SEVERAL CHEMICAL GROWTH SUBSTANC ES WHI CH CAUSE
INITIATION OF ROOTS AND OTHER RESPONSES IN PLANTS'
P. VV. ZIMM E RMAN AND FRANK WILCOXON
In 1933 two papers published from this laborator y (IS, 17) repo rted
th a t ca rbon mon oxide and three un saturated hydroca rbon gases caused
initiation and s timula tio n of adventitio us roo t s on s tems and lea ves of
several varieties of growing plants. Th e same che mica ls caused epinasty
of leaves , pro liferati ons on s tems , and an aesthesia of growing parts (2 , 16).
Went (1 2) repo rted a tes t me thod for rhizo caline , a roo t-formin g sub­
stan ce, whe reby an approxima tion of t he co ncentration co uld be de ter­
mined b y the nllmber of roots t hat were form ed on cutting-s of sweet pea
which ha d been tr eated with this mate rial. Tbimann a nd Went (10), using­
this assa y method, d etermined the compa rative amounts of t his root-fo rm­
ing substa nce occurring na tura lly in plan t and animal mate rial. Laibac h,
MUller, a nd Schafer (8), using- co ncentrates fro m urine and pollen mixed
with lan olin a pplied loca ll y to pla nts, influenced ca llus forma tion , cell
d ivisio n, and inc reas ed ro o t growth of c uttings. Kegl, Haage n-Smit, and
Erx leben ( i ) were the first to isola te a growth substance ("hete ro-auxine" )
as a pure chemical co mpound and ide ntify it as indo leacetic acid CB-in­
dolylacetic a cid ). Hitchcock (5) of this labo rato ry , us ing indoleacetic
acid , indolepro pio nic acid, phen ylacr ylic acid, a nd p henyl pro pioni c acid
disso lved in water, oil, or in lanolin, and appli ed loca ll y to intact plants,
caused initi a ti on of roots , epinasty of leaves , and bending and swelling
of stems of severa l vari eties of pla nts.
\i\1ithin th e last few months nin e new che mical co mpounds have been
fo und to ha ve specia l formativ e effects o n pla nts. They induce adventi­
tious roots, e pinas ty of leaves, bending and swelling of s te ms, and a naes­
thesia. The purpose of t his pa per is t o repo rt th e compara tive effective­
ness of th ese substan ces and th e respo nses made by pla nts.
M ETH O DS AND MAT ERI AL
The co mpounds were used as dis till ed water so lutions Or mixed with
lan olin (U. S. P.) . In a few cases whe re cuttings were involved, dilutio ns
were made wi t h i( nop's so lution. The cuttings were placed in vials o r
Rasks so that th e basal end s \"ere immersed in the solution s. T he wa ter
solutions were introdu ced into the stems and petio les of growing plants
by means of glass tubes drawn t o a ca pillary at one e nd. The capacity of
the tubes varied but held o n th e a ve rage a pproxima tel y 0.3 cc. of solutio n.
The ca pillar:y end of the tube was inserted into th e ste m or petiole a nd
left t u dra in i n to the pla nt. There was considerable va riation in the length
J
T his art icle was preprin ted Jul y 24, 1935.
210
CONTRIBUTIONS FROM BOYCE THOMPSON I NSTITU T E
[VOT - 7
of time required for the tubes to empty. Also the response of the plant
varied with the rate at which the s ubstances drained from the tubes.
Lanolin preparations were applied locally by rubbing the mixture on
stems or leaves with a glass rod. The exa ct amount of each compound used
per gram of la nolin will be reported under the heading of results. The usual
co ncentrat ion range was from o.o ! per cent to 2 per cent.
The fo llowing species of plants were used in the e:«:periments: African
marigold (Tagetes erecta L.), tomato (Lycopersicon esculent"", Mill.), buck­
wheat (Fagopyrum esculentum Moench.), sweet pea (Lathyrus odoratus L.),
Windsor bean (Vicia jaba L.), sunflower (IIelianthus debilis Nutt.)' Jeru­
salem artichoke (Helianthus tuberosus L.), dahlia (Dahlia variabilis DesL),
sensitive plant (Mimosa pudi,ca L.), and Chenopod'ium album L.
SOURCE OF COMPOUNDS TESTED
Indole derivatives. A numbe r of indole derivatives were obtained
through the cou rtesy of Dr. R. H. Manske' of the National Research
Council, Ottawa, Canada. Among them were included the following com­
pounds: tl-[ 2 -ca rbo~y-i nd o l yl- (3) I-propionic acid, {1-[ z-carboxy-6-methoxy­
indolyl-(3)]-propionic acid, tH 7-methoxy-indolyl- (3) ]-propionic acid, "(­
[indol yl- (3) ]-bu tyric acid, {1-[ z-ca rboxY-7-methoxy-indolyl- (3) I-propio nic
acid, IHs- methoxy-indol yl- (3)]- propionic acid , {1-[ z-carboxy-indoly 1- (3)}­
ethyl phenyl ether, indolyl- (3)-succinic acid, ,,(-[z-carboxy-indolyl-(3)]­
butyric acid, (1-[6-methoxy-indolyl- (3)]-propionic acid, iJ-[z-carboxy-S­
methoxy-indo lyl- (3) ]-propionic acid.
Naphthalene der,ivatives. Alpha-naphthaleneacetic acid and iJ-naphtha­
leneace tic acid were prepared from the correspond ing methyl naphthalenes
by bromination, conversion to the nitriles, and subsequent hydrolysis of
the latter as described by Mayer and Oppenheimer (9). The a-compound
melted at 131. C. and the iJ-acid at 139· c. , a fter purification by dissolving
in al kali, boiling with Nori te charcoal and reprecipitating with hydrochloric
acid.
A nthraceneacetic acid , jl1l.ore'neaceM,c acid, and acenaphtheneacetic acid.
These compounds were prepared by heating the hydrocarbons with ch lo­
roacetic acid (13) . The yields were small , but could be improved by add ing
aluminum chloride to the melt, According to the authors c ited above,
acenaphthyl-(s)-acetic acid is formed in this reaction, while in the case
of the antllracene and fluorene compounds, the location of the substituent
group is unknown .
Phenylacetic acid and mandelic ac,id. Phenylacetic acid was obtained
from the Eastman Kodak Compa ny, while mandel ic acid was prepared as
described by Vanino (rI, p. SB9). The latter acid melted at lIB· C.
