close

Вход

Забыли?

вход по аккаунту

?

TUMOR PRODUCTION BY PHYTOMONAS TUMEFACIENS AND INDOLE-3-ACETIC ACID IN CULTURES OF TOMATO ROOTS

код для вставкиСкачать
INFORMATION TO USERS
This material was produced from a microfilm copy of the original document. While
the most advanced technological means to photograph and reproduce this document
have been used, the quality is heavily dependent upon the quality of the original
submitted.
The following explanation of techniques is provided to help you understand
markings or patterns which may appear on this reproduction.
1. The sign or "target" for pages apparently lacking from the document
photographed is "Missing Page(s)". If it was possible to obtain the missing
page(s) or section, they are spliced into the film along with adjacent pages.
This may have necessitated cutting thru an image and duplicating adjacent
pages to insure you complete continuity.
2. When an image on the film is obliterated with a large round black mark, it
is an indication that the photographer suspected that the copy may have
moved during exposure and thus cause a blurred image. You will find a
good image of the page in the adjacent frame.
3. When a map, drawing or chart, etc., was part of the material being
photographed the photographer followed a definite method in
"sectioning" the material. It is customary to begin photoing at the upper
left hand corner of a large sheet and to continue photoing from left to
right in equal sections with a small overlap. If necessary, sectioning is
continued again — beginning below the first row and continuing on until
complete.
4. The majority of users indicate that the textual content is of greatest value,
however, a somewhat higher quality reproduction could be made from
"photographs" if essential to the understanding of the dissertation. Silver
prints of "photographs" may be ordered at additional charge by writing
the Order Department, giving the catalog number, title, author and
specific pages you wish reproduced.
5. PLEASE NOTE:
received.
Some pages may have indistinct print. Filmed as
Xerox University Microfilms
300 North Zeeb Road
Ann Arbor, Michigan 48106
13' H,Vft
LD3907
,G7
Friedman, Bernard A .
f
'
1941
Tumor production by Phytomonas tume.F8
faciens and indole-3-acetic acid in
cultures of tomato roots...
New York,
1941.
iii,93 typewritten leaves, illus.,
tables. 29cm.
Thesis (Ph.D.) - New York university,
Graduate school, 1941.
Bibliography: p.73-93.
AG7940
Xerox University Microfilms,
Ann Arbor, Michigan 48106
T H IS D IS S E R T A TIO N HAS BEEN M IC R O F IL M E D E X A C T L Y AS R EC EIVED .
1.IHRARV
N. Y. U kiv ,
TUDOR PRODUCTION BY
PKYTOTOHAS TUI'tEFACIEIiS AHD IHDOLB-J-ACETIC ACID
IN CULTURES OF TO’iATO ROOTS
BERNARD A. FRIED:"AN
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy at H ew York University
April 1, 19Ul
ACKNOWLEDGMENTS
Acknowledgment Is gratefully given to Professor
Thomas Francis, Jr. for his advice and suggestions
extended during the course of this work; and to
Professor Julius A. Xlosterman, Dr. C. Chester Stock,
and the Staff of the Department of Bacteriology of
New York University for the maqy facilities and
courtesies that were granted.
CONTENTS
ACKNOWLEDGMENTS
I.
INTRODUCTION
II.
HISTORY
III.
EXPERIMENTAL METHODS AND RESULTS
A.
General Procedures
B.
Effect of piytomnnaa
and Various Fractions upon Roots
Culture Media
1. Living bacteria
2. Dead bacteria
3. Frozen and thawed cells of p, Vflflffaclena
4. Filtrate of frozen and thawed cells
5. Sediment of uninoculated broth medium
6. Supernatant fluid of bacterial culture
C.
Effect of Indole-3-Acetic Acid
1. Gross appearance of roots
2. Microscopic appearance of roots
3. Growth of roots
4. Toxicity of indole-3-acetic acid and growth of
galled roots in normal solution
3. Effect of neoprontosil
6. Effect of tryptophane and of indole
?. Effect of acetic acid and hydrogen ion concentration
IV.
DISCUSSION
V.
SUMMARY AND CONCLUSIONS
VI.
BIBLIOGRAPHY
iii
TUMOR PRODUCTION BY
PHYTOMONAS TUMEFACIENS AND IND0LE-3-ACETIC ACID
IN CULTURES OF TOMATO ROOTS
I.
INTRODUCTION
Crown gall of plants Is characterized by large
tumors occurring on any part of the root or shoot, but
frequently at the base of the trunk, i.e., at the"crown.w
These overgrowths are a combination of hypertrophy and
hyperplasia; there may be, in addition, excessive or
abnormal development of organs, particularly adventitious
roots.
The disease, also known as plant cancer, root knot,
broussin, rogna, or krebs, progresses slowly, stunting the
host and may eventually kill it.
It is widely distributed
on a number of suscepts, and is generally agreed to be a
serious disease of peaches, blackberries, plums, grapes,
almonds (Smith, 125; Heaid, 52; Elliott, 38).
The disease has been known for at least twenty-five
years.
It was first considered to be caused hy various
physical factors, as frost,dampness, low temperature, or
mechanical injury.
Later, the infectious nature of the malady
was recognized by Corvo (1885), Cuboni (1891), Cavara (1894),
Selby (1898), and others (38, 125).
Cavara (24) isolated a
bacterium which proved to be infectious upon inoculation
into a vine.
Tourney (1900) considered almond gall to be
produced by a slime mold.
It has also been attributed to
fungi, nematodes, and mites (129).
In 1907 Smith and
Townsend (130) isolated a bacterium from galls on the Paris
daisy and proved its etiologic connection, and named it
Bacterluq
Since then the organism has been
renamed Phortomonas tumefaciens (Smith and Townsend) Bergey
et al. (10).
There have been many hypotheses advanced and experiments
performed to explain and determine the nature of the mechanism
whereby P. tumefaciens is able to incite plant tumors (see
History).
Some investigators believe that substances of the
bacterial cell liberated hy disintegration of the pathogen
in the tissues of the host produce the cancers; others that
microbial metabolites cause the overgrowths; or that changes
in the hydrogen ion concentration, oxidation-reduction potential,
enzyme concentration, or other disturbed factors of the plant
tissues result in gall formation.
In the last two decades, the
role that auxins, indole-5-acetic acid, and many other natural
or synthetic growth substances play in the growth of plants has
been extensively studied (Beysen Jensen et al., 18; Went and
Thimann, 157).
More recently, ty the employment of growth
substances, especially indole-3-acetic acid, galls have been
produced on plants (19, 77, 82, 87, 112).
The similarities
in histology between these chemically-produced tumors and
crown galls have been noted (15, 48, 88).
As early as 1902 Haberlandt attempted to cultivate
plant cells in vitro but failed, and it was not until 1934
(White, 159) that the continuous growth of isolated plant
tissues was achieved (180).
The present investigation was undertaken, upon the
suggestion of Dr. Francis, in the belief that ty means of
isolated plant tissues there is available a technique that
allows a more direct approach to the study of the carcinogenic
properties of P. tumefaciens and its filtrates, and of the
tumor-forming properties of indole-3-acetic acid on tissues
of plants.
II.
HISTORY
After the proof of the bacterial cause of crown
gall was established (130), intensive efforts were made to
discover the means whereby the pathogen induced tumor formation.
Smith and his associates (129) found an excess of oxidases in
gall tissue, and acetic acid and ethyl alcohol in culture medium
inoculated with the organism.
They thought that the stimulus
to overgrowth acting continuously in minute quantities was probably
a by-product of the microbe, possibly a complex colloid, or
even a substance as simple as ammonium acetate, or that these
metabolites might liberate the cancer incitant from the
protoplasm of the micro-organism.
In their next article (128)
there was observed the presence of tumor strands, secondary
galls, and root development.
It was believed that acids
liberated by bacteria within the cells of the host were
germicidal, and that bacterial disintegration was attended by
the release of bacterial endotoxins.
These toxins, in addition
to acids, ammonia, and excess carbon dioxide, which might be
evolved in the process, were said to stimulate nuclear division
and to result in occasional binucleate and quadrinucleate cells.
In crown gall tissue (121) Smith observed hyperplasia
of cells, with thedevelopment of tumor cells whose embryonic
nature was deduced from their small size, large nuclei, thin
protoplasm, staining affinity, and rapid division.
There
were also seen disoriented tissues, with a loss of polarity,
incomplete vascularization, ligniflcation of perenchymous
cells, and various degrees of dedifferentiation.
The galls
were detrimental to the plant locally and systemicaUy.
Inoculation of buds (Smith, 122) incited tumorous flower
shoots, while injected leaf midribs formed leafy intumescences.
Such leafy outgrowths, called embryomata, arose by infection
of an area of totipotent cells (123) with P. tumefaciens.
Aside from microbial products, changes in the osmotic pressure
equilibrium in tissue or removal of local growth inhibitors
were stated (124) to be additional factors in tumefaction
formation.
In 1923 Riker (108) noted that mature galls contained
regions of hyperplasia, hypertrophy, and vascular elements;
that cells tended to divide in the vicinity of the pathogens;
that wounds made in growing tips allowed penetration of bacteria
for more than fifteen nodes in some cases, and accounted for
the migration of the parasites, with the stimulation of tumor
strands and secondary galls.
He was unable to confirm the
intracellular position of the bacteria.
Robinson and Walkden (114)
also discerned the intercellular site of the microbes, the
rapid mitoses in cells surrounding the pathogens, and the
migration of the micro-organisms in tracheae.
6
Levine (79) saw an increased rate of kaiyokinesis
rather than a changed character of cell division in crown
gall.
He discovered neither multipolar spindles nor
multinucleate anaplastic giant cells, although some
quadrinucleate cells were observed.
Smith (127), in 1928, summarized the results obtained
by inoculation of the crown gall organism into various plants.
He stated that there was induced rapid, abnormal cellular
division, even of mature parenchymal cells, which caused cancers
consisting of disorganized cells and vessels, tumor strands in
protoxylem and cortex, pseudo-stem development in leaves, large
localized enlargements of wood and cortex in proximity to galls,
atypically wide medullary rays, misplaced roots, shoots, and
floral organs, and prolepsis of shoots.
In that paper he found
that inoculation of the heads of sunflowers stimulated the
production of ray flowers and galls in the disc among the tube
flowers; stem injections gave rise to cysts and to a vascular
stele in the pith.
Qytologically, Milovidov (96) could not
distinguish between chondriosomes of normal and cancerous tissues.
Leafjr galls were formed by inoculation of the crown
gall pathogen in the midribs of Nicotiana glutlnosa: in addition,
polyploid cells were often found (80).
The earlier studies of plants infected with P. tumefaciens
were mainly of a morphological nature.
In the hope of discovering
the means whereby the crown gall pathogen caused overgrowths, many
workers then turned to a biochemical approach to the problem.
In 1927 Bechhold and Smith (7) reported that
P. tumefaciens elaborated in the culture medium a filterable,
thermostable, inanimate colloid, called "tumefaciens-plastin,"
which upon inoculation of plants gave rise to swellings.
Nemec (101)
smeared the cut surfaces of root cuttings of chicory with a
culture of P. tumefaciens and noted that root formation was
stimulated and bud production inhibited. These effects were
attributed to a substance produced by the bacteria.
In crown gall tissue there was found an increase in
total nitrogen (57), catalase (58), and peroxidase (59).
3y the use of trichloracetic acid Boivin and his
colleagues (11) prepared a lipoid-polysaccharide which was
considered to be the complete somatic antigen (0 antigen),
to be type specific, and to be the principal constituent
of the bacterial endotoxin. Injection into Helianthus brought
about cortical galls resembling those produced by living
microbes. A similar fraction obtained from Salmonen* aertrvcke
caused no outgrowths. In a later paper (12) they employed a
1:1000 solution of "endotoxin" to produce galls.
It was stated
that the histology of these tumors was similar to those of
bacterial origin.
By chemical fractionation of P. tumefaciens. Chargaff
and Levine (25) obtained fat, polysaccharide, and phosphatide
portions. Inoculation of these (83) into sunflower stems
gave different responses; the phosphatide part producing
hyperplasia; the fat constituent, hypertrophy; and the
polysaccharide substance, necrosis with limited proliferation.
Among the obstacles to finding the mechanism whereby
P. tumefaciens oould specifically form plant cancers, were the
observations by numerous investigators that a large number of
other pathogens, physical agents, and chemicals could also give
rise to overgrowths. Thus, for example, various fungi, bacteria,
slime molds, insects, mites, nematodes, and viruses might
induce cell proliferation (8,9, 22, 52, 109, 124, 125).
Tumefaction has likewise resulted from wounding and mechanical
stimulation (14, 127, 141), from frost (49), from excess
humidity (5), from poor ventilation (126), and from damp
chambers (74). Tumors have been induced by lithium
carbonate (115); by copper sprays (150, 151); by vaseline (126);
by permanganate (34); by ammonia, acetic acid, carbonic acid,
ammonium carbonate, hydrochloric acid, sulphuric acid,
phosphoric acid, sodium chloride, sodium carbonate, sodium
hydroxide, mercuric chloride, urea, uric acid, methylamina,
citric acid, malic acid, tartaric acid, alcohol, phenol,
sucrose, dextrose, distilled water, and other substances (124);
by dibenzanthracene, methylcholanthrene, follicular hormone,
and scharlach red (50, 51, 31, 82, 106); by kale extract (4);
and by certain unsaturated gases as ethylene, acetylene,
propylene, and carbon monoxide (32, 94, 148).
The development of knowledge concerning the role
of hormones in the growth of plants has been reviewed in
detail by Boysen Jensen (18) and by Went and Thimann (157).
Although such studies had been undertaken with the aim of investigating
the physiology of growth and the nature of phytohormones,
there had been noticed various pathological effects.
Duhamel du Monceau (37), in 1758, had observed that ringing
of the stem caused swellings, callus, and root formation at
the cut area.
Beijerinok (8, 9), in his work on insect galls,
considered the role of growth enzymes in their formation.
In 1910 Fitting (40) observed that the presence of
pollen or a water extract of pollinia brought about swelling
of the gynostemiua of some orchids after flowering; the action
was ascribed to a pollen hormone.
Molliard (98) found that
sterile filtrates of cultures of flhft
\nq ynHiniftala gave rise
to cortical enlargement, pericyclic divisions, and inhibition
of legume roots.
Loeb (90) saw callus formation only at the basal end
of the stem of Bryophyllum.
In 1924 Choloday (26) noted that
coleoptile tips placed upon coleoptile stumps accelerated their
growth, but retarded the elongation of decapitated maize roots.
While decapitation retarded coleoptile growth, it promoted
root growth, and hence it was deduced that root tips produced
auxin (Cholodny, 27), and the growth substance formed by root
tips retards root growth (28).
Nielsen (102) learned from his experiments that the
medium on which
«uin«g or Absldia ramosa had grown
contained growth substance, that is, it produced negative
Avsna coleoptile curvatures.
The active substance was
called rhizopin, and inhibited root growth (105).
The placing of three maize coleoptile tips on a
root stump by Choloday (29) resulted in marked swelling and
decreased elongation, the enlargement being confined principally
to the cortex.
Heyn (54) decided that growth substance increased
the extensibility of the cell wall of the coleoptile.
Additional
evidence for this was presented by Amlong (5) who removed the
roots of Vicia seedlings, fastened them in a glass tube, and by
an ocular micrometer measured their curvature with the tip left
intact and with the tip removed.
Bobbins and Jackson (111)
studied the effect of 0.2 per cent indole-3-aoetic acid on stem
wall and root materials.
They found it increased the length
of stems and decreased the length of root material.
Therefore,
the growth substance accelerated stem elongation but inhibited
root lengthening.
Stewart (137) repeated Robbins and Jackson's
experiments but could not obtain conclusive evidence as to the
effect of indole-3-ecetic acid on stem or root extensibility.
It was proven by Snow (151) that a substance from
developing leaves and apical buds inhibited axillary buds.
