TOBACCO AS A TEST PLANT FOR COMPARING THE EFFECTI VENESS OF PREPARATIONS CON­
TAINING GROWTH SUBSTANCES A. E.
HITCH COCK
In a previous report (3) methods we re described for determining t he
relative effectiveness of preparations co ntaining synthetic growth sub­
stances in ca usi ng certain formative responses s uch as bending, swelling,'
proliferation, and rooting on tomato, tobacco , and several other plants.
These responses were shmvn to be essentially the same as th ose induced by
carbon monoxide, ethylene, acetylene, and propylene. Although quan t ita­
tive measurements were made of the epinastic (downward bending) re­
spo nse induced by preparations containing these grow th subs tances, the
details of t he lneasurements were not given. It is t he purpose of the pres­
ent paper to give the results of these measurements a nd to show t hat the
tobacco plant may a lso be used to test the effectiveness of similar prepara­
tion s in ca using rooting or in retarding or stimulating th e growt h of axill ary
buds following removal of t he stem tip.
Treatment of one leaf on a tobacco plant w ith a lanolin prepara tion
containing a high concentration of growth substance causes b ending o nly
on th e test Jeaf, and does not indu ce measurable b ending on any other leaf.
In co ntrast to this type of local response , the tomato represents a class of
plants which shows systelnic bending when treated in a simi la r manner­
that js, more than one leaf may s how a m easurable bending response. It
is th us not feasible to apply luore than o ne treatment to a single tomato
p lant, whereas on the to bacco as many treat men ts ma y be made as t here
are leaves which can be measured. Although less sensitive than the tomato ,
the tobacco has a number o f advantages which ma ke it more d esirable as a
test plant than th e tomato, particularly for t he quantitative measurement
o f the epinastic res ponse.
MATERIALS USED
The s ubstances used in these experiments were indoleacetic acid
(hetero-auxine), indol epropionic acid, phenylacrylic acid, a nd phenyl­
propionic acid, which were previous ly used (3), a nd indolebutyric acid,
phenylacetic acid, a nd naphthaleneacetic acid, which ha ve been recentl y
reported by Zimmerman and Wilcoxon (5). These substances were mixed
with anhydrous la nolin, oli ve oil, or with a preparation containin g either
one-half la no lin and one-half Amalie oil (a co mme rcial mineral oil), or
three-fourths lanolin and one-fourth Amalie o il. Lanolin preparations
were nsed in most of the bending and rooting tests. The ingredie nts fo r
lanolin preparation s we re weighed out and mixed in small g lass vials hav­
35 0
CON TRID U TlONS F ROM BOYCE THOMPSON IN STITUTE
IVoL. 7
ing a capacity of IO to 2 0 cc. Thorou gh a nd"quick mixing was a ccomplished
by immersing t h e lower ha lf of the vial for a few seconds in wat er ran ging in
temperature from 30° t o 55° C., t he main purpose of this p ro cedure being
to melt the lanolin. In addition to the c rystallin e acids m ent ioned a bove,
et hyJene and propylene were a lso taken up in la nolin.
The t ohacco (N icotiana tabacum L. vaL Turkish) was the principal t est
pla nt u sed in these experiments.
METHODS
E p·inasty . Preparations of t he growt h substa nce were a pplied WitJl a
sma ll glass rod to the upper side of the base of the petiole over a distance of
ap proximately one-ha lf inch. M easurements of the angl e between the leaf
a nd the port ion of t he stem a bove we re made at the time of treatment and
on each of four successive d ays. The increase in this a ngle due to treatment
was used a s the criterion of the relative effectiven ess of the different prep­
aration s in causing epinas ty .
Distribut ion of the treat ments was according to the Latin Square
method (2, p. 234) . Seven different growth subst ances were applied t o each
t oba cco pla nt, but a different concent ration was used on each pla nt. The
number of plants used in a single t est , t herefore , corresponded to t he num ­
ber of con centrations used. Generall y four or five con centrations were used,
and each test was replicated three or four t imes. Th us, for example, in one
test (F ig. r ) fi ve concentrations of each of seven s ubstances were a pplied
t o five plants in s uch a manner that the same concentra tion of all sub­
stances appeared on t he same plant. Since, in addition, a lanolin control
preparation was ap plied to one leaf on each plant, th e total number of
treatm ents was 40 . This particular t est was r eplicated four times, so that
r60 pre pa ration s were applied on 2 0 to bacco plants. Although a given s ub­
stance appeared at a different level (leaf) on each plant, the same order was
mainta ined on all pl a nts-that is, phenylacrylic acid, for example, a lways
appeared below indolebutryic acid , except when the former appeared at
t h e to p of t he series. The same concentration of each growth s ubstance
a ppeared at four d ifferent levels on fo ur different pla n ts. The plants were
fro m IS to 20 inches in height at the time of treatment.
