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W.R. SCOWCROFT
phase chromosomes from a different species, and enzymes specified by the
donor can be detected in the treated recipient cells (Burch and McBride, 1975).
In plants both observational and experimental evidence indicate that transformationlike phenomena do occur. Most of these studies have involved whole plants,
and these will be reviewed briefly because of their possible relevance to the
development of novel techniques for genetic modification in plants.
A. MODIFICATION BY HOMOLOGOUS DNA
The treatment of mutant plants with wild-type DNA can be associated with
the correction of the lesion at a frequency which is significantly greater than
spontaneous correction in the appropriate control plants. The observations
which have been reported include the correction of anthocyanin deficiency in
petals of Petunia (Hess, 1969), the modification of the effects of the waxy locus
in pollen of barley (Turbin er af., 1975), and the modification of genetically
determined fruiting characters in Capsicum annuum following DNA treatment
(Nawa et al., 1975). This latter observation was similar to graft-induced genetic
alteration observed in red pepper (Ohta and van Chuong, 1975). Pandey (1975)
has also observed gene transfer following the use of “lulled” irradiated pollen to
overcome intraspecific incompatibility in Nicotiana hybrids. Genes which were
apparently transferred from the mentor pollen parent modified the incompatibility genotype or the flower color that characterized the maternal parent. Among
the many reasons Pandey (1975) advanced to exclude rare pollen mentor nuclei,
which might have escaped the very high lethal dose of irradiation, as the cause of
such rare genetic events, is that the transformed plants were otherwise phenotypically maternal and distinctively different from the expected hybrid.
B. MODIFICATION BY HETEROLOGOUS DNA
The uptake, integration, and possible transforming ability of foreign DNA has
been studied using seedlings, seeds, cultured cells, and protoplasts (Ledoux,
1975; Markham et al., 1975). For some years now Ledoux and co-workers (see
Ledoux, 1975) have examined the uptake and integration of bacterial DNA
following its application to germinating seedlings. The evidence for the pseudointegration (covalent binding) of the foreign DNA with that of the host DNA is
based on the occurrence of DNA, isolated from the treated plants, with a
buoyant density intermediate between that of the higher density of the plant
DNA and the lower density of the donor bacterial DNA. This intermediate form
subsequently could be separated into components which corresponded to the
respective buoyant densities of recipient and donor DNA. It has also been
SOMATIC CELL GENETICS AND PLANT IMPROVEMENT
63
claimed that a thamine deficiency in Arabidopsis can be corrected with DNA
isolated from bacteria prototrophic for thiamine (see Ledoux et al., cited in
Ledoux, 1975). However, Ledoux’s buoyant density evidence has not been
confirmed. Kleinhofs et al. (1975) using sedimentation analysis were unable to
confirm Ledoux’s findings. No intermediate peak was found when axenic plants
of pea, tomato, and barley were treated with foreign DNA according to
Ledoux’s procedure. Bacterial DNA covalently bound to recipient DNA was
found when the plants were not treated axenically. Kleinhofs et al. (1975)
argue that the observations of Ledoux are artifactual, resulting from contamination by other bacteria. Lurquin and Hotta (1975) treated Arabidopsis cell
cultures with bacterial DNA but were unable t o find any evidence for the
intracellular presence of bacterial DNA sequences, either in an integrated or in a
free state. With the development of techniques for the characterization of DNA
it has emerged that analysis based solely on buoyant density sedimentation is
not a sufficient criterion to identify the origin of the DNA. It is essential that
base sequence homology be established by DNA hybridization techniques such
as can be done on nitrocellulose filters or in solution. The fidelity of base pairing
can only be established by thermal renaturation studies. Using these more
sensitive techniques, Kleinhofs (cited in Ledoux, 1975) was still unable to
demonstrate the presence of donor DNA sequences in the DNA of treated barley
seedlings. Therefore, until further evidence is provided, the results of Ledoux
claiming integration of bacterial DNA into plant DNA must be viewed cautiously.
