxin-like, and is not a hormone.

The term "auxin" for the whole class of growth-substances

^The acknowledgment given to Dr. T. Eden in the first edition was meant
to thank him for bringing this term to the author's notice — not that Eden has
introduced the term into general use. It is regretted that this was not made
clear.

96

Nomenclature

is undesirable so long as it is also used as a trivial name for
the substances at present known as auxentriolic acid (auxin-a)
and auxenolonic acid (auxin-b),

Williams (1928) has suggested the word "nutrilite" to mean
any substance that affects the growth of organisms, but the
need for such a broad term does not seem to have been
generally felt.

REFERENCES G
Books

Bayliss, W. M. (1924), Principles of General Physiology

(London). See especially the section on hormones, p.

712, and the remarks of Gley and others on nomenclature

and action, p. 713.
Bredereck, H. (1938), Vitamine und Hormone und ihre

technische Darstellung (Leipzig). 2nd edn.
Harrow, B., and Sherwin, C. P. (1934), The Chemistry of the

Hormones (Baltimore).

Periodicals

Allison, F. E., Hoover, Sam R., and Burk, Dean (1933),

Science, 78, 217.
Avery, G. S., Jr. (1937). Ohio Journ. Sci., 37, 317.
Hausen, Synnove von (1936), The role of vitamin C in the

growth of higher plants. Annal. Acad. Sci. Fenn., Ser. A,

46, No. 3. 134 pp.
Havas, L., and Caldwell, J. (1935), Annals Bot., 49, 729.
Lefevre, J. (1939). Compt. rend. Soc. Biol., 130, 225.
Loehwing, J. (1937), Bot. Rev. ,3, 195.
Nicol, Hugh (1934), Biol. Rev., 9, 383, esp. p. 395.
Williams, R. J. (1928), Science, 67, 607.
Williams, R. J., et al. (1933), Journ. Amer. Chem. Soc, 55,

2912.

97

CHAPTER XIII

SYNTHESIS

(With notes on the auxins and vitamins)

*

Putrefaction may, I conceive, be considered as a spon-
taneous analysis, without heat; or a subsidence and laceration
of the particles of bodies, by the weight of their mass, and by
the dilatation of the fluids they contain, but aided by the external
heat of the atmosphere. This spontaneous analysis disengages
the aqueous, oily and saline principles of which the bodies
consist. — Beaum6.

In company with many foul-smelling substances, there occur
amongst the products of decomposition and putrefaction a
number of ring compounds having a pleasant odour. Some of
them, such as phenyl-acetic acid, have a use as perfumery
synthetics, but their discovery in putrescent meat and the like
was made long before any commercial application was found
for them.

Indole-acetic acid has long been known as a product of
natural decomposition of protein. Pathologists have devised
a method of testing — the "urorosein" test — for its supposed
presence in urine (see p. 123). A positive result from this test
has been stated to be suggestive of intestinal disorder. There
is little doubt that indole and indole-acetic acid in nature are
derived from the breakdown of trytophan contained in many
proteins.

It has recently been found that many ring-substituted fatty
acids possess remarkable powers of altering the modes of
growth of plants. The first of these compounds to be chemi-
cally identified after its growth-regulating properties had been
recognized was indole-acetic acid from urine. It was originally
called "heteroauxin" by the plant physiologists. Indole-acetic
acid occurs in urine much more frequently than the old uroro-

98

Synthesis

sein test indicated, and by the sensitive methods of plant
physiology, it can be shown that by virtue of their content of
"auxins", most urines are capable of affecting cell-growth in
plants. Phenyl-acetic acid is also capable of affecting plant
growth, as are a number of homologues.

Effect on Plant Growth

Looking back, it seems a little remarkable that more
curiosity was not shown by agriculturists and plant physiolo-
gists about the effects on plant growth of the products of
protein breakdown. From consideration of the universal
esteem in which composts and animal manures have always
been held, it might reasonably have been deduced that some-
thing was present in them that was not explainable in terms of
N,P,K, and "humus". The fact is that elemental chemistry
(with some quasi-mystical appeals to the unknown humus) has
held this field. Investigation of the ability of certain products
of protein breakdown to regulate growth in plants began
without references to the origin of the substances ; but when
the composition of "heteroauxin" had been ascertained, that
substance was found to be identical with the long-known
decomposition-product, indole-acetic acid.

Already a considerable number of substances shown to
possess growth-regulating properties in plants have been
found to occur naturally, and many have been synthetized. A
description of the chemistry alone of these substances would
fill a small book, which would be out of date as soon as printed.
Therefore, no attempt will be made here even to list the
recorded growth-regulating substances, but an idea will be
given of the synthetical preparation of some representatives
of each complex group, excepting only the active phenyl
compounds such as phenyl-acetic, phenyl-propionic, and
fl//o-cinnamic acids. Interested chemists, having consulted
the standard textbooks, will find more recent details in the
substantially complete references at the end of this chapter.
Being intended to introduce some physiological, as well as
chemical, notions, the account is not presented in an entirely
logical chemical order.

99

Plant Qrowth'Suhstances

Positioning and Nomenclature

The indole molecule consists of one five-membered
(pyrrole) ring (composed of one NH and four CH groups)
having two CH groups in common with a benzene ring.
Numbering of substituents in the pyrrole ring usually com-
mences with the N group as i , proceeding around the pyrrole
ring away from the benzene ring. The pyrrole nucleus some-
times bears the letters a and p in place of 2 and 3, substituents
attached to the nitrogen atom then having the prefix N.
The practice of using Greek letters to denote position in
the indole nucleus is likely to lead to confusion, especially in
considering compounds, like tryptophan (3-indole-a-amino-
propionic acid), in which Greek letters also serve to indicate
positions in a side-chain.^ The prefix Pr has also been used to
indicate substitution in the pyrrole ring. The use of this
prefix, which does not affect the numbering as given above, is
almost extinct.

Most of the naturally occurring indole derivatives are
substituted in the 3-(P) ring position. 2-substituted derivatives
are perhaps less often synthetized than the 3-substituted,
though 2-carboxy-3-methylindole (2-carboxy-skatole ; isomeric
with indole-3 -acetic acid) was made by Wislicenus and Arnold
as long ago as 1887. Recently, indole compounds substituted
in the benzene ring have been synthetized and tested for
activity upon plant growth. The prefix bz- is used to indicate
substitution in the benzene nucleus, numbering then proceed-
ing round the benzene ring consecutively with the number-
ing of the pyrrole ring. The atoms common to both rings are,
as usual, not numbered.

The ring nomenclature of the indolyl and related com-
pounds is not standardized. Sometimes the termination -ole
is used, sometimes -olyl ; in the case of relevant derivatives of
naphthalene and other higher hydrocarbons, the tendency
seems to be to retain the ring name unchanged. This con-
fusion is unfortunate, but no greater degree of precision can
be expected while the Chemical Society (Smith, 1936) write

^ The author has used the a- and ^- notation in discussing the naphtha-
lene compounds, instead of using the more modern i- and 2-.

100

Synthesis

benzenesulphonic acid and phenyl-acetic acid, the simple
CgHg group having two names while fulfilling similar func-
tions. The Chemical Society appear to prefer the form
indole-acetic to indolylacetic, but, to the author, the latter
seems desirable from the analogy of phenyl-acetic. Jackson
and Manske (1935) have introduced the term indylene to
indicate at least some di-substituted derivatives of indole, in
analogy with phenylene.

Typical Formulae of Growth-regulating and Allied
Substances of Physiological Importance.

CH(4)

C3)HC

(6)HC

CM (5)

CH(2)

/\

CH3

\y\/^"'" \/\y

CH
(7)

NH

Indole;

henz-pyrrole.

CH : OH: NH

6 4 2 2

N

Skatole;

S-methyl-indole;

Pr-S-methyl-indole;

fi-methyl-indole.

/\

1/3)

C«)

/\

N-CH3

1-methyl-indole;
N-methyl-indole.

TyQ) (of)

ch2.chCnHj)cooh

N

Tryptophan;

a- {3-indole)-amino-propionic acid

(optically active).

101

Plant Qrowth'Suhstances
Typical Formulae contd.

/\

CHvCOOH

N

Indole-acetic acid;
S-indolylacetic acid.

CH

COOH

N

2-car'boxy-skatole ;
2-carboxy-3-methyl-indole.

CHgfCOOH

Acenaphthyl-5-acetic acid.

CHj'COOH

A nthraceneacetic acid.

/X

CH3
CH-COOH

N

(S-indolyl)-a-propionic acid.
[Presumed configuration of EI-
linger's {1905) indole-meihylacetic
acid]

CHj-CN

a-naphthylacetonitrile.

102

Synthesis

•CH2COOH

Fluoreneacetic add.

Early Work on Indole Compounds

Indole-acetic and -propionic acids bear a close relationship
to the tryptophan constituents of proteins. An excellent
outline of the earlier researches on the constitution and
synthesis of indole-acetic acid and its congeners is given in the
article "Tryptophan," by Martha A. Whiteley, in Thorpe's
Dictionary of Chemistry (191 6 edition). Tryptophan is
3-indole-a-amino-propionic acid. It is somewhat surprising
to learn that tryptophan, discussion of which now occupies a
prominent place in many textbooks on biochemistry, was
discovered so recently as 1901 (Hopkins and Cole), though the
name dates from 1890. Skatole is 3-methyl-indole. A sub-
stance called skatole-acetic acid was isolated by Nencki in
1889. The synthesis of indole-3 -acetic acid was not accom-
plished until Ellinger in 1904 performed it to follow up some
problems of constitution raised by the work of Nencki and of
Hopkins and Cole. Ellinger showed that it was identical with
what Nencki had called skatole-carboxylic acid. In 1905
Ellinger further showed that the substance called skatole-
acetic acid by Nencki was identical with indole-3 -propionic
acid. The preparation by Hopkins and Cole (1903) of indole-
acetic acid from /-tryptophan by the agency of an anaerobic
bacterium has now only a historical interest for chemists, but
as it introduces the time factor, it retains an interest for
those concerned with the biological production of growth-
substances.

103

Plant Qroivth'Suhstances

First Syntheses

Emil Fischer's method of ring-closure was used by Ellinger
(1904) for the first synthesis of indole-3-acetic acid. The
phenylhydrazone of methyl aldehydo-propionic acid, on
heating with zinc chloride, or, better, by prolonged heating
with alcoholic sulphuric acid (Wislicenus), yielded methyl
indole-3-acetate. The aldehydo-propionic acid was prepared

HC=C.COOH
in fair yield by boiling aconic acid | | with water.

O.CO.CH2
The aconic acid was prepared from commercial itaconic acid ;
for details and references, the original should be consulted.
In his next paper, Ellinger (1905) described a preparation of
the now usual indole-propionic ([3-3-indole-propionic) acid by
a tedious and probably uncommercial process, having
y-aldehydobutyric acid as intermediate. In the same paper
Ellinger also described a similar preparation from aldehydo-
wo-butyric acid of the compound isomeric in the side-chain
(a-3 -indole-propionic acid), for which he used the strange-
sounding name indole-methylacetic acid.^

Relation to Animal Physiology

It is of interest that several of the more recent preparations
of indole acids — some of which are now known to have remark-
able effects upon the growth of plant organs — had their
genesis in a study of the role of tryptophan in animal diets.
Jackson (1929) devised a diet deficient in tryptophan, in
substitution for which various indole compounds were tried.
Indole-pyruvic acid was the only one of these compounds cap-
able of replacing tryptophan in rats' diet ; it caused resumption
of growth in animals deprived of tryptophan. A mixture of
pyruvic acid and ammonium pyruvate leads to the formation
(de Jong, 1900, 1904) of the type of amino acids most

^ The name is correct provided that the methyl group has not wandered
into either ring. It suggests an interesting chemical catch. Q. What is
methyl-acetic acid ? Ans. Propionic acid. Q. Is, then, indole-methyl-acetic
acid identical (for any one point of substitution in the indole nucleus) with
indole-propionic acid? — Try it on your friends!

104

J

Synthesis

common among protein derivatives — ^the a-amino acids;
this suggests an explanation of the values of indole-pyruvic
acid as a substitution for tryptophan.

The immediate chemical interest of Jackson's 1929 paper
lies in its preliminary description of new or improved methods
of preparation of several indole compounds, amongst them the
following: indole-3 -butyric acid from application of the
Fischer ring-closure to the phenylhydrazone of ethyl
hydrogen a-ketopimelate (full details of this synthesis were
published by Jackson and Manske (1930) ); indole-3-
propionic acid was prepared according to Kalb, Schweizer,
and Schimpf (1926) ; indole-3-pyruvic acid was prepared
by the method of Granacher, Gero, and Schelling (1924).