R ooting. The same preparations were used for th e rootin g t ests as were
used for the b e nding t ests , and the method of application and the a rra nge­
ment of th e t reatments on the to bacco were essentially t he same, except
that the stem internodes were treated instead of the petioles of leaves.
Lanolin preparations of the growth substances we re applied half way around
t he stem over a distance of approximately t h ree-fo urths of an inch. Each
s uccessive treat ment was placed off of the ve rtical axis so that it would not
lie in a direct line wit h adjacent treatments above or below. The plants
were from IS to 2 0 inches in height at t h e time of t reatm ent .
'9351
HITCnCOCIC-ToDACCO AS A TEST PLANT FOR H ORMONES
35 1
Bud growth. The tips of to bacco plants were cut off a nd a preparation
of growth substance was applied to the cut surface. One series of plants
was from 20 to 30 inches in height and the other series was from 6 to 8
inches in height at the time of treatment. In no case had the axillary buds
shown any s igns of growth when the first treatment was made. The tall
plan ts were treated wHh preparatio ns containing the crystalline acids and
th e short plants were treated I in addition , with lan olin containing ethylene
or propylene. In preparing the lanolin-gas mixtures, several grams of
lanolin were smeared on the inner walls of a salt-mouth bottle from 500
to 1000 cc. capacity. The bottle was then fill ed with water and the water
displaced with the gas. These bottles were then stored at 15 degrees centi­
grade until the preparation was used.
RESULTS
MEASUREM ENT OF THE EPINASTIC RESPONSE
The average change in angle of tobacco leaves receiving different treat­
ments is shown for tests A and B in F igure 1. Each point represents the
average angle of declina tion for four leaves. These two sets of curves were
constructed so as to show the actual direction of movement made by the
leaves. In order to do this it was necessary to change the zero point and
reverse the abscissa and ordin ate values as compared to the normal man­
ner \.vhich is shown in Figure 2. In th e active bending range, an increase in
concentration of three to three and one-half times caused an increase in
declination, except for high concen trations of phenylacrylic and phenyl­
propionic acids. The degree of declination produced by a given growth
substance and the relative effectiveness of the different s ubstances are seen
to be nearly the same in both series of tests, even though the tests were run
at diflerent times. These and other relationships are more readily seen if
the concentrations required to produce different degrees of bending are
plotted as shown in Figure :2. In this case ave rage values for tests A and
B shown in Figure 1 were used to constru ct the curves in Figure :2.
The curves in Figure :2 sho\v that indole preparations a re more eflective
th an phenyl preparations in causing epin asty of tobacco leaves. Indoleace­
tic acid was more effective than indolepropionic acid and phenylacetic acid
was more effecti ve than phenyl propionic acid. For the lower concentration
range (I to 3 mg. per gram of lanolin) indolebutyric acid is seen to be
s lightly more effective than indolepropionie acid. Average values for higher
concentrations were not available, and hence the curve in this range is
based on a single test and is shown by a broad dashed line. Average values
for the phenylacryJic and phenyl propionic curves were likewise not avail­
able for a ll coneentratio ns.
[VUL.7
CONTRIBUTIONS FRO'! BOYCE THOMPSON INSTITUTE
It was stated previously (3) that olive oil and certain other oils were
mo re effi cien t carriers than la nolin for indoleacetic, indolepropionic phenyl­
1
o
r~OOl( !lU"YI~IC
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MG PER CRAM L ANOLI N
FIGURE 1. Relative effectiveness of lanolin preparations conta ining differen t growth
substa nces in causing epinasty of Turki sh tobacco leaves. Plotted so as to show actu al
direction of leaf movement. Average values for these two series of tests are plot ted in the
nsual ma nner in F igure 2. Tests A (above) a nd n (below) were performed a t different times.
acrylic, a nd phenyl propio nic acids. Curves for lanolin and olive oil prep­
arations which conta ined t he same amount of indolepropionic acid are
shown as test C in Figure 2 by a do tted line and dot-dash line respectively .
'9351
HITCHCO CK-
ToBACCO AS A TEST PLANT FOR H O RMONES
353
Th e la nolin curve (do tted li ne) follows very closel y t he curve based on th e
mo re recent tests and shown by the solid line. Th e corres pond ing curve for
t he olive oil preparation (dot-dash line) sh ows an activity from 6 to 8
t imes that of t he lanolin preparation. In fact, the olive oil preparation of
_ _ _ AV ERAGE
or
TE S TS A AND B. - - - - T ES T B. ••• • ••• ---- INDOL E PROP IONIC ACID T EST C - -- - _ . - OLIVE O IL PREPARAT ION TEST C.
