Download Genetic nature of a nontumour mutant isolated from tumour

Received 22 May 1995
Heredity 76 (1996) 335—345
Genetic nature of a nontumour mutant
isolated from tumour-prone amphidiploid
Nicotiana glauca-Iangsdorffii (G G LL): a
critical assessment*
M. R. AHUJA
BFH, Institute of Forest Genetics, Sieker Landstrasse 2, 22927 Grosshansdorf, Germany
The genetic nature of a nontumour mutant, isolated by Izard (1957) following irradiation of
germinating seeds of the tumour-prone amphidiploid Nicotiana glauca-langsdorffii (GGLL-T),
is re-examined in the light of previous and current studies on tumour formation in Nicotiana
hybrids. As there are lingering questions regarding the dominant nature of the nontumour
trait, I shall present experimental evidence and theoretical arguments that suggest that the
nontumour condition does not necessarily involve a single dominant gene mutation, but rather
mutations affecting tumour expression. In the framework of a genetic model of tumour
formation in Nicotiana (Ahuja, 1968), the mutations are probably in those ee loci of N glauca
affecting tumour expression, and not in the I element of N langsdorffii, as previously suggested
by Smith (1972) and Sekine et a!. (1993). As tumour-prone tissues are highly sensitive to
environmental influences, including irradiation, it would be very difficult to obtain a mutation
from tumour to a completely nontumour state, as in all likelihood, the irradiation treatments
would accelerate tumour formation, or induce variation in the tumour expression. Instead, I
propose a different approach for isolating a nontumour mutant, which involves irradiation of
haploid cells (pollen) from N langsdorffii (I species) and then pollinating N glauca (ee species)
with the irradiated pollen. As the cross N glauca x N langsdorffii normally yields a 100 per cent
tumorous progeny, any deviation from this norm, i.e. a nontumour mutant, would be easily
detected in the proposed cross. The reciprocal cross involving irradiated pollen from the ee
parent N glauca may also be attempted to check the validity of the hypothesis.
Keywords: genetic control, genetic tumours, irradiation effects, Nicotiana glauca-langsdorffii,
nontumour mutant, tumour expression.
ving two genomes each of N glauca (GG) and N
langsdorffii (LL) in the amphidiploid (GGLL), and
Introduction
Since the discovery by Kostoff (1930) of nine interspecific tumorous hybrids in the genus Nicotiana,
those with different combinations ranging from one
N glauca genome with two of N langsdorffii (GLL)
to three of N glauca and one of N langsdorffii
almost 20 additional hybrid combinations have been
reported in which the entire F1 is tumorous (Kehr &
(GGGL). However, the tumour expression varies
depending on the genomic combination. For
Smith, 1954; Ahuja, 1965; Smith, 1972). One of
these tumorous interspecific hybrid combinations
involving Nicotiana glauca (2n = 24) and N langs-
example, the tumours are relatively smaller in the
triploid GLL compared with the diploid hybrid (GL)
or the amphidiploid (GGLL) combinations (Ahuja
& Hagen, 1967).
dorffii (2n = 18) has been most extensively investi-
gated from the standpoint of genetic nature and
physiology of tumour formation (see Ahuja &
During the course of his investigations with
amphidiploid N glauca-langsdorfjii (GGLL), Izard
(1957) irradiated germinating seeds of GGLL with a
Hagen, 1967; Bayer, 1982; Smith, 1988). Tumours
also occur in hybrids at different ploidy levels invol-
dose of 1200 r, and isolated a plant that did not
produce any tumours. Although this plant had a
*Dedicated to George L. Hagen on his 70th birthday.
1996 The Genetical Society of Great Britain.
335
336 M. R. AHUJA
chromosome complement of 42, as in the tumorous
GGLL, it showed considerable meiotic abnormal-
ities, including univalents and bridges. By the
process of selfing, Izard isolated two homozygous
lines, which were nontumorous, and showed somewhat normal meiosis. One of these lines (354) was
subsequently employed by Smith & Stevenson
(1961) for determining the genetic nature of the
nontumour mutant (GGLL-NT), and subsequently
by a number of other investigators for characterization of this mutant. As there have been conflict-
ing reports on the behaviour of this nontumour
mutant and its progeny, obtained by crosses with the
tumorous amphidiploid GGLL, under greenhouse
and field conditions in terms of tumour formation,
and with regard to other stress factors such as irradiation and chemical treatments, I think it might be
worthwhile to re-examine this mutant in the light of
earlier genetic and physiological studies, as well as
recent investigations involving Agrobacterium and
progeny from the cross GGLL (T x NT) developed
tumours at the radiation dosage level of 200 r and
above per day. The progeny from the same cross
(T x NT) remained nontumorous under the normal
(field conditions), indicating that the environmental
factors influence tumour expression. However,
another cross involving the nontumour mutant
(GGLL-NT) and N langsdorffii (LL) which yields
the triploid hybrid combination GL(NT)L remains
nontumorous under both normal field and irradiation environments. On the other hand, the
progeny from the triploid hybrid combination GLL
derived from a cross between tumorous GGLL-T
and N iangsdorffii (LL) remains tumorous under the
two sets of environmental conditions.