Thimann and Skoog (14S, 146) brought forward evidenoe that
this substance was a growth hormone.
Rhizopin and other substances have been found to induce
cell divisions in the mesophyll of Bxyophyllum leaves (Mrkos, 99).
In damp chambers and, in nature, on leaves fastened together byinsects, the development of intumescences on poplar leaves has
been observed by La Rue (74).
It was believed that some product
of incomplete oxidation stimulated the cells to produce the
outgrowths (75).
Laibach and Maschmann thought the hormone
that caused enlargement of the orchid gynostemium was
auxin (67, 72).
The application of orchid pollinia to the
decapitated epicolyls of Vicia faba caused a marked increase
in thickness (68)•
Auxin, obtained from extracts of orchid
pollinia and urine, influenced callus formation and stimulated
mitosis and rooting in internodes of Tradescantia and in
hypocotyls of Halianthus (71, 73).
Growth substance of Aspergillus and Rhizopus was shown
to have a molecular weight close to that of indole-3-e.eetic acid (64).
This substance had about the same physiological activity as
auxin a and auxin b in promoting the growth of stems (62).
Auxin a and auxin b, and indole-3-ecetic acid inhibited the
growth of roots of Avena seedlings; concentrations of the
last-named acid of 1.0 to 0.01 mg. per liter retarded elongation,
0.001 mg. per liter had no effect (63).
Beysen Jensen (17)
found that 2 WAS units growth substance per 100 cc. decreased
the growth of roots of Vicia.
Navez (100) grew Lupinus seedlings
In moist chambers, and concluded that the "growth-promoting
substance® of coleoptiles or root tips inhibits the elongation
of decapitated roots*
KBgl and his associates (60, 61) ascertained the
chemical structure of the auxins, and they also showed (64)
that the active substance in yeast was indole-3-ecetic acid
("heteroauxin"), which had been found in urine much earlier
by Salkowski (116).
For reference the formulae (157) are
given below.
°?5
c2%-ch-ch-c-choh.gh2.choh.choh.cooh
I
GH5
auxentriolic acid
(auxin a)
I
CfcHg-CH-CH-G-CHQH.Ci^OCHg .CQOH
Cg%-CH-CH-CH
CH3
auxenolonic acid
(auxin b)
14
H
AC-C-CHp.COOH
HC
I
HI
HC
C CH
*C'Y
H
H
indole-5-acetic acid
(heteroauxin)
Snow and his co-workers (132, 133, 135) demonstrated
that strong cambial growth occurred when strips of hypocotyls
of 8unflowers were placed In solutions containing pure auxin
or indole-3-acetic acid*
Tan Overbeck (149) studied the pathology of dwarf c o m
plants.
These exhibited normal auxin production, transport and
sensitivity.
In the dwarfed forms, however, as compared with
the normal plants, auxin was rapidly destroyed, and therefore
the stems did not elongate in the usual manner.
It was
possible to attribute the excessive auxin destruction to the
high oxidative level of the tissues in which aatalase, oxidase,
and peroxidase activity was higher than in healthy plants.
Tissue proliferation of poplar leaves was brought
about by either fecal matter of insect larvae or 0.0005 per cent
indole-3-aoetic acid (La Rue, 76).
Application of indole-3-acetic
acid resulted in callus formation in epicotyla of Vicia faba (70)}
15.
in. thickenings, callus and root formation in Coleus, V. faba.
and the tomato (69); in cambial cell division and root thickening
(Jost, 55)j in the production of hypertrophied and nearly
isodiamstric cells of Helianthus hypocotyls (Caaja, 35).
With concentrations of indole-3-acetic acid as low as 0.0005 per cent,
stem enlargements developed; microscopically, these consisted of
spongy, fluffy tissue external to the vascular cylinder
(Zimmerman and Wilcoxon, 168). After auxin treatment (Went, 153),
there was first cell enlargement followed by cell division.
The effects of ethylene, propylene, acetylene, and
carbon monoxide on whole plants were stated to simulate the action
of indole-3-acetic acid by producing leaf epinasty, intumescences,
elongation inhibition, and initiation of root formation (Crocker,
Hitchcock, and Zimmerman, 32). Previously, Tan der Laan (148)
considered that the reduction in elongation of etiolated seedlings
by ethylene was to be accounted for by decreased auxin production.
Michener (94) believed that the effect of ethylene on growth was
not direct, but an outcome of change in growth hormone.
Finally, in 1936, the first clear-cut evidence of the
role of growth substances in the formation of galls was brought
forth (19). In that year Brown and Gardner employed the methods
that had been developed in physiological investigations of plant
hormones. They extracted cultures of P. tumefaciens with peroxide-free
ether, allowed the solvent to evaporate, incorporated the residue into
lanolin, and produced tumors upon inoculation of plants. Controls
of indole-3-acetic acid in lanolin similarly formed galls.
The
tumors formed by the latter did not develop as rapidly as those
produced by living cultures, but on the bean plant they were larger.
16.
Decapitated stems of the bean were treated by
Kraus, Brown, and Hamner (65) with 3 per cent indole-3acetic acid. This resulted in tumors that had maty close
similarities to crown gall tissue and to those induced by
growth substance from cultures of P. tumefaciens (see 19).
The epidermal cells underwent a few divisions and some
enlargement, as did those of the cortical parenchyma.
The latter, in addition, had some necrosis on the outer portions.
The endodermal cells were highly responsive with considerable
meristematic activity, formation of xylem and phloem elements,
and the development of large parenchymal cells and adventitious
roots. The phloem parenchyma responded like the endodermal
cells. The cambrium and pith cells also showed much proliferation.
From proliferated ray cells there developed a confused system
of vascular strands intermingled with meristematic areas.
Many
cells were multinucleate.
Cut stems of the tomato were treated with Z per cent
indole-3-acetic acid (Borthwick, Hamner, and Parker, 16) and
exhibited the following histological picture. Epidermal cells
showed slight enlargement with some rupturing; cortical cells
manifested enlargement and increased divisions; marked meristematic
activity was noted in the endodermal cells; pericyclic cells
were not markedly affected, although many were crushed because
of the activity of the adjacent phloem and endodermis; the
external phloem parenchyma was highly meristematic and usually
formed roots; pith cells and internal phloem also formed
adventitious roots. Microchemically, gall tissue had more
protein, but less nitrate and starch.
17.
The action of indole-3-acetic acid on embryos and
embryonic fragments of Ricinus and Fhaseolus (Solacolu and
Constantinesco, 136) resulted in tumor production, increased
thickness, development of internal meristems forming numerous
root primordia, proliferation of superficial tissues, and
suppression of roots, radicle, and gemmule.
Fiedler (39) attempted unsuccessfully the continuous
cultivation in vitro of corn roots.
In root tips grown for
14 days, he observed that down to a concentration of 0.001 mg.
per liter indole-3-acetic acid reduced growth; in the presence
of yeast extract in the medium 0.001 mg. indole-3-acetic acid
per liter also retarded growth, but in the absence of yeast
extract this concentration stimulated elongation of the roots.
In the same year Geiger-Huber and Burlet (43) noted that maize
roots grown in the presence of 10“® molar indole-3-acetic acid
for six days were uniformly thickened with an outgrowth of the
cortex, and in one month the cortex was broken by mary short
-10
secondary roots. With concentrations from about 10
to
-13
n
10
molar (with the optimum at 3 x 10
molar, or 0.000005 mg.
per liter) there was accelerated root elongation.
Nodules of Ehizobium were shown by Thimann (143)
to produce auxin actively. Application of indole-5-acetic acid
induced swellings which resulted from cell hypertrophy and
division in the cambium and pericycle. This investigator
expressed the opinion that legume bacteria caused nodules by
producing auxin in the host.
18
Grieve (46) observed that the injection of indole-3acetic acid into healthy tomato plants produced adventitious
roots while it did not do so in plants infected with spotted
virus. He was of the opinion that the disease symptoms arose
from the effect of the virus on auxin.
La Rue (77) crushed intumescences from poplar leaves*
spread them on fresh leaves* and observed the development of
swellings. Similar results were obtained by ether extracts of
intumescence-bearing leaves* by an extract of Rhizopus suinus.
and by injection of indole-5-acetic acid into leaves.
The following year* it was stated by Link and his co-workers (87*88)
that 3 per cent indole-3-acetic acid and crude extracts of the crown
gall organism caused similar histological responses on the been
hypocotyl.
These consisted of cell enlargement* cell mitosis*
suppression and retardation of normal differentiation and maturation,
genesis of new meristems in abnormal sites* and schizogenous cavities
followed by filling with peripheral callus. It was postulated that
this growth substance* and possibly others* were the means whereby
the pathogen incited crown gall.
E(y 1937 it had become apparent (18, 19, 77, 88, 157),
therefore* that not only were auxins concerned in the normal
growth of plants* but that growth hormones played a role in
pathological processes. Such responses of plants* as hypertrophy*
hyperplasia* meristematic activity* necrosis* root initiation*
epinasty* and bud inhibition* to P. tumefaciens. other pathogens*
chemicals* and physical agents* showed similarities to those
elicited by growth substances. Since that time additional evidence
has served to substantiate this viewpoint.
In such studies
19.
indole-5-acetic acid has been widely employed because of its
intense activity as a growth substance ana because of its
availability in pure form.
Cellular reactions that showed histological structures
comparable to crown gall* but limited in size and development*
were elicited in four species of plants upon treatment by
Levine and Chargaff (83) with a wide variety of chemicals*
including indole-3-acetic acid and fractions of P. tumefaciens.
Leonian and Lilly (78) experimented with fungi, algae*
excised roots* and com shoots which they exposed to various
concentrations of indole-3-acetic acid. These workers concluded
that this substance was a growth inhibitor.
Wheat seedlings were grown by Manner (952) in solutions
of indole-3-acetic acid (concentrations from 50.0 to 0.0000048 mg.
per liter)* and the decreased growth of the primary root was
observed in all concentrations. These roots showed stunting*
thickening* flattening* curling* excessive root hair development*
and an increased number of secondary roots.
By the application of 5 per cent indole-5-acetic acid
paste on the stem interaodes of the bean* Mitchell and Martin (97)
observed galls* increased thickness* adventitious roots*
decreased fresh and dry weight* decreased length, and retardation
of transport of materials from the cotyledons.
Decapitated pea swellings were smeared with indole-3acetic acid paste by Thimann (144).
From the effects seen
on stems* buds* and roots* he concluded that at an optimum
concentration there was acceleration of growth* but that
higher or lower concentrations retarded growth.
The difference
between the organs was quantitative* with roots being inhibited
at lower concentrations than buds or stems.
After spraying blossoms with indole-5-acetic acid*
Gardner and Marth (42) studied the development of parthenogenetic
fruits.
Nobecourt(105) cut off the proliferations arising from
cut sterile slices of carrots and cultured them for three passages
on the surface of nutrient agar moistened with a solution
containing* in addition* 5 mg. indole-3-acetic acid and £5 mg.
cysteine per liter.
of light.
The cultures were kept in the presence
The tissues were stated to have grown ten to thirty
times* developing chlorophyll and consisting of elongate
irregular cells* which were loosely united and formed some
roots.
Gautheret (42a) found the concentration of 0.0006 per cent
indole-3-ecetic acid employed by Nobecourt to be toxic;
0.000001 per cent* however* was reported to stimulate carrot
tissues grown in vitro in light.
It was further noted (42b) that in
higher concentrations (0.01 to 0.0001 per cent) there were cell
hypertrophy* root formation* meristematic activity* and callus
formation.
Spraying of 0.05 per cent indole-3-acetic acid on
tba "double" form of the chiysanthamus was found to inhibit
the m r oiling of the corolla, and an "Incurved" form of
flower resulted (Warns, 152).
Large tumors were produced on cut stems of the bean
and on bean pods by 3 per cent lndole-5-acetic acid paste
(Banner and Kraus, 48).
Secondary galls, I.e., those developing
at uninoculated areas, were found to develop after primary
galls elicited by indole-3-acetic acid (Brown and Gardner, 20).
These were thought to have arisen by a disturbed overbalance
of auxin In the plant.
It was considered also that secondary
galls in plants Infected with P. tuaefaclens might represent
the effect of gall-stimulating substances given off by bacteria
in the primary gall.
It was observed by Link (84) that ether extracts of
legume (Bhizobium) nodules gave a feeble Avena coleoptile test
and also a feeble chemical test for indole-5-aoetic acid.
Hodular roots exhibited indications of having more growth
substance then normal roots.
These facts were believed to
support the hypothesis that tumors were brought about by
local hyperauxoxQr of auxin or hateroauxin-type. Locke, Biker,
and Duggar (89) inoculated several species of plants with a
virulent strain of
+1tpQfaclen8 which not only incited
22
galls but gave responses that suggested an Increase In growth
substance, namely, leaf petiole epinasty, increased adventitious
root initiation, c&mbial stimulation, bud inhibition, and
delayed abscission of senescent leaves.
In addition, by means
of the Arena test it was found that inoculated tissue contained
more growth substance than did uninfected.
The growth substance
appeared to be of the auxin a or auxin b type, which the
investigators believed to be a product of host metabolism
stimulated by the presence of the pathogen.
Lefevre (77a),
however, recorded that by chemical tests indole-3-aoetic acid
was demonstrated in various higher plants under normal conditions.
Ethylene was said by Michener (96) not to be able to
produce swellings unless auxin was present.
Treatment of decapitated cabbage seedlings with
3 per cent indole-S-acetic acid (Goldberg, 44) produced much
callus and root primordia.
The phloem, rays, and pith were
most responsive (hypertrophy, hyperplasia, disorganised strands,
disoriented tracheids, and meristematic areas).
The cambrixa,
cortex, endodernis, and xylem were moderately stimulated, while
the epidermis and pericycle reacted weakly.
At the cut surface,
mounds of callus mere formed from the phloem and pith mainly.
Control plants produced no heaps of callus but a flat phellogen
layer.
or rays.
Some treated plants formed shoots from callus, cortex,
In the cultivation of excised tomato roots it was
found (White, 164; Bobbins and Schmidt, 113) that indole-3acetic acid was neither an essential nor stimulating substance.
Timor production was not mentioned.
The latter authors
observed inhibition down to a concentration of 0.01 mg. per
liter of indole-3-acetic acid.
Etiolated pea seedlings (Scott, 117) responded to
auxin by tumor formation, cell expansion, meristematio activity,
and root development.
The cut stems of Phaseolus vulgaris
were treated with 2 per cent indole-3-acetic acid paste and
harvested seven days later (Alexander, 2).
Compared with
control plant8, they exhibited tumors, decreased dry weight,
translocation of carbohydrates toward the treatment point,
less starch, condensation of sugars to complex polysaccharides,
with increase of acid-hydrolyzable substances.
Went (164, 156, 156) observed that the addition of
indole-3-acetic acid to the apical cut end of pea stems caused
elongation in the lowest concentrations? at higher concentrations
growth in length was inhibited and swellings were induced,
believed that two phases could be distinguished.
be
The first
was redistribution of the normal food factors ("caUnes") of
the plant, which substances other than the auxins could induce.
The second phase was thought to be elicited only by growth
substances «nd was possibly an activation of the accumulated
caline.
Both auxin and caline were necessary for the production
of apical swelling, elongation, and root formation.
On bean plants 3 per cent indole-3-acetic acid
gave the largest galls even in comparison with those formed
by P. tumefaclens. although in other hosts the effect was
reversed (Riker and Nagy, 110).
It was stated by Levine (81)
that the response of Kalanchoft to growth substance was
different from that to the crown gall pathogen.
In a
later, more extensive publication, however, the responses
to the two different agents were not considered unlike
(Levins, 82).
Only inorganic salts, sugar, thiamin, and nicotinic
acid were required for continuous cultivation of pea roots
in vitro (Addicott and Devirian, 1).
