LIBRARY

Michigan .591”
UM

 

This is to certify that the
thesis entitled
THE EFFECTS OF INDOLEACE'RYLAMINO ACIDS
ON CALLUS GROWTH AND SHOOT REGENERATION
IN TOBACCO AND TOMATO

presented by

Michael Douglas Peterson

has been accepted towards fulfillment
of the requirements for

Wdegree in Wild
OF SCIENCE Plant Pathology

{/ZKJWMAaM~— 6L”*EE;\‘

Major professor

Date May 15. 1978

 

0-7639

 

THE EFFECTS OF INDOLEACETYLAMINO ACIDS
ON CALLUS GROWTH AND SHOOT REGENERATION

IN TOBACCO AND TOMATO

BY

Michael Douglas Peterson

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Botany and Plant Pathology

1978

ABSTRACT
THE EFFECTS OF INDOLEACETYLAMINO ACIDS
ON CALLUS GROWTH AND SHOOT REGENERATION
IN TOBACCO AND TOMATO
by

Michael Douglas Peterson

Plant tissue cultures can continue to grow long after
the exogenous indoleacetic acid is gone from the culture
medium. Since it is known that plants conjugate exogenous
indoleacetic acid with L-aspartic acid and store the complex
in their tissues, it was thought that the continued growth
of the cultured tissues might be dependent on some stored
complex as a source of auxin. Accordingly, a number of
indoleacetylamino acids were prepared and their effects on
tissue growth determined.

Indoleacetyl-L-alanine supported growth almost as well
as free indoleacetic acid in tobacco callus tissues and was
very much more active than free indoleacetic acid in supporting
the growth of Marglobe tomato callus tissue. It was also
very much more effective than free indoleacetic acid in
initiating undifferentiated callus development from Marglobe
tomato hypocotyl sections. Consistent with this great auxin
activity was the fact that it inhibited shoot formation in

all tissues.

In contrast, indoleacetyl-D-alanine supported very
little growth of any tissue in any medium. It did, however,
ensure the survival of tissues where the absence of auxin
caused the tissues to die. This persistent survival in
the presence of a severely limited auxin activity favored
the growth of many shoots in tissues already known to be
capable of producing shoots.

Indoleacetyl-B-alanine and indoleacetylglycine had
effects which were intermediate between those of indoleacetyl-
L-alanine and indoleacetyl-D-alanine.

There is evidence that indoleacetic acid and the
indoleacetylamino acids have qualitatively different effects.
The suppression of organogenesis in favor of callus formation,
which occurs in the presence of most of the complexes cannot
be entirely due to a greater availability of free indoleacetic
acid since the addition of a small amount of free indoleacetic

acid itself along with the complex restores organogenesis.

TABLE OF CONTENTS

Page
List of Tables . . . . . . . . . . . . . . . . . . iv
List of Figures . . . . . . . . . . . . . . . . . v
Abbreviations . . . . . . . . . . . . . . . . . . vi
Introduction . . . . . . . . . . . . . . . . . . 1

Methods . . . . . . . . . . . . . . . . . . . . . 14
Results . . . . . . . . . . . . . . . . . . . . . 22

I. Effects of Indoleacetylamino Acids on

Growth of Tobacco and Tomato Cells in
Culture . . . . . . . . . . . . . . . . . 22

II. Effects of Indoleacetylamino Acids on

Regeneration of Shoots and Roots from

Tobacco and Tomato Cells in Culture . . . 32
Discussion . . . . . . . . . . . . . . . . . . . . 44
Literature Cited . . . . . . . . . . . . . . . . . 49

iii

LIST OF TABLES

Table Page

I. Biological Effects of Different Auxin
and Cytokinin Levels on Tobacco Stem
Pith Tissue O O I O O C O O O O O O O O O O 3

II. Growth of Marglobe Tomato Callus on
Solid Media . . . . . . . . . . . . . . . . 30

III. Induction of Callus from Marglobe Tomato
Hypocotyl Segments . . . . . . . . . . . . 31

IV. Shoot Production from Tobacco Callus
with two Different Kinetin Levels
and Various Auxins . . . . . . . . . . . . 35

V. Shoot Production from Tobacco Callus with
Benzyladenine and Different Concentrations
of Various Auxins . . . . . . . . . . . . 36

VI. Production of Shoots and Roots from Marglobe
Tomato Hypocotyl Segments . . . . . . . . . 38

VII. Formation of Shoots from Marglobe Tomato

"Callus" as a Function of the Auxin
Used to Initiate the "Callus" . . . . . . . 41

iv

LIST OF FIGURES

Figure Page

1. The Effects of Exogenous Indoleacetic Acid
and Indoleacetylglycine on the Growth
of Suspensions of Tobacco Cells . . . . . . 24

2. The Effects of Indoleacetyl-L-alanine,
Indoleacetyl—D-alanine and Indoleacetic
Acid on the Growth of Tobacco Pith Callus
Tissue on Solid Media . . . . . . . . . . 26

IBala
IDala
IGly

ILala
ILasp

uM

ABBREVIATIONS

2,4—dichlorophenoxyacetic acid
indoleacetic acid
indoleacetyl-B-alanine
indoleacetyl-D-alanine
indoleacetylglycine
indoleacetyl-L-alanine
indoleacetyl-L-aspartic acid

micromolar

vi

Introduction

A plant is composed of a number of cell types, each
type fulfilling a particular physical or biochemical role.
Certain cells, called parenchymatous cells, are relatively
unspecialized, which is to say they usually are not irreversibly
differentiated into cell types with a single special function.
Occupying internal regions of the plant, such as the pith and
cortex of stems, such cells constitute storage compartments
for substances the plant needs to conserve and sometimes
perhaps as storage compartments for substances the plant
needs to be rid of (9).

Parenchymatous cells, in contrast to their more differen-
tiated neighbors, can often be induced to undergo differen-
tiation as in the restoration of severed vascular connections
and in the formation of adventitious roots or adventitious
shoots. Restoration of vascular connections occurs in two
ways. When a woody plant is girdled, calluses arising from
parenchymatous cells of the bark (phloem) grow from the edges
of the wound. When the two calluses from the upper and lower
sides meet, differentiation of some of the cells into new
cambium and conducting elements takes place. On the other

hand, in herbaceous species, severing of a vascular bundle

can cause the "vascularization" of adjacent parenchymatous
cells to bridge the gap and thus reestablish unbroken
conduits (24).

When entirely new roots are required, parenchymatous
cells are induced to multiply, but in this case they differ-
entiate into the growing points of roots and therefore
ultimately into all of the many cell types Characteristic
of roots. The ease with which new roots are initiated is
very different from plant species to plant species. In
general, however, induction is assisted by the presence of
exogenous auxin, such as indolebutyric acid (13).