'"
"
-- ..-­
.
.-.--..-­
.• ~
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.
·~,~--~--7"o---"---,~,C---"--~=
o
'OC---L--~'~O--~--'~OC- ---L--~ro'
A NGL E OF DOW NWARD MOVEMENT
FIGURE 2. Relationship between concentratio n of growt h substan ce a nd the e pinastic
response of Turkish tobacco leaves. Data ca lculated from c urves in F igure I for tests A a nd
B, a nd from curves for test C which are not shown.
indolepro pionic acid shows nearly the same activity as the lanolin prepara­
tion of indoleacetic acid.
T he effectiveness of the different s ubstances in causing epj nast y of
tobacco leaves may b e judged not onl y from th e standpoint of the mini­
m um active concentrations causin g epinasty , but also fro m the standpoint
CONTRIBUTION S FROM BOYCE TH OMPSON I NSTITUTE
354
IVOL · 7
of the r ange of concentra tions over which th e epinastic res ponse occurs.
In this respect, naphthaleneacetic and indoleaceti c a cids were mu ch mo re
effective t han the o th er five a cids (Figs . I and 2). Phenyl acrylic a nd phen­
ylpropionic a cids were the least effecti ve. Ph enylpro pioni c a cid . for ex­
ample , increased in effectiveness fro m I O to SO milligrams per g ram of
la nolin, which represen ts a maximum range of five times . Ind oleacetic a nd
naphthaleneacetic acids showed a corresponding increase of 10 t o 3 0 times.
Ot her tests for which curves a re not given indicate that th e latter two s ub­
st a nces increase in bendin g effectiveness from o. I up t o a t least 50 milli­
grams per gram of la nolin which represents a range of soo-fold. Higher
concentrations p roduced bendin g , b ut the amount of the increase in d ec­
lination was no t propo rtio nal to th e inc rease in concentration, and t here
was usu a lly some injury acco mpanying t he response. The ran ge for in­
do lepropio nic acid was a pproxima tely 50-fold. The curve for indo leacetic
a cid shows th e closest relationship with respect t o th e increases in declina­
tion caused by an increase in co ncentration , part icula rly over th e range 10
to 40 d egrees. The control lanolin preparations caused only a s light dec­
lination, t he a verage va lues for th e several tests ranging fro m 0. 8 to 1.4
degrees. Leaves receiving no t reatlnen t showed an a verage declination of
I d egree.
On th e basis tha t indolea cet ic acid showed th e closest relationship be­
tween co ncentration a nd the a mount of d eclination, th e correspo nding or
equival ent a mounts of the other substa nces req ui red t o produ ce th e same
degree of bending were calculated from t he curves in F igure 2. These cal­
culated va lues are shown in T a ble 1. Since valu es for a d eclinatio n greater
T ABLE T
C ONCE NTRATIO NS REQ UIRE D TO ( NDUC E A G IVEN D EGREE OF DECLINATION ON T OBACCO
LE AVES, AS CALC ULATED FROM THE DATA IN F IGURE :1
Angle of
declination
-
R ela tive am ount of growth su bstance cal culated to induce the same am ount
of d eclination as indolea cet ic acid
Naph t ha­
leneacetic
Ind ~ lel?ro -
plOnlC
IO
'S
'0
'5
30
35
40
45
SO
0. 80
0 . 67
0. 6 3
0 . 60
0.5 8
SS
0 .64
0· 79
Ir
60
Av.
0 .63
12 .6
0·57
0 ·55
0 .5 8
0·55
23 · 5
1 7 ·0
13 . 8
12. 0
10 · 3
10· 3
Ind ole­
bntyric
13· 0
II . 7
11. 8
11- 0
!4 · 7
22. 8
9·4
8.6
8. I
Phen yl­
acetic
76
83
7'
70
77
9'
P he ~ yl~
propIORlC
Ph enyl·
acrylic
' 3°
" 7
11 5
78
80
83
"0
233
3 00
360
I07
'78
.9
13· 4
! 4 ·2
78
'9351
HITCHCOCK- ToBACCO AS A TEST PLANT FOR H ORMONES
355
than 30 degrees were not available for all snbstances, average valnes ,are
shown in order to give a n approximation of the proba ble relationship for
bending np to 60 d egrees. Also, these average values were nsed as a guide
in making up eqnivalent concentrations that were calcn lated to produce""'a
3o-degree declination . The results of these tests are shown in Table II.