These workers thought
that the high initial auxin content of the root inhibited
maximum growth and that as the cultures were continued, and
the auxin concentration fell, the growth rate increased.
On
the other hand, Bonner and Koepfli (15) reported that between
Q
10
« _
and 10“xu molar indole-5-acetic acid caused slight
stimulation of growth of excised pea roots? apparently these
were only one-week-old cultures.
Ether extracts of c o m meal and Vicia faba shoots
contained a growth-inhibiting substance for the Arena coleoptile
which masked the action of auxin (Goodwin, 45).
From radish
cotyledons and leaves, extracts yielded auxin and an inhibitor.
The latter had no acidic or basic groups, was readily hydrolyzed
to auxin, and in various tests it behaved like auxin to which it
was hydrolyzed in the course of the test (Stewart, 138).
Snow (134) dissected pea seedlings in various manners
and by different treatments with indole-3-acetic acid paste
(IsSOO and 1:300).
From these experiments he concluded that
in order to bring about inhibition of growth, auxin (from the
paste) must react or cooperate with a second factor in the stem
coming from the leaves and cotyledons. (See Went, 154, 155, 156.)
The quantitative extraction of auxin from green tissue
was noted to be difficult (Skoog and Thimann, 119).
The
addition of chymotrypsin increased the yield from Lemma, and
therefore these investigators stated that auxin was bound to
a protein from which it was liberated upon hydrolysis.
Nodules on bean and pea roots were extracted ty Link
and Eggers (85).
These gave greater Avena curvatures than the
normal parts of such roots, which in turn yielded greater
responses than normal roots.
inhibitors was also found.
Evidence of the presence of
In the same year (1940) curvatures
in the Avena test (Link, Eggers, and Moulton, 86) obtained by
the residue of ether extracts of aphids and their parasitized
hosts could be correlated with disturbed auxin relations.
This survagr of the literature emphasizes the extensive
volume of investigations concerning the effect of P. tunefaciens
or growth substances in the production of tumors in plants.
The studies conducted with plant tissue cultures have been
concerned almost entirely with the significance of these
substances as accessory growth factors.
The present study has
been undertaken in an effort to study the production of galls
in artificial cultures of roots by P. tumefaciens or phytohormones,
with primary consideration of the pathological processes involved.
27.
III.
EXPERIMENTAL METHODS AND RESULTS
A.
General Procedures
In order to become better acquainted with the necessary
technique, seeds of the tomato (Lvcoperslcum esculentum Mill.),
variety Bonny Best, were soaked in distilled water for six hours,
then for ten minutes in a 1x1000 aqueous solution of mercuric chloride.
Following this, they were wqshed in several changes of sterile
distilled water and allowed to germinate, under sterile conditions,
upon filter paper in Petri dishes.
When the main root had attained
a length of about three centimeters, the root tip was cut off with
a pair of scissors end transferred by forceps to flasks containing
White's nutrient solution.
The roots were cultivated for several
weeks, but were finally discarded upon receipt of a strain of the
above tomato variety upon which there was available considerable
physiological data (159-164).
This strain was vexy generously
supplied to us by Dr. Philip R. White, of the Department of Plant
Pathology of the Rockefeller Institute for Medical Research at
Princeton.
The roots had been isolated by him on March 1, 1935,
and were received by us on December 22, 1956, from the 294th
weekly transfer.
They have been subcultured in our laboratory
at weekly intervals ever since, and constitute a single clone.
The materials, media, and methods were essentially those
employed by White (159, 161-166).
The medium consisted of yeast
extract solution, stock salt solution, accessory salt solution,
iron, and sugar.
The yeast extract solution was prepared by boiling
10 g. brewer's yeast (Anheuser-Busch) for one-half hour in 800 ml.
28.
redistilled water.
While still hot the preparation was centrifuged
for 5 - 10 minutes at high speed.
The supernatant fluid was decanted
and made up to 1 liter, and autoclaved at 1^ lbs. pressure for
o
20 minutes. The solution was cooled, and stored at -12 C. The
stock salt solution was made up by the addition of 10.03 g.
Ca(N05)2 . HfcO, 6.7 g. KC1, 8.1 g. KNOs, and 1.23 g. KlfeK^ to
800 ml. redistilled water.
In 200 ml. water 7.4 g. MgS0 4 . 7H20 were
dissolved and added slowly with stirring to the salt solutionj this
was then sterilized in the autoclave.
A precipitate formed, but was
not carried over into the final nutrient medium.
The accessory salt
solution consisted of 37.5 mg. KI, 325.0 mg. MnSO^. 4H20, 133.6 mg.
ZnSO^. 7HgO, and 80.0 mg. HgBOg in 1 liter redistilled water.
The
final nutrient medium consisted of 10 ml. yeast extract solution,
10 ml. accessory salt solution, 2.5 mg. Feg (S04)3, and 20 g. sucrose,
with the addition of water redistilled in a Pyrex flask to 1 liter.
When a synthetic nutrient fluid was employed it consisted of
10 ml. stock salt solution, 10 ml. accessory salt solution, 2.5 mg.
ftegtSO^g, 20 g. sucrose, 0.5 mg. thiamin (Betabion Merck), and
1.0 mg. glycine, with the addition of water up to 1 liter.
The nutrient solution was distributed in 50 ml. portions
in 125 ml. Erlenmeyer flasks which were stoppered with gauze-covered
cotton plugs, and was sterilized in the autoclave at 15 lbs. pressure
for 15 minutes.
By means of a pair of scissors root tips about 15 mm. in length
were cut and removed by transfer needles to flasks containing fresh
nutrient solution, which, unless specified otherwise, was the yeast
extract-salt-sugar medium described.
A waxed-paper drinking cup was
inverted over the flask to further reduce contamination.
The flasks
29.
were kept in dim light at room temperature.
the roots were subcultured.
At weekly intervals
In all cases each flask contained
only one root tip.
Growth was measured by the elongation in length, by the
increment in dry weight, and by the increase in the number of secondary
roots formed.
While still in the flasks the length of the roots,
including the branches, was measured by means of a millimeter ruler.
While this method is not accurate, it permits maintenance of sterile
conditions.
In the first horizontal column of table 1, for example,
two root cultures were grown in nutrient solution in the presence of
1.0 mg. indole-3-acetic acid; the figure 47.5 represents the average
increase in length in millimeters, calculated to tenths,of the two
roots.
Similarly, in all tables each figure represents an average
figure for each concentration used.
The number of secondary roots
formed was macroscopically counted.
In the experiments recorded
in table 10, the roots were mounted on glass slides, covered by slips,
and the number of rootlets enumerated under the low power objective (x 100)
of the compound microscope.
It should be noted that the fragment of
the root tip used for subculture rarely possessed rootlets, so that
usually the final number observed represented the increase in growth.
To obtain the dry weight of the roots they were put on weighed watcho
glasses, placed for one week at 58 C. in an oven that was also used
for histological purposes, and then weighed on an analytic balance.
oven was used as no other was available, although it is customary to
perform the operation at 100° C.
Histological preparations of normal and treated
roots were made.
The tissues were killed and fixed by
Navashin’s solution, dehydrated by means of a graded series
of dilutions of ethyl alcohol and of xylol, and embedded in
This
paraffin.
Sections were cut at 10 microns, and stained by
Heidenhain's iron-haematoxylin technique alone, or eosin
was used as a counterstain.
The sections were mounted in
balsam.
Cultures of Ptertomonas tuaefaciens were
received from Dr. E. A. Siegler, of the Bureau of Plant
Industry, end from Dr. A. J. Riker, of the University of
Wisconsin, to idiom we gratefully acknowledge our thanks.
Stock transfers of the organism were made weekly on slants
of potato-dextrose agar, adjusted to pH 5.9.
Potato-dextrose
broth, bean broth, White's yeast extract-salt-sugar or
synthetic solutions were used when large quantities of
bacterial cells or of culture filtrates were required for
experimental purposes; in these cases, seedings were made
from fluid cultures of P. tumefaciens that had been transferred
for at least two consecutive days prior to the inoculations.
At the same time the virulence of the bacterium was proven
by needle inoculations into the stems of young tomato plants,
variety Bonny Best, grown in greenhouse or field at the
Brooklyn Botanic Garden, through the courtesy of Dr. G. U. Heed.
The hydrogen ion concentration (pH) of the
solutions of the root cultures was measured electrometrically
by the use of the Beckman pH meter.
Before readings were
taken, the instrument was calibrated by means of either acid
potassium phthalate (pH 3.97) or standard acetate (pH 4.62)
solutions.
Alteration of the pH of the culture solutions
was made by the addition of small quantities of either
1.0 M or 0.1 M potassium hydroxide or hydrochloric acid.
B.
Effect of phvtomonas tumefaciens. Culture Media, and
Various fractions upon Roots
1.
T.ivinp hacteria.
Immediately after fresh root
tip cultures had been prepared, the nutrient solution was
inoculated with a 24-hour-old suspension of the crown gall
organism (P. tumefaciens).
Resultst
The bacteria grew profusely; the roots did not
elongate, remained white, formed no rootlets, and exhibited
no swellings.
The following variations of the above experiment were
performed*
(a) Root tips were cut in a suspension of bacteria
and then transferred to fresh nutrient solution,
(b) Excised
roots were removed to Petri plates and inoculated by fine
needle punctures with the pathogen and then removed to freshly
prepared flasks,
(c) Isolated root cultures were made and at
varying times up to 48 hours, the medium was seeded with
P. tumefaciens.
Results* In all cases the inoculation of roots or culture
medium with living bacteria resulted in a contaminated root
culture which did not grow and produced no galls.
2.
Dead bacteria. From 48-hour-old broth cultures
a heavy suspension of bacteria was obtained by centrifugation.
Using nutrient solution a set of 44 flasks containing nine
dilutions of bacterial suspension, ranging from 200 mg.
bacterial cells (wet weight) to 10 "* mg. per liter, was
prepared and immediately sterilized.
The first two
concentrations (200 and 150 mg. per liter) were heavy
suspensions of bacteria.
To the flasks, roots were added
in the customary manner.
Results* The tips grew, but not as well as those cultured
in solutions to which no cells had been added; in no case
were any overgrowths observed; see table 1 .
This experiment was repeated with 80 flasks made
19 with suspensions of bacteria which had been dried in the
frozen state in vacuo (41).
The flasks were then sterilized
and the isolated roots added.
Results* See table 2; the outcome of this experiment was
also negative.
TABU
1
The Effect of Killed Celle of Phytoaonas Tuaefaciens
Upon the Growth and the Number of
Secondary Roots Formed by Excised Toaato Roots
tNuaber :
♦
Concentration (as.A* ) of cells of Phytoaonas tumefaciens
: of
-2 : -3 :
-4 x
*
2
2
2
2
: root
{ 150.0 { 100*0 { 10.0 : 1*0 { 10
10
: 10
: 10
:cultures 200*0
. per
: :
: {
:
:
: :
:
t
: {
{
■
J
B. {I. :B.:L. :B.
: cone*
B* {!•* {B* {!•* :B» L. :B*:L* :B.:L* {B U
L.
e e
e e
g
• •
*
e
%
e
:
: *
*
•
t
e
:
2
1 :47.5:7 161.0:7 66.5 7 :12.0: 3:40.0: 6
e e
e
e
:
{
2 2
2 2
2
: :
:
e
34.0:4 77*0:18:47.0:6 ll7.5{2 72.5 6 :66.5:10:
2
:
e
•
2 2
2 2
2
:
: :
:
a
e
a
73*5 6 70.0 {8 40.0:1 20.0:' 4:39.5:7 :18.5:1
:
2
e
• e
•
e
e
e
e
e
a
{
2 2
2 2
2
e
e e
e
e
•
e
a
:
2
37*5 11 41.5 {*7 4.0:1 4.5#: 1:47.5:5 il3.5:2
.e
e e.
i
a
e e
• e
e a
{
2
2 2
2
55*5 8 55.8:7 26.0:2 33.8 : 6:45.4:6 :2T.6:3 *:69.5 6 :39.3: 6:40.0: 6
{Average
e
{Average
2
2
2
2
2
4.54
: dH
4*54 1 4.62
4.67 : 4.81 : 4.88 : 4.82 : 5*09 t 4.94
0
.1
<*
Tot weight
One root culture
* L»* In all tables L*indicates the fined length of roots (including length
of branches) ainus initial length, expressed in millimeters
o B»m In all tables B« indicates the fined number of secondary roots
ainus the initial number
§
x
Control cultures to -which no indole- 3-acetio acid had been added grew
about 8J4. mm., and formed about 9 branches
T S M Jt
2
The Effect of Killed, Lyophilised Celle of Phytoaonas Tumefaciens
Upon the Growth and the Number of
Secondary Roots Formed by Xxeieed Toaato Roots
*
zKuaber :
Concentration (ag./l. ) of cells of Phytoaonas tuaefaciens
Z of
*
: -3x
-1
-2
root
10
10
1.0
: 10
cultures 200.0: 150.0: 100.0 : 10.0
e
e
e
•
e
e
e
e
e
e
:
: per
:
:
B.Z L.
: B. L. : B. zlt. :B.
B. L.
: eonc.
B.: L»
L. B. 1 L. zB. L.
e
60f5
2
3
5 : 54.5 : 5
61.5:
e
:
2
.
t
2
:
86,0
:106.5
11 z 59.5 zlO
77.5 10
45.5
45.5
36.5
8 : 86.5 :17
:
:
2 : 71.5 : 6
:
:
:
2
•
e
2
*
•
•
0 :0
1*0:1
e
:
:
2
2.0:2
e
:
<•0;
e
:
:
»
»
I*
8.0:2
2
e
0 :0 :7.5:2
e
Average
7.0:5
S
e
•
8.0
e
i . o a
e
0 :2
8.0
72.0: 7
1
36.0:10
z
82.5:11
61.0
e
:
•
65.7i 8
Average
1
T.5
:
4.61 ; 4.56 t 4.49
: 4.89
:
4.84
4
z
6 :102.5 :13
:
28.5
:
e
•
z
:
:
:
:
z
:
:
e
*
:
e
e
::
•
e
e
:
#
69.0*: 9 :76.0:4
:
:
:
: 4.94
: S.15
: 4.83
* Yeight of dried, lyophilised cells
x Control cultures to which no indole- 3-acetic acid had been added grew
about 92 mm., and formed about 11 branches
e
:
46.6 ': 4 *: 67.6 *: 9
:
e
:70.0 :10
*
e
e
55.5z 6 :94*5:7
:
:
:
58.5:10 :62*5:4
:
z
e
z
•
2.8:1 :5.1:2 : 4.5:2
e
:
•
1.0:1
e
e
e
93.0: 9 :71.0:2
e
e
12.5 1
e
e
e
.
3.
Frozen and thawed cells of P. tumefaciens.
A 3-day-old culture of the bacterium in the yeast extract—
salt-sugar medium was centrifuged.
The sediment (about 7 ml.)
was sucked 1 9 by pipette and placed in a bottle.
It was
frozen at - 12° C. and then thawed at room temperature; this
was repeated ten times.
Twenty milliliters of a similar
suspension of cells were frozen and thawed four timss, and
the two suspensions combined.
ISader the oil innersion
objective of the microscope the treated material was seen
to consist of detritus and whole cells.
Dilution No. 1
consisted of 27 ml. of a very dense suspension of the
bacterial preparation added to 247 ml. of fresh nutrient
medium.
From it 28 ml. were withdrawn and combined with
247 ml. fresh nutrient solution to make dilution No. 2.
Similarly, dilutions No. 3 and No. 4 were prepared.
Five
culture flasks were made from each dilution and sterilized.
Excised roots were added to the 20 flasks.
Results1
While the roots remained white in the first
dilution there was no growth; in the remainder of the series
there was a moderate amount of growth, with no pathological
signs observed.
Another batch of young, frozen and thawed cells
was prepared, and £0 root cultures were made; with similarly
negative results.
