Dec ..
19401 THIMANN AND SKOOG-EXTRACTION OF Al"XIN
EL'rISlJt:, E. '1'., AXIl H. S. Rt:t:I1. 19-10. The effect of zinc
deficiency upon the root of Lucopersicura esculentu m,
Amer..Jour. Bot. 17: :l31-:~3;;.
Fn:I1u:R, H. 19:1(i. Entwicklungs- und reizphysiologische
L'ntersuchungen an Kulturen isolierter Wiirzelspit-
zen. Zeitschr. f. Bot. 30: 385-436.
HO,\OI.AXII, D. R., AXIl 1. ARSON. 1938. The water-cul-
ture method for growing plants without soil. Cir-
cular 3-l7 Univ, of Calif. College of Agriculture.
- - - , ASD W. C. SXYI1ER. 1933. Nut rltlon of straw-
berry plants under controlled conditions. Proc,
Amer, Soc. Hort. Sci. so, .288-19-l.
Kl:BOWITZ, F. 1937. eber die chemische Zusammenset-
zung del' Kartoff'eloxydase. Bioehem. Zeitschr. '29-
191: i?i?I-
LAXE, H. H. 193(i. The inhibition of roots by growth
hormone.Amer, Jour. Bot. :23: 53i?-.535.
LARSES, P. 1936. eber e inen wuchsstoffinaktivierenden
Stoff aus PhauolWl-K e impftanz e n. Planta i?5: 311-
3H.
LlnIAS, C. B., ASII G. MACKISSt:\". 19:11. Proof of the
essential nature of copper for higher green plants.
Plant Physiol, 6: 593-.59fl.
LrSll}:oARUlI, H. 1939. :\1angan als Katalysator del'
Pftanzenatmung. Planta i?9: -l07--li?6.
On:RBn:K, .J. VAX. 1935. TIH' growth hormone and the
dwarf type of growth in corn. Proc. Nat. Acad.
Sci. :21: :29i?-:29fl.
19:18. Auxin production in seedlings of dwarf
.I1/1i:e. Plant. Physiol, 13: 587-598.
Rt:EII, H. S. 1938. Cytology of leaves affected with little
leaf. Arner. Jour. Bot. '25: 174-186.
1939. The relation of copper and zinc salts to
leaf structure. Amer.•Jour. Bot. :26: :29-33.
THE EXTRACTION OF AUXIN
Kenneth ". Thimann and Folke Skoog
ALTHOl"UH A number of researches have been car-
ried out in recent vears on the extraction of auxin
from plant materiai, the extraction process itself has
been little studied. Following the work of Thimann
( 193-l) on the extraction of auxin from Avena seed-
lings by chloroform, Laibach and Meyer (1935),
Avery (1935), Boysen-Jensen (1936a, H)36b), Lai-
bach and Lotz (1936), du Buy (1938), van Over-
beek (1938a, 1938b, 1910), Linser (1939), Larsen
(19:39a ), Avery, Creighton and Shalucha (1910)
have carried out extractions of man v tissues and or-
gans. using chloroform, etber, and in some cases al-
cohol as solvents, Since it is bv no means clear that
the extractions even approached completeness. the
deductions that can be made from these experiments,
particularly where green tissues were used, are open
to criticism. Such criticism receives support from the
experiments of Linser (1 n3n). who showed that
leaves from which auxin had been extracted with
ether for 2l hours yielded still larger quantities on
subsequent treatment with alcohol in a Soxhlet ex-
tractor, The unpublished experiments of a number of
1 Heceived for publication Aug-ust 7, 19-10.
The authors an' g:ratcful to :\fiss Ava C. Byer for tech-
nica l assis tance.
951
- - - , ASII J. Dt:FRKSOY. 19:15. The effects of zinc and
iron salts on the cell structure of mottled orange
leaves. Hilgardia 9: 113--137.
SClisnIlER, C. L., ASD F. W. WEST. 1938. A photo-
kymograph for the analysis of the Avella test. Bot.
Gaz. 99: -l70--l96.
SKOOG, F. 1937. A deseeded Avella test method for
small amounts of auxin and auxin precursors. .Jour.
Gen. Physiol. :20: 311-:1:~-l.
HI3H. Absorption and translocation of auxin.
Amer . .Jour. Bot. i?;: :llil-37i?
-~-, T. C. BRon:R, ASII K. A. GROSSESBACHER. 1938.
Effects of auxin on rates, periodicity and osmotic
relations in exudation. Amer.•Jour. Bot. :25: HfJ-759.
SO~D[ER, A. 1,., ASII C. B. LIP~[AS. 19:26. Evidence of
the indispensable nature of zinc and boron for higher
green plants. Plant Physiol, 1: i?31-'2-l9.
STO\'T, P. n., ASU D. 1. ARSOX. 1939. Experimental
methods for the study of the riHe of copper, man-
ganese and zinc in the nutrition of higher plants.
Amer.• Jour. Bot. 1(i: H-l-H9.
TJlI~[A"X, K. V. 19:H. The distribution of growth sub-
stances in plant tissues. .Iour. Gen. Physiol. 18: '2:l-
3+.
---. 193(i. Auxins and the g:rowth of roots. Amer.
.Jour. Bot. i?:l: 5(il-;;(i9.
TlIrXBERG, T. 193+. Zink und Kadmium als stimulier-
ende Mittel fii r die Oxydatlonsprozesse in gewissen
Pftanzensamenextrakten. Skand. A rchlv. fUr Physiol.
(i9: 1+7-:25+.
Wux-r, W. F., AXIl K. V. THIl\IAxx. 1937. Phyto-
hormon e s. New York.
FRO~I PLANT TISSUES 1
workers point to the conclusion that quantitative
auxin extraction from green tissues is by no means
simple. '"e have therefore undertaken an extensive
study of the extraction of auxin from green tissues,
and of the related problem of the form in which auxin
is present in the plant.
~fETHoDs,-The use of leaves for work of this
kind is subject to several difficulties. There is good
reason to believe that the auxin relations of the veins
differ from that of the blade; the material is variable
in age and size and samples are not readily compara-
ble. "'e have therefore carried out most of our ex-
periments on Lemna minor. This has the advantage
of heing readily cultured under constant conditions
with a standard nutrient medium; the material is
homogeneous and very thin, so that it does not need
to be sliced; it is nevertheless a flowering plant and
comparable with the larger forms.
The Lemna was grown on Hoagland's solution
(Hoagland and Snyder, 19:3:3) of % strength with
supplementary solution 3A, at :B-25°C, under a
combination of artificial lights of high intensities,
using a 16-hour day. By bringing the solution at the
start to pH -k5, the growth of Chlorella (the chief
952 AMERICAN JOURNAL OF BOTANY [\'01. 27,