To find biochemical genetical differences between
the nontumour mutant (GGLL-NT) and the
tumour-prone amphidiploid (GGLL-T), Bhatia et al.
(1967) employed acrylamide gel electrophoresis.
Their results revealed that the nontumour and
molecular biology. In this report I shall present
experimental evidence and theoretical arguments
that suggest that the so-called nontumour mutant,
obtained following irradiation of seeds from the
tumour-prone leaf tissues showed electrophoretic
variation in proteins and enzymes. Of the three
enzyme systems examined, the nontumour and
tumour-prone genotypes differed at least for two
tumorous amphidiploid GGLL, does not necessarily
involve a single dominant gene mutation, but rather
mutations affecting tumour expression.
esterase loci, and for one of the peroxidases; these
were lacking in the nontumour mutant. The protein
and enzyme patterns of the tumour tissue (normally
tumours develop on the stem and not leaves) were
strikingly different from those in the leaf tissues of
the nontuniour mutant and the tumour-prone genotypes. From these results on the protein profiles and
three enzyme systems, it is rather difficult to draw
Genetic and phenotypic analysis of the
nontumour mutant
Smith & Stevenson (1961) carried out detailed
genetic analysis to determine the nature of the
GGLL-NT mutant. They made a series of crosses
with the tumorous amphidiploid GGLL-T, to study
the inheritance of the nontumour trait. Crosses
between T x NT and NT x T yielded a nontumorous
progeny, indicating that the nontumour condition is
dominant. Backcrosses of the NT-F1 to GGLL-NT
gave all nontumorous progeny, and to GGLL-T
yielded a segregation ratio of 1:1 for the nontumour
and tumour traits, further supporting the dominant
mode of inheritance of the nontumour condition.
However, selfing of the NT-F1 yielded an F2 popula-
tion comprising of more than 90 per cent
nontumour plants by the end of the growing season.
This result would suggest that the validity of a single
dominant gene interpretation is questionable.
Futhermore, Smith & Stevenson (1961) investi-
gated the effects of chronic gamma irradiation on
the tumour and nontumour genotypes. The
nontumour mutant line (GGLL-NT) did not develop
tumours at any of the radiation dosage levels up to
450 r per 20 h day (for 108 days), whereas the
any conclusions regarding the role of specific
proteins/enzymes in tumour formation and/or
suppression.
A detailed cytogenetic analysis by Ahuja & Hagen
(1967) of the hybrid derivatives obtained by selfing
the partially fertile triploid (GLL) or backcrossing it
to N langsdorffii (LL), showed that the tumour
expression was enhanced by a threshold of certain N
glauca chromosomes on the N langsdorffii background. In other words, the lower the N glauca
chromosome number in the hybrid derivatives, the
smaller the size of the tumours. In a hybrid derivative with a full genome of N langsdorffii along with
two glauca chromosomes, very small tumours
developed, compared with those in the triploid
(GLL). These observations were interpreted to
mean that a number of genes on the N glauca
chromosomes enhance tumour expression in the N
glauca-langsdorffii hybrid combination (Ahuja &
Hagen, 1967). Smith (1988) also showed, following a
detailed analysis in the N glauca-langsdorffii hybrid,
that genes on the N glauca chromosomes enhance
The Genetical Society of Great Britain, Heredity, 76, 335—345.
NATURE OF A NONTUMOUR MUTANT IN N/COT/A NA 337
tumour expression, and those on N langsdorffii
greenhouse. Only five of ten were raised to maturity.
chromosomes have an opposite effect on the tumour
Even from this small sample of five plants, two
expression. Smith obtained a tumorous segregant
developed tumours of variable sizes following the
flowering phase. Tumours measured approximately
10 mm in one plant, and 5 mm in the second plant.
These observations also raise questions regarding
with a full genome of N langsdorffii plus a single N
glauca chromosome. However, the tumour size was
reduced in the segregant compared with that in the
diploid (GL) or even the triploid (GLL), consistent
with the observations of Ahuja & Hagen (1967).
We had also received seeds of the tumorous
amphidiploid (GGLL-T) and the nontumour mutant
(GGLL-NT) line No. 354 from Dr Harold Smith,
Brookhaven National Laboratory, Upton, New
York. Fifty plants of the NT line were grown under
field conditions in Philadelphia during the summer
months (temperatures in the high 30°C). Tumours
failed to develop on any of the 50 plants. Several
plants showed slight bulgings on the main stem,
perhaps reminiscent of the incipient tumour condition. Towards the end of the growing season four
entire plants with bulgings were brought into the
greenhouse, generally maintained at 23°C. All four
plants developed tumours (Fig. 1) measuring
approximately 5—10 mm in diameter and approach-
ing in size the tumours in triploid GLL (M. R.
Ahuja, unpublished data).
In another experiment, ten plants from the
nontumour line (GGLL-NT) were grown in the
the behaviour of the nontumour mutant under
greenhouse and the field conditions (M. R. Ahuja,
unpublished data).