2 IL is un derstood that Dr. Manske is in position to synthesize and supply a li mited
quantity of various indole derivatives to anyone interested in these compounds.
1935}
Z IMM ERMAN
& \tVILCO XON- C HEMI CA L GROWTH SUBSTANCES
211
RESULTS
The fo llow ing eight compounds caused unusual ac ti vity w hen applied
to growing plants: a-naphthaleneacetic acid, ~-naphthal e neaceti c acid ,
acenap ht heneacetic acid, indolebu tyric acid, phenylacetic acid, a nthrace ne­
ace tic acid, a-naphthylacetonitrile, a nd Ru oreneace t.ic acid.
Th e principal responses shown by treated plants were local initiation
of roots on stems and leaves, local acceleration or retardation of growth
causing swelling aud bending of sterns, epinasty, hyponasty or twisting
of leaves acco rding to the place the componnds were applied, and possibly
local anaesthesia.
Initiation of roois. Applied t o t he st ems, all of the compounds men­
tioned above definitely caused local ini tiation of roots on growing plants
of to mato, s unflower, marigold, artichoke, b uckwheat, dahlia, and tobacco.
A lpha-naphthale neace tic acid and indolebutyric acid were especially
e ffec ti ve for initia ting roo ts o n both s te ms a nd leaves. If the plants were
kept in a glass case after treatment, t he new roo ts forced their way through
the epidermis and out into the humid atmosphe re (Figs. I and 2) . The
time required for roots to ma ke t heir a ppea rance varied with the che mical
substances used, the concentra tio n, the species of plants, a nd the exac t
place on the plan t where the ma terial was applied . Table I indicates t he
TABLE [
EFFECTIVE CONCENTRATION RAN'GE OF FIVE GROWTH SUllSTANCES F OR T OMATO, EXPRESSBD IN PEa CENT IN LANOLIN Ca using nega tive bending of
stem or epinasty of leaves
Chemicat substan ces
a -na ph thaleneacetic acid
1ndotebutyric acid
1ndoleacetic aci.d
l ndolepropionic acid
Phen y lacetic acid
Fluoreneacetic acid
. . I
0.01 00
0.01 00
0.0003
0. 02 50
0. 02 5 0
0 .0500
-
Inducing adventitio us
roo ts
, .0
, .0
, .0
0.1
0.1
-
a 4
-
2.0
2.0
Z.O
' .0
1.0
1.0
-
3.0
1.0
-
3.0
3 .0
3. 0
z.o
comparati ve effectiveness of several chemical compo unds causing rooting,
swelling, a nd negative (away fro m side where s ubs ta nce was app lied) be nd­
ing of tomato s tems. Positive (towa rd side where s ubsta nce was a pplied )
bending occurred whe n the concentration of the s ubstan ce was high enou gh
to injure t he tiss ue or retard the normal rate of grow th . In gene ra l, it
might be stated t ha t t he most effective concentration for f oot initia tion
falls just below t h at ca llsi ng positive bending of the stem or at the poin t
where there is slight re ta rdation of growth. This, however, has no t been
definitely determined. Wh en t he concentration was high enongh to canse
evident injury , roo ts appeared on the opposite side t or adj oining the place
on the stem where t he sllbstance was applied. This resul t indicates t hat
212
CONTRIBUTIONS FR OM B OYCE THOMPSON INSTITUTE
[VOL. 7
FIGURE 1. Tomato pla nt s treated with grov.,th substances. (A) Control ; (B) P lan t 24
hours after t reatment along t he stem with lanolin pa ste contain ing I % a-naph thaleneacetic
acid . (C) Approximately 0.4 ce. of 0.01% a-naphthaleneacetic acid in water injected by
means oC a glass t ube caused local epin asty and initiation of r oots; appearance after 8 days.
(D) S tem a nd leaves of plant treated wit h lanolin paste containing 2.0% a-naphthaleneacetic
acid. (E) Adventitiou s roots on stem I4 days after t reatment with 2.0% indoleacetic acid.
'9351
ZIMM E RMAN
&
WILCOXON-
CHEMI CAL GROWTH SUB STANCES
213
FrGURE 2. (A) Tomato plants. Left : con trol ; right: 14 days after inject i.ons of 0.01%
iod ole butyric acid which induced e pioasty of leaves a nd initiation of roots. (6) Adven titio us
roots on portions DCtobacco stems cut from intact p lants which had been treated over the
region of elon gation with lanolin preparatious of growth substan ces. Left to right: coutro!;
1.0% O'-naphthaleneacetic acid; 1.0% indolebutyric acid; 0.4% indoleacetic acid; 0.5%
indole propionic acid; 2.0% phe nylacetic acid.
214
C ONTRIBUT IONS FR OM B OYCE T H OMPS ON I NSTITUTE
[VOL 7
as th e substa nce diffused away fro m the poin t of toxic concentratio n , a
range was reached which wa s effective fo r ind ucing roots. Under favora ble
condi t io ns, 5 to 1 0 mg. of indo le buty ric acid or a -na pht ha leneaceti c acid
per g ram of lano li n caused roots to be indu ced and ap pear th rough the
e pidermis on to mato in 6 d ays, marigold in 5 d ays , tobacco in 8 days , and
a r t icho ke in 6 d ays. Twenty m illig ra ms of pheny la cetic acid per gram of
la no lin caused simila r res po nses. On e mill igram of indolebutyric acid or
na phtha le neace t ic a cid per g ra m of la nolin was effective but usually
required two da ys more to ind uce roots.