contaminant) was almost entirely suppressed, while units. Since the extracts were made up to 0.5 cc.,
that of the Lemna was excellent. Heavy inocula were each could produce 50 blocks, so that the yield in
implanted into Pyrex baking dishes, each containing "plant units" (Went and Thimann, 1937, pp. 'H-
two liters of culture solution. Under these conditions ·~3) is 50X3000 or 150,000 plant units. This value,
the growth rate remained constant at about one divi- 3000-'~000 units per 3 grams, has been obtained in
sion every 2.2 days. Samples were removed usually all cases where extraction has been carried to com-
on the fourth day, washed and centrifuged free from pletion by this method, and is, therefore, a repro-
solution and the fresh weight determined. Most ex- ducible measure of the auxin content of Lemna
tractions were carried out with 3.0 grams fresh grown and harvested under the conditions adopted.
weight, or about 0.36 gm. dry weight. There is good evidence, however, that a certain
The auxin was assayed by means of the standard amount of inactivation has still not been excluded,
Avena test (see 'Vent and Thimann, 1937), using an and some experiments with enzymes (to be reported
IDP of three hours and a photographing time of 90 elsewhere) have yielded at least double this amount.
minutes. The samples were kept under ether, allow- Grinding does not promote the extraction and even
ing 15 to 20 cc. per 3 gm. sample, in the refrigerator reduces the final yield. The addition of small quanti-
at about 2°C. 'Vhen the extract was removed, the ties of acid (3 cc. 0.11\1 H CI per sample) has no par-
sample and vessel were rinsed two or three times ticular effect on the extraction and does not affect the
with 5 cc. volumes of ether. In all extractions with final yield (series 3 and .~).
ether, except where dryness is specified, the material B. Comparison of solvenls.-'Vhen chloroform is
remained moist, and aqueous droplets were usually used as extractant, the addition of acid markedly in-
present. All ether was freshly distilled over wet Fe- creases the yield. This might be expected from the
S0-1' The extracts were evaporated to dryness on an fact that the partition coefficient (for those auxins
electric hot plate in a current of air, and at once which have been tested) is lower between chloroform
taken up in 0.5 cc. of melted 1.5 per cent agar. Care and water than it is between ether and water.
was taken to avoid overheating. Serial dilutions were Table 2 shows the increase from acidification. The
made and the preparations cast into blocks of volume total yields, however, are much lower than with
10 mm ' in the usual way. The coleoptiles were cali- ether, although it is to be noted that no further auxin
brated at each test with standard blocks containing was obtained on subsequent ether extraction. In an-
indole-acetic acid, 0.025 and 0.125 mg. per liter of other experiment, on the other hand, eleven extrae-
agar. However, as the sensitivity did not vary wide- tions, lasting a total of 12 weeks, gave with ether
ly, and since the correction to be applied for indole- TABLE 1. A Ilxin from Lemna. Samples extracted with
acetic acid may not be the same as that for the na- successit'e 15 rr. t'olumes of ether, by standing in ire-
tural auxin, the results are quoted uncorrected in box, Yields in au,rin units."
arbitrarv units. These units, as mentioned below, are
equal to about 50 "plant units." In some cases ex- 'J 3 4
tracts were sampled in duplicate and the samples Intact
assayed separately. The agreement between these Dates Ground Intact Intact + acid
duplicates was closer than that between assays of the of tests :2.33 g, 2.33 g. 2.90 g :2.90 g
same extract made on different days. Thus on one
day the duplicate samples gave 75 and 78 units re- 11/13/39 ... '" ti i2
spectively, while on another day duplicate samples .... . . . . . . . . . . 3 4
11/16/39 ...... 50 78 102 84
of the same extract ga\'e 6·~ and 61 units respectively.
11/17/39 ...... 85 48
In all, some 3500-·~000 tests have been made in the 11/:27/39 ...... 5;] 108
course of the work. 1:2/ 7/39 ...... 560 1,920 :2,080 :2,000
THE EXTRACTION OF ArXIN FROM LEMNA.-A. Ex- l:!/14/39 . . . . . . 50 45 l:!O N6
traction with ether.-'Vhen samples of Lemna were 1/ 5/40 ...... 27 93
placed in ether for short periods, and the extracts 1/19/40 ...... 13 24
evaporated and tested, it was found that thorough 1/:25/40 ...... 7 I,;
grinding of the tissue made little or no difference in 1/:29/40 ...... 0 0
:2j;H/40 ...... 0 ~
the amount of auxin extracted. Extraction for 2
2/:26/40 ...... 1
hours ga\'e only a very small yield, but when the ma-
:2 j:29/40 ...... 0 3(i8 315
terial was then left in contact with ether for several 3/ 4/40 ...... 61 108
days, some 20-30 times as much auxin was obtained. 3/ 7/40 ...... 47 35
A further contact of several days yielded an even 3/ 8/40 ...... l:! 1;;
larger quantity. It became clear that in short times :l/14/40 ...... l:! 14
only a minute fraction of the total auxin is extracted. ,;/ ti/40 ...... 99 U6
Table 1 presents a series of extractions on com- ';j:23/40 . . . . . . 37 49
parable samples. It is clear that repeated extrac- Total ..... S,;:l 2,:Hl 2,938 'J,992
tions, spread over a period of some 1·~ weeks, were l'nits per S gm. 1,100 3,(HO 3,040 3,100
necessary to obtain the complete yield of auxin. The
final yield from 3 gill. of Lemna is listed as 3000 • See text for definition of units.
Dec.• 19101 Tll1MANN AND SKOOG-EXTRACTION OF AVXIN 953