Berg, Rose and Marvel (1929-30), who also were interested
in the animal metabolism of tryptophan, reported the prepara-
tion of indole-3 -propionic acid from adipic acid. Diethyl
adipate was prepared and condensed with itself (van Ryssel-
berge, 1926) to give ethyl r^c/opentanone-2-carboxylate.
Indole-3 -propionic ^^id was then formed by the procedure of
Kalb, Schweizer, and Schimpf (1926) as modified by Manske
and Robinson (1927).

The same authors prepared indole-3 -pyruvic ^cid by apply-
ing the Grignard reaction to indole to give indole-aldehyde
(Putochin's (1926) modification of the method of Majima and
Kotake (1922): the method was further modified by Berg
et al. in that di-«-butyl ether was used as a solvent). The
aldehyde was converted to the pyruvic acid according to the
procedure of Ellinger and Matsuoka (1920).

Recent Indole-Acetic Acid Syntheses

Two syntheses of indole-3 -acetic acid and one synthesis
each of indylene-i : 3-diacetic acid and of two other indole
acids, were reported by Jackson and Manske (1935). One of
the indole-acetic acid syntheses was from diethyl-^-cyano-

H2O HCl

propionacetal — >- acid — ►- semi-aldehyde of succinic acid
( +H2O); this, without isolation, was condensed with phenyl-
hydrazine; the product was subjected to the Fischer indole

105

Plant Qrowth'Substances

ring-closure, yielding the required acid. The other was a
variation of that of Piccinini (1899), who used it to prepare
N-methylindole-3-acetic and 2-methylindole-3-acetic acids:
Jackson and Manske (1935) reported that a moderate amount
of ethyl diazoacetate acting upon indole yielded mainly
indole-3-acetic acid and no 2-substituted compound. "The
isolation of the acid proved to be facile enough, and in some
respects this synthesis is the most satisfactory yet recorded."
Details of this synthesis are quoted from the original paper :

Procedure

"A dried ethereal solution of ethyl diazoacetate prepared
from 50 gm. of glycine ester hydrochloride was slowly run
into a Claisen flask (fitted with a long fractionating column)
in which had been placed 19 gm. of indole and a trace of
copper powder. The ether was rapidly distilled off as the
solution was run in, so that the reaction was thus made to
proceed smoothly. Finally the product was heated to ioo''C.
in the vacuum of a water pump, transferred to a Claisen flask,
and distilled at 2 to 3 mm. There was frequently a second
evolution of gas at this stage, and this was particularly pro-
nounced in the experiment in which the proportionate quan-
tity of diazoester was greatly increased. [This second experi-
ment yielded less mono-acetic acid — H.N.]

The almost colourless distillate was hydrolyzed with an
excess of methanolic potassium hydroxide, the solution diluted
with water and the methanol distilled off. Thorough extrac-
tion of this solution with ether yielded 2 gm. of indole. The
alkaline solution was then acidified with hydrochloric acid and
again exhaustively extracted with ether. The combined
extracts were dried with sodium sulphate and the ether dis-
tilled off. The solid, reddish residue was transferred to a
suction funnel and washed with cold ether.

The indolyl-3 -acetic acid readily dissolved in the ether and
was recovered from the fiiltrate by removal of the solvent. It
was recrystallized from a concentrated solution in ethyl
acetate by the addition of benzene, or from hot water. The
latter solvent is preferable when the acid is already of good

106

Synthesis

grade. In either case, the adequately purified product melted
at 167" to i68°C. (corr.), with the evolution of carbon dioxide,
and this melting point was not lowered when the material was
admixed with a specimen prepared from indolyl acetonitrile
or one synthetized by the method to be detailed later [i.e., from
the cyanopropionacetal mentioned above; not otherwise
reproduced here — H.N.], The yield of indolyl-acetic acid was
12 gm. and that of the diacetic 1-2 gm."

Indylene-i : 3 -diacetic acid and the other two acids des-
cribed in this paper (3 -methyl-indolyl-i -acetic and indolyl-3-
succinic acid) are much less effective physiologically than is
indole-3 -acetic acid. The very effective indole-3 -propionic
acid can, however, be obtained by heating the 3 -succinic acid.
Another synthesis of indole-3 -acetic acid, and syntheses of
other indole compounds, were recorded by King and L'Ecuyer
(1934). Several of these compounds had carboxy in the 2-
position. King and L'Ecuyer prepared the first 2-acetic com-
pound (2-carboxy-i-methyl-indole-2-acetic acid). The simple
indole-2-acetic acid is not known. Still another synthesis of
indole-3 -acetic ^^^^ ^^ mentioned on p. 113.

Microbiological Preparation

Biological methods of preparation are suggested by the
work of Ehrlich (1912). In presence of a yeast and much sugar,
tyrosine gives tyrosol (^ -hydroxy phenyl-ethyl alcohol), and
Ehrlich discovered that tryptophan similarly gave the analo-
gous alcohol (named by him tryptophol) : in absence of other
sources of nitrogen, the yeast deaminized the tryptophan. In
the hands of Jackson, (1929) Ehrlich's "elegant biological
method" gave a yield of over 90 per cent, of crude crystalline
tryptophol, but Jackson did not say whether he used the
sugar or alcohol technique. Jackson (1930) has prepared
tryptophol artificially by reduction of methyl (or, ethyl)
indole-acetate. As tryptophol is 3-indole-ethyl alcohol, it is
curious that no laboratory oxidation of it to indoie-acetic acid
has been recorded, though its phenyl ether has been prepared
by Manske (193 1). A physiological oxidation of tryptophol to
indoie-acetic acid has been recorded by Ward (1923).

107

Plant Qrowth'Suhstances

Ehrlich's original method of preparation of indole-ethyl
alcohol involved the use of tryptophan (which is expensive)
and a large excess of sugar. Ethyl alcohol can be employed as
source of energy for the yeast, the purification of the desired
product being thereby made more easy than when sugar is
used. Ehrlich {loc. cit., p. 889) gave details of the preparation
of indole-ethyl alcohol from tryptophan and ethyl alcohol in
presence of a yeast. The requisite amount of alcohol is much
smaller than the massive dose of sugar required for the sugar
preparation.

It is not clear whether the ability to deaminize tryptophan
in laboratory culture is peculiar to strains of yeast similar
to those used by Ehrlich. The biological method, however,
appears to have interesting possibilities and is probably sus-
ceptible of further refinement. It might be worth while to
explore the potentialities of the microbiological deamination
of casein or of its crude products of hydrolysis, the latter
being obtained by either chemical or biological means. See
also p. 103.

A substance probably identical with 3-indole-acetic acid
has been formed in minute amounts by the action of legume
nodule bacteria on tryptophan (Link et al. (1937) ; Chen
(1938); Georgi and Beguin (1939) ); it may help to account
for the auximonic effect of leguminous nodules observed by
Bottomley (191 5- 17, E).

Higher Indole-Acids

Details of a synthesis of 3-indolyl-propionic acid from
CO w-diethoxy-propyl diethyl-malonate (from ^-chlorpro-
pionacetal and sodium diethyl malonic ester ) are given by
Davies, Atkins, and Hudson (1937). The acid prepared in
this way is free from the lower homologue.

Manske and Leitch (1936) have described in considerable
detail the syntheses of 8-(3-indolyl)-valeric acid and the
related 6^-alkyl-p-(5-methyl-indolyl) -propionic acid. Both
these have been shown to possess the plant-physiological
properties of a growth-regulating substance.

The indole-valeric acid was synthetized by the application

108

Synthesis

of the Fischer ring-closure to the phenyl-hydrazone of a-keto-
suberic acid and the subsequent ehmination of carbon dioxide
from the resuhing dibasic acid (5-3-(2-carboxy)-indolyl-
valeric acid). This synthesis was strictly analogous to that of
the two lower homologues (Jackson and Manske (1930);
Manske and Robinson (1927) ), the phenyl-hydrazone being
obtained by means of the Japp-Klingemann^ reaction from
the homologous ethyl cyclo-heptanone carboxylate. This was
prepared following the method first used by Kotz and Michels
(1906) for the synthesis of ethyl cyclo-hexanone carboxylate.
For the 1936 paper, cyclo-heptanone (suberone) was obtained
by the procedure (described as "excellent" by Manske and
Leitch) due to Mosettig and Burger (1930).

In outlining the preparation of the 6^- methyl indolyl deriva-
tive, Manske and Leitch wrote that "the now well-known
procedure, starting with /)-toluidine in this case and ethyl
cyclo-pentanone carboxylate, was employed".

A number of other indole derivatives, mainly of purely
chemical interest at present, have been prepared by Manske
(1930), including the phenyl ether of tryptophol mentioned
above. The "recently much exploited" reaction of Japp and
Klingemann was used in all cases to prepare the requisite
phenyl-hydrazones. Manske also referred to the synthesis by
Barrett, Perkin, and Robinson (1929) of 5- and 6-methoxy-p
(3-indolyl)-propionic acids by methods analogous to those of
Manske and Robinson (1927). Manske himself (loc. cit.)
synthetized the 7-methoxy-3-indolyl-propionic acid and a
more complex 62:-substituted indolyl-propionate.

An isomeric form of indole has been reported by Braun
and Nelles (1937), but it is not known how it or its compounds
affect plant growth.

Other Compounds

For preparation and properties of indole-carboxylic acids,
see the textbooks. The 3-carboxy compound is easily con-
verted into indole.

* The Japp- Klingemann (1888) reaction arises from the addition of
benzene-diazonium chloride in ethyl alcohol made alkaline with KOH.

109

Plant Qrowth'Suhstances

Methyl-ketole (2-methyl-indoIe) and skatole (3-methyl-
indole) converted into Grignard magesium halogenides, form
the respective 2- or 3-methyl-i-carboxy acids when treated
at room temperature with COg. If the CO2 is passed in at the
temperature of boiling toluene, the magnesium halogenide of
2-methyl-indole forms only 2-methyl-3-carboxy-indole. The
magnesium halogenide of skatole persists in yielding the i(N)-
carboxy compound when treated with COg even at 20o°C.,
and a bath temperature of about 310° is necessary for the
carboxy group to become attached in the 2-position (3-methyl-
2-carboxy-indole) instead of in the i -position.

Indolyl magnesium halogenides commonly form 3-sub-
stituted alkyl derivatives from alkyl halogenides, though sub-
stitution in the i -position may occur.

The preparation of many indole derivatives, including
those mentioned in the foregoing paragraphs, and also 3-
acetyl-indole (3-indolyl-methyl-ketone) and homologues, has
been described by Oddo (1921 ). Some of his descriptions are
reproduced in Houben's "Die Methoden der organischen
Chemie," 2nd. edn., Leipzig, 1924; Vol. IV, p. 882.

i-methyl-indole-3-carboxylic acid, from treatment of
methyl-phenylhydrazine-pyroracemic acid with hydro-
chloric acid: Hartley and Dobbie (1899).

i-methyl-indole-3-acetic acid: Piccinini (1899). See p. 106.

Few of the above-mentioned substances have been tested
on plants, but they are mentioned for the sake of complete-
ness in view of their possible synthetic or constitutional
importance.

Still other synthetic compounds have been submitted to the
Avena and other tests by Kogl and Kostermans (1935),
Haagen-Smit and Went (1935 F) and others. Lists of their
results are given in Boysen Jensen's book.

The growth-regulating value of salts of synthetic acids has

110

Synthesis

been very favourably reported by Avery et al. (1937), and by
Zimmerman and Hitchcock (1937), whose paper on "Com-
parative effectiveness of acids, salts, and esters as growth
substances, and methods of evaluating them" provides an
interesting discussion.

^//o-cinnamic acid: see p. 84. Glycerides: see p. 81.

Naphthalene Compounds (See also p. 119).

Zimmerman and Wilcoxon (1935) prepared a- and P-
naphthalene-acetic acids from the corresponding methyl-
naphthalenes by bromination, conversion to the nitrile, and
hydrolysis of the nitrile by the procedure of Mayer and
Oppenheimer (1916). It may be noted that a-naphthalene-
acetic and Y-indolyl-3-butyric acids are the most powerful
root-forming acids known (Zimmerman, 1936). For the
synthesis of indolyl-butyric acid, see Jackson and Manske
(1930). Use of a pure a-naphthyl-acetic acid free from the
P-form and from naphthoic acid present in earlier prepara-
tions, was reported by Zimmerman and Hitchcock (1937).
This was presumably the same as the acid prepared by Wil-
coxon (1937) by a procedure based on Rousset's (1897)
method. Employing the Friedel and Crafts reaction between
naphthalene and ethyl chloroglyoxylate, Rousset obtained
a-and p-ketonic esters in yield 50 per cent, of theory ; reduction
of the ketone acid then yielded the corresponding glycollic
or acetic acid.

