A botanist, Haberlandt,(1) first pointed out the possibilities of the culture of isolated tissues. He suggested that not only could the potentialities of individual cells be determined, but that also some insight might be gained as to the reciprocal influences of tissues upon one another, that is, as to "correlation.

Bonner, James

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PHYSIOLOGY: J. BONNER
PLANT TISSUE CULTURES FROMA HORMONE POINT OF VIEW
By JAMES BONNER
WILLIAM G. KERCKIIOFF LABORATORIES OF THE BIOLOGICAL SCIENCES, CALIFORNIA
INSTITUTE OF TECHNOLOGY
Communicated May 15, 1936
A botanist, Haberlandt,l first pointed out the possibilities of the culture
of isolated tissues. He suggested that not only could the potentialities of
individual cells be determined, but that also some insight might be gained
as to the reciprocal influences of tissues upon one another, that is, as to
"correlation." Haberlandt's many experiments with plant cells did not
succeed. Harrison, Burrows and Carrel, using animal tissue, were able to
realize Haberlandt's ideal, and from the more recent work of Fischer,
Ephrussi, Gaillard and Chambers, it is evident that many different kinds
of problems can be attacked by the method of tissue culture. Since the
time of Haberlandt there have been repeated attempts to cultivate plant
tissue in artificial media, or at least in vitro. Embryonic tissue has been
grown under many conditions. Thus Hannig2 grew immature embryos in
artificial media and eventually raised mature plants from them. LaRue3
has recently cultivated fragments of embryos and has obtained mature
plants from such fragments. It should be clear, however, that the concept
of "tissue culture" does not embrace such experiments. The embryos of
Hannig and others have always differentiated into more or less normal com-
plexes of tissue even though grown on artificial media. The embryonic
fragments of LaRue have similarly differentiatedj and are, in fact, typical
examples of "regeneration" in the sense of Loeb. Isolated root tips were
first cultivated in vitro by Robbins4 and by Kotte,5 but to White6 is due the
credit for having worked out the cultural conditions capable of sustaining
the indefinite growth of these organs. In this case again, as is well shown in
the recent work of Robbins, Bartley and White,7 the root meristem con-
tinues to differentiate in a comparatively normal fashion. Cultures of the
various component tissue of the root have not as yet been possible. In fact,
cultivation of isolated plant tissue has been accomplished only by
Schmucker8 with palisade cells, but his results cannot as yet be confirmed.
This signal lack of success in the cultivation of isolated plant tissues is
undoubtedly in part due to the use of unsuitable tissue. On the other
hand it is also certainly due in part to unsuitability of the media employed.
Because of the work on auxin we are now beginning to realize the great part
special chemical substances or "hormones" play in plant growth and de-
velopment, and it seems probable that such substances must be present in
any medium in which plant tissue is to be successfully cultivated. We
cannot, however, supply these substances as simply to plant cultures as to
426 PROC. N. A. S.
PHYSIOLOGY: J. BONNER
animal, since they are not readily available in embryonic extract. Ex-
perience has shown that extract of plant embryos is in general without
effect or actually toxic for the growth of excised plant tissue. White has
shown, however, that there is a substance (or substances) present in minute
amounts in yeast which is essential for the continued growth of excised
roots. That analogous substances are necessary for the growth of other
organs and tissues, and that these substances may also be derived from
outside sources, seems a fair inference. The objectives of the present work
have therefore been the separation and purification of special substances or
hormones which control growth and development of plant tissues and
organs, and the perfection with the aid of such substances of true tissue
cultures. Only a portion of this work will be reported here, and the re-
mainder will be discussed in greater detail elsewhere.
If a young string-bean pod be split lengthwise along the sutures and the
immature seeds removed, a series of "cups" lined with small iso-diametric
parenchymous cells is exposed. The parenchymous lining of the cup is very
sensitive to injury and responds to a prick by a hypertrophic outgrowth of
dividing and enlarging cells. A similar but larger outgrowth follows in
response to certain tissue extracts, as first described by Wehnelt.9 If small
drops of water are placed in the tissue cup no reaction occurs.'0 Similarly,
auxin is without effect except in concentrations sufficiently high to be toxic,
when a small reaction occurs (see also Jost"). Tissue extracts, however,