2080 units, and with acidified chloroform, carried out
at the same time on a comparable sample, 1815 units.
The extraction was not taken to completion (3g
Lemna would yield in all about 3000 units) but in
this series the chloroform scarcelv differed from the
This represents a relatively rapid rate of extrac-
tion, but the yield to be expected from 5 g. should be
not less than 5000 units. Subsequent extraction of
the material with ether yielded no more auxin. In
17,000 ----,

ether for the first 1800 units. After this time, how- I
ever, the chloroform extractions yielded no more ac-
tivity. Apparently a variable trace of some agent
which inactivates auxin appears in the chloroform.
The experience of Skoog (1935) with the irradiation
.r
of indole-acetic acid in chloroform indicates that this 14,000--;;;

is a decomposition product. Since the chloroform was ~

0-
TABLE ~. The effect of acid on extraction of au.I'in with ~
chloroform .•1 y. Lemna in each series.

Chloroform Chloroform
'I'ime in chloroform alone plus acid
n
~o hours 5 65 10,000

3 days 17 60 E
C>
7 days 16 :15
3 months II :13
6 days 0 o
Total to date .. 49 173
Subsequently extracted
with ether 0 o

always freshly distilled over FeSO-l before use, the

decomposition product must be formed on standing
and is probably chlorine (cf. Chapman, 1935).
Hence, at least for these long-period extractions,
chloroform is less reliable as a solvent than ether.
Absolute alcohol has been used by several workers
and was therefore studied in addition. In one experi-
ment, 3 gram samples, ground with 10 cc. solvent and
0.05 cc, ~/10 HCI gave after 2 hours: ether, 205
units; chloroform, 23 units; alcohol, 58 units.
An experiment with serial extractions is shown in Time in Dc s
i
sc 40
table 3. Even extraction with alcohol in the Soxhlet >0 60 70 00 90 '00

Figure l. The progress of extraction with time. Curve

TABLE 3. Comparison of ether and absolute alcohol as ex- l. Lemna in large volumes of ether; auxin determined
t ractant« for auxin. 3 y. Lemna ill each series, placed by sampling. The ether was changed at arrows. Curve ;2.
in succe.~.~ive 15 cc. uolumee of solvent, startilly 3/6/40. Lemna extracted with small volumes of ether; changed
at each point. The extractions were continued to the 150th
DatI'S of tests Ether Absolute alcohol day, but yielded little increase beyond the highest point
shown.
3/ 7/40 .. 495 o
4/ 1/40 .. 1,:11:~ 60
4/1:1/40 . 1,;2.';0 104- other Soxhlet extractions with alcohol, only from 1
4/15/40 . 788 o to 5 per cent of the total yield was obtained. It will
Total to date . 3,74-6 164- be clear from what follows that owing to its drying
effect absolute alcohol could not be expected to be a
good extractant.
apparatus for periods from 1 hour up to several days Attempts were made to use the Soxhlet method
proved to be less effective than simple standing with with ether also, but in all cases the yields were lower
ether in the icc-box. than by standing in the cold. Probably the continued
On one occasion, with 5 g. Lemna, extracted for heating of the ether solution leads to auxin destruc-
successive 24-hour periods in the Soxhlet with ab- tion.
solute alcohol, the yields were: C. The Progress of Extraction with Time.-The
First day . USO units evidence above indicates that auxin goes on being ex-
Second day . ;280 units tracted for a long time. Further, this slow extraction
Third day , , . 30 units can hardlv be due to a low relative solubilitv of auxin
Total . 1790 units in the soh:ent. I n the first place the volume ~f solvent
9;').t AMERICAN JOURNAL OF BOTANY [\'01. 27,

is many times the volume of the Lemna ; secondly we D. The "ffect of heating.-A numher of attempts
know that the partition coefficient of auxin between have been made to bring the extraction rapidly to
ether and water is very high; thirdly, acidification, completion and to elucidate its nature.
which increases thc fraction of the auxin in the free The effect of heating the Lemna in a current of
ether-soluble form, does not increase the yield in steam before extraction is shown in table 4. In this
ether. The slowness of the extraction must therefore TABU; 4. Effect of «t rn min.q on the e.I'traction of «(Il.1'il/
have some other cause. from Lemna. Two 10 g. su.mple.• extracted b)l •• tan d:
Direct evidence on this point has been obtained by in,'! tcith. succes.•ice portion .• of 100 cc, ether in ice-
following- the course of the extraction with time. One 110,1'. Dnt« reduced to .1 !!' busis. Yields in aU,l'in unit.•.
9 gram sample of fresh Lemna was placed in 100 cc,
ether, kept in the icebox and sampled from day to Time in ether Intact Steamed 10 minutes
day. Another comparable sample was extracted with
successive 20 cc. portions of ether in the usual way :2 days 3(;"
over the same period. The data obtained for about ;;6 days 1,0:IO
four months are represented in figure I. :J.5 days 118
Curve I shows the results obtained bv the sam- 4 days fi
pling method. From the auxin assay of e~eh sample, Total to date. . . .. 1,190 76
and the volume of ether at the time of sampling, the
total amount of auxin present in the ether was com- a These figures confirmed by an assay with slit pea-