Further studies on the nature of the nontumour
mutant (GGLL-NT) were carried out by Durante et
al. (1982). This Italian group also received the seed
stocks for the tumour and nontumour genotypes
(GGLL-T, GGLL-NT, and their hybrid T x NT)
from Dr Smith. Small tumours developed on a
number of wounded plants in the nontumourous F1
(NT x T) plants and apparently on nontumorous
plants derived from backcrossing the F1 to the
tumorous parent (T) in the greenhouse, thus, also
questioning the dominant nature of the nontumour
mutant. They irradiated the seeds of the tumorous
GGLL-T and the nontumourous hybrid T x NT with
X-rays. Tumours were induced at a high frequency
on the seedlings derived from irradiated seeds of the
nontumour T x NT hybrid after 50 kr of irradiation,
whereas the tumour-prone GGLL-T developed 100
per cent tumours on the seedlings at 20 kr irradiation. Durante et a!. (1982) also obtained similar
results following treatment with 6-azauradil in the
tumour (GGLL-T) and the nontumour hybrid
T x NT. In the tumour-prone GGLL-T almost 100
per cent tumours were induced, whereas in the F1
hybrid (T x NT) only about 50 per cent of the seedlings developed tumours following treatment with
6-azauracil. Furthermore, Durante et a!. (1989)
showed that the neoplastic phenotype in the
nontumour GGLL-NT can be restored by treatment
of cells with 5-azacytidine, which is known to block
methylation of the cytosines in DNA. They recovered habituated colonies in cell culture (but with no
shooty differentiation) and the repetitive DNA
sequences of treated cells showed a significantly
lower level of methylation. The habituated clones
obtained by treatment with azacytidine showed
reduced chromosome number, close to the amphidiploid level (2n = 42), but also showed the presence
of dicentric bridges and a breakage-fusion-bridge
cycle. These authors attributed differences between
their results and Smith & Stevenson's (1961) results
regarding the response of the F1 T x NT to irraFig. 1 Tumours on stems of greenhouse-grown triploid
Nicotiana glauca-/angsdoiffii (GLL), amphidiploid
(GGLL-T), and the nontumour mutant (GGLL-NT). The
tumours are from plants of comparable age.
The Genetical Society of Great Britain, Heredity, 76, 335—345.
diation or chemical treatment to the fact that Smith
& Stevenson grew their plants under field conditions, whereas Durante et al. grew their tumour and
nontumour genotypes under greenhouse conditions.
338 M. R. AHUJA
Genetic tumours of diverse origins in Nicotiana
exhibit different responses to environmental conditions. For example, the tumour-prone hybrid deriva-
conditioned or those induced by the crown gall
tives of N debneyi-tabacum x N longifiora, carrying a
single longiflora chromosome or its fragment on the
exogenous supply of essential phytohormones, such
as cytokinin(s) and auxin(s) (Braun, 1958; Schaeffer
& Smith, 1963; Ahuja & Hagen, 1966a). In contrast,
debneyi-tabacum background (Ahuja, 1962), form
bacterium Agrobacterium tumefaciens, are capable of
supporting their growth in vitro without an
tumours in the greenhouse or field conditions.
cells from normal plants require these phyto-
Progenies of another amphidiploid N debneyi-longi-
hormones for their growth and differentiation. In
flora, which forms tumours in the greenhouse, do
not develop tumours under field conditions. The
hybrid N glauca x N longifiora exhibit variable
tumour formation in the greenhouse, but tumours
fail to develop on plants from the same population
grown under field conditions (Ahuja & Hagen,
1967). In a tomato hybrid combination carrying a
dominantly inherited gene frosty spot (Frs) from
Lycopersicon chilense on the genetic background of
the N glauca-langsdorffii hybrid (GL) and amphidip-
bid (GGLL) the tumour-prone tissues are autonomous with respect to kinetin and auxin, whereas the
tissues from the nontumour mutant (GGLL-NT)
had absolute requirements for kinetin as well as
auxin for growth and differentiation of shoots like
those of the parental species (Schaeffer et at., 1963).
Cell lines established from tumour-prone genotypes
exhibit a fully habituated state in culture (Meins,
L. esculentum tumours developed on the ventral
1982, 1994). In a different tumorous hybrid
surface of the leaves when grown in the greenhouse;
however, tumours failed to develop under field
combination involving N deneyi-tabacum x N longiflora the in vitro habituated tumour tissues grow and
conditions (Doering & Ahuja, 1967). Therefore, it
would appear that environmental conditions play a
teratomas on the plants in vivo, and these teratoma-
very important role in the expression of tumours.
differentiate numerous aberrant shoots typical of
tous growths do not generally differentiate roots
with exogenously supplied auxins in vivtro or ex vitro
Cytology
Cytological studies by Smith & Stevenson (1961)
revealed that the tumorous (GGLL-T) and the
nontumorous (GGLL-NT) genotypes, and their
hybrid T x NT have 42 chromosomes, as would be
expected from the parental contributions of N
glauca (2n 24) and N langsdorffli (2n = 18), thus
confirming Izard's (1957) earlier chromosome counts
on the nontumour mutant. The T and the NT
produced 85—90 per cent stainable pollen, while the
F1 T x NT showed 80 per cent stainable pollen.
However, the meiosis was notably more irregular in
the F1 hybrid compared with the parental amphidip-
bids (T and NT). All three amphidiploids had
lagging chromosomes or bridges at anaphase I of
meiosis.