Cultures of P. tumefadens in potato-dextrose broth
that were several weeks old were centrifuged, and 10 ml. of
the sediment were pipetted off and added to 155 ml. of fresh
medium; this dilution was a very dense suspension.
two further dilutions (1x10) were prepared.
From it
In all, there were
nine root cultures.
Results: There was no growth or galls in the first dilution;
in the other two groups the roots elongated and formed secondary
branches in a normal manner.
4.
Filtrate of frozen ami thawed cells. Young cells
(3 days old) of P. tumefadens were frozen and thawed six times.
After centrifugation, the sediment was sucked up with a small
amount of the supernatant fluid and then passed through a
sterile Berkefeld N filter.
Eight milliliters of the filtrate
were added to 100 ml. sterile nutrient solution, and two culture
flasks were prepared.
Similarly, three flasks were set up by
the addition of 9 ml. of the filtrate to 150 ml. of sterile
medium.
Excised roots were transferred to each flask.
Results: The five root tips showed a moderate amount of normal
growth, with no pathological changes observed.
37.
5.
-Sediment of uninoculated broth medium.
Before the negative results of the preceding experiments were
known, a control experiment was performed.
That is, uninoculated
potato-dextrose broth was centrifuged, the sediment obtained was
desiccated in the frozen state in vacuo, and was used to prepare
a series of 44 flasks.
negative.
The outcome of this experiment was also
See table 3.
6.
Supernatant fluid of bacterial culture.
P. tumefadens was grown for three days in 6.5 liters of potatodextrose broth.
The medium was then passed through a Berkefeld N
filter to remove the bacteria.
The filtrate was evaporated under
reduced pressure to a volume of 100 ml.; the temperature, however,
at times went as high as 92° C.
Part of this residue was retained
for further extraction (see below), the remainder was concentrated
further by drying in the frozen state and was employed to prepare
40 cultures ranging in concentration from 200 to 0.1 mg. per liter.
Resultsi
See table 4; the higher concentrations proved to be
somewhat toxic, but no tumors were observed, and the outcome
was the same as that obtained in a control experiment in which
the supernatant fluid of the uninoculated medium was concentrated
and used.
(See table 5.)
TABLE
3
The Effect of the LyophilixeA. Sedinent
Resulting from Centrifugation of Oninoculated Broth Medium
Upon tho Growth and the Number of
Secondary Roots Formed by Excised Tomato Roots
:Namber
of
: root
:cultures
: per
: cone*
t
0
0
.
:
2
2
:
J_
2
i
*
a
o
P H ....
10. 0
:
1.0
a
a
a
a
a
a
a
e
a
a
s
t
t
o
:
3
a
t
:
2
a
t
:
I # " 1
:
a
a
»
a
•
10
“ *
:
•
e
a
a
s
l O * 3 *
a
B.
:
2
o
a
o
a
a
a
a
:
:
:
:6Se5: 8:17.5
:
t
a
a
a
a
a
:
23.5:1
0
:lT«ra£o
:Average
*
200.0 : 150.0 s 100.0
a
a
a aa
a
: :
23.5:1
a
:
:
a
:
a
:11.5:2 :28.0
a
a
a
a
t
e
:
:
a
38.0 : 4
<8.5
a
a
:
8
a
t
:
38.5 : 4
e
:
:
104.0
12
:
34.5
5
41.0
4
59.8
7
a
<3.0: 4
a
: *25.0:3 -8^20:70.0 :11 : 71.0 : 9 : 57.5:12
: : : : : :
:
:
:
:
:
11.0:4 :35.0:4 : 3 4 5 : 5:50.5 : 7 :104.5 :17 :
a t
o
t
t
o
t
t
t
o
*
11.0:4 :30.0:4 :494: 8:41.5 : 5 : 70.5 : 9 : 53.0*. 6
a
4.71
e
t
*
4.59 : 4.48 : 4.92 : 4.54
t
*
: 5.05
: 4.85
x Control cultures to which no indole- 3-acetic acid had been added grew
about 86
, and formed about 11 branches
5.09
,
TABLE 4
The Effect of the Supernatant Fluid, Concentrated
by Vaeuua Distillation, of Broth Medium
Inoculated with Pbgrbomonas Tumefadens
Upon the Growth and the Number of
Secondary Roots Formed by Excised Tomato Roots
X Nuaber
X
of
X root
X cultures
x per
i conc.
i
m
2
9
m
I
i
2
1
2
x
2
I
x
i Averaee
x Average
-L
—
:Concentration (mg./I.)of supernatant fluid of
inoculated medium
>100.0
t 10.0
z 1 .0
x 200.0 s 150.0
x 10 rA x
: :
X
X
X X
X X
X X
X
x L. zB.x L. xB. x L. xB. x L. xB.x L. xB.x L. xB.
e
X
X X
X X
X X
X
s s
X
X
X X
X X
X X
X
f 4,5 x 4* 20.5 > 5 x 18.Ox 5 x 33.5x 8 x 59.0x 6 z 32.6i 5
x z
X
X
X X
XX
X X
X
i $.5 ; 2 ? 0 xl.fix 16.5x 4 x 40.Ox 6 x 40.0x 5x 70.Ox 11
X X
X
X
X X
X X
X X
X
i ?.§ { 3; 5.0x 5 x 34.5s 5 z 70.0x 8 x 34.5z 6 x 56.Ox 6
X X
X
X
X X
XX
X X
X
z 0 I I t 4.6>x 5 x 12.5x 4 X 64.Ox 4x 28.0.x 3x 44.Ox 6
9
9
e
•
•
X X
X
#
•
•
t
X X
X
i 5,4 * 2 * 7.0x 2 x 20/4x 4 x 51.9: 6 : 55.4x 6 x 50.6z 7
•m
X
X
X
X
4.71 x 4.86 x 4*90. X, 4j 70
JL... 4,.82, | .4,73 I
x Control cultures to which no indole- 3-acetic acid had been added
grew about 59 m m . , and formed about 9 branches
X
,
X
x
X
X
x
X
x
X
x
X
z
X
x
•
,
TABLE 5
The Effect of the Supernatant Fluid, Concentrated
ty Vacuum Distillation of Uninoculated Broth Medium
Upon the Growth and the Number of
Secondary Boots Formed ty Excised Tomato Roots
tNumber
s of
s root
(cultures
z per
z conc.
m
: Concentration (mg./l.) of supernatant fluid of
*
™ir»oculated medium
z
z
z
-1 X
z
*100.0
* 10.0
z 1,0
10
z 200-0 i 150.0
e
z z
z
z
z
z
z z
•
z
I
L. zB.z L. zB. * L. zB. z L. zB.z
B, L. zB.
z z
z
z
z
z
z z
z
s
•
e
2
• 8.0 z4 z 6.5z 1 * 17.5* 1 s 78.Oz 8 z 46,5 4 61.5* 8
:
z
z
z z
(
z
z
z z
s
0
zl z
0 z 0 z 8.5* 1 * 55.Oz 6 z 26,0 2 24.0* 6
1 .... 2
1
z
z
z
z
z z
z
z
z z
z
s 2
1 0 :0 : 0 i 0 s 22.5* 2 : 57.5s 6 * 21,0 2 78.5*14
z
z
z z
(
z
z
z z
z
s
5.5s
1
z
55.0s
8 z $4,0 8 48.5* 8
7.0
z4
z
z
z
7.5z
5
i
f
z
z
z z
z
z
z
s
z z
*
z Average t 5.8 z2 z 5.0s 0 * 14.0* 2 * 51.4* 7* 56,9 2 55.1* 9
z Average z
z
z
z
.,.4,69 *
4.57 ,J_...4.?-3_L
5.99 ... 4,61
__ 4,6?
fi M __
9
9
x Control cultures to which no indole- 3-acetic acid had been added
grew about 68 mm., and formed about 10 branches
*
s
z
f
s
s
s
f
s
f
z
•
z
f
•
•
s
z
•
41.
The remainder of the residue concentrated (see 142)
from the inoculated broth was acidified to pH 3, and extracted
seven times with one-half volume of peroxide-free ether.
The aqueous layers were retained as controls.
The ether
extracts were combined and allowed to evaporate at room
temperature.
Twenty flasks were prepared containing amounts
of residue ranging from 150 to 1.5 x 10”1 mg. per liter.
Resultst
The ether residue was somewhat toxic, several of
the cut ends of the roots were swollen, and there was excess
root hair development.
As roots treated with indole-3-acetic
acid (see later) exhibited these responses it is believed
that possibly growth substance was present in the ether
extract; see table 6 .
Twelve root cultures were grown in solution to which
the aqueous layers retained from the extraction ty ether were added.
Resultsi
This fraction was quite toxic, but there were no
responses resembling those elicited ty the ether residue;
see table 7.
With the exception of the ether extract of the culture
medium in which the crown gall pathogen had grown, the employment
of P. tuBefaclens and its products failed to produce galls in
root tips grown in artificial culture media.
TABLE 6
The Effect of Evaporated Ether Extract
of the Cell-free Filtrate of Broth Medium
Inoculated with Phytoaonas Tuasfaciens
Upon the Growth and the Number of
Secondary Boots Formed ty Excised Tomato Boots
{Number
{ of
: root
{cultures
s per
s conc.
:
5
e
b
S
!M
t Concentration (mg./l, *) of ether extract of
inoculated medium
i
{
{
-1 x
t
16.0
£ 1.6 x 10
s 160.0
{
1.6
{
>
S
{
{
{
t
l
s L. { B. x
L. { B. {
L. t B.
L. *_ B. j
*
t
s
{
s
S
{
6
s 50.4 s 4
.1.9. A.Q._ s 10.0 s 7 s 42.8
9
m
9
9
*
Approximate weight
x
Control cultures grew about y2~sn., and formed about
TABLE
s
f
f
t
s
1
1
.JL
7 branches
7
The Effect of the Aqueous Extract
of the Cell-free Filtrate of Broth Medium
Inoculated with Phytoaonas Tumefadens
Upon the Growth and the Number of
Secondary Boots Formed ly Excised Tomato Boots
iNumber
t OI
{ root
{cultures
{ per
t conc.
{ 6
s Average
i
s
s
{
s
(
x
Concentration {%) of aqueous extract of
mocuxawKi aecu.ua
t
t
x
0.4
I 0.04
{
s
{
{
L. { BT f L. t B. s
i
t
s
{
0 x 0 I. 0
s
{
0.004
{
L. x
s
O s
{
{
0.0004 *
i
{
s
B.
L.
s
B.
?
•
{
{
4
..S. .li..3 .1
...I
{
?
:
•
9
Control cultures grew about i+6 m m . , and formed about 8 branches
C.
Effect of Indole-3-Acetic Acid
1.
Gross appearance of roots. Excised roots grown
in the yeast extract or synthetic media are white and translucent,
slender and long, regular in shape, and float on the surface of
the solution.
tip.
Secondary roots fora about 20 mm. behind the
See figures 1, 7, 8 , and 9.
Roots grown in nutrient solution containing indole-3-
acetic acid (Eastman Kodak Co.) in concentrations from 10.0
to 0.0125 mg. per liter, respectively about 6 x 10 ~^ to
n|Q
7 x 10
molar, are completely to slightly brown and opaque,
stunted in length, thickened and irregular in shape, and
submerged in the fluid.
Branches appear even at the tip, and
like the primary toot are stunted in growth.
The higher the
concentration of the phytohormone, the greater the effect.
Con^are figures 1, 2, 5, 4, 5, and 6 .
Well-pronounced galls are found on treated roots.
See figures 10, 11, 12, 13, 14, 15, and 16.
These are generally
found in conjunction with root primordia or branches but
often without the presence of branches or root rudiments.
The
overgrowths may appear on the primary root or branches, at the
tip, medially, or at the cut end.
2.
Microscopic appearance of roots. Fresh unsectioned
roots grown in normal nutrient solutions were mounted in water
and viewed through the microscope.
This method confirms the
regularity and evenness of normal roots.
is readily visible.
The vascular stele
In addition, the root hairs are observed
to be moderate in number and regular in outline.
7, 8 , and 9.
See figures
As seen from histological preparations the cells
of untreated roots axe elongated and rectangular in shape, and
lie together in regular formation.
The epidermis is situated
outermost, and consists of a single layer of cells.
rise to the root hairs.
These give
The cortex is below the epidermis and
consists of about three to five layers of parenclymous cells,
and of a single innermost layer, the endodermis.
tbderaeath
the endodermis, the single layer of pericyclic cells bounds
the vascular stele.
Ey meristematic activity of the pericycle
secondary roots are initiated.
Within the pericycle lie the
xylem and phloem; there is no pith.
See figures 17 and 18.
Treated whole roots observed by means of the microscope
are found to be highly irregular in shape.
There is seen an
excessive production of root hairs, the ends of which are
frequently swolleh at their tips like balloons.
In microscopic sections of roots treated with indole-3acetic acid the enlargement of the cells of the epidermis is
obvious; in the higher concentrations of the hormone there is
much fragmentation of these cells.
The hypertrophy of the
cortical cells is especially noticeable. Necrosis and
irregulariiy give rise to an increased volume of intercellular
space in the cortex, and, in addition, some cellular divisions
(hyperplasia) are present.
Endoderaal cells are hypertrophied.
There is intense stimulation of the meristematic activity
of the pericycle.
Figure 19 is especially instructive,
showing two branches which themselves demonstrate the effect
of the heteroauxin, two evident primordia in advance of
which there is seen typical dissolution of cells and three
other root histogens.
Figures 21 and 22 exhibit typical
thickening in the absence of intense root initiation.
In
the cortex and particularly in the pericycle there occur
multinucleate cells,most of which are binucleate.
lylem
and phloem do not prominently'react*'to treatment.
See
figures 19, 20, 21, and 22.
A comparison of figures 8 and
13 illustrates the action of indole-3-acetic acid upon
the stimulation of callus production.
3.
Growth of roots.
In the previous two sections
the pathological effects observed were incited by dilutions
Fig. 1.
Untreated root,
x 0 .6 .
Fig. 2.
Rootstreated with 0.001 mg.
per liter indola-3-acetic
acid, x 0 .6 .
Fig. S.
Roots treated with
0 .0(1 mg. per liter
indole-3-acetic acid,
x 0 .6 .
Fig. 4.
Rootstreated with 0.025 mg,
per liter indole-3-acetic
acid, x 0 .6 .
Fig. 5.
Rootstreated with
0.05 mg. per liter
indole-3-acetic acid,
x 0 .6 .
Fig. 6 . Rootstreated with 0.5 mg.
per liter indole-3-acethe
acid, x 0 .6 .
Fig. 7.
Normal root,
Fig. 9.
x 24.
Fig. 8 . Normal cut end of
primary root, x 24.
Untreated root,
x 60
r
Fig. 10.
Fig. 11.
Root treated with
0.025 mg. per liter
indole-3-acetic acid.
X 24.
Fig. 13.
Cut end of root
treated with
0.05 mg. per liter
indole-3-acetic acid,
x 24.
Root treated with
0.05 mg. per liter
indole-3-acetic acid,
x 24.
L
Fig. 12.
Root treated with
0.05 mg. per liter
indole-3-acetic acid,
x 24.
Fig. 14.
Root treated with 0.05 mg.
per liter indole-3-acetic
acid, x 24.
A
A
Fig. 15.
Root treated with
0.5 mg. per liter
indole-5-acetic acid,
x 60. (Retouched.)
Fig. 16.
Median section of
root treated with
0.5 mg. per liter
indole-3-acetic acid,
x 60. (Retouched.)
so
Fig. 19.
Histological section
of treated root, x 1 0 0 .
Fig. 20.
Same as Fig. 19.
Fig. 21.
Same as Fig. 19.
Fig. 22.
Same as Fig. 19.
—8
of iadole-5-acetic acid up to about 7 x 10”
per liter).
-6
7 x 10
molar (0.0125 mg.
Up through this concentration, and even to
molar, there is unmistakable inhibition of root
elongation, reduction in the number of branches formed and
in the total dry might.