Plant cell cultures and protoplasts have been used to study the uptake,
expression, and possible integration of foreign DNA. Uptake and maintenance of
integrity of foreign DNA has been reported in plant protoplasts (Vasil, 1976). In
studies such as this, plant nucleases present a real hazard to the integrity of the
donor DNA. Moreover, the enzyme preparations used to prepare protoplasts also
have considerable nuclease activity (Langridge, personal communication). Nuclease activity, particularly exonuclease, is pH-dependent and activity is substantially impaired at pH 9-10. Another precaution to avoid nuclease activity is to
thoroughly wash plant cells or protoplasts prior to treatment.
Studies on the expression and possible integration of bacterial DNA in plant
cells is at best equivocal. Carlson (1973) infected barley protoplasts with a
bacteriophage of Escherichia coli and was able to detect two phage-specific
enzymes, S-adenosylmethionine cleaving enzyme and RNA polymerase. The
expression of these functions was rapid and transitory. Analogous work with
haploid cell cultures of tomato and Arabidopsis suggested that bacterial genetic
information for the utilization of lactose as a carbon source could be transferred
to plant cells using a transducing phage as vector (Doy et al., 1973). Apparent
expression of the transferred genetic information enabled the plant cells to grow
on lactose, whereas untreated cells could not, and immunological studies on the
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W.R. SCOWCROFT
surviving plant cells indicated that the P-galactosidase was of bacterial, rather
than plant, origin. Similar work with the same phage, but with sycamore cells,
was initially confirmatory (Johnson et al., 1973). However, subsequent experiments (Smith et al., 1975) have been unable to demonstrate bacterial P-galactosidase. The growth response of the treated sycamore plant cells showed an initial
burst of cell division which was not maintained. Smith et al. (1975) doubt that
their results provide direct evidence for the uptake and expression of bacterial
genetic information.
Research in our own laboratory by Dr. V. E. Merriam has also examined the
expression of bacterial genes in plant cells, and while some success has been
achieved, the results are still equivocal. Tobacco cells, which are sensitive to
growth inhibition by the antibiotic kanamycin, were treated with plasmid DNA
from Escherichia coli which carried kanamycin resistance. Plant cells were
treated under conditions which minimized the degradation of the plasmid by
plant nucleases and also included concomitant low levels of irradiation, hopefully
to aid integration of the plasmid. Two types of resistant clones were recoveredthose which survived for only a few subcultures in the presence of kanamycin,
and those which have been serially transferred many times with no apparent loss
of resistance. The majority of these stable clones were derived from experiments
where cells were irradiated following exposure to plasmid DNA. Spontaneous
kanamycin-resistant mutants have also been recovered. However, in experiments
using similar numbers of tobacco cells, the frequency of resistant clones was
higher using plasmid DNA than in the controls where plasmid DNA was excluded or replaced by nonplasmid DNA. Similar results have also been reported
following treatment of the green alga Chlamydomonasreinhardtii with bacterial
plasmid DNA and selection for kanamycin resistance (Gresshoff and Hess,
1977).
None of the observations or studies reported in the preceding paragraphs
provide unequivocal evidence that foreign DNA can be utilized to genetically
modify plants. However, other somewhat more sophisticated evidence indicates
that DNA of bacterial origin can be replicated in (Ganem et al., 1976) and
translated by (Wang et al., 1976) eukaryote systems and, as mentioned earlier,
the stable transfer and expression of genetic information of isolated metaphase
chromosomes into mammalian cell cultures of a different species has been
achieved. In plants it would seem that the technology has not yet developed to
effect the transfer and subsequently detect the presence of foreign DNA. The
solution may come from the very recent, and indeed exciting, developments
commonly referred to as “genetic engineering.” In the sense of genetic modification by hybridization, selection, and mutation, genetic engineering has been
widely practiced by plant breeders. These new techniques however involve the
isolation and restructuring of DNA and the reinsertion into a cellular environment where indeed it will function. These techniques are being currently
SOMATIC CELL GENETICS AND PLANT IMPROVEMENT
65
explored as a means of generating genetic variation for use in plant improvement. It is appropriate therefore to consider them in this review.
C. MOLECULAR GENE MANIPULATION
The discovery and utilization of two natural phenomena in bacteria have made
the in vitro rearrangement of DNA possible. First, circles of double-stranded
DNA were found which replicated independently of the bacterial chromosome.