U nder compar able condit ions simi la r con centrations of indoleacetic
acid were not as effective for produ ci ng roots a s eit her ind olebutyric or
a -naphthaleneacetic acid. Hitch cock (5) reported indo lepropionic acid
less effective t han indolea cetic acid . All of t hese chem ica l subst ances, how­
ever, were very effectiv e for inducin g roots a nd w hen t h e mos t favorable
concentration for each was used, th ere were on ly s mall d ifference s in the
final results. The indole and nap hth a lene compounds were effective over a
much wider range than phen y laceti c acid. T he upper a nd lower limits for
effectiveness of the diffe ren t chemicals a re shown in T a ble I. The upper
limit refers to that concen tratio n which does not appea r to kill the t issue
at the point of application; th e lower limit refe rs to t he lowes t concen tra­
tion which with one a p plication induced rooting, t ho ugh t he roo ts mi ght
have been comparativel y few and the time requ ired lo nger t han fo r hi ghe r
concentrations. As a rul e , roo ts a ppeared o n treated s te ms two to five
d ays ea rlier than on t r eated adjacent pe ti oles. T he o ld er o r m idd le-aged
leaves of tom ato pr od uced roots mO re read il y th a n yo ung leaves. W hen
tobacco plant s I S inches ta ll we re treated fro m tip to base of t he ste ms wit h
effective concen tra tio ns of a ny of the five chemical su bs ta nces me nt io ned
above, r oots formed m ost readil y a lo ng t he regio n of elo nga t io n extend ing
fro m near the tip d ow nward fo r a dis ta nce of two t o t h ree inches (F ig.
2B) . T his respo nse resem bles in some res pects t ha t caused by carbo n mo n­
oxide a nd th e three unsatura ted h ydroca r bo n gases as re po rted fro m t his
laborato ry in 1933 ( I S, 17) . The to mato a nd ma ri gold pla nts rooted wher­
ever the grow th s ubstances we re a ppl ied , eve n to t he very t ip . T he yo un g­
est t issue, however , d id no t produce roots as readi ly as midd le-aged or old
tiss ue ( Fig. 2A) . This a lso resem bles t he response t hese pla n ts m ad e to
t he gas t reatments.
W here a generous su pply of lanoli n cont aining two to three per cent
a-naph t h aleneacetic acid wa s applied to stems or lea ves, abT1 o r m ~l roots
were produ ced wit h an ab und an ce of root ha irs down t o th e tip. Also
short, ch u b by roots often resulted from high concentrations of t he sub­
stance (Fig . rD). As a rule, roots induced with specia l substan ces had a
white color a nd ca me out of the stem at an angle of approxima tely 45°,
bu t a -naphthalenea cetic acid brought out a pink shadi ng to brown co lor
•
'9,SJ
ZIMMERMAN
&
WILCOXON-CHEMICAL GROWTH SUBSTANCES
215
of the roots, and they grew downward nearly paralleling the stem. These
qualitative differences were in evidence whether the naphthalene com­
pound was applied as a lanolin paste or injected as a water solution.
\Vater solutions of both indolebutyric acid and a-naphthaleneacetic
acid were effective for producing roots over a range of roo p.p.m. to I
p.p.m. Neither the upper nor lower limits were definitely determined. Fig­
ures I and 2 show the response of toma to plants w here the su bstances were
injected with glass tubes drawn to a capillary at one end.
A convenient method for admitting water solutions of the substances
directly into the vascular system is shown in Figure 4. The stem was slit
upward with a knife so that the resulting overhanging stem piece could
be immersed in the solution. By means of this method, upward transloca­
tion of the substances could be studied.
Ethylene as a root-forming substance. It was reported from this labo­
ratory in 1933 that ethylene induced root initiation when intact growing
plants were exposed to low concentrations of the gas (17). In the experi­
ments at that time the entire plant was subjected to the gas and the effects
were, of course, systemic. It now appears that local effects can be ind uced
if the gas is dissolved in lanolin and applied as described for other growth
substances.
To obtain a solution of ethylene, the inside of a bottle was smeared
with lanolin, after which the air was displaced with the gas and then the
bottle stoppered and set in a cold place for several hours. The concen tra­
tion of ethylene in solution was not determined because it could not re­
main constant when taken to a warm place where plants were treated.
Since the gas escapes from lanolin more readily than the crystalline com­
pounds, applications to the same region of the stems \vere made on three
successive days. Also a larger quantity of the paste was necessary than
for the other substances.
When applied as described above to the middle region of a tomato stem
of a plant 8 to 10 inches in height, numerous roots were initiated locally
and could be seen emerging through the epidermis in six days. This is the
same length of time required for production of roots after treatment with
the best crystalline growth substances.
Local acceleration or retardation of growth. Growth substances applied
to one side of active shoots caused negative or positive bending according
to the concentration of the chemical and the plant species. Positive bend­
ing was assumed to be due to retardation in normal growth rate or injury
to the tissue where the chemical was applied. Negative bending was due
to acceleration of the growth rate on the treated side of the stem. Figure
3 shows the result with artichoke plants of treating one side of the stems
with concentrations which accelerate the growth rate. "YVhitening and swell­
ing of the bark tissue followed within 48 hours after application of con­
TABLE II
EFFECTiVE CONCENTRATION RANGE OF GROWTH SUBSTAN CES CAUSING BENDING OF SWEET
PEA STEMS EXPR ESSED IN PER,CENT IN LANOLIN
Chemical substance
co
For positive
bending Or injury
For negative
bend ing of stems
CH ' COOH
a-naphthaleneacetic acid
0.05
-
1.0
1.0
-
5.0
,"0
0.05
-
1 .0
'.0
0.025
-
1.0
o·s
0 . 0005
-
0·4
,"0
0.025
-
1.0
0.25
-
2.0
0.1
-
0.5
~CH,COOH
VV
{J~naphthaleneaceticacid
W
10 .0
CH 2 CH 2
Acenaphthyl-(s)-a cetic acid
CH,COOI-I
~
CI-I,CH,CH,COOH
VV
IndoJebutyric acid
NH
~CH,COOH
VV
Indoleacetic acid
NH
~
CH,CH,COOH
VV
Jndolepropionicacid
NH
CH ' COOH
O
r
~
Phenylacet ie acid
CH,
[
CH,COOH
Fluoceneacetic aeid
1.0
( Y ' Y 1 1 c H,coOH
[
~
J
1.0
Anthracen eacet icacid
CH'CN
CO
a-naph t hy la cetanitrile
15 . 0
' 9.15 )
Z IMMERMAN
& WILCOXON-ClIEMICAL GROWTH SUBSTANCES 217
centratio ns w hich cause pronounced nega tive bending. Sweet pea seedlings
an inch or more in heig ht showed posi ti ve be ndin g (toward t he side of th e
stem where the subst a nce was applied) when t he stems were treated o n
one side with 1.S per cent in lanolin of a-naphthaleneacetic or 2 per cent
of indol eb utyric acids. One per cent of these s ubs tances caused negative
ben din g. Tabl e II shows a range of e ffective con cen trations for a ll th e
compounds tested.