The production of adventitious shoots from parenchymatous
cells is often more difficult than the production of roots
and has never been accomplished in some plants. On the other
hand, shoot production from the parenchymatous cells of
leaves, stems or roots is an important method of reproduction
in some plants (27). In general, the application of cyto-
kinins favors shoot formation if shoot formation can be
induced at all. Whether roots or shoots are formed from
a given explant is often determined by the ratio of exogenous
auxin to exogenous cytokinin (22). However, if any cell
divisions at all are to occur and therefore if there is to be
any potential at all to produce either roots or shoots, a
minimum level of auxin is usually required. Therefore if the

fragment of plant does not contain any auxin-producting region,

the fragment will fail to grow and ultimately die unless

there is an exogenous auxin source. See Table I.

Table I. Biological Effects of Different Auxin and
Cytokinin Levels on Tobacco Stem Pith

Tissue (15)

Cytokinin Level

 

 

none low high
none death death death of shoots
Auxin low death slow callus shoots
Level growth
high death vigorous callus tight callus
growth, growth
perhaps some
roots

The Culturing_of Isolated Plant Cells and Plant Tissues

In the presence of exogenous auxins, especially in the
presence of stable auxins such as 2,4—D, there is a tendency
for parenchymatous cells to proliferate indefinitely as a
mass of undifferentiated, friable tissue which is similar to
wound callus and can be cultured in a sterile nutrient medium
(33). Such masses of undifferentiated tissue have many uses
and methods for initiating and maintaining them have been
widely explored. In the case of tobacco, tomato, potato,
carrot, soybean, etc., an explant of parenchymatous tissue
(often obtained from hypocotyls or from the pith of the stem
of a vegetative, i.e., not-flowering plant) is placed on a

solid agar medium containing inorganic salts, vitamins, an

auxin, a cytokinin and a carbon source.

Cultured plant tissues can be classified into two groups,
primary and secondary. Primary callus is that which has been
always grown as a block of tissue, usually on agar. In the
case of such primary cultures, it is difficult to be sure
that the tissue organization which was present in the parental
plant has been completely lost. Secondary callus is that
which has, at some point in its history experienced long-term
growth as a suspension culture in liquid medium. In this
case most of the tissue has probably been derived from a
single cell at some stage in the culturing process and, there-
fore, it is probably safe to say that all parental tissue
organization has indeed vanished.

The Regeneration of Plant Organs from Cultured Callus

 

Just as parenchymatous cells can be induced to differentiate
into vascular tissue, roots and shoots in yiyg, so too can
cultured callus sometimes be induced to produce vascular tissue,
roots and shoots in yitrg, Not surprisingly, the high con-
centrations of stable auxins which initiate callus growth from
plant fragments also favor the persistence of callus and
strongly inhibit shoot formation (17). Moreover, with callus
as with stem explants, high cytokinin levels tend to inhibit
root formation and to stimulate shoot formation (15). On
the other hand, a low cytokinin to auxin ratio tends to favor
root formation from either callus or stem explants, provided

the auxin is not so active that only callus growth results.

It has often been possible to generate roots and shoots
and thus whole plants from primary callus tissues derived
from a number of different plant species. However one cannot
be sure that new plants do not owe some of their different
cell types to residual organization carried through the
primary callus. In contrast, secondary callus has rarely
been induced to regenerate new plants and, indeed, retrieval
of plants from secondary callus has been achieved with
regularity only with carrot and tobacco (4,11). No one has
yet been able to regenerate plants from either primary or
secondary callus of most plants (29). Nevertheless, the
fact that any species at all can regenerate entire plants
from single cells means that, at least in these species,
undifferentiated parenchymatous cells (and callus cells derived
therefrom) retain all of the information required for a normal,
mature individual. Whether this "totipotency" is a general
characteristic of undifferentiated plant cells remains to be
seen. The fact that many diverse plant tissues seem to
revert to one common callus cell type implies that there
have been no irreversible changes associated with the develop-
ment of these diverse tissues. The situation is quite
different with many mammalian tissues which retain characteristic
differences when they are cultured. In the mammalian case,
differentiation does seem to have caused heritable changes
which may well be irreversible. On the other hand, we cannot

be sure on the basis of superficial appearances that there

have not also been some irreversible changes in the majority
of cultured plant tissues. The fact that it still remains
impossible to regenerate plants from the cultured callus

of most species may indicate that the cells of such calluses,
like animal tissue cultures, have undergone irreversible
heritable changes which are less apparent than in the case

of the animal tissues. At the moment, we cannot decide
between this pessimistic view and the more optimistic view
that many cultured plant cells are really "totipotent"

but that we have not yet discovered the right signals to
induce organogenesis.

. One reason for the pessimistic view is the fact that
plant tissues often undergo changes in their chromosome number
while they are being cultured. Indeed, there is considerable
evidence that cultured tissues may lose their regenerative
capacity through changes in chromosome number and other
aberrations (30).

Potential Advantages of Plant Regeneration

 

Several promising areas of research depend on our ability
to harness plant cell totipotency. They are: l) Cloning plants
from single cells; 2) Genetic transfer between species using
protoplast fusion and; 3) Mutation formation in single plant
cells and selection for desirable characteristics at the

single cell level (4).

Being able to clone plants is of great importance to the
commercial grower who wishes to produce a great number of
genetically identical individuals. Until the advent of plant
tissue culture techniques, cloning was accomplished using
highly organized tissues such as stem sections and leaves.
However, these methods might allow genetic changes in the
clone to occur and accumulate in the form of chimeras. In
fact, a number of the clones represented by our older cultivars
may be chimeral in nature. For example, the cells of the
apical meristem that give rise to the epidermis (tunica)
may be different from the cells that give rise to the internal
portions of the plant (corpus) (9). Stable chimeras normally
only occur in species where buds arise from places these two
regions of the meristem remain quite distinct and cells of
the two almost never intermingle. It is known that such
chimeras can be initiated as a result of a bud arising at
the graft union between two genetically different individuals
(28).

Culturing plant tissue fragments has in several cases,
such as the orchid, made it possible to reproduce vegetatively
plants that could not be easily propagated using other methods
(17). Such cloning usually does not involve the establishment
of an undifferentiated callus culture but rather the pro-
liferation of an organized tissue, often the apical meristem.
Virus-free stocks of plants have been established in this

manner. An explanation of this might be that apical meristems

sometimes are able to grow faster than the viral infection
can spread (22).