either of the bean or of other plant organs, particularly green parts, cause a
"wart" like protuberance of the parenchymous tissue. This outgrowth is
limited to the area directly under the drop of tissue extract, and may be as
much as 1.5 mm. high. Cross-sections of the wart show that it consists of
cells elongated at right angles to the surface of the cup. Cell divisions
may also be found (see Jost"), their number depending on the variety of
bean used. Thus golden wax and valentine varieties gave no cell divisions,
Kentucky wonder number 1 gave a few, and Kentucky wonder number 2
gave many cell divisions.
Standard conditions for the "bean test" have been established, and it has
been possible to perform semi-quantitative tests for the factor (or factors)
in question by determination of the dilution in which a given preparation is
just active. In this way the activity of a solution may be estimated to
about 3 times. The following chemical properties of the factor involved,
a few of which were previously reported by Wehnelt, have been established:
1. Soluble in water, 75% alcohol.
2. Insoluble in ether, chloroform, butyl alcohol, absolute alcohol.
3. Stable to H202 in both acid and alkaline solution.
4. Relatively heat stable in both acid and alkaline solution.
5. Adsorbable from water by charcoal, elutable in 75% alcohol.
VOL. 22, 1936 42,-
PHYSIOLOGY: J. BONNER
6. Precipitatable by alkaloidal reagents (phosphotungstic acid, picric
acid).
A partial purification of the factor is possible on the basis of the proper-
ties enumerated above, and this purification is being continued. It would
be of interest to know whether or not the bean reaction is due to one factor
influencing both cell division and cell elongation, or to two or more separate
substances. With this in view, pantothenic acid, a "growth hormone" for
certain strains of yeast, and kindly supplied by Prof. Roger Williams, was
tried in the bean test. It was completely without activity over a wide range
of concentrations. Similarly vitamines B1 and B2, shown by Schopfer12 to
be growth substances of Phycomyces, do not come in question here since
they have chemical properties different from those enumerated above. The
conclusion of Dagys"3 that the growth hormones of the lower and the higher
plants are closely related would seem open to some question. One may also
draw the conclusion that the auxins are not the only cell elongation hor-
mones of the higher plants, since we have here cell elongation due to a sub-
stance of quite a different type (insoluble in ether and chloroform).
The fact that the parenchymous cells of the bean pod respond actively
to this extract in vivo suggests that such cells might be suitable for cultiva-
tion in vitro. Accordingly a large number of experiments on their culti-
vation in liquid media have been made. Of the various solutions, used as a
base for the medium, the best thus far has been found to be a somewhat
modified White's6 solution; although this is in all probability not optimal.
The solution was made up as follows:
Ca(NO8)9 230 mg. KH2PO4 12 mg.
MgSO4 35 mg. Fe2(SO4)a 2 mg.
KNO3 80 mg. sucrose 20 gm.
KCI 65 mg. water 1 liter
Tissue extract for the experiments reported here were made by extracting
250 gm. of fresh beans with 250 cc. of 75% alcohol for 8 hours, evaporation
of the alcohol, filtering and dilution of the filtrate to a total volume of 200
cc. This extract was then diluted with an equal volume of the above culture
solutions which had been made up to twice the final concentration.
Cubic pieces of tissue approximately 1 mm. on each side were removed
under sterile conditions from bean pods and placed in petri dishes with 10
cc. of the above medium. The petri dishes were then left in the dark for 6
days. At the end of this period control cultures to which no extract had been
added showed no growth whatsoever. If bean extract had been added a
very striking growth of the parenchymous tissue took place; in some
experiments an increase of volume of as much as 40 times. The originally
cubical tissue fragment rounded out as it enlarged and assumed a spherical
shape. The enlargement depended, in. the main, upon elongation of the
peripheral cells. At the base of this zone of greatly elongated cells was found
428 PROC. N. A. S.
PHYSIOLOGY: J. BONNER
a "meristematic" zone, particularly in cultures of Kentucky wonder number
2. Superficially these cultures resembled strikingly the cultures of fibro-
blasts in vitro. In reality, of course, the cells at the periphery were "mi-
grating" only by their growth and not by any independent active move-
ment.
At the end of six days new fragments approximately 1 mm. on each side
were cut from the central portion of the original cultures and placed in
fresh medium. These "subcultures" grew as did the original cultures.
Subcultures were continued, each time fragments being taken from the