These extrachromosomal particles are called plasmids and they endow their host
with the capacity to adapt to new environments by conferring properties such as
drug or heavy metal resistance, the ability to metabolize long-chain hydrocarbons, and the ability to transfer genetic material by conjugation. Plasmids can
also be integrated into the bacterial chromosome. Some plasmids have a wide
host range and so genetic information can be transferred interspecifically.
Second, was the isolation of a class of bacterial enzymes, restriction endonucleases, which recognize a particular short sequence of DNA and cleave the
double-stranded DNA within this sequence. The enzymatic cut leaves singlestranded ends which are complementary. These endonucleases are normally
produced for degrading foreign DNA which may happen to enter the cell.
Concomitantly, a bacterium can modify its own DNA so that it will not be
degraded when nucleases are produced by that cell. Provided the specific
sequence is present and unmodified, the restriction nuclease will cut the DNA
no matter what its origin, be it prokaryote or eukaryote. Because the singlestrand ends of the staggered cut are complementary, an endonuclease fragment
from one species can be annealed with a fragment from another species that has
been degraded by the same endonuclease to form a ring which can be covalently
closed by incubating in appropriate enzymes (see Cohen, 1975). Hybrid DNA
molecules that have been produced to date usually contain a fragment from a
bacterial plasmid. This fragment has a very specific function, namely, a replication sequence, or replicon, i.e., a nucleotide sequence to which DNA polymerase
can attach so that the hybrid DNA molecule may be replicated. This plasmid
usually carries another gene, e.g., drug resistance, so that bacteria which are then
transformed by the hybrid molecule can be selected. In this way the hybrid
molecule containing the foreign DNA can be multiplied indefinitely and large
quantities of it can be purified.
Using this procedure of DNA fragmentation by endonuclease and hybridization with plasmid DNA, DNA sequences of Drosophila have been multiplied in
Escherichiu coli (Wensink el al., 1974). Some eukaryote DNA sequences specifying a particular function can be isolated, either because of the unique characteristics of that DNA, e.g., ribosomal DNA, or because a specific probe, e.g.,
mRNA, can be used to isolate the particular gene. These nucleotide sequences
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W. R. SCOWCROFT
may also be hybridized with plasmid DNA and multiplied in bacteria. For
example, this has been done with the genes of the toad Xenopus laevis that
specify ribosomal RNA (Morrow et al., 1974), the histone gene from sea urchin
(Kedes et al., 1975), and the gene from rabbit that specifies &globulin synthesis
(Rougeon et al., 1975). While the replication sequence of plasmids is probably
the most versatile, other such sequences have been used. For example, Struhl et
ul. (1976) have hybridized the replication sequence of the bacteriophage h with
yeast DNA, and this hybrid molecule multiplies when inserted into bacterial
cells.
In applying these techniques to transfer specific genetic information t o higher
organisms, replication sequences must be found which will permit the hybrid
molecule to multiply in the eukaryote cell. The purified DNA of the mammalian
virus, SV40, can transform mammalian cells. This has been joined to phage h
DNA and the hybrid molecule can be multiplied in monkey cells (Ganem et al.,
1976).
Because of our interest in developing new techniques for generating genetic
variability of potential value to plant improvement, we are currently examining
the application of DNA hybridization techniques to plants (Langridge, 1977;
Langridge and Scowcroft, 1977). Plant cell and protoplast culture provide a
convenient experimental system since it has been established that plant cells,
particularly protoplasts, can take up macromolecules such as viral particles and
viral RNA which will multiply in the cultured protoplasts. It is more difficult
however to find the appropriate vector since the replication sequences that are
adapted to multiplication in plants tend to be limited. There are a few DNA
molecules which may provide a replication sequence which can be utilized for
the purposes of genetic engineering in plants.
I . Possible Molecular Vectors for Gene Transfer in Plants
The first of the molecular vectors for gene transfer is the DNA of plant viruses.
Most plant viruses are RNA, either single- or double-stranded, and a few have
double-stranded DNA. The latter class comprise the caulimoviruses, of which the
best characterized one is cauliflower mosaic virus (CaMV) (Shepherd; 1976).
Recently, the unrelated potato leafroll virus has also been classified as a doublestranded DNA virus (Sarkar, 1976). CaMV has a molecular weight of 4.7 X lo6
and the purified DNA is infective in plants, but as yet has not been shown to
multiply following “infection” of protoplast cultures.