Tom ato stems showed negative bendin g with 2.0 per cent in lanolin
of indoleacetic aci d , indolepropionic acid, and phenylacetic acid, whil e
t he same concent rations of nap htha leneacetic acid an d indo leb utyric acid
ca used a positive response . It should be possible to determin e inte rm ediate
concentratio ns in a ll cases whi ch do not cause bendin g in either direction .
For example, 0.5 per cent indo leace ti c acid in lanolin, . while increasing
th e di ame ter of the sweet pea a nd ""indsor bean stem tips, did not ca use
bendin g. S lightl y lo wer concentl-ations, howeve r, caused nega tive bendin g.
Sweet pea and \;Vindsor bea n seedlings we re retarded in elo nga ti on
but in creased in diam eter of t he stem tips by various concentra tio ns of
a ny of t he growth s u bstances (Table III, Fig. 3). A detailed microscopic
study has not yet been completed bu t cross sec tio ns o f fres h mate dal ob­
served wit h th e aid of a microscope indicated th at much of the swellin g
of th e s te ms was due to t he production of spongy, fluffy tiss ue ou tside th e
vascul ar cylinder. R etardation of '''/indsor bean treated o n two sides wit h
a seri es of con centra tions was greatest with the stronges t solution , de ­
creasing with dilution down to a place whe re there was no app aren t
e ffect. Mention should here be made of the fact th at Knight a nd C rocker
(6) reported that ethylene ca\lsed re ta rdation in elongation and swelling
at the s tem ti ps of etiolated sweet pea seed lin gs.
TABLE III
E H.ECT OF I NDOLEACETlC AN I) a - NA I'HTf[AI.ENEACET IC A CID A pPLl EI) IN
OPPOS fTE SID ES OF \ V nWSOR BEAN SEEDL INGS
Per cent of substance
Av. height
No. plants in cm . when
measu red
t rea ted
Ap ril
"
0.4 indoleacetic a c id
0 .1 indoleaceti c acid
0. 025 indoleacetic acid
J .0 a- na phthalen eacetic acid
Con trol
Control
Av. increase in height nt three
la ter dates
April 1J
12. r
12· 7
9
9
16. 2
,.' . 3,
·7
"1.1 ·4
' .6
9
.t2
·5
April
.,
'0
'9
.1 8
L ANO LIN TO
" 9
' ·9
,
15
April
17
4·8
8· 3
6.'
1 2·9
8.,
17·5
6 .0
10.0
9 ·9
19·3
10.0
20.6
Measurements fo r rate of g rowth of stems t reated fro m the tip bac k
t o th e base indicated first accele ration and then retardatio n of elo ngatio n
(Table IV) even th oug h th e concentration was low enou gh to cause nega­
218
CONTRIBUTIONS FROM BOYCE THOMPSON I NSTITUTE
(VOl.. 7
FIGURE 3. (A and B) Artichoke plan ts treated a lo ng one side of the stern wit h lanolin
preparations of different s ubsta nces. The upright plant in each pot is th e control. Left to
right the compounds are: 1.0% ind olebutyric acid; 1.0% a-naphthaleneacet ic acid; 1.0%
ind olepropionic acid: 0.4% indoleacetic acid. (C) Windsor bean seed lin gs treated a long one
side with 1.0% a-naph tha leneacetic acid in lanolin. Tall plants arc controls. (D) Sweet pea
seedlings. Same treatment as in "(e)."
1935 1
ZIMMERMAN
&
WILCOXON-CHEMICAL GROWTH SUBSTAN CES
219
FIG URE 4. CA) Tomato plants. Left: control ; right: cut surface treated with 1.0%
a-naph t haleneacetic acid; ph otogra phed after 8 days. (B) Artich okes. Left: control; right:
injected with 0.4 ce. of 0.5 % solution of phenyl acetic a cid. (e) T obacco w ith overhaIlging
slit stem immersed in 0.05% indolebuty ric a.cid. (Note TOots and epinasty a long path of
translocation.)
CONT RIBUTIO NS FROM BOYCE TH OMPSON
220
I NSTITUTE
[VOL. ,
TABLE tV
EFFECT OF A 0.5 PER CENT LANOLIN SOLUTION OF DIFFERENT COMP OUNOS A pPLIED TO A TEN- INCH ZONE FRO:>I THE TIP DOWNWARD Plant
Compound
Heig ht in
em. when
t rea ted
May
Artic hoke
Lanolin cont rol
a - naphth a~e nea ce tic
acid
Indolebutyric acid
Indoleaceti c acid
L'l.noiiu control
"
a- naph thaleneacetic acid
"
Pheny lacetic acid
Indoleacetic acid
"
"
"
"
"
Tobacco
"
"
"
"
"
"
31
I
.0
32 .0
28·5
28·5
28.0
22.0
20,0
2 4·0
22.0
2 6.0
22 .0
25. 0
Increase in heig ht at [our laler dates
May
1. 0
3·5
3·5
3. 0
4·5
, .0
, .0
3 ·5
3. 0
3·5
5 .0
5. 0
2
May 3
4 ·0
6.0
5·5
4· 5
7· 0
3. 0
3·5
6·5
6·5
4 ·5
M ay 5
Ma)'6
6·5
,.
5 0
5
7 ·5
6. 5
6 5
8 5
7. 0
7 ·5
7 ·5
9·5
6.0
[0 .0
{ l.O
I2.0
8.0
11 .0
12,0
6 ·5
5·5
5·5
7. 0
5·5
6 .0
7. 0
9 0
tive bendin g. To prevent bendin g so that mea surements could be made, the
lan olin containing the ch emical was a pplied a ro und the stem so th e effect
would be nea rl y th e same thro ug hout. Some bend ing occurre-d with this
method, probabl y due to failure to ma ke even dist ribution of th e m ate ri a l.
Epin as ty of leaves was induced by app lication of th e s ubstances in
lano lin o r b y inj ectin g the water sol utio n by mean s of g lass tub es. If ap~
plied in la no lin to a narrow zone a ro und the stem of an active plant, leaves
above a nd below this zo ne s ho'lNed down wa rd bending especially at the
base of t he petioles. You ng leaves usuall y showed curling in add ition to
prono un ced bending at the base. Epinasty of a n y single leaf was induced
by ap plying t he che mical lightl y t o a sma ll region on t he u ppe r side of
t he petiole. The epinas tic response of leaves to th e g rowth sli.bstan ces was
like that produced with carb on monoxide and unsaturated hydrocarbo n
gases (2, 16) exce pt that w hen the pla nts we re in an atmosphere con ta in­
in g gas t he en tire system was affec ted in contrast to local effects. Ethy lene,
propylene, a nd acetyl ene gas dissolved in la nolin or in wate r produced
a lso the local e ffect when applied t o t he uppe r side of a to ma to petiole.