TABLE II
ErlNASTY OF TOBACCO LE AVES I NDUC ED BY CON CENTRATIONS CALCULATED FR.OM DATA
I N FIGURE 2 TO PRODUCE A 30-DEGREE DECLI NAT ION
Grow th s ubstance
In doleacetic acid
Phe.ny lacry lic acid
I ndolebutyric acid
Indolepropionic acid
Phenylacetic acid
Naphthaleneacetic acid
Pbenyipropionic acid
Concentration mg . per g.
Average angle of d eclination
for 6 leaves
0·3
84-4
4·8
3. 6
23 ·4
0.2
3°·0
The resnlts in Table II s how t hat five of th e seven calcula t ed conce n­
t rations induced an angle of declination which approximated 30 d egrees.
The bending values for indolebu tyric a nd phenylacetic acids were too high .
All of the calculated concentratio ns in Table II were not exactly propor­
tional to the average values s hown in Table L This is du e to the fact that
some of the preparatio ns a lread y made np we re of nearly the same con­
centration as the calcula t ed values, so that an additional adjustment was
not considered necessary . The fact, h owever, that five of the seven con­
centrations produced bendin g th at approximated 30 d egrees is no do ubt
p urely accidental. The importan t point in this type of test is that eq uiva­
lent concentrations should produce approximately the same amount of
bending and not necessarily a given degree of declination. Since t he degree
of declination for indoleb utyric acid was nearly three times that for in­
do lepropionic acid, it seemed evident that in this particul r concentration
range indolebutyric acid was more e ffective than indolepropionic acid.
This fact is of course substantiated by the curv es fo r these hvo s ubsta nces
shown in Figure 2, but since in the hig her concentratio n ran ge indoleb uty r­
ic acid a ppeared to be less active than indolepropionic acid , it was felt
that the average values fo r eq uivalent concentrations shown in Table I
might be more nearl y correct. In order to verify these results, an add it ional
test was made in the lower concentration range (r to 5 mg.) for th ese two
g row t h substances. The resul ts of t his test are shown in Table III.
The relative differences in bending for indolebutyric and indo lep ro­
pionic acids shown in Table III are in ag ree ment with the data in Figure 2.
It must be concluded, therefore, that in the range from I to 5 m g. per gram
of lanolin, indolebutyric acid is sligh t ly more effective than indolepropio nic
CON TRfR UTI ONS FROM B OYCE TH OMPSON INS TIT UTE
[VOL. 7
acid in causing epinasty. The results of this test a lso show that relati vely
small differences in concentra tion induce measurable differences in dec­
linati on of the leaves.
TABLE III C OMPARATIve EFFECTIVENE SS OF L ow CONCENTRATIONS OF I l'{OOLIlGUTYRIC AND I NDOLIl­
PRO PIO NIC ACIDS I N CAUSlNG EI:' [ NAS TY ON TOGACCO LEAVES Con centrat ion of grow th
s ubstance mg. per g.
Average a ngle of declination fo r four leaves
Indolebutyric a cid
Ind olepropio nic a c id
, .00
£. 50
2.00
31
2. 25
3. 00
46
66
4. 50
73
ROOTING T ESTS
Th e concentra tions used for the rooting tests were as follows: o. I,
a nd 1 0 milligra ms per gram of lanolin for indoleacetic , ind ole­
butyric, and uaphthaleneacetic acids; 3, 10, 3 0, 50, a nd 100 for phenylace­
t ic and phenyipropionic acids; 0.3, 1 ,3. 10, a nd 30 for indolepropionic acid;
a nd 10, 30 , 50, 100 , a nd ISO for phenyi acrylic acid. T he t otal con centration
range for all s ubsta nces was therefore 1500 times. The order of treatments
from top to bottom on the first plant was phenylacetic, jndolepropionic,
na phtha leneacetic, phenylpropionic, lanolin control, indoleacetic, indole­
butyric. and phen y lacry lic acids. Although each growth s ubstance ap­
peared a t a different level (internode) on different plants . t he order of the
substances was the same on each p1an t as in t he case of the be nding tests.
Phen y lacry lic acid . for example. was a lways applied below indolebutyric
acid except when the former appeared on the uppe rmost of the treated
leaves. In the firs t series of tests 160 preparations were applied to the stems
of 2 0 tobacco plants. Each concentration of each of th e seve n growth
s ubsta nces was applied at a different level on four different plants. Two
days la te r the same t reat ments were applied to 20 to bacco plants that had
more succulen t stems than those in the first series.
R es ults of these test s showed th at the roo ti ng response on tobacco
stems varied accordin g to the concentration of growth substance, the kind
of substan ce, a nd the location of the treatment a long t he vertical ax is.