Wilcoxon's paper reviews probably all the existing methods
for the preparation of naphthyl-acetic acids. It is of interest to
note that Wilcoxon was unable to obtain any naphthyl acid
by following the patented procedure of Wolfram et al. (1934)-

His modification of Rousset's method gave a product
(yield 47 per cent, of theory) consisting entirely of the desired
a-form of ethyl naphthalene-glyoxylate.

Reduction of this with sodium amalgam gave a 40 per cent.,
and with hydrogen and nickel catalyst a 88 per cent., yield of
a-naphthalene-glycollic acid. Reduction of the glyoxylate
ester with hydriodic acid and red phosphorus gave a 9a per
cent, yield of a-naphthalene-acetic acid.

Ill

Plant Qroxvth'Suhstances

The ethyl naphthalene-glyoxylate, and the naphthalene-
glycolHc and -acetic acids were stated to exhibit plant growth-
regulating properties in varying degrees, but the details of
the physiological experiments have not yet been published.

Higher Aryl Compounds

These are included in the only set of examples of the pro-
duction of growth-regulating substances protected by patent
(Wolfram et al., 1934).

Anthracene-acetic, fluorene-acetic, and acenaphthene-5-
acetic acids can be prepared by heating the hydrocarbons with
chlor-acetic acid. The yields are small, but Zimmerman and
Wilcoxon (1935) found that they could be improved by adding
aluminium chloride to the melt. The location of the substi-
tuents in the anthracene and fluorene compounds is not
known.

An Important Hint

In this series of indolyl and related syntheses it has been
customary to aim at the preparation of the pure acids, often
via an alkyl ester subsequently hydrolyzed. In view of work
by Zimmerman, Hitchcock, and Wilcoxon (1936) it is doubt-
ful whether the sometimes troublesome isolation of the free
acid is necessary or desirable. These authors have shown that
several esters belonging to the series of phenyl-acetic,
naphthyl-acetic and indole-fatty acids are quite effective in
initiating or otherwise affecting growth of organs and cells in
plants. Methyl 3-indole-acetate indeed, was more effective
in this way than was the free acid: the "natural" "hetero-
auxin". The same authors state that there is no evidence at
hand that hydrolysis of these esters to acids occur in plant
tissues. It has, however, been shown by Zimmerman and
Wilcoxon (1935) that a-naphthalene-aceto-nitrile was not
only not poisonous to plants, but was very effective physiolo-
gically in the same respects as was the free acid derivable from
it by hydrolysis in the laboratory.^

^ The nitrile was more effective in root-production on stems of large
plants than was the free acid at the same concentration.

112

Synthesis

No record of a plant physiological test of indole-3-aceto-
nitrile seems to have been made, but Jackson (1929) secured
13 gm. of the nitrile from 15-6 gm. of indole in the course of
preparing indole ethylamine by the method of Majima and
Hoshino (1925).

The synthesis of Majima and Hoshino represents a quite
practical method of preparing indole-3 -acetic acid: indole
magnesium iodide, treated with chlor-aceto-nitrile in ethyl
ether or in anisole solutions, yields the nitrile. The nitrile is
easily hydrolyzed to the acid.

As some of the acids, more particularly the naphthyl-acetic
acids, are prepared via the corresponding nitrile, Vv^e have an
additional reason for believing that isolation of an acid is not
an essential goal in the manufacture of a potent growth-
regulating substance.

The Auxins

These are true plant hormones. They have not yet been
synthetized. Their structure was established by the work of
Kogl (1935).

The formula of auxin-a (syn., auxentriolic acid) is:
CH CH CH

1^ / N I'

CH-CH-CH-CH CH-CH-CH -CH

^ \ /

CH=C

CisHa.O, " CH-CH,-CH-CH-C0OH

mol. wt., 328 ^ ' OH OH OH

The configuration of auxin-b (auxenolonic acid) is similar,
the side chain (I) being replaced by: (R=Ci3H23)

R-

-CH CH2 CO COOH.

I

OH (C18H30O4; mol. wt., 310)

113

Plant Qrowih'Suhstances

Auxin-a is very stable towards acids, but is readily decom-
posed by alkalis. Auxin-b is decomposed by both acids and
alkalis, whereas indole-acetic acid, though hydrolyzed by
acids, is very stable towards alkalis. These properties were
made use of by Kogl, Haagen-Smit, and Erxleben (1934) as
showing that the growth-hormone in the tips of oat seedhngs
was probably auxin-a.

Auxin-a lactone, distilled with potassium hydrogen sul-
phate, yielded a substance probably identical with auxin-b.
(Kogl, 1935).

Kogl (1938) has pointed out: "The formula of auxin-a
contains seven asymmetric carbon atoms; any organic
chemist knows what that implies. Suppose it were possible
on starting from inactive material to produce an auxin-a of
the correct structure, even then the product would consist of
a mixture of 64 racemates, requiring to be resolved into 128
optical enantiomorphs. And only one component of that
mixture of stereoisomers would be identical with auxin-a."

Synthesis of auxin-glutaric acid (containing four asymmetric
carbon atoms) has been accomplished, and a product identical
with the natural derivative was obtained after about 300 frac-
tionations, so that definite proof of the constitution of a 13-
carbon-atom part of auxin-a and -b has been obtained (Kogl
(1938) ).

Vitamins

Vitamin B^

Although when this book was first planned, the idea of a
synthetic plant growth-substance did not extend beyond the
phenyl, indolyl, naphthyl, and related compounds introduced
into horticultural use about 1935, at least two of the vitamins,
and perhaps other synthetic substances, have or are likely to
have considerable horticultural importance. (P. 80).

The complete synthesis of vitamin B^ was accomplished, or
at least published, almost simultaneously in 1937 by American,
British, and German workers. The question of priority has
been discussed in letters from Robert Robison and A. L.

11+

Synthesis

Bacharach in Chemistry and Industry, 1937, 56, 1141. Vitamin
Bj is called aneurin by many British and French workers, and
thiamin by many Americans. Its structure is much more
complicated than that of the indolyl plant growth-substances,
to which it has no chemical resemblance.

In plants, it is present particularly in the embryo of seeds,
and it appears to act as a hormone within plants. According to
much work by P. R. White and others (summarized in
Chronica Botanica, 1939, 5, 166) vitamin B^ is an essential
ingredient of media for the culture of excised roots and other
parts of dicotyledonous plants^. Its hormonal role in the
intact plant does not seem to have been ascertained. Applied
externally to cuttings, and possibly to seeds, vitamin Bj
behaves as a drug, and, like the synthetic plant growth-
substances of the indolyl group, induces or hastens root-
formation. It is possible that some of the effects observed
from applications of preparations of maize and other seeds to
seeds and cuttings — particularly by Russian workers, were due
to vitamin B^, and not to auxin as was thought. Vitamin Bj
has been found in dung (see p. 68), wherein it is probably
formed by micro-organisms.

The formula of vitamin B^ is :

H

C CI

/ \ '■

N C CH, N C-CH3

il II

H3C-C C-NH, HC C-CH^.CHeOH

N HCL S

vitamin J5, (hydrochloride)

Vitamin Bj is now synthetized in America, Britain, and
Germany by the processes discovered in these respective
countries, and is on sale. The German process has been
patented.

^It is not always necessary to add the complete substance, as some
organisms can synthetize the substance by condensation of its single-
ring constituents, or from an appropriately substituted thiazole alone
(Robbins et al., (1937) ).

115

Plant Qrowth'Suhstances

REFERENCES

Synthesis of Vitamin B^

(Earlier references can be derived from these papers)

Cline, J. K., Williams, R. R., Finkelstein, J., and others
(1937), ^oMrn. Amer. Chem. Soc, 58, 216 (first note), 526,
530 and 1052 — especially 1052.

Todd, A. R., and Bergel, F. (1937), Journ. Chem. Soc, 364.

Andersag, H., and Westphal, K. (1937), Ber., 70, 2035.

Vitamin C

The story of its isolation by Szent-Gyorgyi in 1928 is
sufficiently well-known to those interested. The substance is
widely known as ascorbic acid and by other names. It was
synthetized by Swiss and British workers in 1933, and was the
first vitamin to be synthetized.

Its formula is :

,0.

OH-CH — HC^ ^CO

I \ /

OH-CH2 c c

HO OH

vitamin C

Vitamin B^

This, like nicotinic acid and other substances, is sometimes
useful in culture of excised roots (see White, above). It is
mentioned for the sake of completeness.

REFERENCES H

It is believed that most modern work on the chemistry of
indole-acetic acid and higher growth-substances can be
obtained at first or second hand from the following lists. For

116

Synthesis

earlier work see the article Tryptophan in Thorpe's Dictionary
and the papers of Ellinger. References marked with an
asterisk are of mainly botanical or physiological interest.

* Avery, G. S., Jr., Burkholder, P, R., and Creighton, Harriet

B. (1937), Amer. Journ. Bot., 24, 226.
Barrett, H. S. B., Perkin, W. H., and Robinson, R. (1929),

Journ. Chem. Soc, 2942.
Berg, C. P., Rose, W. C, and Marvel, C. S. (1929-30), Journ.

Biol. Chem., 85, 219.
Braun, J. von, and Nelles, J. (1937), Ber., 70, B, 1767.
*Chen, H. K. (1938), Nature, 142, 753.
Davies, W., Atkins, G. A,, and Hudson, P. C. B., (1937),

Annals Bot., New Ser., 1, 329_.
Ehrlich, F. (1912), Ber., 45, 883.
Ellinger, A. (1904), Ber., 37, 1801.
Ellinger, A. (1905), Ber., 38, 2884,
Ellinger, A., and Matsuoka, Z. (1920), Ztschr . physioL Chem.

109, 259.
*Georgi, C, and Beguin, A. E. (1939), Nature, 143, 25.
Granacher, C, Gero, M., and Schelling, V. (1924), Helv.

Chem. Acta, 7, 575,
Hartley, W. N., and Dobbie, J. J. (1899), Trans. Chem. Soc.y

75, 640.
Hopkins, F, G., and Cole, S. W. (1903), Journ. Physiol., 29,

451-
Jackson, R. W, (igzg), Journ. Biol. Chem., 84, i.

Jackson, R. W. (1930), Journ. Biol. Chem., 88, 659.

Jackson, R. W., and Manske, R. H. F. (1930), Journ. Amer.

Chem. Soc., 52, 5029.
Jackson, R. W., and Manske, R.H.F. (1935), Canadian Journ.

Res., Sect. B, 13, 170.
Japp, F. R., and Klingemann, F. (1888) Ber., 21, 549;

Annalen, 247, 218.
De Jong, Anne W. K. (1900), Rec. trav. chim., 19, 259; ibid..,

1904,23, 131.
Kalb, L., Schweizer, F., and Schimpf, G. (1926), Ber., 59,

1858. L

117

Plant Qrowih'Suhstances

King, F. E., and L'Ecuyer, P. (1934), Journ. Chem. Soc,

1901.
Kogl, F. (1935), Ber., A, 68, 16.
Kogl, F. (1938), Chem. and Ind., 57, 49.
*K6gl, F., Haagen-Smit, A. J., and Erxleben, Hanni (1934),

Ztschr. physiol. Chem., 228, 104.
Kogl, F., and Kostermans, D. G. F. R. (1935). Ztschr.

physiol. Chem., 235, 201.
Kotz, A., and Michels, A. (1906), Annalen, 350, 240. Abstract

in Journ. Chem. Soc. (1907), 92, Ai, 58.
*Link, G. K. K., Wilcox, Hazel W., and Link, Adeline DeS.

(1937), Bot. Gaz., 98, 816; see also Link (1937, E).
Majima, R., and Hoshino, T. (1925), Ber., 58, 2042.
Majima, R., and Kotake, M. (1922), Ber., 55, 3859.
Manske, R. H. F. (1931), Canadian Journ. Res., 4, 591.
Manske, R, H. F., and Leitch, L. C. (1936), Canadian Journ.

Res., Sect. B, 14, i.
Manske, R. H. F., and Robinson, R. (1927), Journ. Chem,

Soc, 240; see also p. i.
Mayer, F,, and Oppenheimer, T. (1916), Ber., 49, 2137.
Mosettig, E., and Burger, A. (1930), Journ. Amer. Chem.

Soc, 52, 3456.
*Nicol, Hugh (1936), Manufacturing Chemist, February.
Oddo, B. (1911), Gazzetta, 41, I, 221; (1912) ibid., 42, I,

361; (1913), ibid., 43, H, 190.
Piccinini, A. (1899), Gazzetta, 29, 363.
Putochin, N. (1926), Ber., 59, 1987.
Robbins, W. J., Bartley, Mary A., et al. (1937). Proc. Nat.