Some spreading occur red when e nough la nolin containing the gas was used
a nd if rep eated ap plications were mad e, the entire pla n t responded as if it
had been placed in an atmosphere conta ining gas. T he res ult was s imila r
to t hat described by Zim merman, Hitchcock, and C rocker ( IS) showing
that gas admitted to any par t of a leaf sp read thro ug ho ut t he entire
system of the plan t .
T he like ness of the growth s ubstances to the gases was furt her empha­
sized by the fact that the d egree of epinastic response in both cases va ri ed
with t he tempe rature. It was previously repo rted from this labo ra to ry
( I4) that tomato plants did not make a typical response to ethyle ne gas
1935]
ZlMMERMAN
&
\ \ ' ILCOKON -
C I-IEMI CAL GROWTH SUBSTANCES
221
a t 50° F. or less; above that temperature, however, t he response increased
with the temperature up to 73 F. Typical responses we re best obt a ined
when the temperature was 60 F. or above. Thi s same variation in epi­
nastic response held also for tomato plants treated wit h la nolin prepara­
tions containing 1.0 per cent of t he grow th s ubs tances.
\iVhen injected as a water solution into the stem of a rtichoke, indole­
buty ric acid was car ried both up a nd down approximately eight inches
fro m the point where it was admitted. The path of translocation co uld
be seen by t he epinastic response of the leaves (Fig. 4). Only leaves associ­
ated wit.h the vascular bund les where the substance was injected sh owed
epinasty. Alpha-naphtha leneacetic acid appeared to move upward a dis­
tance of 19 inches after an overhanging slit portion of an artichoke stem
was immersed in a 0.5 per cent solution , as evidenced by epinasty of
leaves a long the s tem to t he t ip of the plant. H ere again leaves out of li ne
with the vasc ular bundles of the overh a nging stem p iece did not respond
to the che mical. Simila rl y, to bacco plants treated with indolebutyric acid
a ppeared to carry th e substance upward a distance of 16 inches. Th e path
of tra nslocation could be followed first by epin astic response of leaves and
latcr by growth of roots initiated along the stem (Fig. 4). As can be seen
in the picture, there were two distinct rows of roots on a line with th e sides
of the overhanging stem piece. There is evidence of latera l diffusion , since
the s t.em below the pl ace where the solution was ad mitted showed con ­
siderable root production. As t he subs tan ce spread out of the main path
of travel, some was carried dow nward , indu cing roots along the way.
Downward translocation was shown by applyin g the substances t o t he
cu t surface of a tomato stump (Fig. 4). The effect was evidenced by epi­
nasty of leaves all along the stem and production of roots over a two-inch
zone from the point where applied. This dua l response indicates t hat a
higher concentration is necessary for root initiatio n tha n for epinasty.
Th e root response shown in F igure 4 illustrates a lso t hat the e ffect from
the presence of a growth s ubstance overcomes t he influence of polari ty,
t he roots in this case being produced at the t ip instead of at the b ase.
T o mato c ut tings fi ve inches long with basal ends immersed in Knop's
solu tion containing the effective su bstances showed declination of the
uppermost leaves within a few hours' time. Hitchcock (5) reported t his
type of response for tom a to cuttings in solu tions of indolepropionic acid .
The absolute lower limi ts for effective concentrations causing the response
were not. determined for the differen t s ubstances. The following low con­
centrations, expressed jn parts per million of wa ter, we re fo und to cause
epi nasty of tomato cuttings: pheny lacetic acid 125, a -naphthalen eacetic
acid 4. indolebuty ric acid 20.
H ypon asty of individu a l leaves was induced by applying the s ub­
stances to the under side of t he petiole. Carbon monoxide gas caused epi­
0
0
222
CONTRIDUTlONS FROM BOYCE THOMPSON INSTITUTE
IVOL·7
nasty of all leaves, or what might be called a systemic effect. Wh en the
growth substance was app lied to the under side of two petioles and then
the entire plant subjected to an atmosphere containing one per cent car­
bon monoxide gas, two forces worked in opposition to each other. Figure
5 shows the appearance o f tomato plants afte r such a treatment. It can
be seen that the fin a l result is not the typical effect fro m e ither the gas or
the growth s ubstance, but a combined effect. When a wa ter solution o f the
growth substance was injected into the s tem o r leaf, epinasty resulted,
indicating that the tissue on the upper side of the petiole is more suscep­
tible than that of the lower side.
Response of jJ;Iimosa pudica to growth substances. Pulvinal cells of
}'1imosa pud'ica were rendered inactive when trea ted with lanolin contain­
in g one per cen t of a -naph thaleneacetic, indolebu tyric, or indoleacetic ac id.
At first it appeared as though the surrounding tissue had grown and t h us
clamped the pulvinal cell s so they could not respond, but that assumption
did not seem logica l a fter the plant recovered a nd again made a normal
response. Applied to t he upper side of the pulvinal tissue, the petiole
showed a slight declination before it became set o r applied to the lower
side, the pe tiole moved upward. In either case the tissue was rendered
incapable of responding to external stimuli such as touch or sudden change
in temperature. Figure 5 shows two pictures of a plant with the base of
one petiole treated with a-naphthaleneacetic acid. The first shows the
plant in a normal equilibrium position, the second after one leaf had been
stimulated wit h the flame of a match. Note that the angle between t he
petiole of the treated leaf a nd the stem is practically the sa me in both
pictures whereas othe r petioles moved downward. The conditiou o f the
treated pulvina l cells did no t prevent the s timulus from passing through
the petiole and into the lea flets which showed the normal response.
This condition suggests local anaesthesia. Previous reports from this
laboratory concerned induced anaesthesia of l'i1imosa with carbon monox­
ide and unsatura tee! hydrocarbon gases (2, 16). When treated wi th those '
anaesthetics the entire plant lost its irritab ili ty , the gases causing a sys­
temic effect. Wi th a local ap plication of the so-call ed "growth substance"
the effect was local. The result may have been local anaesthesia.