See the experiments comprising
810 root cultures recorded in tables 9 and 10.
In the initial
trials (see table 8 ) with 84 isolated roots indole-3-ecetic
acid appears to accelerate growth at concentrations of
_7
7 x 10
molar and less.
Later work, as shown in tables
9 and 10, does not substantiate this.
As was noted in the survey of the literature, growth
hormones are considered to accelerate elongation of the
coleoptile but to inhibit root growth, except that some
authors report or believe that at low concentrations lndele—
acetic acid stimulates growth of roots.
experiments were performed.
To verily this two
In the first, consisting of
200 root cultures, concentrations of indole-3-acetic acid
-8
-in
-ip
of 1 0 , 10
, and 10
molar were compared with control
cultures to which no pfcytohormone had been added.
are given in table 10, experiment I.
clearly inhibiting, 10
>10
and 1 0
-12
The results
The dilution 10
is
molar have apparently
poorer growth than control roots, the latter seemingly having
16.3 per cent better growth.
TABLE
8
Preliminary Test on the Effect of Indole-5-Acetic Acid
Upon the Growth and the Number of
Secondary Roots Formed by Excised Tomato Roots
1
slfoaber
{
Concentration (me./I.) of indole-3-acetic acid
s of
{
i root
{
s
s
{
s
0.00006 s 0.001
scultures :
: 0.025
s 0.5
z
0
9
•
9
s per
s
s s
s
I
s
s
{
•
L. sB.s L. s B, s L. s B. z L.z B. z
s cone. • L. s B.
s
{
s
{
S
s
s
8
{
s s
z 4
59.5 s6 s 55.0 s6 s 8.0 s 4 {2.0 zO S
I 55.5x 2
•
e
•
9*
s s
s
s
s
8
s
s
{
z &
57.2 {2 { 35.8 s2 slQ.O s 1 s0.9 si z
{ 20.4{ 2
•
s
{
s
s s
•
s
s
s
s
s
s
5
s 59.2s 5
40.5 s5 s 20.6 sO s 5.6 z 1 s0.6 si s
s
t
s
s s
I
s
{
s
{
s
{Average { 51.0s 2
45.7s 4 s 37.1 S3 { 7.9 { 2 si.2 si s
{Total dzy {
s
s
s
s
{ weight 9
2.2
s
2.3
:
0.6
s 0.05 s
m 1.7
s
s
s
.8
__ .S..
______
S-
s
s
;
s
s
•
s
B.
3
s
9
z 0
9
s
9
s1
»
S
s
s0
S
•
s
9
z0
I
S
0.05
S
-t
10 .0
L.
1.0
0.9
1. 0
1.0
9
TABLE
9
Tha Effect of Indole-5-Acetic Acid
Upon the Growth and the Number of
Secondary Roots Formed ty Excised Tomato Roots
X
X
?
X Number
of
!
I root
x cultures
t per
cono.
I
x
5
;
x
&
x
5
I
:
Average
EXPERIMENT I.
Concentration (me./ I . )
X
X
X
£
0
x 0.00125 x 0.0125
x
X X X
X X
X
X
x L.
X B.x L. x B.x L.
x B.X
X
X
X
X X
X X
: 40.8: 5 t 51.6: 5 : 15.0 2 4 t
X X X
X X
X
X
x: 49.2: 4 x 67.8: 6 t 19.2
*5 x
X
X
X X X
X X
x 66.42 4 2 28.0x10 X 25.4 x 8 x
X
X X
X X
X
X
2 52.1X 4 x 45.8* 7 * 18,5 X 4_j
X
X
X
X
of indole-5-acetic acid
£
X
X
X
0.025 1
0.05 X
0.5
£
X
X
X
X
X X
L.
x B. f L. f B, X L.
* Bf
X
X
X
X
* ;
10.4x 6 X 4.4 x 5 X 1.8 X 4t
X
X
X
X X
0.2 x 5f
1 2 .Ox 6 f 1 .8 s 3
X
X
X
X
X s
0.6 x 5,
2 2 .0 x 6
1.4 s 3
X
X
X
X X
14.8x 6 { 2.5: 3
0.9 I & s
0
0
0
0
0
0
0
0
0
0
0
m
0
0
0
EXPERIMENT II.
0
0
0
0
0
.*
0
0
s
X
0
5
5
%
5
f
X
5
f
l
t Average
X
*106.8
X
x 58.4
%
x 53.7
X
X 53.0
X
*15
X
, 7
5
X5
X
2 5
0
0
0
X X X
X X
2108,2x17 x 25.8 * 5 _*
0
0
*
X
x
X
X
•
X
x 58.0 x 7 £
0
0
0
0
21.6s 4
X X
5.6* 5
X X
4,0* 0
X X
54.9 S 6
0
0
, 15,0 s
X
X 18.8 X
X
s 7.0 s
X
£ 16.7 £
0
0
0
0
2
X
7
X
4
X
5
X
X
X
S ;
8.4S 5
M s 4 . 1.4 5 2 ’
X
X
X
I
X s
5.2: 0 { 4.2: 2
0
I Os
X
X
X
X
X X
0
0 x 1 I 0.6 j 2 s
I Os
X
X X
0
0 x 1 f 2 .0 * 2 f
f 0*
X
X
X X
\ .1 4.1t 2 A 0.4 ? Is
x
0
s
0
0
X
0
0
0
0
s
,
0
0
0
TABLE
10
The Effect of Low Dilutions of Indole-3-Acetic Acid
Upon the Growth and the Number of
Secondary Boots Formed ty Excised Tomato Roots
e
|
e
•
X Number
of
z
i
z
X root
X cultures z
z
X per
conc. ef
t
I . 10
e
9
10
s
10
X
s
10
s
10
X
s
s Average 1
*
x Average
pH
Z
z
* '25 flasksz
X each) f
z
e Number
z
•
of
z
z
: root
• cultures
•
per
z
i conc.
10
z
t
10
{
s
10
t
i
10
?
f
10
t
1
10
1
i
10
i
I
10
z
1
t Average 1
: Total
e
•
dry
z weight
-L.. ( » « . ) - s
9
9
9
9
9
9
9
9
*
EXPERIMENT I.
1
s
Concentration (molar)
12
z
0
z _._1 0 .
z
z
z
z B.
z B.
z L.
L.
112.5 Z16.5 z 104.0
Z16.8
72.1 z 8.7 z 33.8
z 3.1
Z16.4
108.8 Z20.8 z 87.4
69.9 Z14.1 z 61.4
zl2.9
zll.4
S5.0 zl0.2 z 71.2
83.5 Z15.8 z 71.6
Z12.1
z
4.49
z
4.72
z
z
EXPERIMENT
of indole-5-acetic acid*
z
-10
:
-8
z
10
z
10
z
z
z
:
z
z L.
z B. z L.
*113.5 *16.8 z 67.8
*
z 52.4 z 6.1 z 48.2
z
z 90.7 zl4.9 z
7.6
*
z 48.8 z 8.1 z 2.4
z
z 52.5 z 9.6 z 1.6
z
z 71.6 sll.l z 25.6
*
z
z
z
4.63
z
4.50
z
z
z
z
II.
Concentration (molar) of indole-3-acetic acid*
-14
-12
z
-10
z
z
t
10
0
10
z
10 z
z
z
z
z
z
z
z
zB. z L.
z 1 . L.
z B.z L.
z B.z
L.
91.1
z 9
71.3 z 8 z 77.9 zlO x
s 11z 61.4
67.3
65.0 z 8 z 59.5 x 6 x
z 8 z 51.2 -- 5. 5
41.6
59.2 zll z 44.9 z 6 z
z 6 z 54.4
zll
120
91.5
98.8 zl6 z 104.0 zl5 z
z 15x 110.8
83.5 zl4 z 64.3 zl2 z
74.5
z 12z 61.9
zl2
48.9 : 8 z 49.2 , 8 z
53.6
z 9z 44.3
z 9
zl2
96.8 zl3 * 105.6 *14 *
75.6
zll z 77.7
82.4
77.5 zlO z 82.0 z 9 z
*16
z lOz 111.7
Zl2
70.9
z 10z 71.7
7 6 ^ *11 * 73.2 *10 z
e
z
z
100.4
:
89.6
z
100.3
z
101.0
z
z
z
*
__________ :________ _.i
In synthetic medium
B.
12.6
8.1
1.7
0.4
0.8
4.7
z
9
s
z
*
.
*
t
:
,
z
.
z
9
s
I
•8
•
10
z
•
L.
zB. y
4.2 z 4 .
26.5 z 1 .
20.2 z 2 z
58.8 zll z
29.3 z 4 s
22.7 z 1 z
27.7 z 2 z
58.3 z 5 z
28.5 z 4 z
z
78.2
z
z
*
In the second testy see table 10, experiment II,
-8
-10
-12
400 roots growing in molar concentrations of 10 , 10
,10
,
-14
and 10
indole-3-acetic acid and control flasks were compared.
At a dilution of 10
-12
molar there appears to be an acceleration
of elongation of 5.9 per cent.
Dry weight measurements, however,
give almost exactly the same readings as the control, 10
and 10
-12
,
molar indole-3-acetic acid treated roots, respectively,
100.3, 101.0, and 100.4 mg.
4.
Toxicity of indoie-5-acetie acid and growth of
galled roots in normal solution.
It may be seen from tables
8 and 9 that in the presence of concentrations of either
10 mg. or 0.5 mg. per liter of indole-3-aoetic acid the roots
exhibit practically no growth.
An additional experiment was
performed in which 5.0, 0.5, 0.05, and 0.025 mg. per liter
indole-3-acetic acid were added to nutrient solution.
Ten
roots were used in each of the firBt three groups, 20 in the
last dilution (0.025).
After cultivation for one week,
the roots were transferred to normal nutrient medium.
The
results are shown in table 1 1 } it may be observed that a
concentration of 5.0 mg. per liter is just about lethal as
there is neither elongation nor root initiation, and, in
addition, only 1 of 10 roots grew upon transfers in normal
solution during a period of several weeks.
In the flasks containing 0.5 to 0.025 mg. per liter
of growth substance a number of roots developed galls.
Six
excised roots bearing prominent swellings were selected and
were cultured for eight weeks in solution containing no
indole-5-acetic acid.
During this period the gall tissue
persisted without apparent enlargement but all new growth
was normal.
5.
Effect of neoprontosil.
In an attempt to determine
whether neoprontosil would selectively1 be "absorbed ty galled
tissue, the toxicity range of this well-known bactericidal drug
(disodium 4-sulphamido-phenyl-2-azo-7-acetyl-amino-l-hydrosynapthalene
3,6 -dieulphonate, ffinthrop Chem, Co.) was tested upon 19 roots.
Resultst It was found that at a concentration of 0.1 per cent
neoprontosil there was good growth of roots and also considerable
dye was picked up by the roots; see table 1 2 .
Therefore, 20 root tips were cultured in nutrient
solution to which had been added 0.1 per cent neoprontosil plus
0.025 mg. per liter indole-e5-acetic acid.
Results:
The dye seems to have no special affinity for nodular
tissue except in so far as such tissue is hypertrophied, has
excess intercellular space, and increased numbers of root hairs;
meristematic areas as tips and root primordia do not absorb the
dye readily; see table 1 2 .
TABLE
11
The Effect of Indole-3-Acetic Acid
and Transfer to Normal Nutrient Medium
Upon the Growth and the Number of
Secondary Roots Formed by Excised Tomato Roots
•Number*
t of
i
: root
s
icultures s
s per
s
: conc. t
* 10
s
• 20-0.025:
Concentration (mg./I.) of indole-5-acetic acid
t
s
:
: 0.025
5.0
s
0.5
: . 0.05
9
s
s
s
s
s
s
B.
L. s B.s L. s B. s L. s B. s L. s
s
s
s
s
:
:
s
0 s 0 s 0 : 2 : 2.6 : 2
s 2.6 ; 2
e
e
:
:Fer cent s
:survival s
Transfer to normal nutrient medium
s
s
10
:
60
s
80
j ......,9P
TABLE
f
s
1
IB
The Effect of Neoprontosil and of
Neoprontosil plus Indole-3-Acetic Acid
Upon the Growth and the Number of
Secondary Roots Formed by Excised Tomato Roots
(Number
s
s
Concentration (£) of neonrontosil
s
s of
s root
s
s
s
s
s
s
t
s
s
:
0.5
s 0.25
:
0.1
s 1.0
s 0.01 : 0.001
scultures s 5.0
S
S t
s
s
s :
s :
s s
s :
s *
s per
•
s L.
sB.s L. sB. s L.
sB.s L.
sB.s L.
sB. sL. sB. sL.
s conc.
iB. f
s s
s i
s
:
s
:
s :
s
s ts
:
s ::
t
: :
S I iS :
1___3_____I__ Q___s 0:10.0 s 0 s 15.5 : 4i 11.0 t Is 25.5 t •«
Kg 28.7s 2t
9
9
0
0.1 %■ neoprontosil plus 0.025 mg./I. indole-5-acetic acid
S
t
s
1—20_____ I______________________________
sAver age
» pH
*
s
s
-____________________________________________ s
Only one culture
s s
s
S 4 ;____________ I
s
s
* .6 *
s_________________ £
; 6 .5
6,
Effect of tryptophane and of indole. As
indole-3-acetic acid is an intermediate product in the
transformation of tryptophane to indole by micro-organisms
(6 , 2 1 , 116), these substances were tested for their possible
carcinogenic effect upon root tips.
A commercial preparation of tryptophane, in
A
-9
concentrations of 10
through 10
moles per liter, was
added to nutrient fluid and 58 root cultures were prepared.
Results: See table 13; a few swellings were noted; no
hypertrophy was seen, nor was there observed initiation
of root histogens.
Twenty isolated roots were prepared to which
indole (Elmar and Amend, C.P.), in dilutions of 200 through
0.1 mg. per liter, was added.
Results: The highest concentrations completely inhibited
root growth but no effects comparable to those elicited by
treatment with indole-3-acetic acid were discovered.
7.
Effect of acetic acid and hydrogen ion concentration.
The addition of acetic acid and of changes in the pH of the
nutrient solution brought about by addition of hydrochloric acid
or potassium hydroxide were studied because acetic acid is part
of the indole-3-acetic acid molecule, and it has been reported to
cause plant tumors (124); acidity gradients have been considered
TABLE
15
The Effect of Tryptophane
Upon the Growth and the Humber of
Secondary Roots Formed toy Excised Tomato Roots
{Number
x of
t root
{cultures
* per
s conc.
2
2
X
t
2
2
9
9
9
9
9
10
:
Ift* B f
:
1
9
L.
8.5
2
9
9
x t
? 2 | 46,0
•
9
0
1.0
0
10.0
0
0
0
•
•
sAvera£e X 0 f 0
tAver.pH X
x6 flasks X.
:
S
5.9
9
9
i Ox 55.5
j t
x 6 i 19.5
s x
r 0 ; 28.5
9
0
9
9
{
L.
9
9
;0 ?
•
•
9 ;
x
x
0 ;
x
0 i
s
o X
9
•
9
Ot 42.5
X
X 2 f 58.0
t
4.59
, X 4.98
t
9
{
•
•
:
t
{
£
:
j
t
t
x
9
B*, I*»
•
•
5 X 28.0
•
•
S X 52.5
9
9
X
10
xB. x L.
|B. I L.