Acad. Sci., 23, 385, and 388.
Rousset, L. (1897), Bull. Soc. Chim. France, Ser. 3, 17, 300.
van Rysselberge, M. (1926), Bull. Soc. Chim. Belgique, 35,

311; Bull. Acad. Roy. Belgique, Classe Sci., 12, 171;

abstracts in Chem. Zbl., (1926), 2, 1846, and in B.C.A.

(1927), A, 1238.
Smith, C. (1936), Journ. Chem. Soc, 1067.
Wilcoxon, Frank (1937), Contrib. Boyce Thompson Inst., 8,

467.
Ward, F. W. (1923), Biochem. Journ., 17, 905.

118

Synthesis

Wolfram, A., Schornig, L., and Hausdorfer, E. (1934), U.S.

Patent, No. 1,951,686.
Zimmerman, P. W. (1936), communication in Manske and

Leitch's (1936) paper.
*Zimmerman, P. W., and Hitchcock, A. E. (1935), Contrib.

Boyce Thompson Institute, 7, 439.
Zimmerman, P. W., and Hitchcock, A. E. (1937), ihid., 8,

337-
Zimmerman, P. W., and Wilcoxon, F. (1935) ibid., 7, 209.
♦Zimmerman, P. W., Hitchcock, A. E., and Wilcoxon, F.

(1936), ibid., 8, 105.

Some plant tests with the acid and its salts, together with a
method of preparation of [3-naphthoxy-acetic acid, are
described by S. C. Bausor, "A new growth-substance", Amer.
Journ. Bot.y 1939, 26, 415.

The following is given for the sake of completeness,
though it does not involve any preparation that is at present
practical :

Fulton, J. D., and Robinson, R. (1939), Derivatives of
P-naphthaldehyde. Journ. Chetn. Soc. 200.

119

CHAPTER XIV

IDENTIFICATION OF GROWTH-SUBSTANCES
AND SUBSTANCES RELATED TO THEM

Animal substances are much more compound than those
we have hitherto examined. Several of the substances
furnished by animals still preserve many of the properties
of the vegetables by which they are nourished. — Beaum6.

The following selected information is given in the hope that it
will be useful to those proposing to undertake the search for
growth-substances in agricultural materials such as manure
and compost. As no work has yet been done along such lines
(except for the early isolations from decomposed meat) the
available information will require to be specially adapted to
suit each worker's need. The existing qualitative tests refer
entirely to the identification of the pure substances in aqueous
solution or as they occur in urine. When urine is mentioned
here, it is usually human urine that is implied.

Critical methods of identifying the growth-substances are
urgently required. No progress can be made in the recogni-
tion and estimation of the substances in organic manures and
decomposition products until simple yet accurate chemical
tests are available. Much of the information that is available
is published in medical and physiological journals difficultly
accessible to agricultural and other chemists. The handbooks
on organic chemistry give very little help of the kind useful
for performing analysis of extracts, and it seems that the
detection of ring-compounds in manure and composts will
remain for some time a matter for the research laboratory
able to isolate the substances for characterization by such
criteria as melting points. (See Tabular Index, Chapter XV).
Such methods are not only laborious, but are seldom suscep-

120

Analytical

tible of quantitative application. Physiological tests can
supply roughly quantitative information in some cases at
high dilutions, but are of little critical value in distinguishing
the constituents of mixtures.

Phenyl-acetic acid. — Steenbock (191 2) has described a
method for the estimation of phenyl-acetic acid in urine in
presence of benzoic acid. He treats the urine with caustic
soda with addition of hydrogen peroxide, extracts the benzoic
and phenyl-acetic acids with ethyl ether, dries, and weighs
after subhmation. The sublimate, of weight S grams, is
titrated with N /20 NaOH, requiring n ml. The phenyl-acetic
acid content in grams is then:

136 {S — o-oo6i n)

The acid is oxidized to benzoic acid by strong oxidizing
agents. It has a beautiful rose-like odour. For other tests, see
the chemical handbooks.

Phenyl-propionic acid. (^-phenyl-propionic acid). — For
tests see chemical handbooks. It has a pleasant, sweet odour.

Phenyl-pyruvic acid (not shown to be a growth substance)
has been found in the urine of a small proportion of mental
defectives by Foiling (1934). Urine containing this substance
gives a deep green colour on addition of ferric chloride solu-
tion. For other properties and quantitative methods, see the
paper by Penrose and Quastel (1937). Like homogentisic
acid (see infra) phenyl-pyruvic acid is a product of incomplete
metabolism; its source appears to be dietary phenyl-alanine.
It is fairly easily decomposed under alkaline conditions in the
presence of bacteria in urine, but the products of such
decomposition are not known.

p-hydroxy -phenyl-acetic acid. — With ferric chloride solution,
its solution gives a faint violet colour, quickly becoming
greyish-green.

p~hydroxy -phenyl-propionic acid (P-(4-hydroxy-phenyl)-
propionic acid). — Said to occur in urine. The cupric salt is
difficultly soluble in water and alcohol, but is soluble in ether.
(Hlasiwetz, Journ. prakt. Chem. (i), 72, 401 ; from Beilstein's

121

Plant Qrowth'Suhstances

Handbuch). The acid gives a deep red colour with Millon's re-
agent.

Tyrosine (/)-hydroxy-phenyl-alanine). — Not a plant-
growth substance while intact. For tests, see textbooks.

For properties and methods of isolation of some aromatic
hydroxy-acids, see the sections by Waser (1923), and by
P. Brigl, in Abderhalden's Handbuch, 1931, Abt. 4, Teil 5(1),
483-512, and Baumann's (1882) method on pp. 510-512 of
the latter.

Homogentisic acid. (2 : 5-dihydroxy-phenyl-acetic acid.) —
The claim of this body to be considered a growth-substance is
not certain, but it may be useful to give a few hints for its
detection and estimation, as a guide to adoption of methods to
search for it in agricultural materials. Readers who are especi-
ally interested cannot do better than consult Weise's mono-
graph (see p. 75).

From urine containing homogentisic acid and shaken with
sodium hydroxide solution and benzoyl chloride, di-benzoyl-di-
hydroxy phenyl-acetamide (C gH 5. CO O )2. C gHg. CHg. CONHg
separates out, which can be recrystallized from hot ethyl
alcohol. M.P. 203-204°. This derivative is characteristic of
alcaptonuric urine, but needs to be differentiated from
tribenzamide, M.P. 201-205°, which is Hable to form in small
amounts when urine is benzoylated. Tribenzamide, however,
is difficultly soluble in hot alcohol.

The lead salt of homogentisic acid is useful for isolation;
it crystallizes in colourless or brownish prisms of M.P. 214°.
It is insoluble in alcohol and ether, and only very slightly
soluble in water.

With ferric chloride solution, homogentisic acid, even in
fairly dilute solution, gives a transient blue colour. With
Millon's reagent, an aqueous solution of the acid gives a
yellow colour; on standing, a yellow precipitate develops in
the cold, which turns red on heating.

A peculiar titration method has been evolved by Baumann
(1891). This depends on the reduction of N/io silver nitrate
solution in urine made alkaline with ammonia. To 10 ml. of
urine are added 10 ml. of 3 per cent, ammonia solution, and

122

Analytical

at once "a few" ml. of standard silver nitrate solution. The
mixture is shaken, and allowed to stand for five minutes.
Five drops of a lo per cent, solution of calcium chloride and
ten drops of a 20 per cent, solution of ammonium carbonate
are then added, in order to form a precipitate of calcium
carbonate to carry down suspended silver. The mixture
is filtered, and the filtrate tested with silver nitrate. If
appreciable reduction occurs, a new experiment is begun,
using 2 to 5 times as much standard silver nitrate solution as
first. The reduction test is repeated, and the whole thing is
gone through several times, if necessary, until no reduction
occurs in the filtrate. This gives an upper limit to the volume
of standard silver nitrate. The experiments must again be
repeated ab initio with successively slightly smaller doses of
standard silver nitrate until no reduction occurs on the
addition of silver nitrate to one portion of the filtrate, and also
no or very slight cloudiness is revealed on the addition of
excess hydrochloric acid to another portion. Each ml. of
standard silver nitrate solution that has produced this limiting
result is equivalent to 4*124 mg. of homogentisic acid. The
method is probably capable of simplification.

Indole-acetic Acid. The Urorosein Test
A number of chemical reactions are available for revealing
the presence of indole-acetic acid. They have been described
in the literature as tests for the substance in urine, or in fairly
concentrated pure solution. Some tests for the pure substance
follow.

E. Salkowski's tests date from 1884 and are the best known.
(i) If a o-i per cent, neutral solution of the acid is treated
with a little very dilute ferric chloride solution, the mixture
becomes cloudy; in transmitted light it appears violet, but
greyish in reflected light. On careful acidification a violet-grey
precipitate is formed, which gives a blue solution in ethyl
alcohol.

If after adding ferric chloride and acid, more ferric chloride
is added and the mixture is boiled, a cherry-red colour is
produced. The colour can be removed by extraction with

123

Plant Qrowth'Suhstances

amyl alcohol, in which the red substance is very
soluble.

(2) If to the neutral solution of the acid is added an equal
volume of hydrochloric acid (D. 1-12) and a few drops of
about I per cent, "bleaching-powder solution", a purple-red
colour is produced. In concentrated solutions a precipitate is
formed, soluble in ethyl alcohol and in amyl alcohol.

EhrlicKs test. — Resembling the usual diazotization reaction
in hydrochloric acid solution, this is performed with alcoholic
/>-dimethylamino-benzaldehyde in hydrochloric acid in pre-
sence of nitrite. With indole-acetic acid a reddish colour is
produced, not very distinct from that given by indole.

Lead acetate gives a crystalline precipitate with a o-i per
cent, solution of sodium or potassium indole-acetate, whereas
other heavy metals give little or no precipitate (Ellinger).

Indole-acetic acid is not volatile in steam, is stable towards
alkalis, and is odourless.

Link et al. (1937, H) used the Salkowski colour test as sole
chemical test for indole-acetic acid. They remark that it is
considered specific for that compound. Evidence for its
specificity is not quite satisfactory. An analytical scheme for
the detection of the principal indole-acids and related com-
pounds is badly needed.

Urorosein is a derivative of indole-acetic acid. It was dis-
covered by Nencki and Sieber in 1882. Opinions differ about
its constitution.

Weise in Abderhalden's Handhuch^ Abt. 4, Teil 5(1), gives
tests for the presence of indole-acetic acid (p. 767) and for the
production of urorosein (p. 768) which, though similar, diff"er
in detail. It has been suggested that urorosein is a derivative
of indolyl-aceturic acid (indolyl-acetoyl-glycine), the conjuga-
tion product (see p. 58) of indole-acetic acid, but it appears
uncertain that the glycine takes part in the production of
urorosein.

The tests given by Weise [loc. cit) are as follows :

"Indole-acetic acid (Salkowski's test). — If a few drops of
pure nitric acid (D. i '2) are added to a very dilute solution

124

Analytical

of indole-acetic acid, and then a few drops of 2 per cent,
potassium nitrite solution are added, a cherry-red colour
appears ; if the dilution is not too high, a red colouring-
matter is precipitated, which is easily soluble in ethyl acetate
and in amyl alcohol. Caustic soda solution discharges the
colour from ethyl acetate solution and acquires a yellow
tinge. The alkaline solution is rendered colourless after
addition of zinc dust and remains colourless on exposure
to air. (Distinction from indole; nitroso-indole after
reduction becomes blue on standing — Salkowski.)"

The same test is given by Ellinger on p. 792 of Teil 7, Abt.
I, of Abderhalden's Handbuch. (Urorosein may often be
formed from urine by simply adding hydrochloric acid; its
formation requires the presence of nitrite, which is often
present in urine owing to bacterial activities). The following
will now be understandable.

"For the demonstration of urorosein, 5-10 ml. of con-
centrated hydrochloric acid are added to 50-100 ml. of
urine. If no red colour appears after a few minutes, a little
0-2 per cent, sodium nitrite is cautiously added, drop by
drop. The red colour then appears if the chromogen is
present.

"The dyestuff thus formed can be separated out by
shaking with amyl alcohol: this operation is especially
necessary when other red or yellowish-red colouring-
matters are present. The amyl alcohol solution should be
examined spectroscopically.

"Urorosein dissolves to a reddish solution in water, ethyl
alcohol, amyl alcohol, but is insoluble in ether and in
chloroform (distinction from indirubin and skatole red).
The colour of the solutions is discharged by addition of
alkaUs, and restored on acidification. The amyl alcohol
solution shows a sharp absorption band in the green,
about 557 A.U."