puted. To this was added the amounts which had stems,

been removed in the previous samples. The values
therefore represent the total amount of auxin which experiment 10/1:. samples were used, but the data are
has been extracted from the Lemna. Curve 2, on the reduced to apply to 3 g. in order to be comparable
other hand, shows the total amount of auxin which with the other tables. Steaming evidently has no ap-
had been obtained by repeated extractions up to the preciable effect on the first extraction, but therca fter
same dates, i.e. the sum total of the successive extrac- it prevents almost completely the liberation of auxin.
The same conclusion was arrived at by experi-
tions.
ments in whieh the Lemna was plunged into boiling
In the early stages, the amounts obtained by re-
peated extractions (curve 2) compare favorably with TABLt: 5. Tico experimrnt .• Oil the eff ert of boilil/gin
those obtained by simple standing in the lar~e volume water on the extradion of au:";n from Le mna. Yield.
in allxin units,
of ether (curve I). Thereafter, however, curve I lags
A, Two .j g. sample.• (olle bein q boil('(T) eet rnct ed b)l
and finally reaches what appears to be a true equilib- .•lrnul iru] willi. .surressire portion» of ether ill ice-box,
rium. The fluctuations in this region of the curve are
not unduly large when it is considered that the values Time in ether Auxin yields
are based on analyses of 1 cc, samples from a large
volume, and are also not corrected for changes in the Boiled in water
sensitivity of the test plants, On the ·13rd day the Water extracts Hesidue
Intact
whole of the ether remaining in this flask was re- material 1st ;.?nd
moved and the material rapidly extracted by wash-
ing with several small volumes of fresh ether. As will I hour 8;; 3 ..5 6" 78
be seen, there was an immediate release of a large 3 days 730 17
3 days .;70 10
quantity of auxin (about 7500 units) to the ether,
that is, this amount of auxin was already present in
1.5 days 171 o
extractable form. A fresh 100 ee. of ether was now Total to date.. 1,.;5(. 11.;
added, and sampling continued as before. \Vithin a B. Four it !!. .• ample.• (three b ehu] boiled) ext rart ed by
few days equilibrium was again reached, and on st.andiiu] tcith. succeesiue portions of ether or ab-
changing the ether once more on the 92nd day a solute alrohol in ice-box.
further amount of auxin at once appeared. In the Days in Fresh; Boiled; Boiled; Boiled;
third 100 ceo of ether only a small concentration was solvent ether ether ether ale. abs.
reached, indicating that the extraction was about ·H :2H ;'?8 ;.?:l
complete. The total yield by the two methods was not dried dried
very different. s 1:10 10 0 1
wetted wetted
It is evident that the equilibrium reached was due
1 1:18 8 1:1 ];;
to the partition of free auxin between the ether and
l:l ·n:l 0 0 0
the Lemna, both phases containing some water, but 4 0 0
that auxin continues to be liberated independent of 7 :100 0
any such equilibrium. Hence there must be a process :28 ;.?Ofi 0 0 0
at work in the tissues slowly setting free extractable Total .. 1,;'?:ll ·~6 50 S!)
auxin. It is this process which explains the extreme Activity in water extract equivalent to :l units per sample.
slowness of the extraction procedure.
Dec .• 1910] THIMANN AND SKOOG-EXTRACTION OF AFXIN 955

water for a few minutes. In these, the water extract with indole-acetic acid solution. It was then extract-
was evaporated and tested, and found to contain ed with ether in the usual way:
small quantities of auxin. The residue, on extraction Auxin added: 0.125 gamma.
with ether, at first gives up as much auxin as the un- From the sensitivity of the Aven«
treated material (table 5). Subsequently the yield of tbis corresponds to :l., 0

auxin comes rapidly to zero, in striking contrast to Auxin obtained in 1st extraction
the behavior of the unboiled controls. In table 5B it (10 days in ether) 20.5°
mav also be noted that alcohol is no more effective as Auxin obtained in 2nd extraction
(4- days in ether ) 15.;,°
an ~xtractant than ether.
Auxin obtained in 3rd extraction
Since the prolonged evaporation of the water ex- (40 days in ether ) 0.2°
tracts is undesirable, the auxin in these extracts was Total 36.2°
determined by extracting it therefrom with solvents.
Thus in another experiment a 3 g. sample was boiled There is thus complete recovery, but it is impor-
five times, in an attempt to increase the amount ex- tant to note that even here two extractions are neces-
tracted bv the water. Of the water extracts, two sary.
equal portions were acidified and extracted, one with Another experiment with unboiled material which
chloroform and one with ether, as a check on the two had also been extracted to completion gave similar
solvents. The yields were .51 and 57 units respective- results. In this case 26 units were recovered in the
ly, i.e, they showed very good agreement. The total first extraction, 7.5 in the second and 2 in the third.
yield from the first three water extracts was 93, from Thus the rate of removal of added auxin is exactly
the next two, 78 units. eomparable with the removal of the plant auxin from
I f the liberation process were a simple hydrol- boiled material in table 5. Hence in the boiled Lemna
ysis, boiling with acid or alkali might be expected to no more auxin was liberated after boiling.
promote it. However, table 6 shows that this is not In fresh Lemna the auxin must be present in a
TABU: 6. Effect of boiling in acid and basic solutions on
free and a bound form, the amount extractable at any
the amount of au;rin extracted b!/ the alJueou .• time being only a small fraction of the total amount
medium. Each extraction for 10 minute•• ; S g. fresh present. It is also clear that the auxin which is ini-
Lemna in earh sample. tially free cannot be determined satisfuctorily, since
two extructions are needed to recover it. In the
Auxin yields in the extracts course of these extractions further quantities will be
Extraction
numbers Acid (pH 3) Base (pH 9) Water liberated. If uny determination of free auxin is pos-
sible, it must be from the heated material. Here the
1 and 2 .. ... .... 2R IS 50 extraction yields what was initially free, together
:1 and 4- ......... 1 () 8 with uny auxin set free during the heating up of the
..i and Ii .. - ...... 5 () :1 tissue. In view of the very rapid heuting to which
7 and S ......... 0 :1 :2 the samples were subjected. it is probable that this
!} and 10 ........ 1 40 latter quantity is small and thus the yields from boil-
Total ......... 35 25 (i4o ed Lemna may be a measure of the auxin present in
free form.
R. The influence of dr.'linrJ on the extraetion.-A
the ease. In this experiment threeL5 g. samples
eons ide ration of the inefficiency of absolute alcohol
were eaeh extracted with ten successive 20 cc. por-
for successive extractions suggested thut this might
tions of boiling dilute H'Cl (pH 3), dilute KOH
(pH 9), and distilled water respectively. The ex- TABLE 7. Effert of dryill.'! on the yield of au.l'in from
tracts were eombined in pairs as shown, and tested Lamna, Samples 3.0 g. each.
for auxin by bringing to pH 3 and extracting five
times with half the volume of chloroform, followed Dried Dried
bv four times with half the volume of ether. Subse- Days in ether Fresh in air in oven
quent extractions showed that the auxin had been
completely removed from the aqueous layer. Both (:1 hours ) .... 40 1
add and base had reduced, rather than increased, :1 days 170 () 0
40 days 1i()(J () 0
the total yield of auxin in the extracts.
wetted wetted
All these experiments show that high temperature :1 days :11;, 1:1 ·1:1
stops the process which sets free extractable auxin. 15 days ;,.;0 1.50 110
The question arises as to whether the relatively small 7 days ;!H:l Ill! H:l
amounts of auxin obtained in the later extractions of Ii days I:1S 79 110
boiled material (sec table 5) arc really due to a small 7 days 117 ;!O ~:;