Rooting was induced by t he three highest co ncen t rations of a ll growth
su bstances. Phenylacetic and phenylacry lic acids were the only two s ub­
stances for w hich rooting was induced by the lo"\vest con centration used
in th ese tests. The next lowest concentration of all s ubstances except in­
dolepropionic acid caused rooting. The to ta l number of treatments which
caused rooting was 46 for the highest concentration, and 42, 38, 18, and
0·3, 1.0, 3.
,
•
1
'935 1
HITCHCOCK-ToBACCO AS A TEST PLANT FOR HORMON ES
357
4 res pecti vely fo r the next lo west concentrations. T h ese are the averag"e
values fo r t he two series of tests jn which r60 t reatments were applied to
20 plants iu each of the two seri es. Each treatmen t was therefore replicated
eight times.
Rooting induced by the highest concentratio ns was dela yed on some of
the plan ts o r in some cases was a ccompanied b y visible injury such as
d iscoloration or a marked in crease in diameter of the roots. Indolebu t yric
and indoleaceti c acids 'were the least inju rious in t he high concentrat ion s.
From the s ta ndpolnt of a good rooting respon se , indolebutyric acid was
the most effective , followed in decreasing order by indoleacetic and phenyl­
acrylic, ph enylacetic, naphth aleneacetic, phenyJpropionic, and indole­
propionic acids. If judged from t he standpoint of th e total number of
treatments which induced ro oting, the decreasing o rd er of effec ti veness
was as follows : phen ylacetic a nd phen ylacrylic ac id s, naphthaleneaceti c ,
indolebuty ri c, phen ylpropioni c, indo leacetic, and indo lepropionic a cids.
Preparations 'w hich induced bending of the st em , a s illustrated in
Figure 3, we re us ually very eflecti ve in producing roo ts. The plant on th e
right was t reated with the high est concentrations of th e seven growth s ub­
stances, and th e plant on th e left with the Im.vest concentrations of th e
same materi a ls. After several weeks r oo ts usuall y appeared from tissue ad­
jacent to the treated areas in th e case of the most effecti ve treatments.
The plant o n th e right in Fig ure 3 illus trates this t ype of response. The
extent t o which one treatment wi ll a ffect other nearby t reatments \v ill
therefore d epend to a certain degree upon the duration of the experiment.
During a p eri od of two weeks this effect was not noticea ble.
Treated intern odes on the sa m e plant varied in th eir capacity to form
roots . The tota l number of ro ot ed internodes from to p t o base was as fo l­
lows: r6, 22, 25 , 24, 23, r6, 14. a ud 7. These results show that the portion
of the tobacco stem beginning fro m o ne to two inches be low the tip a nd
extending do wn fo r four to six inches, roots more readily than the very
young tiss ue near th e tip or the o lder tiss ue on the lower half of the stem .
It was observed t ha t the same t reatm ents were equa lly effective farther
down on su ccul ent stems as compared to stems of less a ctively growing
plants. The m ost prono unced rootin g responses occurred o n t he second and
third in ternod es.
nU D GR OWTH AFTER DECA PITATION OF ST E M TIP
W hen tips of to bacco stems were cut off and the cut su rface was treated
with a contro l lanolin preparation , one or more of th e upp er three buds
commenced grow th . Generally at least two shoots d eveloped to a length
of several inches, a nd then one of th ese a ssumed leadership a nd the other
one ceased growth . There was no g row t h of side buds o n control tobacco
CO~TRIBUTIOl.'\S FR01\f BOYCE THOMPSON INSTITUTH-
plants \vhich were not decapitated and the!T '.vas no evidence of flmver bud
fornlation at the time these treatments ,:ver€' bfgun.
l
FIGURE 3. Rooting response on citeal of Turkish tob&cco after 5S days, Lowest Con(ell~
trations of seven di~ereJTt groWI:1 subst.ancesapplied sepamlely and to different internodes
of plant on left. Hig'hest cor:centrat ions of same growth SUl:st.aHce:: applied to plant on rig;11.
Leaves n:~moved at time of photographing.
If the cut surface of decapitated stems \vas treated \\-'ith a lanoHn prep­
aration containing a high concentration of any OIle of the seven growth
substances used in the bending tests! marked inhibition of the growth of
I9351
HITCHCOCK-ToBACCO AS A T EST PLANT FOR HORMONES
359
the upper two or three buds occurred. The degree of inhibition was pro­
pOl-tional to the concentration of the substance. In the case of indoleacetic
FIGURE 4. Bnd growth aft er decapitation of stem tip. A. Control~plants=afterhl days.