Alpha-naphthylacetonitrile. The response of plants to treatment with
this compound needs specia l mentio n. The s ubs tance is a product resulting
fro m the reaction between sodium cyan ide and naphtho-benzyl bromide,
one of the steps in making a-naphthaleneacetic acid , which compound
causes epinastic response of tomato leaves in less than two hours. How­
ever, this nitrile does not canse an evident response until the second day
after its applica tion, and then the response comes on slowly. It is effective
when applied in lanolin or injected with th e glass t ube method with water.
The ·final results were sim ilar to those obtained with a-naphthaleneacetic
[9351
ZIMMERMAN
&
WILCOXON-
C HEMICAL GROWTH SUBSTANCES
22 3
FIGURE 5. (A) Tomato plants given com bined treatmen t of ind oleacetic ac id (0.4%
in lan olin) and LO% carbon monoxide gas . Left: upper side of two midd le leaves treated
with the acid; r ight : lowe r side of t wo middle leaves t reated with th e acid. (B) Mimosa
pudiC(}. 20 hours after base of on e petiole (noted by arrow) had been treated with 1.0 %
a-naph thalen eacetic acid in la nolin. Left: a ppea rance before flame test; right : o ne minnte
after flame of ma tch was a pplied to t ip of leaf on left. (Note change in position of leaves
except ing one treated. )
224
CONTRIllUTIONS FROM BOY(" E 7HOMPSON INSTITUTE
IVoL. 7
acid. The type of response made by the plan t suggests that the raw prod­
uct, the nitrile, undergoes hydrolysis in th e living tissue , forming a n effec~
tive growth substance-possibly a-naph thaleneacetic acid.
Gaseous ema.na.tions from treated plants. Since the chemical compounds
in question acce le rate growth and since plant tissu es are known to give
off physiologically effective gases, the question arose as to whether treat­
ment with th ese chemical substances would has ten the rate of me ta bolism
and thereby increase th e rate of production of em a nation s from the tissues.
Three t est pla nts, African marigo ld , Chenopodiwn album, and sunflower,
previously reported as more sensitive to ethylene than tomato (2). were
used in th ese ex periments to d e tect gaseous e manation s from plants .
Trea ted and control tomato plants were inclosed with appropriate test
plants in IS-liter bell jars. Ch.enopodium album inclosed with one Is-inch
high tomato plant treated a long the stem with 1.0 per cent indoleacetic
acid in lanolin showed a pronounced epinastic res ponse in 16 hours. Also
th e test pla nt inclosed with two large tomato plants sho\ved a prono unced
epin as ti c response. The tes t plants with the comparable control lots did
not so respond in 72 hours. In o ne case where th e test pl a nt made agood
response in 16 ho urs, a ma rigo ld plant was substituted for Chenopodium .
Four hours later th e marigold showed evident epinas ti c response, resem­
bling plants tr eated with ethylene , propylene, ace tyl ene, or ca rbo n mon­
oxide gas.
In th e next set o f experiments plants were t reated with 0.2 per cent
water solutions of indoleacetic acid injected along the stem by the glass
tube method . M a rigold pla nts were so sensitive to the emanations that
they made a slight epinastic res ponse with control plants. They m ade,
however, a pronounced epinastic response w here inclosed with treated
plants . Chenopodium plants respo nded only where associated with treated
plants. Mimosa pudica appeared normal with control plants, but where it
was 'inclosed with two la rge treated to mato plants, the leaves of this sensi­
tive plant lost th eir normal orientation to gravity, assumed a ruffled ap­
pearance , and lost their irritability to externa! stimuli. In every res pect
these plants appeared like those treated with carbon monoxide gas, where
the res ponse was described as anaesthesia ( I6). When inclosed with only
one treated tomato plant, 111imosa plants became sluggish but had not
entirely lost th eir irritabili ty .
\;'\lith proper selection of plants for variety, size, and a ctivity. it should
be possible to find typ es whi ch as controls would not cause a res ponse when
inclosed with the most sensitive tes t plants. In this way the work could
be standardized so that quantitative measurements could be made for
th e effectiveness o f various concentrations of the different growth sub­
stances. D e terminations are now being made of the change in respiratory
ra te due to ac celeration of metabolism by means of growth substances.
f 9351
Z IMM E RMAN & WILC01C01, -CHE MI CAL GROWTH SUIlSTANCES
22 5
Wh en pla nts are treated with kn own concentrations of ethylene very
defini te fo rma tive effects resulted. Reco un t ing some of them we find t hat
ethy lene induced epinasty of leaves, g rowth ri gor, anaesthesia, a nd initi­
at io n of roots. Nm·v that chemical co mpo unds are found to hasten th e
produ cti o n of ethy lene or a like ga s in pla nts, some of the effects a tt ribu ted
to so-called "growth subs tances" mi ght Qe du e indirectly to th e unsat­
nra ted hydrocarbo n gas produced in t he t iss ue.
The con ceptio n that eth ylen e e ma nates fro m plants is well sup po rted
b y previo us published wo rk. In 1933 Botj es ( I) found by inclosin g an a p ple
with a to mato pla nt that the frui t gave off a physiologically effective gas
which caused epinasty of leaves. Kn owing how t omato plants respond to
traces of ethy lene in air, he concluded t hat ethylene emanated from th e
apples . Cane (4) went a step furth er by ac tu a ll y isolating ethylene from
air surrounding a pple tissue. Denny a nd Miller (3) recently have found
that prod uetton of emanations caus in g epinas ty of leaves is not res trict ed
to fruit bu t they a re given off also by va rio us t y pes of plant tissue includ­
ing leaves, s te ms, and flowers. \Vith s uch facts cons tantly increasing a nd
with th e act ua l identification by Can e of ethy lene as the gas emana ting
from a pple tissue, it a ppears as tho ugh e th y le ne might be associated in
genera l with t he no rmal metabolis m of pla nts. If so , increase of th e rate
of me tabo lis m wo uld at the same time incr ease t h e I-ate of producti on of
emanatio ns fro m the tissue.