There is currently much interest in the possibility of
obtaining hybrids between plant species that cannot hybridize
in nature. To date, protoplast fusion with subsequent plant
regeneration has been achieved only in the case of pairs of
species that do hybridize in nature (4,16,21). Protoplast
fusion and heterokaryon formation have been accomplished
between many other more distantly related species, but no
plants have been regenerated from such fused cells (6).
Experience with somatic animal cell hybrids indicates that,
at least in fusions between cells of very distantly related
species such heterokaryons are likely to be unstable. Usually
part or all of one of the genomes is excluded. However there
is reason to believe that, in cases where there seems to have
been a complete loss of one genome by cytological criteria,
there may be in fact a retention of some of the genetic infor-
mation from that genome. For instance, in one example, an
enzyme coded for by the apparently missing chromosome comple-
ment continued to be produced (7). Thus there is reason to
hope that it may some day be possible to transfer genes
between widely different species which certainly could never
cross using the regular sexual process and probably could not
make successful heterokaryons.

The considerable advances of mutagenesis and screening

techniques in microorganisms in recent years are beginning

to be applied to single plant cells (4). Single cells are
better suited than are whole plants for the recovery of some
desired mutant types for several reasons. Whole plants are
large organisms with long generation times; the large nutrient
pools make difficulties in assessing biochemical mutations
since failure to synthesize substances may become apparent
only after some time; whole plants are composed of cells in
many differentiated states; they are diploid, or often,
polyploid, and again this presents grave problems in the
analysis of mutations. In contrast, single haploid cells
are small with short generation times and small nutrient
pools; they are usually composed of uniform cells in an
undifferentiated state; genetically there is one copy for
each trait, so that recessive traits can be expressed.

Cell lines with disease resistance (12), cell lines
that have herbicide tolerance (19), cell lines that over
or underproduce certain amino acids (20), and cell lines
that are tolerant of high levels of different elements (8)
have already been produced. However only in tobacco has
a trait selected at the single cell level been expressed
on the whole plant level (3). Unfortunately, a major draw—
back to the application of all single cell techniques in the
selection of desirable plant characteristics is that we are
usually unable to distinguish these desirable characteristics

in their single cell manifestations.

10

Auxins in Plant Tissue Culture

 

Currently 2,4-dichlor0phenoxyacetic acid (2,4-D) and
indoleacetic acid (IAA) are the most widely used forms of
auxin for the growth of tissue cultures. Of the two, 2,4-D
is the more persistent in tissue culture media and it is pro-
bably for this reason that it seems to be about 100 times
as active as indoleacetic acid in the tobacco callus growth
bioassay. Since it is often necessary to incubate plant
fragments on a culture medium containing a high level of
auxin activity to induce friable callus formation, 2,4-D
has sometimes been used routinely as a constituent of callus
induction media, especially when the plant species being
investigated does not respond well to the more labile IAA
(29). As expected from the fact that it is more active
in supporting callus growth than IAA, it is easy to show
that 2,4-D is much more inhibitory to shoot formation than
is indoleacetic acid. Furthermore, friable callus initiated
using 2,4-D frequently cannot be induced to produce shoots,
even after it has been transferred for some time to IAA.
Perhaps in providing enough auxin activity to induce callus
we have been providing too much auxin for retention of an
organogenic potential.

In many cultured tissues that do form shoots, a very
low level of indoleacetic acid is either required for or

greatly stimulatory to shoot production (26). Usually a

11

certain auxin to cytokinin ratio, rather than the auxin
level per se promotes the most bud formation. But might
not the concentration of indoleacetic acid that is optimal
for shoot induction be somewhat less than the concentration
of IAA initially present in the culture medium. This would
be true if the initial concentration needs to be high in
order to allow for the rapid disappearance of IAA. That is
to say, the rapid disappearance of auxin may make it necessary
to apply an ititial level which produces a long-term inhibition
of shoot production. Thus the situation with indoleacetic
acid in tissue cultures may be analogous to the situation
which we suspect may exist in 2,4-D-initiated callus, that is,
in order to provide enough auxin activity for the growth
of parenchymatous cells, it may be necessary to supply too
much auxin activity for shoot induction. Therefore it seemed
possible that the presence of a large reservoir of a stable
but relatively inactive auxin or auxin precursor might permit
cell multiplication without inhibiting bud formation.

Whole plants may be regulating internal levels of free
indoleacetic acid in this way, by the reversible formation
of auxin complexes which in turn may or may not have some
auxin activity in their own right. Free IAA does not seem
to occur in plants except in extremely low concentrations.
Most of the IAA is bound, presumably either by rather stable
amide linkages or by more labile ester linkages. The best

characterized of these IAA complexes are various naturally

12

occurring isomers of indoleacetyl-mesg-inositol (2). When
plant tissues are supplied with generous amounts of indole-
acetic acid, the IAA is taken up and converted into indole-
acetyl-L-aspartic acid, indoleacetyl-l-glucose and into

much smaller smounts of other complexes (1,25,31). The
laboratories of Hamilton (10) and Rekoslavskaya (23) have
shown that these stored forms of auxin may be used to support
the growth of tissue cultures.

The fact that auxin-dependent tissues will often undergo
several mitotic divisions in the absence of exogenous IAA
before ceasing to grow suggests that the indoleacetic acid
initially presented to the cultures may have been taken up
and stored as IAA complexes. We say "as complexes" rather
than "as free IAA" because it has been shown that the uptake
of exogenous indoleacetic acid rarely results in any accum-
mulation of free IAA in the tissues (1). If an appreciable
amount of the exogenous IAA is stored as complexes in the
tissues, it would indeed seem to be serving as a reservoir
for the slow release of an auxin, hence the continued growth.

My research dealt with how well various indoleacetic
acid-amino acid complexes could serve as exogenous sources
of auxin for plant tissue cultures. This work was especially
concerned with the development of improved regeneration of

whole plants from callus.

13

Summary of the Rationale for this Research

 

Indoleacetylamino acid complexes might have the double
advantage of constituting a long lasting source of a stable
but low auxin activity, thus perhaps preserving organogenic

potential while permitting continued cell division.

I.

Cell Culture Medium.

A.

Basal medium:

medium in Murashige and Skoog, 1962:

Constituent

 

KNO3

NH4NO3
MgSO4'7H20
CaC12°2HZO
KH2PO4
MnSO4°H20
H3BO3
ZnSO4°7H20
NaMoO4'2H20
CuSO4'5H20
CoC12°6H20
KI
FeSO4'7H20
NaZEDTA°2H20
Thiamine HCl
Myo-inositol
Sucrose

Agar

Methods

14

slightly revised version of the

 

Concentration

(mg/l) (Molar)
1900 1.88 x 10'2
1650 2.06 x 10‘2
370 1.50 x 10’3
440 2.99 x 10’3
170 1.25 x 10’3
16.8 9.94 x 10"5
6.2 1.00 x 10’4
10.6 3.69 x 10'5
0.25 1.14 x 10'6
0.025 1.57 x 10’7
0.025 1.05 x 10'7
0.83 5.00 x 10’6
27.8 1.00 x 10'4
37.3 1.00 x 10’4
1.0 2.96 x 10‘6
100 5.55 x 10’4
30,000 8.76 x 10‘2
9000

II.