From Weise's account of the indole-acetic acid tests with
acid and nitrite, it looks as if nitroso-indolyl-acetic acid

125 g^

Plant Qrowth'Suhstances

is the product. Apparently, urorosein and the indole-acetic
acid nitritation compound behave differently with alkalis.
On account of the apparent absence of information regarding
the behaviour of urorosein in ethyl acetate, the question of
the identity of the two substances in question must be left
open. Plimmer (1918) wrote that urorosein is most probably
nitroso-indole-acetic acid. Ellinger and Flamand (1909) and
Riesser (191 1) believed urorosein to belong to the group of
tri-indolylmethane dyes discovered by Ellinger and Flamand ;
Weise {loc. cit., p. 768) imputed to H. Fischer (1923) the
view that it is a di-indolyl-methene derivative. Urorosein has
also been studied by Herter (1908).

Schmitz (193 1 ), apparently quoting Riesser (191 1), wrote:
"Eine Darstellung des Uroroseins in Substanz ist bisher
nur auf synthetischem Wege durch Oxydation von Indole-
essigsaure mit Eisenchlorid oder mit Natriumnitrit und
Salzsaure versucht worden."

Further pursuit of the subject is left to those particularly
interested.

With the modified Adamkiewicz-Hopkins (tryptophan)
reaction of Winkler (1934), indole-acetic acid gives a reddish
violet colour. The best ratio of reagents is i mol. of indole-
acetic acid, I mol. of glyoxylic acid, 2 mol. Cu (sulphate or
acetate) and excess of concentrated sulphuric acid — added in
that order. (Winkler and Petersen, 1935).

For titration-curves of 3 -indole-acetic, -propionic and
-butyric acids in distilled water, see the paper by Albaum
and Kaiser (1937).

A study of three colorimetric methods of estimating indole-
acetic acid in aqueous solution has been made by Mitchell
and Brunstetter (1939).

See also Lefevre (1939, G).

Methods of Isolation of indole-acetic acid

The Avena (oat) test has been used by Kogl et al. (1934)
as a method of assay of indole-acetic acid in fractions of urine
during the process of isolation. The normal urinary excretion

126

Analytical

of growth-substance was estimated by them at about 2 mg.
per day, of which only about a fifth is indolyl-acetic acid, the
remainder consisting chiefly of auxin-a. In the case of their
special patient, "E.W.V.", who excreted 8-10 mg. of growth
substance per day, at least three-quarters of that amount
consisted of indole-acetic acid.

The oat assay of the successive urine fractions showed very
satisfactory agreement with the determinations of indole-
acetic acid by weight, allowance being made for losses and for
the presence of auxin in the original urine. (The oat-test
activities of the two auxins and of indole-acetic acid are very
similar).

Weise (loc. cit. ) gives the following method of preparation
of indole-acetic acid from urine. It may perhaps serve as a
basis of a method of isolation from agricultural materials.

Several litres of urine are evaporated down, and the residue
extracted with absolute alcohol. The extract is distilled, and
the residue is dissolved in a little water and acidified with sul-
phuric acid. It is then shaken with ether. The ether extract
is separated, and is shaken with a sodium hydroxide solution
to remove the indole-acetic acid. The alkaline solution is
evaporated down, and the residue is repeatedly extracted with
absolute alcohol to a total volume of about 100 ml. Ether is
then added, and a precipitate forms. The precipitate is
separated from the liquid, the latter is evaporated down, and
the residue is acidified with hydrochloric acid. After an ether
extraction of the acid mixture, the ether is evaporated off.
This gives an impure product (containing traces of hydro-
chloric acid) which can be dissolved in hot benzene and re-
crystallized.

Kogl and others (1934, and other papers) have improved on
Weise's method in several respects, notably by taking up the
indole-acetic acid on an absorbent, thus avoiding the tedious
evaporation. They have also made use of a chromatographic
technique to separate the indole-acetic acid from auxin. Their
papers should be consulted for details.

E. Salkowski's original methods of isolation of indole-
acetic (1884) and indole-propionic acid (1899) from decom-

127

Plant Qrowth'Suhstances

posed meat, on account of the presence of indole and other
substances in the mixture, are too complicated to be given
here, but they should be borne in mind.

Indole-propionic acid. — Ferric chloride added to a dilute
solution of the acid gives a white turbidity, which becomes red
on heating. Concentrated potassium nitrite and acetic acid
added to a saturated solution in water form fine yellow needles
of a nitroso compound, which melts at 135° with evolution of
gas.

Tryptophan. — Not a growth-substance^ when intact. For
usual tests see textbooks.

An important modification of the well-known Adamkiewicz-
Hopkins reaction has been made by Winkler (1934). This
reaction depends on the blue-violet colour developed when
glyoxylic acid (often present as an impurity in acetic acid) and
concentrated sulphuric acid are added to free or combined
tryptophan solutions. Winkler has shown that the test is
inadequately given, or even fails, unless a trace of copper
salt (conveniently, sulphate or acetate) is added before the
sulphuric acid. The glyoxylic acid should be roughly equi-
molecular with the tryptophan. Under conditions described,
the modified reaction is quantitative for free tryptophan at
least.

For a method of detection of free and combined tryptophan
in plants by nitration, see Roth (1939).

Creatine and creatinine. — For properties and tests, see text-
books on human biochemistry. Creatinine was recognized by
Schreiner et al. (191 1, E) by means of its zinc chloride com-
pound.

Oxal-acetic acid. — This is not known to be a growth-
substance in the current sense, but it is assumed by Virtanen
and Laine (1937) to be an intermediate in the biological
fixation of nitrogen, particularly in leguminous plants. In
either aqueous or alcoholic solution, both forms of the acid
give an intense red colour with ferric chloride solution, where-
fore it may perhaps be confused with indole-acetic acid.
Oxal-acetic acid, however, has a peculiar reaction : in presence

128

Analytical

of aniline, both of its forms produce a lively evolution of
carbon dioxide (Wohl, 1907), even at temperatures as low as
10°. Both forms are insoluble in chloroform and benzene
(distinction from indole-acetic acid), but the higher-melting
form is said to be more soluble in ether than the other is.

Vitamins

Chemical methods for the estimation of vitamin C are
reviewed by Gstirner (1939), who considers the foremost
method to be the standard one of J. Tillmans [Chem. Ahs. 25,
5692; variously modified since). What appears to be a more
reliable method for determining ascorbic acid in urine has
been described by Roe and Hall (1939).

For a biological method of determination of vitamin B^ in
urine by its effect on growth of the fungus Phycomyces, see
Villela (1938) ; for the original Phycomyces method, see
Schopfer and Jung (1938). For a fluorescence method of
estimation of vitamin Bi, see Marrack and Hollering (1939).

Wenfs Pea Test
A simple test for the presence of at least some growth-
substances has been described by Went (1934). It was
devised to test for the presence and rough quantitative
estimation of auxin, but works equally well with indolyl-acetic
acid.

Peas are germinated in any convenient manner (on damp
cloth, sand, or blotting paper) in a darkened cupboard or
room maintained at about 25° C. No nutrient solution is
required, and small amounts of light do not matter so long as
the shoots grow and remain straight. When the shoots are
from 5 to 20 cm. long they are ready for use. The variety of
peas mentioned by Went was Alaska.

The description that follows is given in Went's words:

"Pieces of stem 2 to 20 cm. long are cut about 5 mm.
below the terminal bud, and are used either directly or
4 to 8 hours after cutting. Immediately before application
of the auxin solutions, the top of the cut piece of stem is
split lengthwise by an exactly median cut with a razor

129

Plant Qrowth'Suhstances

blade over a length of i to 3 cm. If the cut is very far from
median the curvatures are smaller. Upon subsequent
immersion in the solution the two halves will bend out
because of the tissue tension. If no auxin is present in the
solution these original outward curvatures do not change
any more and are fixed, since they do not go back upon
plasmolysis. In the presence of auxin, however, about an
hour after immersion (at 25°) the free ends of the two halves
will start to bend inwards; the higher the concentration
the further they bend. After about 6 hours the final stage is
reached, and the originally most rapidly growing zone will
be convex at the outside. If the median slit is long enough,
the halves will each show an S-shape. Also, these curvatures
are not changed by plasmolysis. For the appearance
of the curvatures it is only necessary to have the apical cut
surface in contact with the auxin-containing solutions.
So either the shoots are reversed and placed upside down
in a small amount of solution (5 plants together in o-2-i
cc.) or they are left floating on the surface of the solution in
a shallow dish. If the auxin can enter only through the
surface of the radial cut no curvatures appear.

"The simplest quantitative auxin determination with the
pea test is to place a number of pea shoots, apically slit
lengthwise over a distance of + 10 mm. in a whole range
of dilutions of the solution to be tested ; 5 plants per dilution
will be enough. After 12 hours it is determined in which
concentration a well-recognizable reaction occurs. In this
way either directly or by intrapolation the critical concentra-
tion may be calculated with an accuracy of 50 per cent."

This critical concentration, or difference of concentrations,
corresponds to about 480 AE (Avena units), or i'4Xio~5
mg. of auxin-a, per ml. For purposes of comparison, it may be
mentioned that ordinary human urine contains, per litre,
about 2 mg. of auxins and indolyl-acetic acid together, each
having an activity of about 50,000,000 AE per mg. Hence,
Went's test is sensitive to the amount of growth substance
present in about one-hundredth of a millilitre of urine.

130

Analytical

Went says: "The only work involved in this method is the
making up of the dilutions and the splitting of the peas; the
estimation of the reaction is done at a glance."

The curvatures can also be classified. Went's scheme is:
Degree of reaction Appearance of Split Stems.

0 No auxin reaction, both halves concave at
the outside over their whole length.

(as A, Fig. 6.)

1 Slight auxin reaction, trace of convexity
in limited regions.

2 Definite auxin reaction, ends of both
halves approximately parallel.

3 Fair auxin reaction, ends bent inwards.

(as B, Fig. 6.)

4 Strong auxin reaction, ends just touching.
e Very strong auxin reaction, both halves

crossing at end.
6 Very strong auxin reaction, halves cross-

ing midway or at base (as C, Fig. 6).

A

B

Curvatures in split sections of pea

Fig. 6. (After F. W. Went, (1934) )•
stems, immersed in :

B Tn/ C, auxin-containing solutions producing curvatures of degrees 3
(fair) and 6 (very strong) respectively.

With acknozvledgments to Dr. F. W. Went and to the Editors of the

Verslag. Kon. Akad. Wet., Amsterdam.

131

Plant Qrowth'Suhstances

The crux of the preparation Ues in the apparently simple
recommendation to germinate the peas on a damp material.
Germination is easy to accomplish, but peas are very prone
to invasion by micro-organisms, often borne on the seeds
themselves, and unless suitable treatment is applied, the
experimenter is liable to the disappointment of finding the
seedlings as a pulpy, stinking, mass. It is hoped that the follow-
ing suggestions will anticipate for once the reproach that text-
books give all the details except the essential ones.

The pea seeds (Alaska) should be externally sterilized.
This may be accomplished by shaking the peas in any easily-
removable disinfectant, such as free chlorine, or by washing
in 95 per cent, alcohol followed by 0*2 per cent, aqueous
mercuric chloride solution (not in reverse order !). A simple
and effective method is to cover the peas with concentrated
sulphuric acid (pure oil of vitriol), and leave them in it for
five minutes with occasional shaking. A conical flask of large
capacity is best. Whatever method of sterilization is adopted,
rinsing should be performed with several changes of water.
Tap water will do, as absolute sterility is not necessary. If the
sulphuric acid method is used, the first addition of water to
the peas should be made rapidly and in great excess, with
avoidance of local overheating (for the sake of the peas), and of
spurting (for the sake of the operator).

Sand is perhaps more conveniently freed from unwanted
organisms than are cloth and paper, which are best sterilized
by autoclaving or baking. Heat-sterilization of large amounts
of sand is a tedious process of uncertain effect. A sufficient
degree of freedom from micro-organisms can be attained by
the use of strong chemicals, such as are available to many
workers not possessing an autoclave. Here again, any reason-
able method can be adopted. The following technique gives
satisfactory results:

Sand q.s. is placed in a stout-walled shallow vessel. Better
than the usual laboratory ware, a glazed "china" pie-dish
fills all requirements. The sand is treated with a hot solution
of potassium permanganate acidified with sulphuric acid
(5 or more grams per litre of each substance, and 60° C.) and

132

Analytical

well stirred with a rod or strong spatula; the solution is
renewed if necessary. After washing out the solution until
only a faint pink colour remains, a slight excess of powdered
sodium bisulphite is added, and the sand is intimately mixed
under warm water — most effectively by using clean hands. It
is important that no brown stain should be left on the sand ;
securing its removal demands some care. The sand is finally
washed under a slow stream of running tap water, care being
taken to manipulate so that every part of the sand is washed.
The washing must be intimate, and need not be prolonged:
mere running water will not penetrate far. Most of the excess
of water being quickly drained off, the sand is ready for use.
The peas should be sparsely placed, uncovered, on the more
than saturated sand, and should be sprinkled with water, and
inspected twice daily for the first few days of germination.