Three plants on left not decapitated; three on right decapitated and coIltrollanolin~pr epara·
don applied)o cat surface. B. Lanolin-Amalie oil preparation of indolepropionic acid (50
mg. per g.) applied to cut snrface alter decapitation.
CONTRIBUTIONS FROM
BOYCE
TH OMPSON
I NSTITUTE
[VOL. 7
and indolepropionic acids, noticeable inhibition res ulted from treatment
with preparations containing from 3 to 100 m g. per gram of lanolin. No
attempt was made t o co mpare the effectiveness of t he different substances
in t hese t ests. In addition to inhibiting the growth of some of the upper
buds th ese same preparations induced the grmv th of many of the buds
lower down the s te m as illustrated in Figure 4 B. One of t he most effective
preparations contained So milligrams of indolepropionic acid per gTam of a
mixture consisting of equal parts of lanoliu a nd Am a lie oil.
j
FIGURE 5, Young tobacco plants 20 days after decapitation. Lanolin pre parat ions CO U ~
ta ining the following substances we re appli ed to the cut s urface, from left to right: none
(co ntrol), indolepropio uic acid , et hy!ene, and propylene.
As a general rule, at least three of the buds b elow the second one and
abo ve t he eleven th bud , developed into shoots that were several inches in
length after a period of 20 t o 30 days. I n one case, for example, the third ,
seventh, and ninth nodes produced shoots 10 , 6, and 16 inches in length ,
respectively. III another case the fonr th, eighth, and tenth buds developed
into shoots 4, S, a nd 7 iuches long, respectively. Eventually, however , one
of the upper sh oots became the leader and th e other s hoot s ceased t o elon­
gate. In addition to the shoot s which atta ined a length of several inches,
there were othe rs which formed one or more leaves from one to three inches
'935J
HITC HCOC K-TO BA CCO AS A TEST P LANT F OR HO RMONES
lo ng bu t failed to exceed a stem length o f one in ch . Shoots of this ty pe a re
sho wn in F ig ure 4 B. All of t he pl ants in th is series of t ests we re fro m 20
t o 30 inch es in height a t th e time of t reat me nt.
La nolin preparat ions con ta inin g eth yJe ne o r propy lene we re tested in a
s imilar ma nner on yo ung ac t ively growin g tobacco plants from 6 to 8 inches
in heig ht. E ssentia ll y t he same res ponse resulted f rom treat ment with t he
la noJin-gas mixtu re as with th e prepa rations con t a ini ng th e crystall ine
acids (Fig. 5). W hen the lano lin -gas mixtu re was applied on alternate d ays
unt il fo ur applications had been given . th e response was m uch more pro­
no un ced th an w hen only on e or t wo applications ,vere given . I n these tests
ethyl ene was more effective t ha n p ropylene. T hese res ults appea r to be
simil ar to th ose p revi ously re ported for t he fo rcin g effect of eth ylen e gas o n
latent rose buds (4) in whic h it was s how n t hat 70 p er cent of all b uds on a
trea ted pla nt fo rmed shoots.
DISCUSSION
The results of t ests relating to th e epinastic response of to bacco leaves
showed t hat the decli natio n caused by t he c rystalline grow th su bs tan ces is
a measura ble response. D ifferences in concentration of o ne and one-half
to t hree an d o ne-ha lf ti mes p rodu ced m eas urable d ifferences in the degree
of declina t ion of t he leaves. The fact th at t he bendin g response of o ne leaf
o n a to bacco plan t is independent of other t reated leaves o n th e sam e plant,
ma k es it possi ble to a ppl y seve ra l t reatments to a sing le p la nt. T hus a
large number of pre paratio ns may be tes ted o n a rel atively sma ll n umbe r
of plants. In prelimin a ry t ests it was fo un d th at t he capacit y of leaves to
bend d ownward varied wi t h t he age of the leaf. T he d egree of decl ination
d ecrea sed wi th increasing age of the leaf. Vari ations du e to t he age of th e
leaf a nd to individ ua l differences a mong plants were co mpensated for w hen
th e d i ~tri buti o n of treatments was made acco rdin g to t he La ti n Squa re
method. F o ur replicates of each treatment proved s uffi.cien t to de monstrate
measu rab le di ffere nces in t he epinastic respo nse. E ach of t he four replicate
t reatments, fo r exa mpl e, \va s ap plied to a di ffe rent level on a di fferent
plan t .