15

B. Preparation of media:

1.

Tissues.

Before autoclaving, all media were adjusted

to pH 6.0 with KOH.

When agar was used,sucrose was autoclaved
separately from the other constituents of the
medium.

Volumes of media greater than or equal to 250 mls
were autoclaved for 15 minutes at a steam pressure
of 15 lbs. Volumes less than 250 mls were auto-
claved for 12 minutes.

No constituents were filter sterilized.

Auxins and cytokinins were added to the media
before autoclaving.

Culture media were stored at room temperature

in the dark.

A. Tobacco cultivar "Wisconsin 38":

l.

Callus which had always been maintained on
solid medium: a one year old pith culture
initiated and grown in the dark at 27 i 1°C
on solid basal medium to which were added

17 uM indoleacetic acid and 1.4 uM kinetin.

l6

Callus which had been through a liquid
suspension phase: a one year old pith culture,
initiated and grown in the dark at 27 i 1°C

on solid basal medium to which were added

17 uM indoleacetic acid and 1.4 uM kinetin;
then transferred, after the establishment of
rapidly growing callus, to liquid basal medium
also containing 17 uM indoleacetic acid and

1.4 uM kinetin and grown in the dark.

B. Tomato cultivar "Marglobe":

l.

Hypocotyl sections from seedlings, grown
sterilely in the dark at 27 i 1°C for six

days on solid basal medium plus 1 mg per liter
each of nicotinic acid (8.12 x 10'6 M) and
pyridoxine HCl (4.86 x 10'6 M).

Callus which had been through a liquid suspension
phase: a two year old culture derived from
anthers; callus initiated on solid basal medium
to which were added 1 mg per liter each of
nicotinic acid and pyridoxine HCl, 29 uM
indoleacetic acid, 2.3 uM 2,4-D and 1.4 uM
kinetin. After initiation of vigorous growth,
callus was transferred to a liquid medium with
the same constituents. After about one year

as a suspension culture, growth was continued

for another year on a liquid medium from which

17

the 2,4-D had been omitted but which contained

all the other constituents.

III. Growth conditions during experiments.

A. Suspension cultures were grown in the dark at

27

1°C in 50 mls of culture medium in 125 m1

Erlenmeyer flasks with foam stoppers. Flasks

were shaken at 125 revolutions per minute on

a rotary shaker.

B. Callus cultures:

1.

Calluses were grown in the light at 22 i 1°C
on a 16 hr light- 8 hr dark cycle under GE
Delux cool-white fluorescent tubes at a dis-
tance to give 550-750 lux of illumination.
Calluses were grown in the dark in controlled-

temperature incubators at 27 i 1°C.

IV. Biological variables studies.

A. Growth:

1.

Growth of callus was recorded as (final fresh
weight-initial fresh weight)/initial weight

or as increase in volume/initial volume.
Growth of cell suspensions was recorded as

the settled volume of cells after settling

for 30 minutes at one times gravity. Settled
volumes were measured using graduated conical—

bottom 50 m1 test tubes with screw caps.

18

B. Organogenesis:

l. "Shoots" were defined as centers from which
at least one green, leaf-like structure
emanated, distinguishable at 6x magnification.

2. The roots counted were those arising directly
from the callus, as descernable at 6x magni-
fication. In this procedure, lateral roots
were not counted.

V. Syntheses of IAA-amino acid complexes.

The complexes of indoleacetic acid with glycine, D-alanine,
L-alanine and B-alanine were prepared in the following manner
which is a modification of the method of Weiland and HOrlein
(32). The reaction sequence is shown below. Ten millimoles
of IAA were dissolved at 0°C with stirring in 30 m1 of tetra-
hydrofuran plus 1.4 ml of triethylamine (10 millimoles). Over
a period of 30 seconds 0.96 ml of ethylchloroformate (10 milli-
moles) were added dropwise with rapid stirring (I). After
another 5 minutes of stirring at 0°C, 10 millimoles of the
sodium salt of the amino acid were added in 8 m1 of H20.

The mixture was allowed to warm to room temperature with

continuous stirring (II).

19

 

o .0
fl fl +
1. R-C\OH + N(C2H5)3 -————» R-c\0_ + HN(C2H5)3
o 0
H + h
2. R-c\0_ + HN(C2H5)3 + fC-OCZH5 ——————+
C
(C2H5)3 NHCl + R-fi-O-S-O-CZHS
o
o o I
n n - +
3. R-C-O-C-O-C2H5 + HZN-CH(R')-C02 Na 4
o
n _ +
___ I
R C NH CH(R ) co2 Na + CO2 + CZHSOH

II

R = indole

R' = side chain of the amino acid

The aqueous lower phase was then adjusted to pH 3.6 with

HCl and extracted 3 times with 25 ml of chloroform in order

to extract any unreacted indoleacetic acid. The pooled
chloroform phases were extracted twice with 25 ml water

to remove any product carried over into the chloroform and
these washings were back-extracted once with 20 ml chloroform.
The chloroform washed original aqueous phase and the aqueous
washings of the chloroform washings were combined and acidified
to pH 2.9 with HCl in order to convert the product into its
non-polar protonated form. The acidified combined aqueous
phases were then extracted 3 times with 25 m1 of l-butanol.
The pooled butanol extracts were washed once with 50 m1 of

water. The water wash was back-extracted twice with 5 ml

20

of 1-butanol. The butanol phases were pooled and the butanol
was removed by distillation at reduced pressure (about 15 mm Hg).
The viscous residue was dissolved in approximately 15 ml of

50% 2-propanol in water (v/v). The crude product was then
passed over a Sephadex LH-20 column, 4 cm x 30 cm, using more
50% aqueous 2-propanol as the solvent. The products were

eluted after about 450 to 500 m1 of solvent had passed over

the column. The residual unreacted IAA was not eluted until

630 to 670 ml had passed. The three 20 ml fractions containing
most of the product were pooled and concentrated by distillation
at reduced pressure (about 15 mm Hg). The products were then
crystallized from ethylacetate by adding hexane. Note,

however, that the indoleacetylglycine crystallized from the
water as the 2-propanol was removed by distillation.

At all stages in these operations the distributions of
the product and the unreacted IAA were followed by thin layer
chromatography of the solvent phases and location of the
indoles on the chromatogram using the Ehrlich indole reagent.
Yields in most cases were somewhat under 40%.