liberation of auxin after boiling, or only to incom- 17 days 20S

plete extraction of that which was already free. To :1:1 days :n:l HI 2:l
settle this point, a 3 g. sample of Lemna which 21 days S·l 0
had been boiled, and from which serial extractions Total ~.771 4010 :191
showed that all auxin had been removed, was treated
956 AMERICA:-J JOURNAL OF BOTA:-JY [Vol. 27,

be related to its dehydrating effect, i.e. it might be in the tissues due to the high temperature. There is
essential to maintain water in the tissue. A compari- good evidence, however, that the destruction of auxin
son was therefore made between the cther extraction itself is not the principal cause of loss. In fact, the
of samples of fresh Lemna and of similar samples effect of drying in preventing the extraction of auxin
dried in a stream of hot air or in the oven. It was at can be achieved without any loss in activity. This is
once found that when the samples were dry, extrac- demonstrated by an experiment in which drying was
tion with ether yielded only traccs of auxin in the carried out by grinding with anhydrous sodium sul-
first extraction, and none thereafter, while the con- fate. Table 8 shows that the material so trcated
trol samples gave thc normal quantities. As soon as yielded no auxin, while when water was subsequently
water was added auxin could be again extracted. A addcd auxin was set free as before. The total vield
complete experiment, involving 11 serial extractions, from exhaustive extraction of this sample agrees
is given in table 7. It is clear that auxin is set free closely with that from the fresh control, while the
from the dried samples only after water has been yield from the sample dried in hot air is, as before,
added. much lower.
This experiment, as well as the data on rl uena be-
TAHI.E 8. Comparison of yields after dr.'ling u'ith N a~S04
an d. air. :/ ,fl. Lemna in each sample. low, demonstrates that the auxin in bound form may
be quantitatively "fixed" within the plant materi~l
1" '1 s- by drying. It will now be shown that it is possible to
Dates Fresh Dried with Dried with set the auxin free and then again to "fix" it in non-
of tests ether hot air ~a~S04 extractable form. To a sample of dried Lemna, water
was added, and, after standing, the control portion
3P8/40 o o showed that ,1,0·1, units of auxin had been set free (see
wetted wetted table 9). Nevertheless, on re-drying no auxin could
312 9/40 .. 54 <20 <20 be extracted. \"hen water was again added, within 2
4/ 1/40 . 59 12 4 hours ,1,88 units were obtained. This extremely rapid
4/11/40 . 405 200 128
liberation did not occur in subsequent extractions of
7 extr. up to 6128 . 1,514 268 1,929
the same sample, nor is it observed generally in the
7/18/40 . 5:2 o 325
first extraction of dried samples, in which auxin first
Total . 2,130 500 2,406 has to be set free from its original state." In other
words, auxin which has been liberated can be again
a Extractions from samples 1 and 3 are not yet com-
"fixed," but is not converted to its original "bound"
plete.
form.
The total yield from the fresh material closely ap- These observations as to the forms in which auxin
proximates the 3000 units which we have usually ob- may be present have an important bearing on the
tained, and the extraction may still not be quite com- technique of extraction. Four factors must be clearly
plete. The yields from the dried samples are much distinguished:
less, however. In another series in which six extrac- (1) The irreversible inactivation discussed above,
tions were made over a period of 33 days, two sam- and exemplified in table 7.
ples which had been dried and rewetted yielded 588 (2) Some physical effect of drying which prevents
and 779 units respectively, against 31,80 units from auxin extraction. This is suggested by the improve-
the fresh control. ment in yield which resulted in one experiment from
The reduction in total yield after drying and re- grinding the dried material to a fine powder. A 3 g.
wetting has varied with the plant material used. sample, dried, rewetted and extracted to completion
Thus, in Avena (as is shown in the following sec- (20 extractions spread over 4 months) yielded a
tions) it was observed with the roots but not with the total of 706 units, while a comparable sample which
coleoptiles. It might be brought about in several e It will be noted in table 5 R that this type of fixation
ways. There is probably some inactivation of auxin also occurs on drying boiled material.
TABLE 9. Fixation of auxin by drying. 24 grams dried Lemna extracted tuiice 'with ether. Yield
26 units. Water added, material left one 'week under ether, then divided into two equal
parts.