DISCUSS ION
Previously eight chemical substances were kn o wn to induce initiation
of roots: carbo n mo noxide, ethylene, propy len e, acetylene, indolepropioni c
acid, phen y lacry lic ac id , and phenylpropio nic ac id reported from this
laboratory , a nd ind oleacetic acid isolated b y K ogi a nd others (7). The
present pa per desc ribes eight mo re che mical co mpounds which also induce
initiation of ad ve nt itio us roots. This ma kes a tota l of sixteen substances
thus effecti ve. O f t he ma ny thousands of kno wn che mical compounds,
there are pro ba bly ma n y o thers which would be equa ll y e ffective with those
known t o date. Consid ered from that an g le, th e recent disclosures militate
against th e idea of specificity for a part icul a r Ilg rowth substance" or
flhormone " unl ess we choose to put new m eanin g in to those terms. It
wouid seem m ore logical to speak simply of th e res ponse of plants to cer­
tain chemical compounds. If the plants manufact ure t heir mvn growth­
regulating substances , it is not likely that an yo ne pla n t would make all
of those known to be effective. Neither is it logical to ass ume that all
plants naturall y ma ke and use one and the same gro wth s ubs tance. The
delayed r esponse of pla nts to a-naphth y lace to nitrile re po r ted under "re_
sults" s nggest s th a t th e raw ma terials at hand may d etermin e the kind of
substances th e tissues manufac ture. Perhaps th e g row th of a given species
226
CONTRIll UTlON S FROM BOYCE THOMPSON INSTITUTE
IVOL · 7
is not a lways regulated by the same s ubstance but it ma y var y w ith t he
env ironmenta l conditions of the pla nt.
T here was a qu a li tat ive difference in t he response of pla uts to the
different s ubstances which deserves further consideration. Ro ots prod uced
after treatment with indoleacetic acid were of a w hi te color and grew away
from the stem at an ang le of approximately 45'. Alpha-naphthaleneacetic
acid, especially in the higher concentrations, caused roots to develop a
pinkish-br own color and grow downward, nearly paralleling the stem. Also,
if the supply of the latter s ubst a nce either in water or lanolin was m a iu­
tained for five to ten days, som e a bnormall y th ick s tubby roots appeared
a nd the majority of roots were completely covered with root hairs. It has
bee n show n ( IS, 17) that carbon monoxide and the unsaturated hydro­
carbons change the OI-ien ta tion of roots and ind uce a bnormal prod u etion
of root hairs.
A characteristic response of sweet pea and 'VVindsor bean seedli ngs
treated with growth substances was abnormal enlargement of th e stem
tip (Fig. 3) and retardation in elongation. Growth is t hus increased in one
direction a nd retarded in a nother. From the standpoint of concentrations,
indo leacetic acid was t he most e ffective s ubstance caus ing this response.
As low as 0.0005 per cent was effective . The other substances caused t he
response, but with higher concentrations. Iu general, a concentration
which ca used bending of the stem also caused abnormal enlargement at
the tip .
Through the kindness of Dr. R. H. Manske who supplied the material
from his labo ratory, it has been possible to test I2 new indole compounds
for possible e ffectiveness as g rowth s ubs tan ces. The only ac tive one of the
list was indo le buty ric acid, though , fro m the stand point of mo lecular make­
up, it is hard to believe that the slig ht difference in structure could account
for the vast difference in effectiveness. As a root-forming substance, indole­
butyric is more active than the oth er tw o effective indole compounds. As
a grow th-promoting substance for producing epinasty of leaves or nega­
tive bendin g of stems from local app lication, fi-indo ly lacetic acid was the
most effective and indolepro pion ic the least. In contrast wi th t he highl y
active fi-indolylacetic acid made by Dr. Manske, a-indolylace tic acid made
in o ur labo ratory was ineffective.
ln the naph t ha lene series a- a nd fi- naphthaleneacetic acid were bo th
active but the a lpha compound was approximately 100 t imes more effec­
ti ve than beta. There was a possibility of co urse, that the beta compound
was slightly contaminated with the alpha. Compared with indole deriva­
tives the a -naph thaleneacetic acid rates equally with indoleb utyric for
producing epinasty of toma to leaves and as a roo t-forming substance.
Acenaphtheneacetic acid and fi-naphthaleneacetic we re muc h a like in
effectiveness. Wit h these two co uld be classed a lso anthrace neacetic acid
'9351
ZIMMERMAN
&
WILCOXON-CHEMICAL GROWTH SUBSTANCES
227
for mild activity. Fluoreneacetic acid fall s somewhere between t he last
three na med compounds and a -naphthaleneacetic acid from the sta nd­
point both of ro ot init iation a nd epinasty of leaves.
Leaves treated with high concent rati ons of the most effective sub­
stances made a very pronoun ced epinastic response from which t hey did
not recover. If, on the other hand, the leaves resp onded to lowe r concen­
trations, recovery followed within a few hours or days , var ying with the
amount of material in solution available to the plant. This response indi­
cates either that the plant utilizes the substances or that they deteriorate
after entering the tissue. \Vater solutions of all the substances deteriorated
on standing a few days ; lan olin preparations, however, retained their
effectiveness indefi nitely. Leaves were slower to recover if a large amo unt
of la nolin containing a low concentra tion of the substance was a pplied
than where only a thin film was employed. In t he latter case the ent ire
s uppl y proba bly entered the plant where it d eteriorated or was consumed
b y the living tissue.
E thylene, propylene, and acetylene dissolved in lanolin caused t he
same responses when applied locally a s the lower concentrati ons of the
other growth substances. Repeated applications of t he ethylene or propy ­
lene preparations t o the same region of t omato stems for three s uccessive
days a lso caused local initiation of roots.
SUMMARY
1. Sixteen growth substances have been considered as follows: ,s-indolyl­
acetic acid isolated by Kogi and others and tested by Went a nd others ;
incioiepropionic acid , phenylacrylic acid, and phenylpropionic acid re­
ported by Hitchcock; carbon monoxide, ethyl ene, propylene, and acetyl­
ene re ported fro m this laboratory as root-forming substances; and the
following eight compounds rep orted in the present paper: a -naphthalen e­
acetic acid , Ii-naphthaleneacetic acid, acenaphthyl-(s)-acetic acid , indole­
butyric acid, phenylacetic acid, ftu oreneacetic acid , anthraceneacetic acid,
and a-nap hth ylacetonitrile.
2. The principa l plan t responses induced b y the growth s ubstances
were local initiat ion of adventitious roo ts on stems and leaves, prolifera­
tions, swelling and bending of stems, acceleration of grow th, and epi­
nasty of leaves.