Possibly owing to the production of a volatile growth-
substance, a decided curliness tends to become manifest in
crowded pea shoots, hence a ventilated cupboard or dark room
is better than a closed incubator, but the temperature should
be maintained at about 25" C, and if small amounts of light
gain entry, the vessels should be turned frequently. Some few
of the peas are almost sure to turn mouldy ; as soon as their
condition is noticed, these should be removed with flamed
forceps, and dropped into a bath of disinfectant of low surface
tension. The germination cupboard should be disinfected
after each crop has been grown. Time for a crop, about ten
days.

Those who are thinking of performing this pea test as a
research method will not need advice about its application,
but those who desire merely to make a demonstration may be
grateful for the hint that salt action and other effects may
interfere with the test. For example, the author has found
that after floating the split fragments of pea-stem on 20 ml.
of liquid in a Petri dish, there was no certain response with
concentrated urine, or with stale urine at any dilution.
Fresh urine gave the maximum response of which it was
capable — not, of course, the maximum of Went's scale — when
about 4 ml. were diluted to 20 ml. Here, however, is a dislocat-

133

Plant Qrowth'Suhstances

ing snag for the unwarned non-biologist. Tap water (if not
chlorinated) or water from a silver-lined or glass still should
be used as diluent. Water from a copper still contains enough
copper to ruin many experiments with even intact plants, and
these tender pea preparations are very easily hurt. My
parting word to my fellow chemist is : Remember in doing any
work with plants, that you are handling a structure of wonder-
ful complexity, and alive.

REFERENCES J

Book

Plimmer, R. H. A. (1918), Practical Organic and Biochemistry
(London). (New edition, 1938.)

Periodicals

Albaum, H. G., and Kaiser, S. (1937), Amer. Journ. Bot., 24,

420.
Baumann, E. (1882), Ztschr. physiol. Chem., 6, 191.
Baumann, E. (1891), ibid., 16, 268.
Ellinger, A., and Flamand, C. (1909), Ztschr. physiol. Chem.,

62, 276.
Fischer, H. (1923), Ber., 56, 2313.
Foiling, A. (1934), Ztschr. physiol. Chem.y 227, 169.
Gstirner, F. (1939), Pharm. Zentralhalle, 80, 1-5, 22-27,

34-38 (from Chem. Abs., 33, 2928).
Herter, C. A. (1908), Journ. Biol. Chem., 4, 253.
Kogl, F., Haagen-Smit, A. J., and Erxleben, Hanni (1934),

Ztschr. physiol. Chem., 228, 90.
Marrack, J., and Hollering, Helga F. (1939). Lancet, I, 325.
Mitchell, J. W., and Brunstelter, B. C. (1939). Bot. Gaz.,

100, 802.
Penrose, L., and Quastel, J. H. (1937), Biochem. Journ. y 31,

266.
Riesser, O. (191 1), Zur Chemie des Uroroseins. Inaug.

134

Analytical

Diss., Konigsberg (Berlin) ; Zur Kenntnis des Uroroseins,

Konigsberger med. Inaug-Diss., (Berlin) : both as quoted by

Ellinger in Abderhalden's Handbuch, Abt. i, Teil 7.

Roe, J. H., and Hall, J. M. (1939). Journ. Biol. Chem., 128,

329-
Roth, H. (1939). Angew. Chem., 52, 149.

Salkowski, E. (and H.) (1884), Ztschr . physiol Chem., 8, 417;
(1899) ibid., 27, 302.

Schmitz, E. (193 1), Die Harnfarbstoffe, Sect, in Abder-
halden's i7«n</6Mc/i, Abt. 4, Teil 5 (i), 513-544.

Schopfer, H., and Jung, A. (1938). Ztschr. Vitaminforsch.
7, 143. {Chem. Abs., 32, 6298).

Steenbock, A. (1912), Journ. Biol. Chem., 11, 201.

Villela, C. G. (1938). Compt. rend. Soc. Biol, 129, 989 (from
Chem. Abs., 33, 2928).

Virtanen, A. I., and Laine, T. (1937), N-fixation by excised
root-nodules. Suomen Kemistilehti, B, 10, 24.

Waser, E. B. H. (1923). In Abderhalden's Handbuch, Abt. i,
Teil 7, 567-778.

Went, F. W. (1934). Versl. K. Akad. Wet., Amsterdam, 37,

547-
Winkler, S. (1934), Uber die Tryptophan-Reaktion von

Adamkiewicz-Hopkins, Ztschr. physiol. Chem., 228, 50.
Winkler, S., and Petersen, S. (1935), Ztschr. physiol. Chem.,

231, 210.
Wohl, A. (1907), Ber., 40, 2282.

A review of chemical methods of determining carotene and
vitamins A, B^, Bg (lactoflavin), and C, including some
methods adapted for urine analysis, has been given by
K. Hinsberg in Zeitschr. anal. Chem., 1939, 117, 284. See
also p. 68 for work with vitamin B^.

135

ENVOI

As to author's afterthoughts and the more or less sincere
apologies that often find place in a preface, I have little to
say. Qui s'excuse, s^ accuse. Only one point should be men-
tioned that cannot be fairly introduced elsewhere. It is that
but little reliance has been placed on abstracts and references
at second-hand except as regards some analytical data; most
of the sources quoted have been seen in the original by me,
though possibly a smaller proportion for the second than for
the first edition.

Another function of a preface is to be the expression of the
author's acknowledgments, a sort of grace before meat : often
scamped by the reader anxious to get to the meal — though, to
be sure, it is the author's grace and not the reader's. I think
that the following acknowledgments will be better noted
because they come at the end:

To Dr. George T. Moore, Director of the Missouri
Botanical Garden, St. Louis, Missouri, U.S.A., for kind
permission to reproduce Fig. i from the "Bulletin" of the
Garden.

To Dr. A. E. Hitchcock, of the Boyce Thompson Institute
for Plant Research, Yonkers, New York, U.S.A., for kind
permission to reproduce Figs. 2, 3, 4 and 5 from the
"Contributions" of the Institute.

To some senior colleagues of Rothamsted Experimental
Station, for having read and criticized portions of the manu-
script of the first edition (the blame for any mistakes remain-
ing mine, of course), and finally:

To Dr. H. L. Pearse and the Imperial Bureau of Horticul-
ture, both of East Mailing, Kent, for courtesies in connexion
with the supply of references.

136

CHAPTER XV

TABULAR INDEX

oj Principal Substances Mentioned {other than intermediates)
with Outline of Tests and some Physical Properties

The late M. GeofFroy the physician was the first who
thought of comprizing in a table the fundamental relations
or affinities in Chemistry. — Beaum:§.

This table has been arranged in alphabetical order under
each main heading of Miscellaneous, Phenyl, Indolyl,
Naphthyl, and Other Aryl Compounds.

Solubilities are indicated qualitatively as follows: in
water (w...), ethyl alcohol (a), ethyl ether (e), acetone (t),
ethyl acetate (s), benzene (b), chloroform (c), methyl alcohol
(m), usual fat solvents (f) — :

Almost insoluble in cold solvent: w
Appreciably soluble in cold solvent: w
Appreciably soluble in hot solvent: W '

Very soluble in hot solvent: W
Almost all the substances are solid or oily at ordinary
temperatures, and only one boiling-point has been given.
*An asterisk indicates corrected melting-points.
(With acknowledgments to Beilstein's Handbuch, Heil-
bron's Dictionary, and other authorities.)

137

Plant Qrowth'Suhstances

Miscellaneous Compounds, including Inorganic and Aliphatic
Substances, those of Unknown Constitution, and some trivial names

Those marked * are hypothetic substances.

Effects on

Chemical and

<•

plants, etc

.

other tests.

Acetic acid

page

page

"Adco" manure

9

Amino-acids (general)

26, 62, 104

Aneurin (see Vitamin B^)

Ascorbic acid (see Vitamin C)

127, 129 seq.

Auxins (general)

5-9, 53, 71,
91,96,113

85,

Auxin-a

5-7, 77, 78.
"3, 127

88,

114, 129 seq.

Auxin-b

5. "3

114

Caulocaline*

Charcoal

36

—

Co-enzyme R

92

—

Creatine

63, 88

Creatinine

63,88

128

Dihydfo-folliculin

56

—

Dihydroxystearic acid

65

—

Ethylene

28,77

—

Florigen*

54, 96

—

Formic acid

57

—

Heteroauxin

10, 27, 37,

91,

—

(syn. indole-acetic acid, q.v.)

98, 99, 112

Honey

45

—

Magnesium compound

45

—

Manganese compounds

43-45

—

Mercurial seed disinfectants

46

—

Nicotinic acid

88, ii6

— ,

Ornithine and compounds

71, 75

— 1

Oxalacetic acids

128

128

Pantothenic acid

91

—

(composition unknown)

Phenol(s)

72

—

Phyllocaline*

— ^

Picoline compounds

75

—

Potassium permanganate

43-45

—

Pyridine compounds (see picoline)

Rhizocaline*

26, 44, 60

26

Rhizopin

62

—

(syn. indole-acetic acid, q.v.)

Sugars

43, 44. 81

—

Talc

46

—

Thiamin (see Vitamin Bi)

Urorosein

—

98, 123

(see also indole-acetic acid)

Uvitonic acid

65

—

Vitamins Bj

55, 61, 68,

80,

68, 129, 13s

81, 115, 116, 1

- B,

116

—

— C

33, 68,80,93,

116

129, 135

138

Tabular Index

Phenyl Compounds

(R^CeH^)
A=acid; AA=acids

Prepara-

Effects on

M.P.,

tion, etc.

plants, etc.

Solubilities

°C

page

page

Benzoyl-glycine

R.CO.NH.CH2.COOH

71, 73

—

A;W

187

(hippuric A)

- oxide

(R. C0)20

—

87

/

—

- peroxide

R. CO. 0.0. CO. R

—

87

f

Cinnamic AA

R. CHaiCH. COOH

- ordinary (trans)

—

84

w

133

— triglyceride

81

?/

III

- alio- (cis)

99

84, 86

68

— m-o-methoxy A

84

—

■ — trans-o-methoxy A

—

84

—

—

Hippuric A

(see benzoyl-glycine)

Homogentisic A

74,75

64, 122

b;c;w;a;e

152-154

Hydroxy-phenyl-acetic A

65,74

65,74

—

:

Hydroxy-phenyl AA

74

—

(general)

Phenaceturic A

(see phenyl-glycine)

Phenol, urinary

72

59

—

Phenolglucuronic A

72

—

Phenyl-acetic A

73, 74, 98,

13, 28, 9,

R. CHa. COOH

99

32,33,37,
39,56,86,

99, (112,
esters)

tv; W

76

— glycerides

81

81

f

oil

-acetoyl-glutam||ne

73

—

—

—

— glycine

73

74

f; a; A

146?

(phenaceturic A)

-alanine

65,73,74

65

—

-butyric A

21, 8s

33,85,86

(various

isomers)

-propionic A

73,74,99

33,34,39

—

— triglyceride

Si

—

/

oil

-pyruvic A

R. CHOH. COOH

121

121

P-phenylenediamine

—

45

—

—

Tyrosine (/)

64,75,107

64,65,87

a;e;«)

270
decomp.

Tyrosol

107

—

—

Chemical tests for some phenyl compounds will be found
on pages 1 21-122.

139

/

Plant Qrowth'Suhstances

Indole, and Compounds
Indole =C6H4. C^U^, NH

Prepara-
tions,
page

EflFects on
plants,
page

Chemical
tests,
page

Solubil-
ities

—

vv; a;
b;e

123 seq.

w; a;
c

—

f

—

w; a;
e; W

126

«;?/

—

w;f;a

126, 128

dil.a

W

B

—

—

M.P.,
°C

Indole

-fcs'-substituted cpds.

-carboxylic cpds.
— esters

-2-acetic compound
-3-acetic A

— 3-acetonitrile
-3 -acrylic A

— 3-alanine {see
tryptophan)

-3 -butyric A
(normal)

-3-ethyl alcohol

{see tryptophol)
— 3-ethylamine
— hydrochloride
-3-lactic A {dl)
-3-methylacetic A

-3-methyl-indole

{see skatole)
-3-propionic A

(normal)

-3-pyruvic A

-3-succinic A

-3-valeric A
(normal)
Indoxyl
Indylene compounds

109

108,

100

109

107-110

ICO, 108,

109

107

73.75.88,

98-108,

113, 126,

127 (100,

isomer)

113

86
108

84, III
112, Fig. 3

6-(),\oseq.,

31,37-43,
^bseq., SI
seq., bo,']'!
s^g.,86,93
113

52

87 (Bauguess and
Berg, 1934)

105

"3

87

I02, 104

102, 104,
105, 108,
127
104, 105

107

108

75

lOI

12,28,29,

32-34 40,
41,46,48,

53,78,79,
85, III

87

87 (Kogl,
1938)

26,31,32,

33,40,78,

79

84

107

108

75

140

164-5*
167-8*

B.P. 160

(o'2mm.)