Indoleacetic acid was obtained from Aldrich Chemical
Company, 940 West Saint Paul Avenue, Milwaukee, Wisconsin
53223. Ethylchloroformate and B-alanine were obtained from
Eastman Organic Chemicals, 1187 Ridge Road West, Rochester,

New York 14650. Glycine, D-alanine and L-alanine were
obtained from Sigma Chemical Company, P.O. Box 14508, Saint

Louis, Missouri 63178. Already prepared indoleacetyl—L-

21

aspartic acid was obtained as the dicyclohexylammonium salt

from Calbiochem, P.O. Box 12087, San Diego, California 92112.

Results

The D—alanine, L-alanine, B—alanine, glycine and
L-aspartic acid complexes of indoleacetic acid were used
as auxin sources in growth and regeneration experiments with
tissue cultures from tobacco and tomato. Some experiments
in which there was no organogenesis provided data on growth
of callus. In those experiments where "shoots" and roots
appeared in addition to callus, measurements of growth
included contributions of mass made by organized tissues.
Probably in some cases the organized tissues themselves
contributed endogenous hormones which permitted further
growth of callus and therefore the factors contributing
to organogenesis cannot always be separated clearly from
the factors contributing to callus growth.

I. Effects of Indoleacetylamino Acids on Growth of Tobacco
and Tomato Cells in Culture

 

 

A. Effects of indoleacetylglycine and indoleacetic acid
on the growth of suspensions of tobacco cells.

Indoleacetylglycine persists for much longer in the
suspension medium than does indoleacetic acid itself. One
week after the beginning of the experiment, when indoleacetic
acid had long since disappeared from the medium, about 30%

of the indoleacetylglycine remained, as detected by the

22

23

Ehrlich indole reagent on thin layer chromatograms of
chloroform extracts of the media. Nevertheless, the total
amount of growth supported by IAA is about the same as that
supported by indoleacetylglycine at all concentrations tested,
down to less than 1 micromolar.

As shown in Figure 1, there seems to be a considerable
carry-over of auxin from the inoculum since growth persisted
in these auxin-dependent cells for 8 days even in the absence
of exogenous auxin. Indoleacetylglycine at the concentration
used supported the same amount of growth as did the slightly
lower concentration of IAA. Moreover, indoleacetylglycine
did not inhibit growth for the first few days as IAA always
did at almost any concentration (down to 0.6 uM). In fact,
indoleacetylglycine did not have this inhibitory effect at
any concentration tested (up to 10 uM).

The persistence of growth in the absence of any exogenous
auxin should be noted. As was pointed out in the introduction,
this phenomenon suggests that auxin is somehow stored in the
cells, and that the stored auxin is capable of supplying all
of the auxin needs of the cells for many days.

B. Effects of indoleacetyl-L-alanine, indoleacetyl—D-
alanine and indoleacetic acid on the growth of
tobacco pith callus tissue on solid media.

Figure 2 shows some of the effects of maintaining tobacco
tissue cultures on indoleacetylamino acids through several
passages. Indoleacetyl-D-alanine had very little effect on

either callus growth or "shoot" initiation except at the

24

Figure l. The effects of exogenous indoleacetic acid and
indoleacetylglycine on the growth of suspensions of tobacco
cells. Growth was measured at two day intervals as milli-
liters of settled cells after 30 minutes at 1 x g. The
cells were taken from basal liquid medium contain 1? uM

IAA and 1.4 uM kinetin and grown for two days on a similar
medium containing no indoleacetic acid. The cells were
then transferred to the test media, all of which contained
1.4 uM kinetin. Although these cells had an absolute re-
quirement for an auxin and would have ultimately died in
the medium lacking indoleacetic acid, they continued to
grow well for a total of 10 days after their removal from
an external source of auxin. This implies that the tissues
were able to store indoleacetic acid or a product of indole-

acetic acid which served for a time as an internal auxin source.

 

cell volume

in millililers ol sellled

Grow”:

50

A.
0

w
0

N
O

O

 

q

25

4 quM

. no auxin

 

 

 

4
Time in

IAA Glycine
IAA

J
6

days

26

Figure 2. The effects of indoleacetyl-L-alanine, indoleacetyl-
D-alanine and indoleacetic acid on the growth of tobacco pith
callus tissue on solid media. Five callus inocula, each
weighing about 60 mg, were placed in a 10 cm petri plate con—
taining basal medium plus 1.4 uM kinetin and the indicated
auxin. Incubation was in the dark for 4 weeks, whereupon one
piece of carefully selected undifferentiated callus from each
block of tissue was sub-cultured for another 4 weeks on the
same medium as the parent block. The initial callus was
sub-cultured in this manner 2 or 3 times giving a total of

3 or 4 "passages". At the end of each passage the callus
block the small piece used for transfer, was weighed. Growth
data represent increase in fresh weight/initial weight.
Between 4 and 10 plates were used per treatment. Numbers
before the names of the various auxins are concentrations
(micromolar). The numbers in brackets indicate the number

of "shoots" per plate arising as a result of each passage.
Standard deviations among the weights of the blocks grown

on 17 uM IAA are shown in the upper part of Figure 2a.

Means of growth on 1? uM IAA for the 4 passages were

35.2, 38.6, 36.9 and 30.3.

 

 

7
2

.6. a _
6...... a 72...
as. A
.va ..
6.6.: t 1
+6.62 3

  

 

5: t. .w
.23. 2

.614. _. .N

m _
m.

 

   

0.0... D
+0.00. u—

<<. n—
+ 0.00. n—

 

.

0.00. h—

 

 

..an 0: 00030;

4 m cm. .nw.

       
    

O
(V

O
V

O
'O

0.0... A.

o
co
WI uo"q;mo:6 go abortion“! :0 qgnwg

O
O

as

<<. n. A

28

highest concentration where there was a slight stimulation
of callus growth without any decrease in the number of
"shoots" formed. Both indoleacetyl-L-alanine and IAA
itself supported vigorous callus growth and suppressed all
"shoot" formation. The D-alanine complex had very little
effect on the growth without organogenesis when it was
combined with either the L-alanine complex or with IAA.
The high auxin activity of the L-isomer and the very low
auxin activity of the D-isomer is consistent with the idea
that these complexes are serving as sources of IAA after
their hydrolysis by the cell's peptidases which are likely
to be almost entirely specific for the L-isomer.

On the other hand, as we shall see, there is some
reason to believe that these complexes may be auxins in
their own right. If so, hormone receptor sites must also
be specific for L-isomers.