Turkish t obacco was selected as a test plant primaril y because m a ny
comparisons co uld be made wit h th e use of a sm all numbe r of plants. The
results o bta in ed with to bacco wi tl not necessaril y be th e same as th ose re ­
su)tin g fro m t he use of a nother kind of t est pl ant. It is believed, however,
t h a t th e o rder o f re1ative effe ctive ness of the d ifferent growth substan ces
in causing epin as t y will be a pproxi mately t he same for other test plants
such as the t oma to , African marigold, and su nflow er. \~l ith resp ect to th e
mini mum active concentrat ion , th ere w iH no do n bt be differe nces acco rd­
ing to t he kind of test pJa nt used . The m inimum concentratio n of indo leace ­
tic acid w hich ca used epin,!-st y on t omato leaves was 0.003 mg . pe r gra m of
CONTRIBUTIONS FROM BOYCE THOMPSON I NSTITUTE
[VOL. 7
lanolin, whereas for tobacco the minimum concentration was approxi­
mately 0.1 mg. (Fig. 2). Th e same stock preparation of naphthale neace tic
acid used in these tests was slightly more effective than indoleace ti c acid
on tobacco (Fig. 2), but th e minimum active concentration for the tomato
was between 0.03 and o. I mg, or the same as for the tobacco. Th ese res ults
are similar to those reported for the relative effectiveness of ethylene and
certain other gases in causing epinasty on different test plants (I). Addi­
tional data on the minimum active concentrations of the different growth
substances are given by Zimmerman and Wilcoxon (5).
The relative effectiven ess of the different growth substances depends
not only upon the kind of test plant used, but also upon the carrier or
solvent. Olive oil and certain other oils .were reported as' more effective
carriers than lanolin (3) . The quantitative difference for an olive oil prep ­
aration of indole propionic acid is shown in Figure 2. The use of water as
a solvent for the growth substances would also be expected to give different
results. Lanolin has bee n used as the principal carrier because of the ease
with which the pre paration may be applied to the plant and because it does
not appear to injure the plant. All lanolin preparations were made with
anhydrous lanolin .
Tests per fo rmed at different times with the same preparations did not
produce the same d egree of bending, but the order of relative effectiveness
of the different growth substances was essentially the same (Fig. I ). For
this reason comparisons should be made simultaneously or under standard
conditions with respect to atmospheric conditions, the age and activity of
the t est plants, and the age of the preparations used. Afte r having dete r­
mined the relative effectiveness of several growth substances, one of these
may then be used in all future tests as a standard of comparison. If , for
exampl e , standard preparations of indoleacetic acid are used in a ll tests,
the relative effectiveness of other preparations will be on a comparable
basis. Any loss in activity of a preparation of growth substan ce could be
readily detected by the method described. This would apply mo re particu­
larly to water preparations of the growth substances. The me thod used for
injec tin g water preparations has been described in an earlier report (3).
In the rooting tests it was evident that a pronoun ced and uniform
rooting response was limited to the upper part of the st em, and hence ap­
proximately half of the treatments in these tests were placed on tissue
which was not capable of exhibiting an optimum rooting response. The
tobacco is therefore less e fficient as a test plant for rooti.ng than it is for
the bending response. It is believed that, if the treatmen ts are applied only
t o the portion of the stem which is capable of bending, more accurate com­
parisons could be made. This would of course necessitate the use of fewer
treatments per plant.
In will be noted t hat the order of relative effectiveness in rooting was
'9351
HITCHCOCK-ToBACCO AS A TEST PLANT FOR H OR MONE S
not the same as for bending. No doubt t he relative effectiveness of the
different growth substances in causing rooting will vary accord ing to the
kind of plant to which they are applied. Certain unpublished data relating
to the treatment of shoots of hardwood plants would appear to support
this idea. Indolepropionic acid, for example, was more effective than the
other six acids in causing rooting of t he Japanese maple. In si milar tests
\vith the Grimes Golden apple, indoleacetic was more effective than indole­
propionic acid, and these \-vere the only two of the growth substances
tested which induced rooting. Diphenylacetic acid which caused rooting
on the African marigold but not on th e tomato fail ed to induce epinasty on
either plant. For this reason, diph enylacet ic acid has no t bee n repo r ted
as an active growth substance.
Tests with the decapitated tobacco plants s howed th at ap ical d omi­
nance was interfered with by the application of g rowth substan ces to th e
cut surface. The irregularity of shoot growth be low t he c ut s urface of
treated plants indicated that there was no unifo rm influence exe r ted by t he
buds or subsequently formed shoots on the upper part of t he stem as was
the case on the control plimts. The fact that a la no lin prepa rat io n of ethy­
lene caused the same type of response is a furth er indicatio n of thesimila ri­
ties previously mentioned (3) between th e crystalline g rowth s ubstances
and the gases. In some of the bending tes ts, th e la no lin preparat ions con­
taining growth substance were sometimes applied in s uch a ma nner th at
the axillary bud was covered. It was o bserved that in th e case of t he hi gh
concentrations the axillary buds st a rted g rowth a nd then sto pped a fter
reaching a length of one-fo urth t o three-eighth s of a n inch . The fact t hat
latent buds were .induced to grow as a res ult of treatm ent directly or in­
directl y with crystalljne o r gaseo us growth s ubstances s uggests t he possibil­
ity that oth er ty pes of d o rma nt buds may undergo cha nges of a similar
na ture w hen their d orm a ncy is broken natura ll y or by artificial m eans.