195-196

123-4*

146?

251-3*

144-145

133-4*

211

indef. ?
198

decomp.
105*

Tabular Index

Indole and compounds — contd.

Prepara-

Effects on

Chemical

Solubil-

M.P.,

tions,

plants,

tests,

ities

°C

page

page

page

-1 :3-diacetic A

los

107

—

a; e;

242*

/50-indolinone-3-

—

86

—

—

—

acetic A

i-methyl-R-3-acetic A

io6, no

84

—

—

—

3-methyl-R- 1 -acetic A

107

107

—

—

—

5-methyl-R-3-acetic A

—

84

—

—

—

2Veo-indole(?) and

compounds

109

—

—

—

—

Skatole and cpds.

75, 101,

33,75,87

—

a\f

95

103, no

(skatole)

(skatole)

Tryptophan

76, lOI,

—

76, 126

—

—

(indole-alanine)

103

128

^

Tryptophol

107, 109

dil. a ;
/

59*

j^OTE. — For absorption spectra of some indole compounds,
see Hare and Kersten (1937, F); Ward (1923, H; and
ihid., p. 907); and Ellinger, in Abderhalden's Handbuch,
Abt. I, Teil 7, p. 805.

141

Plant Qrowth'Suhstances

Naphthyl Compounds

Preparation,
page

Effects on

plants,

page

Solubilities

M.P.,

°C.

Naphthyl-acetic

AA

HI, 113

112

—

'"

— esters

30, 54, 112,
Fig. 2

"■

—a A

1

III

12, 28, 29,

30, 34, 37-
39, 41-43,
46-48, 55,
III

si. W;
a; b; e

131

-PA

III

Now unim-
portant com-
pared with
a-A

si. W;
a; c; s

142

-a -acetonitrile

102, III

28, 112

a;b; t

79-81

-glycollic AA

III

112

—

—

-a -glyoxylic A

III

112

W

98

(glyoxalic)

- ester

III

III

— ""

Napthoxy-acetic

A, p

119

119

Acetic Acid Derivatives of Other Aryl Compounds

Preparation,

Effects on

01

formula,
page

plants,
page

Acenaphthene, CijHio

102

, 112

29,

112

(ethylene-naphthalene ,

Cio He: C2

H4)

Anthracene, Cn Hio

102

, 112

29,

112

Benz-furan

{see coumarone)

Coumarone

82,

83

82,

83

Fluorene, C13 Hio

103

, 112

29,

112

Indene, C9 Hg

82,

83

82,

83

Thionaphthene

82,

83

82,

83

(thio-indene ?)

142

INDEX OF AUTHORS

Note. — The occurrence of names in the lists of references
given at the end of chapters is not indicated here unless
the names are not specifically mentioned in the text.

Abderhalden, E. (Handbuch), 75,

76, 122, 124, 125, 141
Adamkiewicz, A., 126, 128
Afanasiev, M., 40
Aikin, J., xii
Albaum, H. G., 126
Allison, F. E., 92
Ambrose, A. M., 76
Amlong, H. V., 39, 44, 48, 49. 55,

78,79
Andersag, H., 116
Armstrong, E. F., 57
Armstrong, H. E., 57
Arnold, E., 100
Atkins, G. A., 68, 108
Avery, G. S., Jr., 23, 82, 96, 11 1

Bacharach, a. L. , 115

Barrett, H. S. B., 109

Barritt, N. W., 67

Bartley, Mary A., 118

Bauguess, L. C, 87, 140

Baumann, E., 122

Bausor, S. C, 119

Bayliss, W. M., 92, 97

Beaum^,A.,xii, 1,71,91,98, 120,137

Beguin, A. E., 108

Beke, A., 36

Bennett, J. P., 55

Berg, C. P., 26, 87, 105, 140

Bergel, F., 116

Bonner, D., 68, 86

Bonner, J., 68, 81

Borgstrom, G., 80

Bottomley, W. B., 65, 68, 94, 95, 108

Bowden, R. A., 36

Boysen Jensen, P., 4, 23, 24, 25, 95,

no
Bradley, R., 17, 62
Braun, J. von, 109
Breazeale, J. F., 66
Bredereck, H., 71
Brigl, P., 122

British Drug Houses, Ltd., 11-12
Brown, B. E., 63
Burger, A., 109
Burk, Dean, 97
Burkholder, P. R., 23, 117
Butenandt, A., 56, 91, 93

Caldw^ell, J., 91
Carlson, Margery, G., 60
Chailakhian, M. H., 54
Chen, H. K., 108
Cholodny, N., 49
Chouard, P., 53, 56
Cline, J. K., 116
Cole, S. W., 103
Constantinesco, D., 90
Cooper, W. C, 30, 32, 86
Creighton, Harriet B., 117
Crocker, W., 77
Crook, E. M.,83
Culpeper, Nicholas, 10
Curtis, O. F., 44

Darvi^in, C, I
Darzens, G., 81
Davies, W.,168, 83, 108
Deuber, C. G., 45
Doak, B. W.,38
Dobbie, J. J., no
Duhamet, L., 82
Dyddwna, M., 69

Eden, T., 96, 105, 124, 125, 126^ 141
Ehrlich, F., 107, 108, 124
EUinger, A. ,76, 87, 102, 103, 104, 125
Ellis, M. M.,49, 92
Erxleben, Hanni, 89, 114
Evenari, M., 32, 43, 44, 52
Eyster, W. H., 49, 92

Ferman, J. H. G., 56
Finkelstein, J., 116
Fischer, Emil, 104, 105, 109
Fischer, H. 126
Flamand, C, 126
Foiling, A., 21
Friedlander, M., 51
Fulton, J. D., 119

Gardner, F. E., 54, 55
Garner, R. J., 38
Gautheret, R., 81
Geoffroy, St.-F., 137
Georgi, C, 108
Gero, M., 105
Gillett, S., 41

^It is not certain whether this entry refers to one or two authors.

143

Plant Qroivth'Suhstances

Gley, E., 97
Glover, J., 86

Grace, N. H., 32, 46, 47, 80
Granacher, C, 105
Greene, J., 68, 81
Greenwood, M., 67
Grumbein, Mary L., 77, 79
Gstirner, F., 129
Gustafson, F. G., 54
Guthrie, J. D., 54

Haagen-Smit.A. J.,84, 89, no, 114
Hall, J. M., 129
Hamilton, T. S., 76
Hammett, F. S., 87
Hardisty, F. E., 36
Hare, Dorothy, 79, 141
Harrow, B., 76, 91
Hartley, K. T., 67
Hartley, W. N., no
Hausdorfer, E., 119
Hausen, Synnove von, 93
Havas, L., 91
Heilbron, I. M., 83
Herter, C. A., 126
Hinsberg, K., 135

Hitchcock, A. E., 18, 26, 27, 28, 30,
31, 33,66, 68,77, III, 112, 136

Hlasiwetz, , 121

Hollering, Helga F., 129
Hoover, Sam R., 92
Hopkins, F. G., 103, 126, 128
Hopkins, J. W., 50
Hoskins, T., 113
Houben, J., no
Hubert, B., 45, 86
Hudson, P. C. B.,68, 108

Jack, W. R., 80

Jackson, J. R., 57

Jackson, R. W., 26, loi, 104, 105

106, 107, 109
Jackson, T. H., 32, 41
Japp, F. R., 109
Johnston, S., 38
Jong, Anne W. K. de, 104
Jung, A., 129

Kahl6wna, M., 69
Kaiser, S., 126
Kalb, L., 105
Kaufmann, E., 81
Kersten, H., 79, 141
King, F. E., 107
Klingemann, F., 109
Knop, W., 27, 28
Koepfli, H. W.,86
Kogl, F., 6, 7, 8, 9, 23, 78, 87, no,
n3, n4, 126, 127, 140

Komisarov, D. A., 38, 39
Konis, E., 32, 43, 44, 52
Kostermans, 7, no
Kotake, M., 105
Kotz, A., 109
Krijthe, Eltien, 38

Laibach, F., 27, 51
Laine, T. , 128
LaRue, C. D., 81, 82
Lathrop, E. C, 63
Laurie, A. L. 44
Lawrence, W. J. C, 17, 18
L'Ecuyer, P., 107
Leffevre, J., 53, 95, 126
I^eitch, L. C, 108, 109
Lek, H. A. A. van der, 38
Leonian, H., 68, 77
Liebenberg, A. R. von, 67
Lilly, V. G., 68, 77
Link, G. K. K., 64, 94, 108, 124
Link, Adeline DeS, 118
Loehwing, J., 95

Macht, D. L, 77, 79

McRostie, G. P., 47

Maimonides, Moses, 51

Majima, R., 105, 113

Manske, R. H. F., loi, 105, 106,

107, 108, 109
Marmer, Dina R., 78
Marrack, J., 129
Marrian, G. F., 91
Marshall, L. C, 47
Marth, P. C, 54
Marvel, C. S., 105
Matsuoka, Z., 105
Mayer, F., in
Michels, A., 109
Mitchell, H.H., 76
Mockeridge, Florence A., 68
Moore, George T., 136
Moore, J. A., 59
Mosettig, E., 109
Miiller, H., 84
Myers, M. C, 45

Naundorf, G., 43, 55
Nelles, J., 109
Nencki, M., 103, 124
Newell, J., 17, 18
Newton, R., 80
Nicol, Hugh, 64, 68, 87, 95
Niklewski, B., 66
Nixon, R. W., 55
Nowosad, F. S., 42

Oddo, B., 45, no
Oliver, R. W., 45

144

Index

Oppenheimer, T. , 1 1 1
Overbeek, J. van, 85

Pearse, H. L., 2, 23, 26, 32, 38, 77,

85, 136
Penrose, L., 121
Perkin, W. H., 109
Petersen, S., 126
Piccinini, A., 106, no
Plant, W., 56
Plimmer, R. H. A., 126
Pollacci, G., 45
Putochin, N., 105

QuASTEL, J. H., 121

Rappaport, J-, 45
Reed, H. S., 64
Reichstein, T., 84
Riesser, O., 126
Robbins, W. J., 57, "5
Robertson, I. M., 44
Robinson, R., 105, 109, 119
Robison, Robert, 114
Roe, J. H., 129
Roodenburg, J. W. M., 56
Rose, W. C, lOS
Roth, H., 128
Rousset, L., Ill
Rowe, F. M., 86
Rysselberge, M. van, 105

Salkowski, E.^, 76, 123, 124, 125,

127
Salkowski, H.,'' 76
Schelling, V., 105
Schimpf, G., 105
Schlenker, G., 23
Schmitz, E., 126
Schornig, L., 119
Schopfer, H., 129
Schreiner, O., 63, 64, 65, 128
Schwarz, W. (See Evenari)
Schweizer, F., 105
Shackell, EthelM.,82, 83
Sherwin, C. P., 76, 91
Shorey, E. C, 63, 65
Sieber, Nadina, 124
Skinner, J. J., 64, 69
Skoog, Folke, 53, 55, 85
Smith, C, 100
Smith, G., 65
Snov^r, A. G., Jr., 40
Snow, R., 87, 96
Solacolu, T., 90

Statkovskaia, E. I., 49
Steenbock, A., 121
Stewart, W. S., 57
Stoughton, R. H., 56
Sullivan, M. X., 69
Szent-Gyorgyi, A., 116

Tallarico, G., 51

Templeman, W. G., 33

Thimann, K. V., 23, S3. 82, 83, 84,

85, 86, 94, 95
Thornton, H. G., 65, 68
Tiddens, B. A., 56
Tilford, P. E., 56
Tillmanns, J., 129
Tincker, M. A. H., 12, 13, 37, 43.

86
Titoff, V.,84
Todd, A. R., 116
Tolkowsky, S., 51
Tovarnitzky, V. I., 49
Traub, H. P., 41, 52, 54

ViLLELA, C. G., 129

Virtanen, A. I., 128

Ward, F. W., 107, 141

Warne, L. G. G., 56

Waser, E. B. H., 122

Webster, M. E., 44

Weise, W., 75, 76, 122, 124, 125,

126, 127
Went, F. W., 23, 26, 30, 44, 52, 56,

60, 83, 84, 86, 94, 95, 96, 110,

129, et seq.
Westphal, K., 116, 129, 131
White, P. R., IIS, "6
Whiteley, Martha A., 103
Wilcox, Hazel W., 118
Wilcoxon, F.,27, 28, 29, 30,68, III,

112
Williams, R. J., 9^. 97
Williams, R. R., 116
Winkler, S., 128
Wislicenus, J., 100, 104
Wohl, A., 129
Wojciechowski, J. , 66
Wolfram, A., 119

Zhdanova, L. R., 54

Zimmerman, P. W., 18, 26, 27, 28,

29. 30. 31, 33. 54. 66, 68, 77, in,

112
Zirkin, D., 58

'Correct attribution between these is sometimes difficult to attain.