center of the old culture. After the first two cultures, however, the growth
steadily declined until it became practically 0, as is shown in table 1.
TABLE 1
No. of subculture 0 1 2 3 4 5
Av. diam.,mm. 1 4.5 0.6 4.5 .0.7 3.3 0.3 2.8 0.3 1.8 0.3
6 7
1.5 & 0.3 1.3 i 0.2
It was at first supposed that this decline might be due to the exhaustion
of some other essential factor originally present in the tissue. The addi-
tion of other substances to the later cultures, as well as to the culture during
its entire history, was therefore attempted. Peptone, yeast extract, hetero-
auxin and malt extract were tried in this way, each over a considerable
range of concentrations, but no substantial improvement of the later cul-
tures was found. It seems more likely that there is a cumulative toxic effect
of the crude preparation employed, but a test of this hypothesis must await
further purification of the active principle.
The chief interest of the cultures described here is that they represent
a somewhat closer approach to true plant tissue cultures than do other at-
tempts thus far reported, even though they are not, as yet, capable of in-
definite subculture. They show, in the first place, both cell division and
cell enlargement, and not merely cell enlargement as did the cultures of
Haberlandt, Bobilioff-Preisserl4 and others. In the second place, paren-
chymous tissue in this case continues to grow as parenchymous tissue, and
there is no differentiation into root and shoot as in the cultures of LaRue,
or into vascular tissue as in the root cultures of White. It is necessary to
note, however, that the present cultures are not completely without a
progressive differentiation, since the greatly enlarged cells of the periphery
are incapable of further division, and subcultures must be made from the
central portion of smaller, dividing cells.
I Haberlandt, G., Sitzungsb. Akad. Wiss. Wien, Math. Natur. Ki., 3, 69 (1902).
2 Hannig, E., Bot. Zeitung, 62, 45 (1904).
8 LaRue, C., Proc. Nat. Acad. Sci., 22, 201 (1936).
4 Robbins, W., Bot. Gaz., 73, 376 (1922).
A Kotte, W., Beitr. z. aUg. Bot., 2, 413 (1922).
Voi,. 22, 1936 429
PSYCHOLOGY: C. I. HOVLAND
I White, P., Plant Physiol., 9, 585 (1934).
7 Robbins, W., Bartley, M., and White, V., Bot. Gaz., 97, 554 (1936).
* Schmucker, Th., Planta, 9, 339 (1930).
' Wehnelt, B., Jahrb. wiss. Bot., 66, 773 (1927).
10 Wehnelt experienced difficulty in that pure water sometimes caused a reaction.
If the pods are allowed to wilt slightly before use this difficulty can be avoided.
11 Jost, L., Ber. d. d. bot. Gesell., 53, 733 (1935).
12 Schopfer, W., Ibid., 52, 308 (1934).
13 Dagys, J., Protoplasma, 24, 14 (1935).
14 Bobilioff-Preisser, W., Beih. z. bot. Zentralbi., 33, 248 (1917).
'INHIBITION OF REINFORCEMENT' AND PHENOMENA OF
EXPERIMENTAL EXTINCTION
By CARL IVER HOVLAND
DEPARTMENT OF PSYCHOLOGY, INSTITUTE OF HUMAN RELATIONS, YALE UNIVERSITY
Communicated April 25, 1936
In the literature on conditioned reflexes, two contrasted varieties of
curves of experimental extinction have been described. The first type,
illustrated in the experiments of Pavlov,l Kleitman and Crisler,' Switzer'
and others, is characterized by a continuous decline in the magnitude of
the conditioned response on successive unreinforced elicitations. The de-
cline is rapid at first, then more and more gradual. The second type, re-
ported by Switzer,4 Scott,6 Hudgins6 and Hilgard and Marquis,7 shows a
larger response on the second or third extinction trial than on the first,
demonstrating the initial rise characteristic of these curves. Hull,8 in his
summary of studies of conditioned reflexes, points out that "as yet the ex-
perimental conditions which bring about these two types of curve have not
been determined" (p. 438).
The initial rise in the extinction curves of the second variety has been
attributed to a "practice effect" or "improvement through use." This
fails to explain why the rise is not uniformly observed. Research by the
writer9 on the irradiation of conditioned responses has suggested an ex-
planation based upon the experimental conditions of the reinforcement
process. There appears to be a negative adaptation to continuous rein-
forcement, an effect which may be labeled, for descriptive purposes, the
'inhibition of reinforcement."0 Following protracted reinforcement the
omission of the unconditioned stimulus might be expected to produce
'disinhibition,' resulting in an augmentation of response on the second
or third extinction trial. This explanation for the rise in their extinction
curves has been recently proposed by Hilgard and Marquis :7 "The first
extinction trial is followed by altered conditions. . . This unexpected
430 PROC. N. A. S.


Plant tissue cultures from a hormone point of view

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Plant tissue cultures from a hormone point of view

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