Decapitation of the rapidl y growing canes of the Crimson Rambler rose
a nd th e ap plica tion of the lanolin preparations of growth s ubstance to the
c ut surface resulted in responses that we re essentially the same as those
induced o n th e tobacco_ In this case, h owever , the high concentrations
prevented the growth of all buds on canes that were three to five feet long.
Slig htl y lower concentrations induced the growth of three shoots nea r the
cut s urface, but not necessarily the first three. At t he end of seven weeks
these s hoots had attained a length of one to three feet. :f\.1 uch lower con­
centI-ations ac ted the same as the control lanolin preparation. On control
canes a shoot grew from the first node and in a few cases also from the sec­
o nd node. E x periments with decapitated plants are presented primarily
to show that the Turkish t obacco may prove useful as a test plant for d e­
terminin g the effects of growth substances on bud growth! as "veIl as for
th e epinastic and rooting responses.
CONTRIBUTI ON S FROM B OYCE THOMP SON IN STITUTE
(VOL. 7
SUMMARY
1. Five concentrations of each of seven different g rowth substances
were tested simultaneously and at different t imes on leaves of Turkish
tobacco in order to determine the relat ive effectiveness of t he preparations
in causing epinasty. The decreasin g o rder of effectiveness was as foll ows:
naphthaleneacetic and indoleacetic acids, indolebutyric and indolepro ~
pionk acids , phenylacetic, phenyl propionic, and phenyl acrylic acids .
2 . Th e relative effectiven ess of the same prepa rations was also d eter­
mined for rooting on stem s of th e tobacco . From the standpoint of a good
rooting response the decreasing orde r of effectiv eness was as follo ws :
indolebuty ric acid, indoleacetic and phe nylacry l-ic acids, pheny lacetic,
naphthaleneacetic, phenylpropionic , and indolepropionic acids. Since there
were markr d differences in th e ca pacity of different portions of th e stem to
form roo ts, the order of effectiveness given represents th e average respo nse
of internodes at different levels and is not based on the optimum root ing
respon se fo r each s ubsta nce.
3. A pplication of la nolin prepara tions of growth substance to the cu t
s urface of d ecapitated tobacco st ems Iet a rded o r inhibited the growth of
the upper buds and stimulated the growth of many buds on the middle and
lowe r part of th e stem . Although t he normal apical do minance was dis­
turbed fo r a pe riod of several weeks , eventuall y one of th e upper shoots
beca me th e leader. Lanolin prepa ration s of ethylen ~ a nd propy lene ca nsed
similar respon ses.
4. Tobacco was found to be better adapted for testi ng the epinastic
response th a n it was for the rooting response.
LI T ERATURE CITED
WILLIA~I , P. W. ZH.J.M ERMAN, and A. E. HIT<":HCOCK. Eth ylene-induced epi­
nasty of lea ves a nd the relation of gravity to it. Cant-rib. Boyce Thom pson lnst.
1. CR OCKER,
4: 1 77- 2 ( 8 . f93 2.
2. FISHF.R, R. A. Statisti cal methods for research wor kers. 3rd eel. 283 pp. Oli ver & noy d ,
Edj nburgh . 193.0.
3. HIICHCOC K, A. E. Indo le- 3 ~ n-pro pi o nic acid a s a gro\..,th hormone a nd the q uan t it ative
measureme nt of pla nt response. Contrib. Boyce Th om pson lnst. 7: 87 -95 · 1935 ·
4. ZIMMl~ RMAN, P. W ., A. E. I-ilTc HCOCK , and WI LLIAM" C ROCKF.R. Gas injury to roses in
greenh ouses. Fl or. Exch. 78(9): Il , 3 3-34. 193 I. (A lso in TIo)'ce Th om pson 1nst. Prof.
Pap. 1(20): 198-- 209 . J93 1.)
5. ZI MM ER MAN, P. W.o and FRANK WILCOXO N. Several chem ical growt h subs tances which
ca use initiation of roots a nd other respo nses in plan ts. Contrib. Boy ce Thompson
lnst . 7: 2<><.)-2:29 · 1935.