145

Plant Qrowth'Suhstances

INDEX OF PRINCIPAL SUBJECTS
(For substances, see Tabular Index, Chapter XV)

Abstracting journals, 22
Adventitious roots, 2, 26-30, 33, 60,

79, Figs. 2, 3
Aerial roots, 60, Fig. i
Alcaptonuria, 64, 74
Amino-acids as nutrients, 62, 104
Animal physiology, 104
Applications (see Methods)
Artificial farmyard manure

("Adco"), 9
Artificial silk, 57
Auximones, 66, 68, 93, 94, 96
Axillary buds {see also Lateral buds),

56

BiPHASic phenomenon of rooting,
60, 86

toxicity, 77

Buds, axillary, 56

dormant, 55

lateral, 58, 85

terminal, 52, 53, 55, 85

Callus-formation, 31, 35, 40, 44,

52, S3
Cell-elongation, 4, 5, 7

growth, 4, 82

"Chemical messengers", 3, 92

Coleoptile, 4

Composts (manures), 66, 99

propagating, 17-18

Copper in water, 1 34
Cotyledons, excised, 82

Decomposition (by microbes), 8,

62-66, 98-99
Detoxication, 72, 76
Diet, 7, 71, 121

Diphasic phenomenon of toxicity, 77
Dormancy, breaking of, 53-56, 85, 86

, effect of, 40

Dormant buds, 55

Dry matter of treated plants, 2, 26, 33

Dusting techniques (see Methods)

Effectiveness of substances, re-
lative, 12, 112

Elongation of cells, 4, 5, 7

roots, 60, 78, 79

stems, 56

Embryos as sources of growth-
substance, 39, 49, 115

Enantiomorphs, 87

Epinasty, 25, 28, 29, 32, 33, 54

Excised cotyledons, 82

hypocotyls, 8i

roots, 78, 81, 82, 115

Experiment Station Record, zz

Fertility of soil, 8, 9, 63-68
Field experiments, 46-48
Flowering hormone, 54, 56
control of, 53-55

Grafting, 51-53
Greenhouse soil, 64
Green-manuring, 64
Growth, inhibition of, 77-82

modes of, 4, 26, 104

promotion, 2, 25, 77, 81, 96

regulation, 2, 25, 96

Growth Hormones in Plants, 23

Hormones, classification, 91-96

definition, 94, 97

general, 3, 20

animal, 87, 92-95

plant, 3, 54, 93-95, "5

"Hormonization", 49
Horticultural Abstracts, zz
"Humus", 66, 99
Hypocotyls, 81
Hyponasty, 25

Inhibition of growth, 77, 86
Injections {see Methods)
Inorganic substances, 43-45
Inversion of cuttings, 41
Isomers, optical, 87, 114
spatial, 84

Lanolin, use of (see Methods)
Lateral buds and shoots, 53, 56, 85
Leafiness of cuttings, 15, 32, 39, 41
Leaves, roots on, i, 29, Fig. i
Legumes, 64
nodules of, 68, 108

Manure, farmyard, 62, 63, 64, 66,
67, .68, 75, 99

artificial, 9

Manures, organic, value of, 8-9, 60
Metabolic products, human, 7,

71 seq., 121
Metabolism, error of, 74-75
Meristem, 82
Methods of application :

Crystals, pure, 52

Dusting, cuttings : charcoal, 36

mercurial dusts, 46, 47,

48
talc, 32, 42, 46

146

seeds, 46, 47

Index

In formalin, 47

Lanolin smear, general, 27-29,

31.32 .
to cuttmgs, apical ends,

32' 85 , J o
tocuttings, basal ends, »S

to grafts, 31,51

to intact plants, 27, 28,

29, 31

Solid (pure) substance, 52

Solutions, mixed, 43, 49

to cuttings : cut ends dipped

cold, 28, 30-32, 37 seq.,
42, 43, 45, 49, S3, 85-86

cut ends in warm bath, 55

inversion, 41

submersion, 53

to seedlings, 46, 77 seq.

to seeds, 47-49

to soil or growing plants, 32,

33, 48, 49, 55, 56

Spraying: cuttings, 53

petals, 56

plants, 32, 33, 46, 49, 55, 50

sand or soil, 32, 46, 48

Microbial actions (see Decom-
position, Nodules)
Mixed cropping, 64
Mixtures of substances, 43

Nastic responses, 25
Nodules, leguminous, 64, 68
Non-biological applications, 57
"Nutrilite", 97

Oat test, 4, 25, 79
Optical isomers, 87, 114
Organic manures {see Manure)
Ovary, development of, 54

pH, effect on activity, 86
pK, effect of, 86

Parasitism, 59

Parthenocarpy, 54

Patents, x8, 112

Pea test, method, 129

Petioles, 27, Fig. 2

"Phytamin", 95, 96

Phytohormones, 92, 94-95

Phytohormones, 23

Polar transport, 85

Prices, 11 -12

Pronunciations, 10-11 \

Pruning, 85

Purines, 63

Root, definition of, 59

elongation of, 68, 78, 79

Roots: adventitious, 2, 26-30, 33,
34, 60, Figs. 2, 3

adventitious, 60, Fig. i

aerial, 60, Fig. i

excised, 78, 81, 82, 115

normal (underground) 47, 60,68

Root-cap, 59
Root-hairs, 68
Root-meristem, 82
Root-nodules, 64, 68
Rotting-down, 8, 66, 98-99

Sandalwood as parasite, 59
Seeds, treatment of, 46
Shoots, formation on roots, 60

growth of, 4

Sickness, soil, 63
Silk, artificial, 57
Soil fertility, 8, 9, 63-68
Spraying {see Methods)
Stems, bending of, 4

roots on, i, 29, 34, Fig. 3

Stereoisomers, 84, 87, 114
Sterilization, partial, 64

soil, 18, 64

"Sub-auxin", 96

Terminal buds, 52, 53, 85

Tests for growth-substances, 120 seq.

vitamins, 68, 135

Textile fibres, 57
Thorns, underground, 60
Thyroid gland, 87
Tissue culture, 81, 115
Toxicity of growth-substances, 34,
46, 61, 64, 65, 77-80, 82

soil, 63-65

water, 134

Transplanted trees, 56

Transport of hormones, etc., 52, 54, 85

Trees, treatment of, 39, 40, S6

Units of activity, 25, 85

Urine, 5, 6, 7, 37, 39, 71, I29,i33, I35

Vernalization, 49

Vitamins (general), 6, 22, 80, 92

93. 96 ^ ^ ^

Bi, 61, 68, 80, 81, 115, 116

Be, 116

C, 68, 80, 93, 116

tests for, 68, 135

Warm bath treatment, 39

Woody plants, 30-32, 37-41, 44, 45,

51 seq.
Wuchsstoffe der Pflanzen, Die, 23

Yarovization, 49
Yeast, 7, 55

147

Plant Qrowth'Suhstances

INDEX OF PLANTS MENTIONED

And tested with treatments likely to be of agricultural,
horticultural, or instructional importance.

Acer, 31, 39,40
Alfalfa, 42, 48, 49
Algae, 45
Allium, 80
Apple, 30, 52
Artichoke, Jerusalem, 28
Aspen, 40
Azotobacter, 68

Maple, hard, 40
Maple, Japariese, 31
Medicago sativa, 42, 48, 49
Mimosa pudica, 28 (Also used by

Bausor, 1939)
Myrtus, 44, 52

Nasturtium, 46

Barley, 46, 47, 67
Betula, 40
Bignonia, 41, 44
Blackcurrant, 38
Blueberry, 38
Bougainvillea, 57

Cactus, 44

Carica spp., 41

Carnation, 44

Cherry, 52

Chrysanthemum, 44, 45, 56

Cissus sicyoides, 28, Fig. i

Citron, 52

Citrus spp., 66 {See also common

names)
Clover, 42
Coffee (arabica), 41
Conifers, 30, 39, 45, 52
Cupressiis, 30

Date, 55 (Nixon et al., 1939)

Elder (?), 55
Euglcena, 65
Euonymus, 44

Fig, 38, 52

Heliotrope, 44
Hibiscus, 52

Holly, American, 31, 54
Holly, Japanese, 31

Kalanchoe, 30, Fig. 3
Kudzu, 42, 45

Larix, 39
Lemon, 32
Lettuce spp., 46, 81
Ligustrum, 52
Lilac, 44, 55
Lucerne, 42, 48, 49

Maize, 48

Oak, 39
Oak, red, 56
Oats, 49
Olive, 52
Orange, 52

Pandanus, 59
Papaya (papaw), 41
Passiflora spp., 41
Pear, 38, 52, 55
Pelargonium zonale, 37
Perilla, 54
Phleum pratense, 42
Phy corny ces, 129
Pinus, 39
Plum, 38

Pogonia ophioglossides, 60
Potato, 54

Quercus, 39

Ribes spp., 38, 39
Rosaceae, 52

Salix, 39, 68, 85
Sea-kale, 56
Sensitive plant, 28
Stilt-plant, 59
Stravi^berry, 55, 56
Sugar-beet, 48
Sweet orange, 52

Taxus cuspidata, 31

Thuja, 39, 45

Timothy, 42

Tomato, 28, 29, 32, 34. 37. 46, 54

Viburnum carlesii, 37
Vine (Vitis vinifera), 52, 53

Wheat, 46, 47, 48, 63, 64, 66, 80

Yew, Chinese, 31

Zea mays, 48

BOOKS on PLANT GROWTH

Growth Hormones in Plants

P. Boysen Jensen — translated and revised by G. S. Avery
and P. R. Burkholder. 1st edition 1936. 268 pages. 23s. Od.
Practical Organic and Bio-Chemistry

R. H. A. Plimmer. 6th edition 1938. 623 pages. 24s. Od.

Phytohormones

F.W. Went and K.V. Thimann. 1st edition 1937. 294 pages.
21s. Od.

Dictionary of Organic Compounds

Editor in Chief — I. M. Heilbron. Three volume 6 guineas
each volume. The constitution and physical and chemical
properties of the principle carbon compounds and their
derivatives together with the relevant literature refer-
ences.

Outlines of Fungi and Plant Disease

F. T. Bennett, 1924. 254 pages. 7s. 6d.

Bacteria in Relation to Soil Fertility

Joseph E. Greaves, 1928. lOs. 6d.

Diseases of Economic Plants

F. L. Stevens and J. G. Hall, 1933. 18s. Od. 507 pages.
Science of Soils and Manures

J. Allan Murray. 3rd edition. 298 pages. 12s. 6d.

Propagation of Horticultural Plants

G. W. Adriance and F. R. Brison, I st edition 1 939. 3 1 4 pages.
20s. Od.

Soil Conservation

H. H. Bennett, 1st edition 1939. 993 pages. 40s. Od.

Soilless Culture — Simplified

A.Laurie, 1st edition 1940. 205 pages. 12s. 6d.

Scientific Principles of Plant Protection with special reference to
chemical control

H. Martin, 2nd edition 1936. 379 pages. 22s. 6d.
Practical Plant Breeding

W. J. C. Lawrence, 1937. 155 pages. 5s. 6d.
Use of Fertilisers

A. S. Barker. 1935. 204 pages. 7s. 6d.
Growing Plants in Nutrient Solutions

W. 1. Turner and V. M. Henry. 1939. 154 pages. 18s. Od.
Soilless growth of plants

C. Ellis and M. W. Swaney, 1938. 155 pages. 16s. 6d.

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ViNEY Research Nursery

H. VINEY. A.M.I.Mech.E., F.R.H.S.

Hon. Organiser of The Hydroponic Fellowship

HYDROPONIC SERVICE STATION
WHITCHURCH VINEYARD. BRISTOL

npHE VINEY RESEARCH NURSERY was founded to give growers
proof of the utility of Hj^droponics applied to Commercial
Crops, and to forward amateur effort.

It sprang from the necessities of the hour and is the outcome of
that service and goodwill which exist between researchists
universally.

It is in a position to advise intending growers on the best layout
of Tanks, Aeration, Heating and Circulatory Systems, Nutrient
Solutions and General Equipment. Their special requirements
can be adapted to both existing and new glass-houses. Supplies
and Tuitional Correspondence are available at reasonable terms
on application.

Hydroponics solves the problem of how to increase the market
value of crops without increasing their cost of production. It
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readily operated with semi-skilled labour and there is more time
available to attend to the crops.

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