Part 1 Part 2

Dried in hot air, then extracted. Extracted directly.

Yield: 0 units Yield: 404 units
Water added, re-extracted after two hours.

Yield: 488 units

Five extractions made over a period of 120 Five extractions made over a period of 110
days days
Yield: 8,620 units Yield: 11,200 units
Dec., 19401 THIMANN AND SKOOG-EXTRACTION OF AUXIN
had been finely ground after drying and' then re-
wetted yielded 1500 units, or about double the
amount.
(3) The phenomenon of reversible "fixation," i.e,
the auxin is in some way held in a non-extractable
form by drying. This is exemplified by tables 8 and 9.
(4) The process which, using water, liberates
auxin from its bound form in the plant tissue.
The relative importance of these factors may dif-
fer with different plants, although obviously it is the
last two which are of principal interest. They must
be considered in detail for each material studied.
Examples of the behavior of some materials other
than Lemna will be given below.
THE EXTRACTION OF AUXIN FROM AVENA SEED-
LINGs.-The,slow release of auxin from Lemna, and
the influence of water on the extraction, made it de-
sirable to reinvestigate the extraction of auxin from
Avena seedlings. These experiments brought to light
two interesting contrasts: (a) the contrast between
the behavior of Avena coleoptiles and of Lemna, and
(b) that between the behavior of the coleoptiles and
the roots in Avena itself.
In table 10 are given the results of four experi-
ments on 2-2.5 mm. tips taken from coleoptiles 30
mm, long, in each of which duplicate samples were
extracted, one fresh and one dried in the hot air
stream. Three conclusions may be drawn. Firstly,
except in one experiment (where the test itself is
open to question) no auxin is extracted from the
dried tips. On wetting auxin immediately appears.
Secondly, the total yield from the dried sample is in
each case not significantly different from that of the
fresh sample, so that, in contrast with Lemna, dry-
ing evidently causes no auxin destruction. Thirdly,
there is a striking difference from Lemna in the rate
at which extraction takes place. All the auxin is ob-
tained from the coleoptiles in a single extraction. In
other words, there is no prolonged liberation of
TABLE 10. Extraction of auxin from Avena coleoptile tips. In each experiment one set of tips
was dried, then wetted after one or two extractions; the other set was used [resh, Four
successive extractions by standing in ether. Lengths of tips in experiments 1 ~: 2, 2 mm;
in experiments 3 <S. 4, ca 4 mm. Each extraction time 24 hours or more.
Experiment No. of tips in
No. each group Treatment
1 ........
t284
84 Dried
Fresh
{257 Dried
2 ........
257 Fresh
3 ••••••• 0 f26 Dried
126 Fresh
4 ........ {120 Dried
120 Fresh
957
auxin under ether in the coleoptiles, comparable with
that in Lemna. However, an experiment with larger
samples left in ether for three months after the ini-
tial extraction did yield an additional 10 per cent.
It is of interest that coleoptile tips do, however,
continue to liberate auxin in vivo. Since it is well
known that excised tips yield auxin to agar over a
period of several hours, the results of extractions
were compared with those obtained by placing the
tips 'on agar in the usual way. From such diffusion
experiments with one set of tips in experiment 1, the
yield in 1 hour's diffusion was 2.6 plant units per tip
per hour, a figure in good agreement with the litera-
ture (see Went and Thimann, 1937). This corre-
sponds to an expected yield in the first extraction ex-
periment of 13.7 of our arbitrary units. Extraction
thus gives about the same amount of auxin as one
hour's diffusion. The same result was obtained bv
Thimann (1934). It follows that extraction recove;s
only a small part of the auxin which the tips could
have produced had they remained alive (ef. also van
Overbeek, 1940).
Extraction of sections below the tip gave results
similar to those with tips. Thus 240 sections of the
first 6 mm. below the tip yielded, when dried, one
unit in the first extraction and nothing in the second,
while the fresh material gave ten units in the first ex-
traction and nothing thereafter. On adding water to
the dried sections, eight and zero units were obtained
in the first two extractions respectively. The more
basal sections behaved similarly although giving
lower yields. Here again the amounts obtained were
less than those found by Skoog (1937) in diffusion
experiments lasting four hours.
The behavior of the first leaf of Avena, removed
from inside the coleoptile, differs from that of the
coleoptile in that four successive extractions, spread
over about a week, are required to remove all auxin
from it. Theyield from 250 leaves, as a mean of four
Total
Yield of auxin in extraction number: yield
1 2 3 4
wetted
0
12
0
0
14
wetted
0
0
14
12
(12?) 0 8 0 (20)
17 1 1 19
wetted
0 15.5 0 15.5
14 0 14
wetted
0 9.5 0 9.5
10.5 0 10.5
958 AMERICAN JOURNAL OF BOTANY [Vol. 27,

TABLE 11. Auxin [rom Avena roots. Each sample the roots [rom 250 plants, 3 daY8 old, [resh.
weiJht about 6 gram8.