C. Growth of Marglobe tomato callus on solid media.

Tomato tissue, unlike tobacco tissue, seems to lose
the capacity to produce roots and shoots in culture soon
after callus growth is initiated. We wondered whether
the responses of tomato tissues to indoleacetic acid and
its amino acid complexes might differ depending on the
level of organization of the tissues used. For this
reason tomato cell suspensions and tomato stem hypocotyls,

representing a very wide range of organization, were grown

29

on these various auxins.

The tomato cell suspension, totally lacking the ability
to form any organized structures, was tested for growth on
agar. See Table II. Interestingly, in this case, the
L-alanine, glycine and B-alanine complexes all supported
more growth at every concentration than did IAA. In fact,
the L-alanine complex supported more growth at the lowest
concentration than IAA did at the highest. There were
no marked synergisms and no marked inhibitions by excess
auxin even in the case of the L-alanine complex where the
highest concentration (57 uM) gave almost the same growth
as the lowest concentration (0.57 uM). The D-alanine complex
supported very little growth at low concentrations but at
57 uM it was almost as good as IAA.

The media used in Table II were also used to study the
responses of hypocotyl sections. See Table III. All of
the complexes except indoleacetyl-D-alanine were responsible
for initiation of unorganized, fast-growing callus. Roots
and "shoots" were produced in many of the treatments and
these data are reported in Table VI. The D-alanine complex
and IAA itself did not support the development of much
callus but instead supported the growth of "shoots" or
roots. In general there was a good correspondense between
callus initiation in the hypocotyl sections and growth of

callus from the suspended cells.

30

Table II. Growth of Marglobe Tomato Callus on Solid Media

Suspended cells (about 0.5 g fresh weight) were
removed from liquid medium by means of a mesh scoop and
inoculated onto 6 cm petri plates containing basal medium
to which were added 1 mg per liter each of nicotinic acid
and pyridoxine HCl, benzyladenine (8.9 uM) and the indicated
concentrations of indoleacetic acid or indoleacetic acid-
amino acid complexes. Data represent increase in fresh
weight/initial weight after 37 days growth in the light.
Weight after 37 days in the absence of any auxin was
arbitrarily chosen to represent the initial weight.

Callus Growth
(fresh weight gain/Initial weight)

 

 

Kind of Auxin 57 uM auxin 5.7 uM auxin 0.57 uM auxin

IAA 13.1 6.4 1.6

IDala 11.2 0.74 0.49

IDala + IAA 11.1 3.7 0.23
9 : l

ILala 15.2 17.1 14.1

ILala + IAA 14.0 15.4 12.3
9 : l

IGly 15.3 13.8 6.6

IGly + IAA 15.0 15.4 7.7

9 : l
IBala 13.5 17.8 2.5
IBala + IAA 15.2 12.8 2.2

9 : l

31

Table III. Induction of Callus from Marglobe Tomato
Hypocotyl Segments

Data taken from the experiment also reported in Table VI.
Sterile hypocotyls from seedlings grown 6 days in the dark
were cut into sections 0.5 cm long and placed on a solid basal
medium to which were added 1 mg per liter each of nicotinic
acid and pyridoxine HCl, benzyladenine (8.9 uM) and the
indicated concentrations of indoleacetic acid or indoleacetic
acid-amino acid complexes. There were 6 replicates, 3 on
each of two 6 cm plates. Callus formation was assessed
after 53 days growth in the light. At this time, 3
calluses from each treatment were photographed and paper
tracings of the photographs were cut out and weighed.

The weights were taken to the 3/2 power as estimates of
tissue volume, and the average volumes were calculated.
Data represent increase in volume/initial volume. Volume
after 53 days in the absence of any source of auxin was
arbitrarily chosen to represent the initial volume, since
the hypocotyl sections denied auxin failed to grow at all
and died.

Callus Initiation
(volume gain/initial volume)

 

 

Kind of Auxin 57 uM auxin 5.7 uM auxin 0.57 uM auxin

IAA 29.3 26.1 3.6

IDala 3.8 3.8 6.2

IDala + IAA 10.0 4.5 4.4
9 : l

ILala 69.9 67.5 15.2

ILala + IAA 67.0 75.6 18.8
9 : 1

IGly 75.1 69.4 0.86

IGly + IAA 96.0 75.5 1.8

9 : l
IBala 85.6 9.3 0.37
IBala + IAA 82.7 50.8 2.5

9 : l

32

II. Effects of Indoleacetylamino Acids on Regeneration
of "Shoots" and Roots from Tobacco and Tomato Cells
in Culture

 

 

 

The use of auxins in tissue culture is usually thought
of in terms of the support of growth of undifferentiated cells.
Nonetheless, auxins are often necessary components of media
designed to induce "shoot" formation from callus. Because
these tissues are not able to make their own auxin until the
appearance of differentiated structures which can serve as
sources of hormones, a low level of auxin is required to
keep the cells alive. One of the difficulties in achieving
regeneration of plants from cultured plant tissues may be
the problem of supplying the tissue with auxin over the long
periods of growth which are often necessary to induce "shoot"
formation. IAA would not seem to be a satisfactory auxin
source for regeneration experiments because of its rapid
disappearance from the culture medium. The persistence of
the indoleacetylamino acids and the long-term auxin activity
associated with this persistence would seem to make them
more useful than IAA in attempts to provide the stable very
low levels of auxin which might be expected to help in the
initiation of "shoots".

In order for an IAA complex to be useful in regeneration
experiments, IAA must not be released from the complex at a
rate that is inhibitory to "shoot" formation. One might
expect the organogenic capacity of cultured tissues to be

more suppressed by those IAA complexes that have high activity

33

in supporting growth, than by those IAA complexes that have

a limited auxin activity in this regard. One might also
expect that the activity of the complexes in supporting growth
would be related to the ease with which they can be meta-
bolically converted into free IAA. For instance, the L-isomer
of the alanine complex should be much more active in supporting
growth than the D-isomer and as we have seen, such is the
case. This argument implies, of course, that the complex
serves as an auxin only by breakdown to IAA. However, it is
difficult to understand the behavior of IAA itself in either
growth or regeneration experiments because the auxin activity
of IAA relative to the IAA complexes seems to vary from one
plant species to another and, as we shall see later on, from
one kind of medium to another.

A. Effects of indoleacetylamino acids on the regeneration
of "shoots" from tobacco callus

When tobacco tissues were grown on a kinetin-containing
medium lacking nicotinic acid and pyridoxine HCl, "shoot"
formation was inhibited most by IAA itself. See Table IV.

All IAA complexes inhibited "shoot" formation less than did

IAA. The glycine and L-alanine complexes, which supported

good cell growth (Fig. l and 2), inhibited "shoot" initiation
more than did the D-alanine complex, which only weakly supported
callus growth. Indoleacetyl-D—alanine strongly promoted "shoot"
formation even at the highest levels. It was better for this

purpose than the other complexes, or than indoleacetic acid

34

itself, at any concentration tested. It was also better
than no auxin at all.