Nevertheless,
amphidiploids
carrying
complete genomes of two parents provide a buffering effect and adequate functional pollen and egg
cells are produced, even if there are some meiotic
irregularities. From the cytological observations it
would appear that the nontumour mutant has not
lost any chromosomes, but probably carries chromosome rearrangements, including deletion(s), or gene
mutation(s).
Genetic control or hormones in tumour and
nontumour genotypes
Generally plant tumour cells, which are genetically
in a rooting bench (Ahuja & Hagen, 1966a; Ahuja,
1971), suggesting that the balance is heavily tipped
in favour of kinetin in these tumour tissues. It was
indicated by Schaeffer et at. (1963) and Feng et a!.
(1990) that kinetin is an essential requirement for
the differentiation of shooty morphology in the
nontumour mutant GGLL-NT, resembling that of
the tumorous GGLL-T. However, it is unlikely that
kinetin alone will lead to the growth autonomy and
habituated state in the nontumour mutant tissues, as
auxin is also essential for the early growth phase.
Our tissue culture studies showed that stem discs
from plants with small tumours (ST) and nontumour
plants in the same nontumour mutant (GGLL-NT)
line did not grow on a modified basal Murashige &
Skoog's (1962) medium (Ahuja & Hagen, 1966a),
unsupplemented with hormones. The tissues from
the ST genotype, however, showed slight enlarge-
ment of the cells in the first week in culture. But
subsequently the stem discs turned brown and there
was no further growth. Perhaps this may result from
the slightly higher amounts of the free auxin in the
ST compared with NT genotypes, which may have
provided the initial trigger. Subsequent cessation of
growth of the ST tissues is dictated by its genotype,
and is reflected in its inability to synthesize continuously the necessary growth-promoting substances.
Both ST and NT tissues grew when the medium was
enriched with 0.1 and 1 mg/L 1 indole-3-acetic acid
(IAA). However, the differences in the growth rates
The Genetical Society of Great Britain, Heredity, 76, 335—345.
NATURE OF A NONTUMOUR MUTANT IN N/COT/A NA 339
of the ST and NT tissues were striking indeed. The
growth of the NT was only slightly enhanced by the
two concentrations of IAA, and further supplemen-
prone tissues from diploid hybrid GL and
tation of the medium with both phytohormones,
higher in the GL compared with GGLL, and the
IAA and kinetin, would be necessary for obtaining
optimal growth, as was shown by Schaeaffer et al.
(1963). By contrast, the growth of the ST tissue was
GLL combination had approximately the same auxin
levels as in N langsdorfii, but three times lower than
greatly enhanced by both levels of IAA, and the
growth of callus increased linearly with increasing
concentrations of IAA (Fig. 2). At each concentration, comparatively the ST tissues grew ten times
that of the NT tissues as determined by the dry
weight increase (M. R. Ahuja, unpublished data). It
may be pointed out that the aim of the investigation
was to demonstrate differences in the auxin requirement between NT and ST segregants in the same
population, and not to bring out the optima for their
growth in vitro. It would appear that the plants with
ST may be somewhat similar to the tumour-prone
triploid GLL, which also requires auxin for its
growth (Ahuja & Hagen, 1966b). Besides, tumourprone GGLL-T stem pith tissues may also require
an initial treatment with auxin to trigger habituation
in culture (Cheng & Smith, 1973), compared with
constitutively habituated teratomatous tissues.
The inherent growth requirements are also reflected in the endogenous auxin levels in the tumour•OmgiLJM
UOlmgLIM.
180
DIOm9IUAA
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.5 120
__________________ ____________________________
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__________________
60•
40
N glauca (Bayer, 1967). The amphidiploid GGLL
showed auxin levels only slightly higher than in N
glauca. In another hybrid combination involving N
debneyi-tabacurn and N. longifiora and their tumour-
prone hybrid derivatives, Bayer & Ahuja (1968)
showed that the progeny from tumour-forming F1
hybrids and the early tumour segregants from the
backcross generations had very similar auxin levels,
but the auxin levels were much lower in the parents
and the late tumour-forming segregants. The
isozyme analysis of Ahuja & Gupta (1974) revealed
that a peroxidase locus from N longifiora inherited
in the tumour-prone hybrid derivatives of the latter
backcross generation of the cross N debneyi-tabacurn x N longifiora was only activated in the tumorous
tissues. These studies suggest that the endogenous
auxin levels are determined by the genotype and the
genomic relationships between parental species
entering tumorous combinations, and these may be
critical for the development of tumours in the Nicotiana hybrids. Studies by Ames and his associates
(Ames & Mistretta, 1975; Ames, 1981) have shown
that there may be a close correlation between
200 -
0.
amphidiploid GGLL as determined by Bayer (1967).
The extractable auxin (IAA) levels were several-fold
____________ ___________________
20
0
I
NT
I
I
ST
Fig. 2 Effect of indole-3-acetic acid (IAA) on in vitro
growth of tissues from small tumour-prone (GGLL-ST)
and nontumour-prone (GGLL-NT) segregants derived by
selfing the nontumour mutant. Stem explants were grown
on modified Murashige & Skoog (1962) medium, supplemented with 0.1 mg/L 1 IAA, and 1 mg/L 1 IAA for 5
weeks. Growth is represented by an increase over the
initial dry weight.