Times for extraction

Yields in auxin units of f.?5% of the auxin
Days in ether Dried Fresh from the fresh material

1 14 3f.?8 1 day
4 o
wetted
(3 hours) 3f.?8 ::}... I week
3 84
1 f.?3
3
3
f.?1
19
::1
. 73
1 14 15~ ..............•. f.? weeks
3 13 ggj
3 16
16J
1 6 f.?1
7 19 7f.?
5 3 93
1 3 14
3 5 14
7 15 16 ...........•.... If.? weeks
10 o f.?8
14 7 f.?8
o f.?3
13 o 10J
Total 105 days 589 1,309

experiments, was 187 units, or about ten times that THE EXTRACTION OF AUXIN FROM PHASEOLUS NOD-
from the coleoptile tips. The behavior with respect ULES.-Still another type of behavior is presented
to drying was again the same, only traces of auxin by the root-nodules of Phaseolus vulgaris. These ob-
being obtained when the material was dry, but the jects, as table 12 shows, contain large quantities of
full yield being set free on wetting. auxin, the bulk of which can be very rapidly ex-
Table 11 gives the results of extraction of the tracted. This agrees with the previous finding (Thi-
roots of Avena seedlings, taken at the same age as mann, 1989) that auxin diffuses out of such nodules
the coleoptiles above. It may be seen that again auxin with great rapidity. However, as in the other cases
is obtained only when water is present. In this ma- studied, drying by hot air almost completely pre-
terial, however, the total yield is greatly reduced by vents extraction with ether, and it also greatly re-
the dry process, and, furthermore, very long pe- duces the total yield.
riods of extraction are needed to remove all the ac- THE EXTRACTION OF AUXIN FROM EXCISED CALLUS.
tivity. In these two respects the roots differ from -Cultures of NicoticLna callus were propagated
coleoptiles and resemble Lemma. from a specimen obtained from Dr. White (see
TABLE 12. AWlJin from Phaseolus root nodule», 200 halves White, 1989), and their auxin content determined by
of nodule8, total [resh weight ca 0.4 gram, placed in ether extraction. Results from young cultures were
8ucceB8ive 10 CC. oolumes of ether. comparable to those with Lemna, with the exception
of the fact that the activity of auxin obtained in
Days in ether Dried sample Fresh sample ether extracts is generally somewhat masked by ac-
companying inhibitors (Skoog, 1940). Some results
1 5 5,940 with old cultures are included here because they are
1 0 650 in direct contrast to those obtained with nodules. In
wetted the first place, the cultures yield no auxin at all by
(5 hrs.) 420 diffusion into agar blocks. In the second. place, as
3 1,625 68
18
shown in table 18, no activity is obtained in the first
3 625
1 107 26 ether extracts, but on subsequent extraction auxin is
3 96 49 gradually released over a long period.
7 54 71 DISCUSSION.-The results make clear that plant
4 0 0 tissues differ widely in regard to extraction of their
f.?0 0 0 auxin by solvents. The materials we have studied
Total 43 f.?,932 6,8f.?2 provide examples of four different types of behavior :
1. Auxin yielded almost entirely in a single short ex-
Dec.• 1940] THIMANN AND SKOOG-EXTRACTION OF AUXIN 959