The effects of the various auxin complexes on the
regeneration of "shoots" from tobacco callus were investigated
in the presence of a different cytokinin, benzyladenine,
nicotinic acid and pyridoxine HCl. Results are shown in
Table V. In this experiment, the inhibitions of "shoot"
formation by indoleacetyl-L-alanine, indoleacetylglycine
and indoleacetyl—D-alanine were similar to the inhibitions
of "shoot" formation by these complexes in the previously
used media. In addition, the effects of two other complexes,
indoleacetyl-L-aspartic acid and indoleacetyl-B-alanine
were studied. Indoleacetyl-L-aspartic acid caused about
the same degree of "shoot" initiation as did indoleacetyl-D-
alanine, while indoleacetyl-B-alanine inhibited "shoot"
formation about as much as did indoleacetylglycine.

Note that indoleacetyl-D-alanine and indoleacetyl-L-
aspartic acid caused the initiation of more "shoots" than
did any of the other complexes, and more than occured in the
absence of any exogenous auxin, but not more than did
indoleacetic acid itself. In contrast to the earlier results
with a different medium (see Table IV), indoleacetic acid
promoted "shoot" formation at concentrations of up to 57 uM.
Perhaps this inability of IAA to inhibit "shoot" formation

is causally related to the inability of IAA to initiate and

35

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37

support growth of unorganized callus at low concentrations
in tomato (See Table III), since in the tomato experiments,
the same benzyladenine-nicotinic acid—pyridoxine HCl medium
was used.
B. Effects of indoleacetylamino acids on the
regeneration of "shoots" from tomato stem
hypocotyl tissue and in the callus derived
therefrom
One of the prime aims of this research was to explore the
possible uses of the amino acid complexes of indoleacetic
acid in helping to bring about the regeneration of plants
from undifferentiated callus. Since we had no way of knowing
the organogenic potentials of already established homogeneous
callus cultures, we decided to measure the effects of the
various auxin sources on organogenesis at various stages in
the transformation of differentiated hypocotyl tissues into
relatively undifferentiated callus. And, since we had already
investigated the effects on established tobacco cultures,
we decided to study organgenesis in another species where
it is more difficult to achieve, namely in tomato. We looked
at the effects of the auxin complexes both on the maintainance
of organogenic potential and on the expression of this
potential in the form of "shoots" and roots.
As shown in Table VI, those IAA complexes which favored
undifferentiated callus formation from the hypocotyl sections

also tended to suppress "shoot" and root formation. The

better the callus growth, the stronger was the inhibition

38

Table VI. Production of "Shoots" and Roots from Marglobe
Tomato Hypocotyl Segments

Data from the experiment reported also in Table III,
quod vide. Numbers of "shoots" and roots were recorded after
53 days. Numbers to the left of the slash refer to "shoots"
and numbers to the right refer to roots. Unbracketed numbers
record the individual "shoots" or roots, while bracketed
numbers record hypocotyl segments giving rise to at least
one "shoot" or one root.

 

"Shoots" or Roots Formed by 6 Hypocotyl Sections

Total Auxin
Concentration
(micromolar) 57 5.7 0.57 0

Kind of Auxin

 

 

IAA 0/50(6) 7(3)/31(5) 10(3)/0 no growth
of any kind
IDala 7(1)/0 l(l)/0 23(2)/0 "
IDala + IAA 10(3)/0 8(3)/5(1) 8(2)/0 "
9 : 1
ILala 0/0 0/0 0/l(l) "
ILala + IAA 0/2(1) 0/3(3) 0/0 "
9 : l
IGly 0/3(2) 0/0 l(l)/0 "
IGly + IAA 0/0 0/5(2) 6(2)/0 "
9 : l
IBala 0/0 0/0 0/0 "

IBala + IAA 0/2(1) 6(2)/11(2) l(1)/0 "

39

of "shoots" and roots whereas the better the growth of "shoots"
and roots, the worse the growth of callus. In the absence

of any source of auxin, there was no growth at all and the
hypocotyl segments eventually died. As expected (18), low
concentrations of IAA caused "shoot" formation and high
concentrations of IAA caused root formation; there was also
considerable growth of a not very friable kind of callus at
high levels of IAA. At all concentrations tested, indole-
acetyl-D-alanine maintained the sections in a healthy con-
dition and allowed the segments to produce "shoots", but no
roots and no callus until "shoots" were initiated; many
sections failed to produce either organs or callus and failed
to grow although they seemed to remain healthy. In contrast,
indoleacetyl-L-alanine supported good callus growth at all
concentrations and organogenesis was almost entirely abolished.
Indoleacetyl-B-alanine suppressed all organogenesis and also
allowed the sections to produce large amounts of callus at
higher concentrations. Interestingly, a small amount of IAA
in the presence of larger amounts of indoleacetyl-B-alanine
restored the organogenic potential which the B-alanine complex
alone seems to have abolished. Indoleacetyglycine, like the
B-alanine complex, supported massive callus growth at the
higher concentrations but did not abolish organogenesis quite

so effectively.

4O

Callus tissues which had been initiated from the
hypocotyl sections on media containing IAA, indoleacetyl—L-
alanine or a combination of these were subcultured on media
which had promoted "shoot" formation from the original
hypocotyl sections (See Table VII). In general, tissues
taken from hypocotyls which had produced no organs at all
in the first passage, seemed to have lost organogenic
potential in that they produced no organs even when they
were transferred to media which had allowed organogenesis
in the first passage. On the other hand, whenever the
medium used in the first passage supported any organogenesis
at all, the "callus" derived therefrom always retained
organogenic potential. In other words, the degree of
organization in the first passage played a large role in
organogenesis in the second passage. Thus, there was a
good deal of cryptic organization remaining in the first
generation callus grown on indoleacetyl-L-alanine, but
only if the complex was augmented with a low concentration of
indoleacetic acid. This residual organization was very
marked when IAA was the sole auxin. Indoleacetyl-L—alanine
seemed to abolish organization in the first generation and
in so doing to make organogenesis very difficult in the
second generation. This observation has two profound
implications. First, it would seem that indoleacetyl-L-alanine
and indoleacetic acid are somehow doing different things since

the observed results could not have been observed if the

41

Table VII. Formation of "Shoots" from Marglobe Tomato
"Callus" as a Function of the Auxin Used to
Initiate the "Callus"

"First generation" refers to tissues growing from the
original hypocotyl sections (see Table III). "Second
generation" refers to tissues growing from selected relatively
undifferentiated regions of the first generation tissue. For
the second generation studies, pieces of tissue about 80 mg
in size, which had been initiated from hypocotyl sections
on media containing the indicated first generation auxin source,
were transferred to a second generation medium containing the
indicated second auxin source. Five pieces were used per
second generation treatment. Both the first and second
generation media contained 8.9 uM benzyladenine and 1 mg
per liter each of nicotinic acid and pyridoxine HCl. "Shoots"
in the second generation were recorded after 53 days growth
in the light. Unbracketed numbers refer to total "shoots",
while bracketed numbers refer to pieces producing at least
one "shoot".