3. G row th substances a p plied in lanolin paste caused loca l responses
where applied though tending to become systemic' when high concentra ­
tions were used. "\i\Tater solution s of the substances caused systemic re­
sponses when the concentration was high, but local eHects with lower con­
centra tions.
4. The substances moved both upward and downward in stems. When
injected by means of glass tubes drawn to a capill ary at one e nd or ad­
CON TRIBUTIONS FROM BOYCE Tf-lOMPSON INSTITUTE
IVOL·7
mitted into an overha nging sli t ste m, the substances moved upward for
a distance of [9 inches in the artichoke stem and 16 inches in the tobacco
stem. Th e distance was indicated first by e pinastic respo nse of leaves and
later by adve nti t io us roots which were induced al o ng the way.
s. All of the substances retarded elonga tion of sweet pea seedlings but
iucreased the growth in diameter of t he stem especially when applied at
the tip. This response was like that induced by carbon mon ox ide gas and
the unsaturated hydrocarbons.
6. There was some indication that the s ubs tances iuduced local anaes·
thesia in pulvin al cells of 111imosa pu.dica.
7. Alph a -naphtha leneacetic acid and indolebuty ric acid are the most
effective root·formin g substances yet discove red; th ey are no t as effective
as indoleace tic acid fo r epinas tic response of lea ves. Ethylene or pro py·
lene dissolved in lanolin and a pplied locally for three s uccessive da ys in­
duced local initiation of roots in six d ays.
8. There was an indica tion tha t plan ts can use a-naphthylacetonitrile
as raw mate rial from which it can make an effec tive growth substance.
9. Th e production of e manations, thought to be ethylene, from growing
plants was increased by treat ment with growth substances. These chemical
compounds accelerated grow th , hastening metabolism, which in turn was
though t to in crease the production of ethylene by the living tissue . In­
creased growth resultiug from the applica tion of the special subs ta uces, as
evidenced by the epinastic respo nse, in creased with th e te mperatu re, b e~
gin ning at 50° F.
LITERATURE CITE D
OORn .... I.1N . Aelhylee n als v ermoedelijke oo rzaak van d e groeirenunende
werking van rijpe appels. Tijdschr. Plante nziek. 39: 207- 2 1 (, 11)3 3.
2. CROCKER, WILLl A."', P. W . ZIMMERMA N, and A. E. HITCHCOCK. E thylene·induced epi·
nasty o{ lea ves a nd th e relat ion of gravity to il. Contrib. Boyce Thompson [nsl.. 4 :
I. I3 0 T)iiS, JE .
177- 2J8. I93 2.
3. DENNY, F. E., and LnvRENcE P. MILLER. Production of e th ylene by plant tissue as
indicated by the ep inastic response of leaves . Contrib . Boyce Thompson Inst. 7:
0 7- 102 . 1935·
4. G"~E, R. Prod uction of et hylen e by some ripen ing fru it.s. Na t ure ILondon ] 134: loo~t
193 4·
5. HITCHCOCK, A. E. Indole- 3-n-propion ic acid as a growth hormooe an d t he quantitative
mea.surement of plant res ponse. Cont.rib. Boyce Th ompson In st. 7: 87-9 5· 1935.
6. KNIGHT, LEE I., and W~J. CROCKER. Toxicit y o f s moke. Bot. Gaz. 55: 337- 371. IQ I 3·
KOGL, FRITZ, A. J. HAA.GEN·SJ.IJT, und HANNI ERXLEBEN. Ober ein neues Auxin ("Het­
ero·auxin") aus Harn. II. Mittei lung libel' ptlanzlich e Wachstum sstofFe. Hoppe­
Sey ler's Zeitschr. Physio!. Chem. 228: 90-r03. 193 4.
8. L AIBA.'c"H, F., A. M OLLER , \V. SC HAFER . Ober wurze lbildende Stoffe. Naturwi ss. :z:z:
7-
588- 589. 1934·
9. MAYER, FRITZ,
und TIWDI OPPENH E IMER. tlber Naphthy l-essi gsauren . BeT. Deutsc h.
Chem. Ges. 49: 2 []7- 2141. 19I6.
L935 1 ZIMMERMAN
&
V\' ILCO XON- C HEM] CA L GROWTH SUBST ANCES
2 29
KENNETH V., and F . \V. WENT. On the c hemica! nature of t he rootforming
hormone. Proc. Akad. Weten sch . Amsterdam, Sec. Sci. 37: 4 56-459 · 1934.
1 t. V ANINO, Lu nwlG. hrsg. Handbu ch cler pra para tiven Chemie. :2 Aun. Bd. 2. Orga nischer
Teil. 887 pp. Ve rlag v on fo'erd inand Enke, St uttgart. 19 23.
12. "'V1.<;ln, F.
A test method for rhizocaline, the rootforming su.bstance. Proe. Akad.
Wetensch . Amsterda m, Sec. Sc i. 37: 445- 455· 1934·
13. W OLFRAM, ARTHUR, a nd EM IL HAUSoORFER. Nev{ process of preparin g a ry lace tic acids
a nd new produ ct obra inable t he re by. U. S. Pat en t No. 1,95 1,686. 193 4.
14. Z lMM E Rl-JAN , P . W ., WILLIAM CR OC KER, a nd A E. HITCHCOCK. Th e res ponse of plants
to illuminating gas. Proe. Amer. Soc. Hor t. Sc i. 27 (1930): 53-56. 1931.
15. - - - - Ini tiation and s tjmulation of roo ts from exposure of pla nts to carbon mono
ox ide gas. Co ntrib. noyce Thompson Inst. 5: 1- 17· 1933.
16.
The effect of carbon moooxide on plants. Con trib. Boyce Th ompsoo I nst. 5:
195- 2 1 1. 1933·
17. ZIMMERMAN, P. W ., and A E. HITCHCOCK. Initiation a ud stimula tion of adventitio us
roots caused by un sat nrated hydroca rbon gases. COJltrib. noyce Thompson In st. 5 :
35 1-369. 1933·
IS. ZlMMER MAN, P. W., A. E . HITCHCOCK, and WILLtAM CROCKER. The movement of gases
into and throu£"h olan ts. Contrib. Boyce Thompson Inst . 3: 3 13- 320. 193 1.
10. THIM."NN,
""T.