•
xB. x
X
X
X
X
9
9
-9 X
9
9
9
9
9
i
9
9
-4
9
9
9
9
t
•
9
9
Concentration (molar) of tmrtouhane
•
-6
-5
-7
x
-8
X
•
3,0
I 10
10
10
9
9
9
9
t
X
x
x
X
9
9
s
t
X
X
X
X
X
X
9
9
9
x 4 ,50.0 X 5 X
9
9
9
9
9
9
x
•
X
e
X
x 9 X56.5 X 6 x 105.5 • 15;
•
X
X
X
X
e
4 :... 25r0 X 6 x48.0 X 7 x 91.5 x 17x
•
X
X
X
X
X
I
•
65.5
12;
2 X 38,5 x 5 x52.5 x 3
9
9
9
9
9
9
m
X
X
X
X
9
9
9
9
•
9
s * 27.6 x 5 x48.5 1 9 X 27.0 X 2 x
*
X
9
X
9
•
•
•
9
9
9
9
5 f 55.9 : 6 ,45.1 X 6 X 71.4 x 1 2 ;
X
X
4*81
X
X
4.66
X
X
x Control cultures to which no indole- 3-acetic acid had been added
grew about 122 m m . , and forced abovt li; branches
5.06
X
X
as growth factors (139* 140)* and we have observed in various
experiments* see table 14* that the addition of indole-3-acetic
acid raises the concentration of hydrogen ions in the medium.
Nutrient solution to which acetic acid (10-2 through
-8
10
molar) had been added was used to prepare 20 culture flasks.
Results: The appearance of these roots was normal; no swellings*
root histogens* excess root hairs* or other effects were noted;
even when roots were completely stopped in growth there was
neither browning of the tissues nor root stimulation; see table 15.
Ey the addition of either hydrochloric acid or
potassium hydroxide* changes in the pH of media were obtained
ranging from 0.95 through 11.5.
A total of 121 cultures was
studied.
Results: No galls were observed; some roots exhibited increased
root hair production; some were irregular in shape; some showed
excess root primordia initiation; growth was mmrinmi between
pH 3.4 and 5.0; see table 16.
TABLE
14
The Ejrdrogen Ion Concentration (pH) of Nutrient Medium
Containing Various Concentrations of IndoIe-3-Acetic Acid
Before and After Root Cultivation
*
Fresh
{Concentration of •
:indole-5-acetic I
tacld (m e . /I.)
{
{
•
0
{
{
0.00126
i.
;
•
9
i
9
i
(uncultured) solutions
Number of {
{
cultures t Average
{
tested
pH
f
f Ranee of p H
{
13
4.48
{ 4.38 - 4.68
t
9
9
9
9
s
16
0.0125
i
f
4.50
9
9
•
z
•
9
16
1
4.33
{
9
m
0.025
i
}
17
•
9
9
0.05
9
17
0.5
9
4.32
•
9
4.30
{
9
{
i
9
9
4.64
-
4.89
? 3.90 - 4.60
{
4.01 - 4.69
9
*
9
9
9
{
9
9
t
{
{
{
{
{
:
9
9
t
9
9
9
3.92
21
-
9
9
9
9
9
f 3.98
{
3.93
t
•
•
3.79 - 4.69
9
{
9
9
Cultured solutions
9
9
9
0
9
{
J
9
9
128
9
9
9
0.00125
9
f
0.0125
9
19
0.025
18
9
9
i
s*
s
f
21
*
0.5
A
s
4.29
{
;
I
9
19
?
4.24
9
___
{
4.28
?
4.21
{
9
{
3,58 . 5.58
9
9
•
3.68 - 4.93
9
9
3,76
-
4.60
3.39
-
4.59
3.96
-
4.55
9
9
I
t
I
{
18
••
4.12 - 5.99
t
9
S
i _______
4.59
9
9
(
0.05
9
9
S
9
9
9
9
t
4.98
9
9
I
9
9
9
9
9
9
9
S
pH readings were made prior to sterilization
--- *
TABLE IS
The E ffe c t o f A cetic Acid
Upon the Growth and the Number o f
Secondary Roote Formed by Excised Tomato Boots
:Number
z of
z root
scultures
: per
: conc.
t
z
2
s
z
Concentration (molar) of acetic acid
s
t
•
-2
—3 z
—4
j
-6 s
-6
z
-7 z
—8 x
:
z
•
•
10
z 10
s 10
s 10
i 10
z 10
z 10
z :
z z
z z
z s
s z z s
s
•
zB.z L
(
1 L,
: :
z
z z
z z
z z
z z z z
z
z
z *15.5 z 7 z 74.5zlSz68.0 zl6z97.5zl7zll6.0 *11 s
z z
;
z
z z
z z
z z
z z
z z z z ;
z
I
t 0
z
2
z Oz 8.5 z Oz 0 z o Z 2.5* 0*55.0 ; 9z
t z
z
f
: Average m
z
z
z
z
z
z
Z
2.86
8.28 z 4.07
: 4.76 z 4.84 z 5.78 : 5.44
z DH
*
A
9
9
9
9
.9
•
x Control cultures to which had been added no indole-J-acetic acid
grew about 121 mm., and formed about 16 branches
63.
TABLE 16
The E ffe c t o f D iffe re n t Ifcrdrogen Ion Concentrations
Upon the Growth and the Number o f
Secondary Boots Formed by Excised Tomato Boot Tips
o H of culture medium
2
sNumber
after s
1
s
sof roots
Initial
« pH after
Average growth
s culture
sper H
B.
sconc.
L.
2 sterilizations oeriod
2
2
•
•
1.45 *
: 1.45
2
s
•
0
0
12
J (0.95 - 1.98) 2 (1.16-1.80) 2
—
1.2
0
&
2.16
£
2
*
•
1.93
0
2.20
0
2
2
?
2
•
0
0
2.63
2.50
6
2
*
—
2.97
0
5
1
2
2
s
•
2.77
2.2
2
6
3.13
s
2
—
28.5
6
2
3.42
2
t
|
s
—
3.54
0
5
1.8
2
(
2
•
»
_
4
4
3.58
39.0 •
:
2
•
5.30 _
44.8
4
6
4.09
2
•
4
6
4.22
4.07
54.2 2
{
2
•
40.4 2
5
4.59
4.65
3
i
{
2
29.1
2
6
4.77
2
f
I
s
4.87
4.86
4.6 ••
6
1
;
2
2
2
5.00
2
5.65
5.7 2
3
2
2
2
5.04
1.7 2
1
4.57
6
2
2
*
5.54
1
6
5.29
2
_ .5,7 2
I
2
*
6.75
6.12
2.4
„
1
6
I
2
6.52
3.7 2
0
5.79
3
s
2
6.54
7.46
0
1.7 2
3
2
2
•
6.42
5.88
4.7
1
6
I
2
5.79
6.49
0
1.7
6
S
2
2
2
.
6.87
1.0
0
6.67
6
::
2
2
2
*
7.66
1.3 2
6.92 #
0
3
2
3.2
7.04 #
0
6
7.27
i
2
2
2
1
7.14 #
0
— 2
2
|
2
2
6
7.59 #
6.33
2.5 2
1
I
f
2
6.41
6
1
I
7.77 #
3.8 •
2
2
2
6
6.16 #
7.82
1
1.8
I
2
2
•
•
6.5 •
8.21
•
3
1
8.19 #
2
2
•
2.5
4
1
f
8.21 #
I
2
2
10.9 * #
•
8.34
**2
s
2
2
0.6 ,1
0
(9.3 - 11.5) .^9*55-8.96) ••
.ft i U
m
9
9
t
.
2
2
2
9
9
9
9
m
9
9
9
9
9
£
9
9
*
9
9
9
9
9
9
9
9
9
9
•
9
2
9
9
9
9
9
9
9
1
9
9
*
z
9
9
.
£
z
£
9
9
9
9
99
9
1
Cultures were performed at different times of the year and
hence conditions, as temperatures, were different. Therefore,
growths were only comparable.
#
These solutions contained a precipitate after treatment
adjustment.
*
pH readings taken before sterilization.
**
Two roots in the lower pH range showed some growth.
2
2
9
9
9
■ ,f
IV.
DISCUSSION
A search of the literature has disclosed no previous
publication on the effect of Phytomon&s tuaefaciens on plant
tissues growing in vitro.
According to White (158),
E. F. Smith felt the desirability of studying plant cancer
by means of tissue culture technique and conducted an
investigation with the Lewises, but the outcome of this
unpublished work was negative.
The failure in the present experiments to produce galls
by inoculation of excised roots with a virulent culture of
the crown gall organism might be explained in that the roots
cease their growth upon contamination of the medium by rapidly
growing bacteria.
The latter probably stop the growth of the
roots by utilization of the nutrients of the solution,
especially the sugar.
Various constituents of P. tumefaciens obtained by
chemical fractionation, as endotoxin (1 1 , 1 2 ), phosphatide,
lipoid, and polysaccharide (25, 85) portions, produce galls
upon inoculation into susceptible plants.
The inability in
the present study to produce tumors by the addition to the
culture solution of dead bacteria and fractions of the pathogen
liberated by freezing and thawing might have resulted from the
fact that such substances were not able to diffuse into the
root tissues.
It is well known, for example, that smearing
tomato plants with a virulent culture gives rise to no galls
unless the host is wounded (107).
The additional possibility
that a thermolabile tumor?dLncitant present in such dead or
disrupted bacterial cells was destroyed in the sterilization
of the nutrient fluid, was precluded by those experiments
in which a sterile Berkefeld N filtrate of frozen and thawed
cells was added to the fluid medium.
The one suggestive result occurred when an ether
extract of the culture fluid in which P. ttanefaciens was
grown was added to the plant cultures.
Some slight swellings
and excess hair formation on the roots indicated the presence
of growth substance in the solution.
These substances have
been shown to be produced by many bacteria and fungi (6 , 21 ,
102, 142) and by P. tumsfaciens (10a, 19, 88 ).
The yields
of growth substance were, however, vexy small.
Thimann (142)
obtained only 1 mg. from 140 liters of medium, and this may
have been the reason why there was such a weak response in
our experiment in which only 6,6 liters of potato-daxtrose
broth were extracted.
The presence of growth substance in
potato tubers has been demonstrated (18).
While uninoculated
potato-dextrose broth was not subjected to ether extraction,
an experiment was performed in which such medium was concentrated
by vacuum distillation (see table 5).
The fact that so many diverae physico-chemical stimuli
(as humidity, frost, wounding, acids, alkalies, inorganic
and organic compounds, gases, etc.) and pathogens (bacteria,
fungi, nematodes, and viruses) have been able to elicit galls
in plants, has pointed to the possibility that these agents are
able to induce tumors by interference in the normal balance of
growth hormone in plants.
Brown and Gardner (19) first clearly
demonstrated that structures arising from inoculation of
growth substances into whole plants resembled those produced
by the crown gall organism.
The present study was the first
primarily concerned with the production of galls in plant
tissue cultivated continuously in vitro in the presence of
accessory growth factors.
In the present investigation, overgrowths were shown to
be incited only by concentrations of indole-5-acetic acid which
actually inhibited growth.
The technique of plant tissue
cultures has permitted a quantitative study of the relationships
of growth and tumor formation to the concentration of growth
substance, and adds confirmation to the role of hyperauxoxy in
the formation of galls (20, 76, 77, 84, 88 , 101).
The gross and microscopic responses of isolated roots
to indole-3-acetic acid resembled those induced on intact plants
by this substance as shown by various workers, namely, gall
and callus formation, inhibition of elongation of roots,
hypertrophy, hyperplasia, root initiation, necrosis, excess
root hair formation, and the presence of multinucleate cells.
Similar responses of plants to inoculation with the crown
gall pathogen have been recorded (79, 89, 108, 121, 128, 129).
Crown gall tissue has been shown to contain more growth
substance (Avena coleoptile test) than uninoculated tissue,
and such hormone appeared to be of the auxin a or auxin b type
rather than of the heteroauxin type, and it was believed the
additional growth substance was more likely formed by the host
under the stimulus of the bacteria rather than being a
bacterial metabolite (89).
The question whether very dilute solutions of growth
substance accelerate the growth of roots is a moot one.
Acceleration of root elongation has been reported (3, 15, 37a,
39, 42a, 45, 56), while others have noted Inhibition (17, 26, 63,
73a, 78, 92, 103).
Excised root tips have been grown under
conditions of continuous cultivation in vitro in synthetic
media to which no indole-3-acetic acid had been added (1, 113, 164).
tfader such experimental conditions, there are no reports in which
the presence of added growth substance has been shown to be
essential.
In the present study it was not possible to
demonstrate clearly either stimulation or inhibition of
tomato root growth with very dilute concentrations of
indole-3-acetic acid (1CT^0 through 10"^molar).
In these
experiments a synthetic meditsn was employed as there is
growth substance present in yeast (61, 104, 111) and possibly
in highly purified sugars (111 ).
Carter (23) has dyed corn seedlings with solutions of
prontosil.
From our results it would appear that this drug is
not selectively picked up by galled tissue; it seems that
young meristematic tissue with little or no vacuolar or
intercellular space hardly absorbs the dye, while older tissue
with larger vacuoles does.
In the present work tryptophane appears to have been
inactive in inducing gall formation as the few slight swellings
observed were unaccompanied by hypertrophy or stimulation of
root primordia.
This possible slight activity of tryptophane
may have been due to the toxic concentration employed, or to
growth activating impurities in the preparation (157) as it
is reported to be inactive in the Avena test (18).
other hand, it is said to be active in the Pisum
test (157,).
On the
root formation
Furthermore, minoculated agar remained inactive
69.
with the exception of tryptophane agar which showed same
activity following sterilization (21 ).
Consistent with our observations, indole has not
been reported as an active substance (18, 157).
We have found acetic acid to be negative in gall
formation.
This substance has been reported to cause tumors (4, 124);
to be inactive in the pea test (47); to have no effect on the
growth of roots and root hairs (93).
Alteration of the pH of the nutrient solution was not
observed by us to induce overgrowth in excised root tips of
tomato.
Varioios acids have been recorded as producing
overgrowths (4, 124).
Strugger (139, 140) noted that the
growth rates of Halianthus roots were dependent upon the pH
of the buffer solutions, and exhibited a bioodal curve with
a minimum at pH 5.1.
Bonner (13) believed the acid effects
observed by Strugger were caused by the conversion of growth
substance already present in the plant from an inactive salt
to an active non-dissociated form.
The alteration of the
hydrogen ion concentration by acetic acid had no influence on
the growth of root hairs and roots (93), nor did pH changes
by hydrochloric acid affect inhibition as indole-3-acetic acid
did (73a), and growth was observed to depend upon the concentration
of growth substance in buffered solution (92).
From a survey of the literature and from our own
experiments, it is possible that the crown gall organism
(P. tumefaciensK
other pathogens, various physical and
chemical agents, and growth substances elicit tumors by
raising the concentration of growth hormones in the affected
tissues, or possibly by lowering the concentration of inhibitor
substances.
Such augmented auxins might arise by direct
formation as a metabolite of the invading micro-organism, by
stimulation of the host tissues to more active auxin production,
by conversion of metabolic acids of the pathogens of plant
growth substance from an inactive to active form, by action of
microbial enzymes in liberation of bound auxin, or by the
accumulation of growth substance in the disturbed tissue as
a result of interference with the normal streaming of plant
hormones.
V.
SUMMARY AMD CONCLUSIONS
1.
The addition of living cultures of Pfartomonas
ttmafaciens. bacteria killed by heat or by freezing and
thawing, and filtrates of frozen and thawed bacteria elicited
no galls when added to the nutrient solution in which tomato
root tips were grown*
2.
3 y means of ether extraction of culture broth in
which the organism had been grown indications were obtained that
growth substance was produced by the crown gall pathogen in the
course of its cultivation.
3.
Excised tomato root tips growing in nutrient solution
to which indole-3-acetic acid had been added responded tgr the
production of galls.
Microscopically, the tumors were
characterized by hypertropiy of the cells of thB cortex and
epidermis, by marked meristematic activity of the pericyclic
cells resulting in the abundant formation of root primordia, by
some multiplication of cortical cells, by the occasional
development of multinucleate cells in the pericycle and cortex,
by excess root hair production with frequent swellings of the
tips of the hairs, and by stimulation of callus formation at the
cut ends of the roots.
In the higher concentrations of the growth
substance there was much fragmentation of the epidermal and
cortical cells, and, in addition, the roots were irregular
in shape and thickened.