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42

 

 

 

 

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43

complex was simply serving as a source of IAA. Second,
the observation points up the fact that "callus" tissues
can have very different regenerative capacities depending

on the hormones used in their initiation.

DISCUSS ION

The purpose of the research in this thesis was to explore
whether indoleacetylamino acids could act as auxin sources in
growth and regeneration experiments with cultured plant tissues.
The specific aims were two. The first was to find an auxin
source that did not suppress organogenic potential as much as
did 2,4-D. The second was to find an auxin that would persist
in tissue culture media so as to provide a continuous low
level of indoleacetic acid to tissues when the production
of shoots was desired. Both these aims were based on the
hypothesis that the indoleacetylamino acids would act as bio-
logically inert storage forms of indoleacetic acid. An early
experiment showed that free indoleacetic acid does not remain
in the medium in the presence of tissues, while indole-
acetylglycine does remain. It was our thought that indoleacetic
acid in complexed form might be safe from oxidative enzymes
secreted by the tissues, since it had already been shown that
all of the complexes used in our experiments were insensitive
to horseradish peroxidase, under conditions where indole-
acetic acid is extremely labile (5).

The auxin activity of indoleacetic acids may be due
to two things: the ease with which they can enter the cell,

and the susceptibility of the peptide bond to cleavage by

44

45

peptidases. Indoleacetyl-L-alanine, indoleacetylglycine
and indoleacetyl-B-alanine appear to be available sources
of auxin, that is to say, they support, to varying degrees,
unorganized callus growth in both tobacco and tomato which
usually depends on a good supply of an active auxin. The
other auxins which were tested, indoleacetyl-D-alanine and
indoleacetyl-L-aspartic acid do not seem to be very good
sources of available indoleacetic acid. However the fact
that they often promoted "shoot" production better than no
auxin shows that they do have some auxin activity even at
concentrations less than micromolar.

It may be useful to introduce here the concepts of a
complex's auxin activity relative to indoleacetic acid.
Relative auxin effect is defined as: the concentration of
indoleacetic acid that is optimal in a particular bioassay/
the concentration of indoleacetylamino acid that is optimal
in the same bioassay. A relative auxin effect of less than
one means that the auxin complex has a lower auxin activity
than indoleacetic acid. A relative auxin effect of greater
than one means that the auxin has a greater auxin activity
than indoleacetic acid.

In tobacco tissues, when kinetin was the cytokinin, all
complexes had relative auxin effects of less than one in the
callus growth and "shoot" production bioassays. (It should
be remembered that, the more stimulatory an auxin is to callus

growth, the more inhibitory it becomes to "shoot" formation.)

46

Tobacco callus grew best on indoleacetic acid of all the
auxins tested. As expected, "shoot" production was inhibited
most by indoleacetic acid. The order of auxin activities of
indoleacetic acid and the complexes was the same whether
"shoot" induction or callus growth was measured. The order
is: indoleacetic acid, indoleacetyl-L-alanine, indole-
acetylglycine and indoleacetyl-B-alanine. However, there

is some evidence that this order may be affected by the
nature of the culture medium. See Table V.

Tobacco was chosen as an initial test species because
of the ease with which plants can be regenerated from
undifferentiated callus. Much work has been devoted to the
discovery of a culture medium that would support such callus
growth. A systematic search of this kind has not been
carried out with many other species. The lack of an ideal
culture medium for callus growth may be one of the problems
confronting attempts to regenerate plants from callus.

Several experiments were conducted with the tomato
cultivar Marglobe, in order to extend the application of
indoleacetylamino acids to a species in which it is more
difficult to regenerate organs from callus than in tobacco.

Tobacco and tomato are both members of the family, Solanaceae.

 

They both form adventitious roots easily from cuttings.
Both will also regenerate plants from plant fragments.

However, tomato tissue loses its capacity to produce "shoots"

47

or roots as soon as, or soon after it is transformed into

a friable callus culture. As mentioned earlier, it was
suspected that perhaps the medium used to initiate callus
formation might be at fault. The work described here
investigated the roles of auxin complexes in initiating and
maintaining callus growth and in the unfortunate abolishing of
organogenic potential of the recently initiated callus.

Any serious attempt to optimize either the basal medium or
the cytokinin level for callus growth was outside the scope
of my research. Thus, a slightly amended tobacco growth
medium was used as the growth medium for tomato.

In studies of the induction of callus growth and support
of undifferentiated callus growth of tomato, benzyladenine was
the only cytokinin tried, thus precluding any cross-cytokinin
comparisons.

In stimulating the growth of primary callus (immediately
derived from hypocotyl sections), indoleacetyl-L-alanine,
indoleacetylglycine and indolacetyl-B-alanine all had relative
auxin effects greater than one, while the relative auxin effect
of indoleacetyl-D-alanine seemed to be less than one. The
same trend was observed with secondary callus tissue of
Marglobe, which did not regenerate plants. These data suggest
that the responses to auxins involved in tissue growth did
not change appreciably with succussive subcultures, even

though organogenic potential was lost during this period.

48

The data discussed thus far can be explained on the
basis of a slow release of indoleacetic acid from the
complexes. However not all of the data can be so explained.
As has been noted, indoleacetyl-L-alanine is particularly
active in initiating callus and in maintaining callus growth
in tomato hypocotyl sections. It also abolishes "shoot"
initiation completely. By these criteria, one would suppose
that indoleacetyl-L-alanine is an excellent source of
available indoleacetic acid. This supposition is quite
consistent with the fact that it is the L-isomer and pre—
sumably susceptible to hydrolysis by peptidases. However,
the addition of a small amount of free indoleacetic acid
to the very "available" complex restored organogenesis--a
result which is in no way consistent with the argument that
indoleacetyl-L-alanine is already providing the tissues with
abundant indoleacetic acid. It would seem that indoleacetic
acid and indoleacetyl-L-alanine must somehow be doing different
things.

This possibility opens the door to entirely new areas of
investigation. Thus, by varying the kinds of auxins used in
generating "callus" we may be able to preserve organogenic
potentials that are now being lost. And in attempting to
regenerate shoots, we may find that different complexes elicit
quite different responses. Certainly the number of possible
auxin complexes is almost limitless and some of the complexes

may have ideal properties for use in regeneration experiments.

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49

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