The Genetical Society of Great Britain, Heredity, 76, 335—345.
decline in IAA levels and onset of tumour formation
in hybrids grown in the greenhouse. Recently, SyOno
and his group (Ichikawa et al., 1989; Fujita et a!.,
1991; Ichikawa & SyOno, 1991) have provided
support for this observation that tumour induction
may be associated with a temporary decrease in
endogenous levels of auxin in the plants, and after-
wards there is a dramatic surge of some 30- to
80-fold in the auxin level within 5 days after tumour
induction. These differences in the endogenous
levels, observed by different investigators, before
tumour formation may result from the techniques
employed for auxin assays, for example bioassay of
free or bound auxin, the genotype, in vitro or & vitro
derived tissues, type of tissues employed (stem
segments or apical tissues), and the time tissues
were analysed before the flowering phase, which
triggers tumour formation under normal conditions,
that is, whether tissues were analysed at the elonga-
tion phase or just before the flowering phase.
However, auxin levels alone do not confer growth
autonomy to tumour tissue in vivo or in vitro, but
other biosynthetic systems, including cytokinins,
must also accompany the characteristic teratomatous
tumour growth. I tend to favour the hypothesis that
340 M.R.AHUJA
the initial levels of auxins or cytokinins in the
tumour-prone genotypes may be in a flux at the time
of flowering, which triggers tumour formation under
normal conditions. Accordingly, it is the inherent
capacity of tumour-prone cells to turn on, among
other things, auxin and cytokinin biosynthetic
systems, sequentially or simultaneously at the flow-
ering phase, or at the critical time in response to
stresses before flowering, for the initiation and for
the continuous and proliferative formation of the
tumour tissue.
Agrobacterium T-DNA genes and genetic
only in the presence of the wild-type plasmid; ipt
alone did not induce shoots in N langsdorffii. The
wild-type and ipt + plasmids induced shoots at a low
frequency (up to 6 per cent) in the nontumour
mutant GGLL-NT, whereas in the tumour-prone
GGLL-T, a high frequency of shoots (above 80 per
cent) was induced by the wild-type and insertion
mutant plasmids. Nacmias et a!. (1987) interpreted
these results as implying that there are different
genetic controls for initial cell proliferation and for
continued autotrophic growth in genetic tumours.
On the other hand, Feng et al. (1990) obtained the
fully habituated phenotype in the nontumour mutant
nontumour tissues have been further investigated by
(GGLL-NT) following transformation with the Ti
plasmid carrying the ipt gene. They showed that
shooty morphology of the nontumour mutant was
not only restored with kinetin, but also with the ipt
employing T-DNA genes from the Ti plasmid of
gene. The transformed NT tissues exhibited a shooty
Agrobacterium tumefaciens. Genetic transformation
by Agrobacterium in the genetic tumour system in
phenotype, which was indistinguishable from the
tu mou rs
The
growth requirements of the tumour and
wild-type untransformed genetic tumours on GGLL-
Nicotiana has revealed that certain wild-type or
mutant genes from the T-DNA can be used for
understanding growth autonomy in this system.
Nacmias et al. (1987) transformed N glauca, N
T. However, some questions remain unresolved
regarding the transformation studies in the Nicotiana genetic tumour system with the Ti plasmids
langsdorffii, tumorous amphidiploid GGLL-T, and
the nontumour mutant GGLL-NT with wild-type
by kinetin and the ipt gene are also not quite
and mutant genes iaaH, iaaM (tms), and ipi
from the Ti plasmid of Agrobacterium tumefaciens.
The iaaH and iaaM loci are involved in enzymes
which code for indole-3-acetic acid synthetase, and
the ipt locus encodes for isopentyl transferase, which
is a key enzyme in cytokinin biosynthesis. The
results of Nacmias et al. (1987) showed that fully
habituated calli (23 per cent) were obtained in N
langsdorffii following transformation with only wild-
type tms ipt + genes, but in N glauca the habituated calli (up to 90 per cent) were obtained either
by the tms — ipt + gene or tms + ipt gene constructs,
—
indicating that the two parental species have
different requirements for attaining a habituated
state. The nontumour mutant GGLL-NT could also
be habituated by the same insertion mutants (tms
ipt + or tms + ipt ) gene constructs as in N glauca,
but at a significantly lower frequency (up to 20 per
cent). On the other hand, high incidences (100 per
cent) of habituated calli were obtained in the
tumour GGLL-T with wild-type and mutant gene
constructs. However, differentiation of the shooty
phenotype was under a different genetic control.