I4.
Galls were formed only in those concentrations of
indole-J-ecetic acid which caused inhibition of growth.
-9
5. U p to dilutions of about 7 * 10
moles per liter
indole-J-acetic acid inhibited root elongation, reduced the
number of branches formed, and decreased the total dry weight.
Concentrations of
10-^ , 10“^ , and 10“ ^
molar did not seem
to accelerate the growth of isolated roots growing in
continuous cultivation in v i t r o .
6.
(about 3 x 10
Five milligrams indole- 3-acetic acid per liter
”5
molar) was the approximate lethal dose,
killing about 90 % of the roots.
7.
The hormone galls persisted u p o n transfer to
fresh nutrient solution, but all new growth in such media
v.ns normal.
8.
Ueoprontosil did not appear to be selectively
absorbed by nodular tissue.
9.
The addition of tryptophane, indole, acetic acid,
or variations in pH in control cultures failed to induce the
formation of tumors.
VI.
1.
BIBLIOGRAPHT
Addicott, F. T., and P. S. Deririan.
A second growth
factor for excised pea roots > nicotinic acid.
An. J. Bot.,
2.
Alexander, T. R.
£6 s 667-671, 1959.
Carbohydrates of bean plants after
troatnsnt with indole-5-acetic acid.
PI. Physiol.,
5.
Anlong, H. U.
15t 845-858, 1958.
Der Einfluss des Wuebsstoffes auf die
Wanddehnbarkeit der Vicia-Faba-Wurzel.
Ber. Deut. Bot. Ges.,
4.
Aimand, P. H.
5.
Atkinson, G. F.
§ ± i 271-276, 1956.
Tunors in kale.
Science, 6 6 s 665-664, 1927.
Oedena of the tonato.
Ck>rnell Univ. Agr.
Exp. Sta., Bel. 65, 1895.
6.
Ball, £.
Heteroauxin and the growth of Escherichia coll.
J. Baet.,
7.
56s
549*666,
Bechhold, H., and L. Saith.
1958.
Das Tunefacians-Plastin.
Zeitschr. f. Krebsforsch., 26s 97-104, 1927.
8.
Beijerinck, M. W.
Uber das Cecidiun von Neaatus Capreae auf
Salix asygdalina.
1888.
9.
Bot. Zeitsehr.,
46 s 1-11, 17-27,
Cited by Went and Thiaann (157).
Beijerinck, H. V.
Sur la ceeidiogeneae et la generation
alternants ches le Qyaips calicia.
is 199-252, 1897.
Vers. Geschr.,
Cited by lent and Thiaann (167).
74.
10.
Bargey, D. H. and others.
Bacteriology.
Bergey 's Manual of Determine,tire
Baltimore, Will Inna and Wilkins,
1939, 1032 pp.
IDs.
Berthelot, A., and Germaine Anoureux.
Sur la formation
d'acide indol-5-acetique dans 1'action da Bacterium
tumefaoiens sur Is tryptophane.
Conpt. rand. Acad,
sci., g£&t 637-640, 1938.
11.
Bolrin, A., M. Marba, Iydia Masrobaanu, and P. Justar.
Sur l'axistanca, dans la Bacillus tuoafaciens, d'une
endotoxins capable da proroquer la formation da tumeurs
ches las vegetaux.
Compt. rand. Acad, sci.,
201i 984-986, 1936.
12.
Boivin, A., Iydia Masrobaanu, M. Marba, P. Justar, and
T. Sauulesco.
Sur la production da tumours chez la
plants, au noyen da l'andotoxine non proteique du
B. tuoafaciens.
Arch. Roun. Path. Microbiol.,
iQ: 67-78, 1937.
15.
Bonner, J.
The relation of hydrogen ions to the growth rate
of the Arena ooleoptile.
14.
Bonner, J.
rlaw.
16.
Protoplasaa,
21 1 406-423, 1934.
Plant tissue cultures from a hormone point df
Proc. Hat. Acad. Sci.,
Bonner, J., and J. B. Koapfli.
by auxins.
An. J. Botany,
2&: 426-430, 1936.
The inhibition of root growth
261 867-666, 1939.
76
16.
Borthwick, H. A., K. C. Banner, end M. W. Parker.
ElBtologic&l and nlcrochamlcal studies of the
reactions of tomato plants to indoleacetic acid.
Bot. Gas.,
17.
i§: 491-619, 1967.
Boysen Jensen, P.
Die Bedeutung dee Vuchsstoffes
ftr das Hachstum und die geotropische Krtamung
der Wurzeln von Ticia Faba.
18.
Boysen Jensen, P.
Planta,
20: 688-698, 1966.
Growth Hormones in Plants.
Trans, and Rev. ky G. S. Avery and P. B. Burkholder.
New fork, McGraw-Hill, 1936, 268 pp.
19.
Brown, Nellie A., and F. £. Gardner.
Galls produced ty plant
hormones, including a hormone extracted from Bacterium
tumefaciens.
20.
Phytopath.,
Brown, Nellie A., and F. E. Gardner.
of a secondary type.
21.
26: 708-715, 1936.
Burkholder, P. R.
Indoleacetic acid galls
Phytopath., 2£: 1110-1113, 1937.
Production of growth substance by bacteria
in media containing specific organic and inorganic
nitrogenous compounds.
22.
Butler, E. J.
Carter, V.
26: 422-428, 1939.
Some aspects of the morbid anatomy of plants.
Ann. Appl. Biol.,
23.
Am. J. Bot.,
i£: 176-212, 1930.
The use of prcmtosil as a vital dye for insects
and plants.
Science,
90: 394, 1939.
76.
24.
Cavara, F.
1894.
Aperou somm&ire de quelques maladies
de la Vigne parues on Italle en 1894,
de Viti, e D ’Oenologie,
is 447-449.
Bar. Intern,
Cited fay
Elliott (58).
25.
Chargaff, E .t and M. Levine.
Bacterium tumefaciens.
Chemical composition of
Proc. Soc. Exp. Biol, and lied.,
Ji: 676-677, 1936.
26.
Cholodny, N.
Uber die hormonal* Wirkung der Orgaaspitze
bei der geotropischen Kz^fammung.
Ber. Deut. Bot. Ges.,
566-362, 1924.
27.
Cholodny, H.
Beitrag zur Analyse der geotropischen Beaktioa.
Jahrb. siss. Bot.,
28.
Cholodny, N.
Biol.
29.
Planta,
50.
Coma8 , 0.
Wuchshomooe und Tropisaen bei den Pflamzen.
Zeatralhl.,
Cholodiy, N.
6£t 447-469, 1926.
42: 604-626, 1927.
Zur Pbysiologie des pflanzlichen Vuchshormons.
14* 207-216, 1931.
1891.
Bacillus ampelopsorae, Ires., Batterio
della rogna della vita.
Crittogamia Agaria,
1: 622-626.
Cited by Elliott (38).
31.
Corvo, A.
Sur le role des bacilles dans les ravages attributes
au Phylloxera Testatrix.
1 0 1 : 628-530, 1886.
Compt. rend. Acad, sci.,
77.
52.
Crocker, V., A. E. Hitchcock, and P. W. Zimmerman.
Similarities in the effects of ethylene and the
plant auxins.
Cont. Bqyce Thomp. Inst.,
2: 251-248, 1955.
55.
Cuboni, G.
1889.
Sul bacteri della rogna vite.
Atti R.Acc&d. Maz. Lineal Rend. Cl. Sci. Fis.,
Mat. e Hat.,
54.
Curtis, 0. F.
5s 571-575.
Cited by Elliott (58).
Stimulation of root growth in cuttings
by treatment with chemical compounds.
Cornell Univ. Agr. Exp. Sta.,
55.
Czaja, A. Th.
Folaritlt und Wuchsstoff.
Ber. Deut. Bot. Ges.,
56.
Doldge, Ethel M.
55: 197-220, 1955.
The role of bacteria in plant diseases.
So. African J. Sci.,
57.
Mem. 14, 1918.
^ 8 : 65-92, 1919.
Duhamel Du Monceau. La physique des arbres.
Paris. Cited by Went and Thimann (157).
57a. Duhamet, L.
1758.
Action de l'h^tero-auxine sur la oroiseance de
racines isolles de Lupinus albus.
Compt. rend. Acad, sci.,
208: 1858-1840, 1959.
58.
Elliott, Charlotte.
Manual of Bacterial Plant Pathogens.
Baltimore, Williams and Wilkins, 1950, 549 pp.
59.
Fiedler, H.
Eatwicklvnge- und reizphysiologische Qxtersuchungen
an Eulturen isolierter Wurzelspitzen.
£2* 585-456, 1956.
Zeitschr. Bot.,
78.
40.
Fitting, H.
Welters entwicklungsphysiologische Untersuchungen
an Orchideenbluten.
41.
Zeitschr. f. Bot., £s 226-267, 1910.
Flosdorf, E. W., and S. ttudd.
Procedure and apparatus
for preservation In "lyophile" form of serum and other
biological substances.
42.
J. Immunol.,
Gardner, F. E., and P. C. Marth.
29; 589-425, 1955.
P&rthenocarpio fruits
Induced ty spraying with growth-promoting chemicals.
Science,
8£: 246-247, 1937.
42a. Gautheret, R. J.
Sur la possibility de realiser la culture
indefine des tissue de tubercles de carotte.
Compt. rend. Acad, sci.,
42b. Gautheret, R. J.
208a 118-120, 1939.
Action de l'acide indol-^s -ac^tique sur
les tissus du tubercle de carotte.
Compt. rend. Soc. biol.,
130: 7-9, 1939.
43. Gelger-Huber, 1U, and E. Burlet.
Ober den hormonalen Binfluss
der ^ -Indolylessigsaure auf das Wachsti* isolierter
Wurseln in keimfreier Organkultur.
Jahrb. wiss. Bot.,
JJ4* 233-253, 1936.
44. Goldberg, Ethel.
Histological responses of cabbage plants
grown at different levels of nitrogen nutrition to
indole (3) acetic acid.
45.
Goodwin, R. H.
Bot. Gaa.,
100; 547-369, 1938.
Evidence for the presence in certain ether
extracts of substances partially masking the activity
of auxin.
Am. J. Bot.,
2§>; 130-135, 1939.
79.
46.
Grieve, 5. J.
Spotted wilt virus and the hormone
heteroauxin.
47.
Nature,
158: 129, 1956.
Haagen Salt, A. J., and F. W. Vent.
analysis of the growth substance.
Akad. Wetensch., Amsterdam,
48.
Hamer, K.C., and E. J. Kraus.
A physiological
Froo. Kon.
5g: 852-857, 1935.
Histological reactions
of bean plants to growth promoting substances.
9£
Bot. Gas.,
49.
Harvey, R. B.
j
755-807, 1937 .
Hardening process in plants and develop­
ments from frost injury.
J. Agr. Res.,
85-111, 1918.
50.
Havas, L.
Follicular (oestrus) hormone and plant
tuaors.
51.
Havas, L.
Nature,
Growth of induced plant tumours.
Nature,
52.
1361 516, 1935.
145: 769-791, 1939.
Heald, F. D.
Introduction to Plant Pathology.
New York, McGraw-Hill, 1937, 579 pp.
53.
Hedgeock, G. G.
The cross-inoculation of fruit trees
and shrubs with crowngall.
U. S. Dept. Agr.,
Bur. PI. Ind., Bui. 131, 1908.
54.
Hsyn, A. N. J.
Der Mechanismus der Zellstreckung.
Rec. Trav. Bot. Neerl.,
2gt 113-244, 1931.
80.
55.
Jost, L.
Wuchsstoff und Zellteilung.
8 er. Deut. Bot. Ges.,
£51 733-750, 1935.
56.
Jost, L., and Elizabeth Reiss.
Zur Physiologic der
Wuchsstoffe. II. Einfluss des Heteroauxins auf
Langen - und Dickenwachatua.
Zeitscbr. Bot.,
j&s 335-576, 1936.
57.
Klein, 6 ., and E. Kqrssner.
pflanzlicber luooren.
Biochea. Zeitscbr.,
58.
Beitrage
I, Mitt.
zub
Chemlsaus
Stickstoffbilanz.
254i 251-255, 1932.
Klein, G., and V. Ziese.
Beitrage
zub
pflanzlicber Tumoran. III. Mitt.
Cbeaismus
Der Katalasegebalt
ron pflanzlicber Tuaoren in Tergleich zua
Katalasegebalt gesuaden Pflanzemgeeebes.
Biochea. Zeitscbr.,
59.
60.
Klein, G., and V. Ziese.
Beitrage
zub
Cheaisaus
pflanzlicber Tuaoren.
IV. Mitt.
pflanzlicben Tuaoren.
Biochea. Zeitscbr.,
Kogl, F., and H. Srxleben.
Auxine a und b.
Wachstumsstoffe.
61.
2£4; 264-285, 1932.
Uber Peroaydaae in
Uber die Konstitution der
X. Mitteilung uber pflanzliche
Zeitscbr. physiol. Cham.,
Kogl, F., H. Erxleben, and A. J. Haagen Sait.
Phytohormon der Zellstreckung.
krystall!sierten Auxins.
Chea.,
267* 22-25, 1933.
2^6: 31-44, 1933.
227 1 51-73, 1934.
Uber ein
Zur Cheaie des
V. Mitt.
Zeitscbr. physiol.
81
62.
Kogl, F., A. J. Haagen Salt, and H. Erxleben.
XI. Hitt.
Uber ein neues Auxin (Ifateroauxln)
aus Ham.
Zeitscbr. physiol. Chen.,
228i 90-103, 1934.
63.
Kogl, F., A. J. Haagen Salt, and H. Erxleben.
XII. Hitt.
Uber den Elnflues der Auxine auf das
Wurzelwachstum und uber die chanisehe Natur des
Auxins der Graskoleoptilen.
Zeitscbr. physiol. Chen.,
228i 104-112, 1934.
64.
Kogl, F., and D. G. F. R. Kostermans.
XIII. Hitt.
Heteroauxin als Stoffwecbselprodukt niederer
pflanzlicber Organisnen.
Zeitscbr. physiol. Chan.,
66.
Isolierung aus Hefe.
226: 113-121, 1934.
Kraus, 1. J., Nellie A. Broun, and K. G. Hamer.
Histological reactions of bean plants to indoleacetic
acid.
66.
Kuster, E.
67.
Laibacb, F.
Bot. Gaz.,
98* 570-420, 1936.
Die Gallon dHr Fflanzen.
Pollenbomoo rod Vucbsstoff.
Bar. Deut. Bot. Ges.,
68.
Laibacb, F.
Laibacb, F.
30: 385-390, 1932.
Wuchsstoffversucbe nit lebenden Orcbideenpollinien.
Ber. Deut. Bot. Ges.,
69.
1911, 457 pp. Leipzig.
5^: 536—340, 1933.
fiber die AuslSsung ran Kailua - und
ffurzelbildung durcb^S -Indolylessigs&ure.
Ber. Deut. Bot. Ges.,
35: 359-364, 1933.
82.
70.
Laibach, F., and 0. Fiscbnich.
Uber eine Testaethode
zur Priifung der ka 11 ugblldanden Wirkvsag Ton
Wuchsstoffpasten. Ber. Deut. Bot. Gee.,
£3: 469-477, 1935.
71.
Laibach, F., 6. Mai, and A. Muller.
ZelTteilwgshormon.
72.
Uber ein
Haturwiss.,
Laibach, F., and £• Maeehnann.
den Orchldeenpolllnlen.
221 288, 1934.
Uber den Wuehsstoff
Jahrb. else. Bot.,
2§: 399-430, 1933.
75.
Laibach, F., A. Miller, and W. Scb&fer.
wurzelbildeade Stoffe.
75a. Lane, R.E.
Naturwies.,
Uber
22 z 588-589, 1934.
The inhibition of roots by growth hormone.
An. J. Bot.,
74. La Rue, C. D,
2£t 532-535, 1936.