TABLE 13. Extraction of auxin from tobacco callus. (Cul-

tinue to completion." In the majority of cases, this
tures 19 weeks old groWlt on White's agar medium.) would require a long time. Many of the data in the
literature are based on extractions limited to very
Yields in auxin units short times. With the ·exception of coleoptiles and
Sample A SampleB perhaps certain other etiolated tissues, we have
Days in ether ~.5 gms. 5gms. found no material whose auxin content could be
quantitatively determined in this way.
1 -0.1 It might be possible to obtain results of compara-
1 0.6 tive significance by a single extraction carried out un-
6 6.4 1.4 der rigidly controlled conditions. For comparative
1 3.3 -0.8
4.3 ~.8
purposes, however, as in correlation with growth
6
7 1.8 16.0 measurements, the diffusion method, which measures
4 3.7 6.8 the free auxin together with that made available in a
7 6.3 40.0 limited time, is probably more reliable. This method,
3 1.8 3.8 of course, is mainly applicable to tissues with polar
4 ~.3 ~.~ transport.
1 -0.3 -o.~ When it is further considered that in some cases
34 -1.~ o.s inhibitors are present (as with Nicotiana callus) and
Total 75 30.5 73.~ that auxin may be inactivated during the extraction,
it is essential that in all cases conclusions as to the
absolute amounts of auxin present be drawn only
traction period; no loss on drying. (Avena coleop- with great reserve.
tiles) . The experiments of Avery, Creighton and Sha-
2. Auxin yielded only slowly over a long period of lucha (1940) on corn endosperm lead them to the
time; large loss on drying by hot air; evidently only conclusion that more auxin is extracted by successive
a small fraction of the auxin is in free form. (Lemna, treatments with different solvents tha~ when one
A vena roots.) There is reason to believe that green alone is used. Of the 6 extractions cited, two show
leaves and other mature tissues belong in general to that extraction with several solvents gave yields
this type. greater than the best results obtained with a single
3. Auxin yielded very rapidly at first, followed by solvent. Assuming that these extractions were com-
subsequent slow release; large quantities yielded on plete, this would mean that a number of compounds
short time diffusion. (Phaseolus nodules). with auxin activity, but different solubilities, are
4. No auxin obtained in first extraction, but subse- present. A priori, however, one would not expect a
quent behavior as with Lemna; the activity is in part substance insoluble in ether to be extracted by chlo-
masked by relatively large amounts of inhibiting ma- roform at the same pH or vice versa; such behavior
terial. (Nicotiana callus; cf. also the tomato, studied might occur, on the other hand, with two solvents as
by Laibach and Lots, 1936, and by Larsen, 1939b). different as ether and alcohol. It is true also that
This outline is not intended as a rigid classifica- Larsen (l939b) has shown the presence of both an
tion. For instance, the leaves of Avena seedlings, acid and a neutral auxin in plant material, but both
which yield little auxi~ by diffusion (Skoog, 1937) of these are ether-soluble. In our experience with
but from which large amounts are obtained in rela- Lemna, when extraction with one solvent was carried
tively few extractions, fall between classes 1 and 2. to completion, the use of another solvent produced no
A true classification would have to be based on de- further yield. While our experiments provide no evi-
terminations not only of the relative amounts of free dence as to the presence of more than one active sub-
and bound auxin present, but also of the activity of stance in the tissues we have examined, they do show
the enzyme system which sets the auxin free. that the auxin is present in more than one form,
In ail thes'e types water is necessary. for the ex- namely, naturally "bound," free, and reversibly
traction. Even in coleoptiles and nodules (tables 10 "fixed." The first of these is apparently an auxin-
and 12), from which auxin diffuses rapidly into agar protein complex.
and is rapidly extracted by ether, almost no activity This investigation was undertaken in the hope of
is obtained in extracts unless water is present. The developing a simple quantitative extraction method.
evidence indicates that water participates in two It must be admitted that this has not been attained.
ways. It enables free auxin to be removed by the On the other hand, the results elucidate the several
solvent, and it is required for a process which re- factors on which a successful procedure must be
leases auxin from the original bound form. This lib- based, and show that the total auxin content of a
erating process is a hydrolysis, which our experi- variety of materials can be determined satisfactor-
ments have shown can be carried out by proteolytic ily.
3 The rate of release, i.e., the units of auxin set free per
enzymes (see Skoog and Thimann, 1940).
day, often increases for the first few days, suggesting
When extraction is made by solvents, it is clear that contact with. ether promotes the activity of the
that if the total amount of auxin is to be determined, enzyme system. This behavior is very clear in the fresh
the solvent must allow the hydrolytic process to eon- intact plants of tables 1, 5b and 7.
960 AMERICAN JOURNAL OF BOTANY
SUMMARY
The extraction of auxin by solvents from a num-
ber of plant materials has been studied.
In Lemna, grown under controlled conditions, re-
peatable results can be obtained by successive ex-
tractions with ether, but the process requires several
months to reach completion. Chloroform, ethyl alco-
hol and water are less satisfactory solvents. If chlo-
roform is used, the material must be acidified, and
there is danger of inactivation. The process cannot
be hastened by steaming, boiling at different pH, ex-
traction in the Soxhlet apparatus, or grinding. All
these treatments reduce the total yield.
With Avena coleoptiles the auxin is almost com-
pletely extractcd in 24 hours, but the roots, and to a
LITERATURE CITED
AVERY, G. S., JR. 1935. Differential distribution of a
phytohormone in the developing leaf of Nicotiana.
Bull, Torrey Bot. Club 6'2: 313-330.
- - - , H. B. CREIGUTON, AND B. SUALUCUA. 1940. Ex-
traction methods in relation to hormone, content of
Maize endosperms. Amer. Jour. Bot. '27: '289-300.
BOYSEN-JENSEN, P. 1936a. trber eine Mikromethode zur
quantitativen Bestimmung der Wuchsstoffe der A-
Gruppe. Planta '26: 584-:-594.
- - - . 1936b. Dber die Verteilung des Wuchsstoffes in
Keimstengeln und Wtirzeln. Kg!. Danske Videnskab.
BioI. Melt 13: 1-31.
CHAPMAN, A. T. 1935. The peroxidation of chloroform.
Jour. Amer. Chern. Soc. 57: 419-4'2'2.
Du Buy, H. G. 1935. A method for extracting growth
substances from pigmented tissues. Jour. Agric. Res.
56: 155-15S.
HOAGLAND, D. R., AND W. C. SNYDER. 1933. The nutri-
tion of strawberry plants under controlled conditions.
Proc. Amer. Soc. Hort. Sci. 30: '2S8-'294.
LAIBACH, F. AND R. LOTZ. 1936. Methodisches zur
Wuchsstoffuntersuchung. Biochem. Zeitschr. '288:
'250-'256.
- - - , AND F. MEYER. 1935. uber die Schwankungen
des Auxingehaltes bei Zea Mays und Helianthu« an-
nu1tS im Verlauf der Ontogenese. Senckenbergiana
17: 73-86.
LARSEN, POUL. 1939a. Untersuchungen tiber den thermo-
labilen wuchsstoffoxydierenden Stoff in Phaseolus-
Keimpflanzen. Planta, 30: 673-68'2.
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lesser degree the leaves, behave like Lemna. Phaseo:
lus root-nodules and cultured Nicotiana callus show
still other types of behavior.
Water is essential for the extraction process in all
these materials. On drying, the auxin, whether previ-
ously bound or free, becomes "fixed" in a non-ex-
tractable form. This "fixation" mayor may not be
accompanied by a partial destruction. Water is also
necessary for the hydrolytic liberation of auxin from
its bound form in the tissues. This process determines
the rate at which auxin can be extracted.
BIOLOGICAL LABORATORIES,
HARVARD UNIVERSITY,
CAMBRIDGE, MASSACHUSETTS
- - - . 1939b. Skototenln, ein neuer Streckungswuchs-
stoff in hoheren Pflanzen. Naturwiss. 3'2: 549-550.
LINSER, HANS. 1939. Zur Methodik der Wuchsstoff-
bestimmung II. Die Extraction von Pflanzenmaterial.
PJanta '29: 39'2-408.
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extraction. Proc, Nat. Acad. Sci. '24: 4'2-46.
- - - . 1938b. Auxin distribution in seedlings and its
bearing on the problem of bud inhibition. Bot. Gaz,
100: 133-166.
- - - . 1940. In press.
SKOOG, F. 1935. The effect of X-irradiation on auxin
and plant growth. Jour. Cell. and Compo Physio!.
7: '2'27-'270.
- - - . 1937. A deseeded Avena test method for small
amounts of auxin and auxin precursors. Jour. Gen.
Physiol. '20: 311-334.
1940. Synthesis of auxin in excised tobacco
callus. (In prep.)
- - - , AND K. V. TUUfANN. 1940. Enzymatic libera-
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