The N glauca tissues did not differentiate any
shoots when transformed with the wild-type plasmid,
but differentiated shoots (some 18 per cent) only in
the presence of the ipt + gene, while the N langsdorffii tissues differentiated some shoots (4 per cent)
from Agrnbacterium. The shooty phenotypes induced
comparable. The shooty phenotype induced by Feng
et al. (1990) in the nontumour mutant tissues in vitro
was not induced by kinetin alone; the media also
contained auxin for growth of the callus. It is also
not clear in this study whether nontumour tissues
were preincubated in an auxin-containing medium
before transformation with the ipt gene. The levels
of cytokinins in the tissues before and after transformation of the nontumour mutant are also not
comparable to those in the untransformed wild-type
GGLL tumour. Of the three cytokinins measured,
only isopentyl adenosine (IPA) levels showed significant differences between the GGLL-T (85 units)
and the untransformed GGLL-NT (6 units) genotypes. However, after transformation of NT with the
ipt gene, the IPA levels increased three times to 18
units, but way below the IPA levels in tumour
GGLL-T. The levels of two other cytokinins, transzeatin riboside (t-ZR) and dihydrozeatin riboside
(DHZR), are also puzzling. There are no significant
differences in the levels of t-ZR (T = 2.1; NT = 1.2)
and DHZR (T 8.7; NT = 7.3) between the tumour
GGLL-T and nontumour GULL-NT. However, after
transformation with the ipt gene the levels of these
cytokinins are more than 100 units in the NT genotype. The total levels of the three cytokinins are at
least two and half times higher in the transformed
NT compared with the T genotype. Based on their
results, Feng et a!. (1990) concluded that genetic
The Genetical Society of Great Britain, Heredity, 76, 335—345.
NATURE OF A NONTUMOUR MUTANT IN NICOTIANA 341
tumours are caused, at least in part, by the elevated
levels of cytokinin in the interspecific Nicotiana
species hybrids. But that seems to be only part of
Conclusions and theoretical considerations
Taken
together these observations question the
the story.
The role of Agrobacterium rhizogenes T-DNA from
validity of a single dominant gene interpretation of
the mutation from tumour to nontumour state in N
the Ri plasmid has been extensively investigated in
gtauca-langsdorffii. It would appear that by X-
hairy root disease, growth and differentiation in
irradiation of Nicotiana glauca-tangsdorffii germinating seeds, Izard (1957) may have produced genetic
variability affecting growth and tumour expression,
and these genetic variants may have included cryptic
plants (see Constantino et a!., 1994). Recent studies
have shown the presence of rol gene sequences
designated Ng rot (Furner et a!., 1986) and more
recently as cT-DNA (cellular T-DNA; Aoki et al.,
1994) from the Ri plasmid in the untransformed N
glauca, one of the parents involved in the tumourprone hybrids. Furner et a!. (1986) found more than
80 per cent homology between the endogenous N
glauca Ng rol sequences (rolB, and rotC) and the TL
region of the Ri plasmid. As the Ng rol sequences
are not transcribed in N glauca, Furner et at. (1986)
considered that they may be pseuodogenes.
However, recent studies by Ichikawa et at. (1990)
have shown that the Ng rolB and Ng rotC gene
sequences are transcribed in the tumour tissue of
the hybrid N gtauca x N langsdorffii; the tran scription of rolB increased, whereas rolC decreased, with
an increase in the endogenous auxin level in the
tissue. However, transcription of these cT-DNA
genes was completely suppressed by exogenous
application of auxin. In addition to rolB and rotC
sequences, two other genes homologous to open
reading frames 13 and 14 (ORFs 13 and 14) of Ri
plasmid are also present in the cT-DNA of untransformed N glauca, which are also expressed in the
genetic tumours on N glauca x N langsdorffii, but
not in the leaf tissue of the tumour-prone hybrid
(Aoki et a!., 1994). According to Aoki et al. (1994)
ORFs 13 and 14 may be involved in the synthesis of
growth substances similar to cytokinin. If untrans-
formed N glauca has all the necessary cT-DNA
genes for auxin and cytokinin autonomy, the question arises why does it not develop tumours when
treated with chemicals or with irradiation? What is
the contribution of N langsdorffii in the N
glauca x N langsdorffii tumorous combination? Does
N tangsdorffi contribute gene(s) for an additional!
different cytokinin, or for deregulation of hormone
synthesizing genes, or possibly demethylation of the
Ng cT-DNA, at the time of initiation of tumors in a
hybrid system? The main questions, however, still
need to be addressed: what is the specific role of the
Ng cT-DNA in the development of tumours and do
other Nicotiana species involved in the tumorous
combinations also carry oncogens that are derived
from Agrobacterium?
The Genetical Society of Great Britain, Heredity, 76, 335—345.
genetic aberrations. Subsequently, he isolated two
nontumour lines under field conditions. No gross
structural chromosome abnormalities were observed
by Izard (1957) and Smith & Stevenson (1961) in
the nontumour line 354, which carried 42 chromosomes (the summation of two parental contributions) as does the tumorous amphidiploid GGLL.
Subsequent studies with the nontumour mutant
showed that it behaves differently under field and
greenhouse conditions, and that small tumours
develop on the nontumour mutant under greenhouse conditions, and in the F1 progenies derived
following crosses between T x NT. Tumours also
developed on nontumour derivatives following irradiation (Smith & Stevenson, 1961; Durante et al.,
1982). However, one critical cross involving
NT x NT, and its F2 progenies have not been
analysed so far. As the first breeding results
suggested that the nontumour condition was transmitted as a dominant trait, the NT x NT cross was
perhaps considered unnecessary.