Intumescences on poplar leaves.
and development.
75. La Rue, C. D.
An. J. Bot.,
I. Structure
2 0 * 1-17, 1933.
Intumescences on poplar leaves.
II. Physiological considerations.
Am. J. Bot.,
76. La Rue, C. D.
2gs 159-175, 1933.
The role of auxin in the development of
intumescences on poplar leaves? in the production of
cell outgrowths in the tunnels of leaf-niners; and
in the leaf-fall in Coleus.
Am. J. Bot., 22 * 908, 1935.
83.
77.
La Rue, C. D.
Intumescences on poplar leaves.
III. The role of plant growth hormones in their
production.
77a. Lafevre, J.
Am. J. Bot.,
25s 520-624, 1936.
Stir la presence normale d ’acides lndoliques
et particulierement de l'hcide indoleS■acetlque
dans diverse plantes superieures.
Compt. rend. Acad, sci.,
206i 1676-1677, 1938.
78.
Leonlan, L. H., and V. G. Lilly.
growth-promoting substance?
79.
Levine, M.
Is heteroauxin a
Am. J. Bot.,
A comparative cytological study of the
neoplasms of animals and plants.
Biol, and Ued.,
80.
Levine, U.
Q i 606-608, 1924.
16t 1410-1494, 1931.
Levine, 11. Crown gall-like tumors induced with scbarlach
red in the plant, Kalanchoe.
Med.,
82.
Proc. Soc. Exp.
Studies in the cytology of cancer.
Am. J. Cancer,
81.
24s 136-139, 1937.
Levine, M.
Proc. Soc. Exp. Biol, and
4gs 599-603, 1939.
Plant responses to carcinogenic agents and
growth substances; their relation to crown gall and
cancer.
83.
Bui. Torrey BOt. Club,
Levine, M., and E. Chargaff.
67s 199-226, 1940.
the response of plants to
chemical fractions of Bacterium tumefacians.
Am. J. Bpt.,
461-472, 1937.
84
84.
Link, G. K. K.
Bole of heteroauxones in legume nodule
formation, beneficial host effects of nodules, and
soil fertility.
85.
Mature,
140* 807, 1937.
Link, G. K. K., and Virginia Sggers.
Avans coleoptile
assay of ether extracts of nodules and roots of
bean, soybean, and pea.
86.
Bot. Gas.,
101* 650-657, 1940.
Link, G. K. K., Virginia Eggars, and J. E. Moulton.
Arena coleoptile assay of ether extracts of aphids
and their hosts.
87.
Bot. G&z.,
101* 928-939, 1940.
Link, G. K. K., and H. W. Wilcox.
Tumor production
by hormones from Fhytomonas tuoafaciens.
Science, 8 6 : 126-127, 1937.
88.
Link, G. K. K., Hazel W. Wilcox, and Adeline Des. Link.
Reponses of bean and tomato to Fhytomonas tumefaciens,
P. tumefaciens extracts, {£ - indoleacetic aoid, and
wounding.
89.
Bot. Gaz.,
98* 816-867, 1937.
Locke, S. B., A. J. Riker, and B. M. Duggar.
Growth
substance and the development of crown gall.
J. Agr. Res., £1* 21-39, 1938.
90.
Loeb, J.
Regeneration - From a Physicochemical Viewpoint.
New York, McGraw-Hill, 1924, 143 pp.
91.
Magnus, W.
Wtad-callus und Bakterien-Tumore.
Ber. Deut. Bot. Ges.,
36: 20
1918.
86.
92*
Manner, Dina R.
Growth of wheat seedlings in solutions
containing growth substances.
An. J. Bet.,
£i* 159-145, 1957.
95.
lieesters, A.
The influence of heteroauxin on the
growth of root hairs and roots of Agrostemna
Githago L.
Proc. Kan. Akad. Wetensch., Amsterdam,
3jj>: 91-97, 1936.
94.
Michener, H. D.
hormone.
95.
Science,
Michener, H. D.
An. J. Bot.
96.
Milovidov, P. F.
Protoplasma,
97.
Effects of ethylene on plant growth
821 551, 1955.
The action of ethylene on plant growth.
2£: 711-720, 1958.
Zur fytologle der Pflansentumoren.
Jgs 294-296, 1980.
Mitchell, J. W., and V. E. Martin.
Effect of indoleacetic
acid on growth and chemical composition of etiolated
bean plants.
98.
Molliard, M.
Bot. Gaz.,
99: 171-185, 1937.
Action hypertrophiante des produits elabores
par le Rhizobium radicicola Beyer.
Compt. rend. Acad.
sci., i£5t 1881-1555, 1912.
•(
99.
Mrkos, 0.
Uber den Einfluss des Wuchsstoffes auf die
Regeneration und Wundgewebebildung. Plants,
Q l 206-210, 1955.
86
100.
Kavez, A. E.
of roots.
e
m
e
c
,B.
. N
101
"Growth-promoting substance" and elongation
J. Gen. Physiol.,
16: 733-739, 1933.
Bakterielle Vuchsatoffe.
Ber. Deut. Bot. Ges.,
j& t 72-74, 1930.
U
b
e
rStoffe, die das
Wachstum der Avena-Koleoptile b
e
schlaunigea.
102. Nielsen, N.
Untersuchungan
Planta, £: 376-378, 1928.
103.
Nielsen, N.
Untersuchungen uber elnen aauen
wachstumsregulierendan Stoff: Rbizopln.
Jahrb. wiss. Bot.,
104.
Nielsen, N.
Sber Wuchsstoffe der Safe.
Biochem. Zeitscbr.,
105..
Noblcourt, P.
73: 125-191, 1930.
237: 244-246, 1931.
Cultures en eerie de tissue vegetauz
sur milieu artificial.
Compt. Mad * Acad, sci.,
205: 521-623, 1937.
106.
Patton, R. L., and B. R. Nebel.
Prelialnaiy observations
on physiological and cytological effects of certain
hydrocarbons on plant tissues.
Am. J. Bot.,
S it 609-613, 19*0.
107.
Biker, A. J.
Some relations of the crowngall organism
to its host tissue.
108.
Biker, A. 3.
J. Agr. Res.,
25: 119-132, 1923.
Some morphological responses of the host
tissue to the crowngall organism.
2&: 425-435, 1923.
J. Agr. Bes.,
87.
109.
Riker, A. J., end T. 0. Berge.
Atypical and
pathological multiplication of cells approached
through studies on crown gall.
Am, J. Cancer,
g > t 310-557, 1935.
110.
Riker, A. J., and R. Nagy.
Cell stimulation by
chemicals in relation to crown gall.
Phytopath.,
2§: 18-19, 1958.
111.
Robbins, W. J., and Mary A. Bartley.
growth of excised tomato roots.
Vitamin
and the
Science,
85* 246-247, 1957.
112.
Robbins, W. J., and J. R. Jackson.
Effect of 3-indole
acetic acid on cell walls of stem and root.
Am. J, Bot.,
113.
24s 83—88, 1937.
Robbins, V. J., and Mary B. Schmidt.
roots of the tomato.
114.
Robinson, W., and H. W&lkden.
crowngall.
115.
Bot. Gaz.,
Ann. Bot.,
Rumbold, Caroline.
Growth of excised
99; 671-928, 1938.
A critical study of
57t 299-524, 1923.
Pathological anatony of the injected
trunks of chestnut trees.
Proc. Am. Phil. Soc.,
ffet 485-493, 1916.
116.
Salkowski, S.
faber das Verhaltaa der Skatolcarbonslure
im Organlsmus.
23-53, 1885.
Zeitscbr. physiol. Chem.,
88.
117.
Scott, Flora M*
Anatony of auxin treated etiolated
seedlings of Pisum sativum.
Bot. Gaz.,
100i 167-186, 1958.
118.
Selby, A. 0.
Preliminary report upon diseases of the
peach. Ohio Agr. Exp. Sta. Bui.,
119.
Skoog,F., and K. 7. Thiaann.
auxin from plant tissues.
120.
Smith,
C. 0.
92: 208-217,1898.
Enzymatic liberation of
Science,
92: 64, 1940.
Further proof of the cause and
infectiousness of crown gall.
Qaiv. Calif. Agr.
Exp. Sta., Bui. 255, pp. 551-657, 1912.
121.
Smith, E.F.
Studies on the crown gall of plants.
Its relation to human cancer.
J. Cancer Bes.,
1* 251-510, 1916.
122.
Smith,
E.F.Crowngall structures showing changes in
plant structures due to a changed stimulus.
J. Agr. Bes*,
125.
Smith,
6 .: 179-182, 1916.
E.F.Babiyomas
inoculations.)
in plants.
(Produced bybacterial
Bui. Johns Hopkins Hosp.,
277—294, 1917.
124.
Smith,
E.F.Mechanism
J. Agr. Bes.,
125.
Smith,
of tumor growth in crowngall.
8 : 165-186, 1917.
E.F.BacterialDiseases of Plants.
Philadelphia, V.B. Saunders, 1920, 688 pp.
89
126.
Smith, E. F.
Production of tumors In absence of
parasites.
Arch. Dermatol, and Syphil.,
£; 176—180, 1920.
127.
Smith, E. F.
Tumors, cysts, pith-bundles, and floral
proliferations in Helianthus.
Mem.. Bat. Acad. Sci.,
£2: 1-50. 1928.
128.
Smith, E. F., Kellie A. Broun, and Lucia Me Culloch.
The structure and development of crown gall; a
plant cancer.
U. S. Dept. Agr., Bur, PI. Ind.,
Bui. 255, p. 60, 1912.
129.
Smith, E. F., Kellie A. Brown, and C. 0. Townsend.
Crown-gall of plants: its cause and remedy.
U. S. Dept. Agr., Bur. PI. Ind., Bui, 213, p. 218, 1911.
130.
Smith, E. F., and C. 0. Townsend.
bacterial origin.
131.
Snow, R.
Science,
A plant tumor of
£5: 671-673, 1907.
Experiments on growth and inhibition.
increase of inhibition with distance.
I. The
Proc. Roy. Soc.
Lond. B. 108: 209-223, 1931.
132.
Snow, R.
Mature,
133.
Snow, R.
Activation of cambial growth by pure hormones.
155: 876, 1935.
Activation of cambial growth by pure hormones.
New Fhytol.,
34: 347-360, 1935.
90
154.
Snow, R.
A second factor involved in inhibition by
auxin in shoots.
155.
Snow, R., and B. Le Fanu.
Nature,
156.
Activation of cambial growth.
Solacolu, T., and D. Constantinesco.
Stewart, W. S.
Compt. rend. Acad, sci.,
Stewart, W. S.
inhibition.
Strugger, S.
205« 457-440, 1956.
Extensibility of cell wall material
in indole-5-acetic acid.
159.
Action de
-indolylao^tique sur le diveloppement des
plantules.
158.
581 210-225, 1959.
155i 149, 1955.
l'acide
157.
New Pbytol.,
Am. J. Bot.,
25 1 525-528, 1958.
A plant growth inhibitor and plant growth
Bot. Gaz.,
101 1 91-108* 1959.
Die Beeinflussung des Vachstums und des
Geotropismus durch die lasserstoffionen.
Ber. Deut. Bot. Ges.,
140.
Strugger, S.
5Q,; 77-92, 1952.
Uber das Hachstum dekapitierter Keiapflanzen.
Ber. Deut. Bot. Ges.,
141.
195—209, 1955.
Gylwegter, E. P., and Mary C. Countryman.
a
comparative
and histological study of crowngall and wound callus
on apple.
142.
20s 528-540, 1955.
Thiaann, K. V54 On the plant hormone produced by Rhizopus
auinus.
145.
Am. J. Bot.,
J. Biol. Chem.,
Thimann, K. Vi
109s 279-291, 1955.
On the physiology of the formation of
nodules on legume roots.
22s 511-514, 1956.
Proc. Nat. Acad. Sci.,
91.
144.
Thimann,
K. V.
by auxin.
145.
On the nature of Inhibitions caused
An. J. Bot.,
24* 407-412, 1937.
Thiaann, K. V., and F. Skoog.
hormone of plants.
111.
Studies on the growth
The inhibiting action of
the growth substance on bud development.
Proc. Nat. Acad. Sci.,
146.
lg: 714-716, 1935.
Thiaann, K. V., and F. Skoog. On the inhibition of bud
development and other functions of growth substance
in Vieia faba.
147.
Tourney, J. W.
crowngall.
148.
Proc. Bey. Soc. Lond. B. 114* 517-359, 1934.
An inquiry into the cause and nature of
Ariz. Agr. Exp. Sta. Bui., 55 : 7-64, 1900.
Van der Laan, P. A.
Der Einfluss von Aethylen auf die
Wuchsstoffbildung bei Avena und Vicia.
Bot. Neerl.,
149.
Van
Bee. Trav.
690-742, 1934.
Overbeek, J. The growth hormone and the dwarftype
of growth incorn.
Proc. Nat. Acad. Sci.,
£i: 292-299. 1935.
150.
Van
Schrenk,
H.
On the production of wart-like
intumescences produced by various fungicides.
Science,
151.
Von
iZ* 263, 1903.
Schrenk,
H.
Intumescences formed as a resultof
chemical stimulation.
Missouri Bot. Gdn.,
Sixteenth Annual Report,
pp. 125-148, 1906.
92.
152.
Warne, L. G. G. Effect of spraying solutions of growth
substances on the inflorescence of the florists'
Chrysanthemum.
153.
Kent, F. W.
Nature, 140x 1065, 1937.
ti igAiantrm Betrachtungen 'uber das Auxin-Problem.
Biol. Zentralbl.,
154.
Went, F. W.
56x 449-463, 1936.
Special factors other than auxin affecting
growth and root formation.
Plant Physiol.,
55-80, 1938.
155.
Went,F« W.
The dual effect of auxin on root formation.
Am. J. Bot., 26i 24-29, 1939.
156.
Went,F. W.
in plants.
157.
Went, F. W.
A case of correlative growth inhibition
Am. J. Bot.,
2£x 505-512, 1939.
and K. V. Thimann.
Phytohormones.
New Tork, MacMillan, 1937, 294 pp.
158.
White,
P.
R.
Plant tissue cultures.The history and
present status of the problem.
Archiv.exper.
Zellforsch., 10: 501-518, 1931.
159.
White,
p.
R.
Potentially unlimited growth of excised
tomato root tips in a liquid mediw.
Plant Physiol.,
£x 585-600, 1934.
160.
White,
P.
R.
Plant tissue cultures.Bot. Rev.,
£x 419-457, 1936.
161.
White,
P.
R.
excised root tips.
Seasonal fluctuation ingrowth rates of
Plant Physiol., 12x 183-190, 1937.
93
162.
White, P. R.
Separation from yeast of materials essential
for growth of excised tomato roots.
Plant physiol.,
A&: 777-791, 1937.
165.
White, P. R.
Amino acids in the nutrition of excised
tomato roots.
164.
White, P. R.
White, P. R.
Plant Physiol.,
White, P. R.
roots.
167.
Plant Physiol.,
13: 391-598, 1938.
Glycine in the nutrition of excised tomato
Plant Physiol.,
Wolf, F. A.
12: 803-811, 1937.
Accessory salts in the nutrition of
excised tomato roots.
166.
12; 793-802, 1957.
Vitamin Bj_ in the nutrition of excised
tomato roots.
168.
Plant Physiol.,
14: 827-338, 1939.
Intumescences, with a note on mechanical
injury as a cause of their development.
J. Agr. Res.,
168.
1JS: 283-289, 1918.
Zimmerman, P. W., and F. Wilcoxon.
Several chemical
growth substances which cause initiation of roots
and other responses in plants.
Inst.,
2: 209-229, 1938.
Library
N. V. U nty.
Cont. Bcyoe Thomp.
Документ
Категория
Без категории
Просмотров
0
Размер файла
3 110 Кб
Теги
sdewsdweddes
1/--страниц
Пожаловаться на содержимое документа