We might ask the question: if the nontumour
mutant derived from the tumorous state does not
involve a single dominant gene mutation, then what
is the nature of this mutation? There are at least
three other possibilities which present themselves:
(i) that the nontumour mutation is deficient in transmission; (ii) that the nontumour condition results
from an incomplete dominant mutation; and (iii)
that the nontumour state involves more than one
gene mutation, which control tumour expression.
The first possibility does not appear to be tenable, as
the entire F1-NT x T remains nontumorous under
field conditions, but tumours can be induced by
wounding in the greenhouse, and under the irradiation environment. The second possibility that the
nontumour state is controlled in inheritance by a
partially dominant gene is also questionable, as the
F1 is nontumorous, and the F2 segregation ratios are
skewed towards the nontumour condition (about 90
per cent of the progeny is nontumorous in the F2).
This leaves us with the third alternative, which states
that more than one mutation is involved in the
342 M.R.AHUJA
origin of the nontumorous state with variable
expression of tumours under different sets of
environments. The question arises whether the
N. longiflora contributes a relatively simply inherited
gene(s) on the N glauca or N langsdorffli genomes
have mutated to account for the nontumour state?
The following is an attempt to answer this question.
factors (more than one locus) from N debneyi-
It has been shown by earlier studies that some
tumour genotypes in Nicotiana and Lycopersicon
respond differently to two sets of environments, that
is, field and greenhouse conditions (Ahuja & Hagen,
1967; Doering & Ahuja, 1967). As some of the
plants from the nontumour mutant line form small
tumours in situ, but do not grow on the hormone-
free medium in vitro, it would appear as though
genes affecting tumour growth and development
may have been impaired. The differential growth
rates of the tissues from the tumour-forming and
nontumorous plants from the same population in
vitro also strengthen the view concerning genetic
variability in the nontumour mutant line. In many
respects the tumorous plants in the nontumour
mutant line resemble the triploid GLL (with one
genome of N glauca and two genomes of N langsdorffii): they both form small tumours and require
exogenous supply of phytohormones for their growth
and shooty differentiation (Ahuja & Hagen, 1966b).
Based on detailed cytogenetic studies, Ahuja &
Hagen (1967) suggested that a number of genes on
certain N glauca chromosomes enhance tumour
expression in the N glauca-langsdorffii hybrid
genetic component designated I, essential for
tumour initiation, which interacts with the ee genetic
tabacum to form tumours. Earlier Näf (1958) had
proposed that Nicotiana species entering tumorous
combinations may be divided into two groups: one
arbitrarily designated the 'plus' group consists
largely of the species of the section Alatae with nine
or ten chromosome pairs; whereas the 'minus' group
consists of a variety of species coming from diverse
sections of the genus, with 12 chromosome pairs or
a multiple thereof. Barring a few exceptions, most
species of Nicotiana entering tumorous combinations
can be classified according Naf's scheme. According
to Ahuja's hypothesis, the 'plus' group species such
as N longiflora and N langsdorffii are the I carriers,
whereas the 'minus' group species such as N debney,
N tabacum and N glauca are the ee contributors
involved in enhancement of tumour expression.
In his review on plant genetic tumours, Smith
(1972) wrote: 'If the hypothesis of Ahuja (1968) is
correct, the mutation for the nontumorous condition
may be expected to involve an I factor in the
chromosome of N langsdorffii'. A similar statement
was recently made by Sekine et a!. (1993). I would
have agreed with Dr Smith if the nontumour condition had been transmitted as a single dominant trait
in the selfed and backcross progenies, and tumours
did not develop on the NT mutant or its nontumor-
combination. It is tempting to suggest that by irradiation of tumorous amphidiploid N glauca-langs-
ous progeny under different conditions. But that
dorffii seeds, the genes on the N glauca genome may
have been impaired.
In another interspecific tumorous hybrid combination involving N debneyi-tabacum and N longifiora a
nontumour condition does not seem to be transmitted as a dominant trait in the crosses, and the
detailed cytogenetic study, accompanied by a
repeated backcross programme, revealed that a
specific N longifiora chromosome or its fragment in
an otherwise N debneyi-tabacum background was
adequate for the development of tumours (Ahuja,
1962). The tumour condition was transmitted as a
dominant trait in this hybrid combination and, there
was no diminishing of tumour expression in the
backcross derivatives, compared with the tumorous
F1. These results suggested that gene(s) on a single
N longifiora chromosome are sufficient, whereas
from N deneyi-tabacum a number of genes distributed over several chromosomes are required for
tumour formation. Based on these observations,
along with cytogenetic and tissue culture studies
involving hormone requirements in the N debneyitabacum-longifiora and N glauca-langsdorffii hybrid
complexes, Ahuja (1968) proposed a hypothesis that
does not seem to be the case. Because the
tumour expression is greatly modified by the
environmental factors and irradiation, it is tempting
to suggest that ee genes on the N glauca parent in
the amphidiploid N glauca-langsdorffii may have
been impaired by irradiation. That I factors on the
N langsdorffii genome have not mutated comes from
the cross involving the nontumour mutant and N
langsdorfjii (GGLL-NT x LL) from Smith & Steven-
son (1961), which gave a nontumour triploid
(GLL-NT), indicating that addition of a normal
genome of N langsdorffii did not initiate tumours,
and that the radiation-induced mutations are probably in the ee factors of N glauca. It would be interesting to investigate if the mutation/deletions are in
the cT-DNA, involving any specific rol or ORF
genes, in the genome of N glauca.
Stresses of diverse nature, such as wounding,
chemical treatments, irradiation, etc. are known to
initiate and enhance tumour formation at any stage
of tumour-prone Nicotiana hybrids or its tumourThe Genetical Society of Great Britain, Heredity, 76, 335—345.
NATURE OF A NONTUMOUR MUTANT IN NICOT/ANA 343
prone hybrid derivatives. With this kind of a germ-
Parent
line-conditioned tumour-prone system, which is
highly sensitive to environmental stresses, it would
be rather difficult to induce a mutation from tumour
to nontumour state by irradiation in a tumour-prone
amphidiploid, or a diploid, because this will in all
Nicotiana langsdorfihi
(LL)
Nicotiana glauca
(GG)
7
Irradiation
Gametes
likelihood enhance or alter tumour expression.
Pollen
Experimental data are consistent with this hypothesis. Many investigators (Sparrow et a!., 1956; Hagen
et al., 1961; Smith & Stevenson, 1961; Ahuja &
Cameron, 1963; Conklin & Smith, 1969; Durante et
a!., 1982) have irradiated tumour-prone Nicotiana
hybrid seeds, or plants at various stages of development, but so far no one has been able to induce a
mutation in the nontumour direction in this system.
Izard also attempted it, but in my opinion may have
Fl
GL
Nontumorous, if I mutated
succeeded in introducing genetic variability for
tumour expression, possibily impairing the ee genes
on the N glauca chromosomes.
It would appear, therefore, that in the germlineconditioned tumour-prone hybrid system in which
the whole plant is a 'potential tumour', the somatic
tissues or seed are not suitable material for the
induction of a nontumour mutation, in particular in
the complex amphidiploid. Radiation may influence
plant tumours in at least two different ways, but the
distinction between these may be operationally
somewhat difficult. The first type of effect is by the
disturbance of metabolism, in particular the
hormone biosynthetic systems, which would then
trigger tumorous growth. This mechanism appears to
be more relevant to tumour-prone Nicotiana hybrids
which maintain a precarious balance between
normal growth, before flowering, and neoplastic
development, after flowering. The balance can be
easily disturbed at any stage of vegetative growth
before flowering by a variety of diverse stresses,
including irradiation. The second type of radiation
effect may involve mutations in the nuclear genes
(including chromosome aberrations or gene mutations). The mutations may affect tumour expression,
ranging from new genotypes with nontumour state,
to those with variable tumour incidence, as in the
case of the nontumour mutant in the tumour-prone
amphidiploid Nicotiana glauca-langsdorffi produced
by Izard (1957).
Theoretically, it should be possible to induce a
mutation in the I element in a diploid, but it would
be more likely to induce mutations in the ee
elements, as more of them are available as targets. It
is a numbers game involving probability events.
Instead, I propose a slightly different approach to
the problem of mutability in the Nicotiana tumour
system. I suggest irradiation of haploid cells (pollen)
The Genetical Society of Great Britain, Heredity, 76, 335—345.
Chromosome doubling
GGL'L'
Nontuinorous amphidiploid
Fig. 3 Diagram showing a crossing scheme involving irradiation of pollen from Nicotiana langsdorffii (I species),
and then pollinating N glauca (ee species) with the irradiated pollen to produce the F1. As the cross N
glauca x N langsdorffii normally yields a 100 per cent
tumorous progeny, any deviation from this norm (a
nontumour mutant) would be easily detected, in which
case an I element has been impaired by irradiation. The
chromosome number of the nontumour mutant could
then be doubled to produce a homozygous and fertile
nontumorous amphidiploid (GGL'L'). The reciprocal
cross utilizing the irradiated pollen from the ee parent N
glauca should also be attempted to test the validity of the
hypothesis.
from one of the normal Nicotiana species, which
enters into tumorous combination, and then producing specific hybrids utilizing the irradiated pollen. In
the test systems, I suggest irradiation of pollen from
an I carrier species, N langsdorffii, with appropriate
doses, and then pollination of the ee species, N
glauca, with the irradiated pollen (Fig. 3). As the
cross N glauca x N langsdorffii normally yields a 100
per cent tumorous progeny, any deviation from this
norm, that is, a nontumour mutant, would be easily
detected in the proposed cross, in which case an I
element has been impaired by irradiation. To test
the alternative hypothesis, the reciprocal cross utiliz-
ing the irradiated pollen from the ee parent N
glauca should also be attempted. But it is anticipated
344 M.R.AHUJA
on the basis of my hypothesis, that the latter cross
may produce variation in the tumour expression in
the hybrids. Both these possibilities are testable. The
F1 (GL) progeny showing a nontumour mutant
(GL'-NT), if produced, could be maintained in
tissue culture, or its chromosome number doubled
by colchicine to produce a nontumour amphidiploid
(GGL'L'-NT), which would be homozygous for the
nontumour trait, and at the same time fertile.
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BRAUN, A. C. 1958. A physiological basis for autonomous
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