Chloroplasts and mitochondrial DNA may also provide a suitable replication
sequence. They have respective molecular weights of 90 X lo6 and 40 X l o 6 .
Current research is attempting to isolate the replication sequence of the chloroplast DNA.
A third possible molecular vector is a plasmid of Agrobacteriurn rurnefaciens.
This bacterium is a plant pathogen responsible for a neoplastic disease, crown
SOMATIC CELL GENETICS AND PLANT IMPROVEMENT
67
gall. The plasmid of A . tumefuciens may represent the first documented case of a
natural example where genetic information is tranferred from bacteria to plants,
and in the context of this review warrants further discussion.
Infection of wounded plants by A . tumefuciens leads to the transformation of
the host cells to the neoplastic, crown gall state. Once transformation has been
established the neoplastic growth is self-proliferating in the absence of the
inciting bacterium. Ever since this fact was firmly established, considerable
effort has been devoted to attempting to understand the molecular nature of the
tumor-inducing principle (TIP) (see recent reviews by Drlica and Kado, 1975;
Lippincott and Lippincott, 1975). The evidence for a transmissible TIP, although
indirect, is compelling. Secondary tumors appear on some infected plant species
and these may be free of the inciting bacterium. Bacteria-free crown gall cell
cultures can also induce nonself-limiting growth when grafted onto normal,
healthy plants.
The experimental evidence that bacterial DNA, RNA, or bacteriophage DNA
of A . tumefaciens was tumorigenic has been challenged and, indeed, unconfirmed by others (for discussion and appropriate references, see Drlica and Kado,
1975; Lippincott and Lippincott, 1975). Because of the lack of an unequivocal
assay for the phenomenon of transformation, most recent studies on the role of
bacterial or phage nucleic acids in tumor induction have utilized nucleic acid
hybridization techniques. The initial experiments which claimed sequence
homologies between tumor DNA and bacterial or phage DNA were based on the
hybridization of tumor cell DNA with RNA complementary to A . tumefuciens
DNA or PS8 phage DNA. Confirmatory evidence that A. tumefaciens sequences
were represented in tumor cell DNA was based on DNA-DNA filter hybridization experiments. However, in these studies the fidelity of base pairing, which
can be evaluated by examining the thermal stability of the presumptive DNADNA duplexes, was not determined. When this was done, it was found that no
more than 0.02% of the crown gall genome could be homologous with A .
tumefuciens DNA. This amounts to less than one bacterial genome per diploid
tumor cell. Similarly, little or no homology was found between bacteriophage
PS8 DNA and A . tumefuciens DNA.
There is now compelling evidence that a large plasmid is involved in the
tumor-inducing process. There is a high, though not absolute, correlation between virulence and the possession of a large plasmid (Zaenen er d., 1974). The
early demonstration of the transfer of virulence to avirulent Agrobucterium
strains by an unknown mechanism of genetic exchange in plunru (Kerr, 1969),
now appears to be due to plasmid transfer (van Larabeke er ul., 1974; Watson et
ul., 1975). Moreover, many virulent strains lost oncogenicity following growth at
high temperatures (Watson er ul., 1975; Bomhoff et ul., 1976) and this was
correlated with loss of a large plasmid. Different strains of A. tumefuciens carry
different plasmids, and these vary in molecular weight from 96 X lo6 t o I56 X
lo6 (Zaenen et uL, 1974). From DNA hybridization studies Matthysse and
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W.R. SCOWCROFT
Stump (1976) suggest that approximately 0.1% of bacteria-free tumor cell DNA
is A. tumefaciens plasmid DNA. This amounts to approximately 10 plasmids per
diploid tumor cell.
In addition to virulence there are a number of other properties which now
appear to be plasmid linked and they include bacteriophage exclusion (Schell,
1975), sensitivity to a bacteriocin produced by a nonpathogenic species, A.
radiobacter (Kerr and Htay, 1974; Schell, 1975), octopine or nopaline synthesis
and degradation (Bomhoff et al., 1976). These two unusual arginine derivatives,
octopine and nopaline, are normally found in tumorous tissue, and the production of one or other of these compounds is A. turnefaciens strain specific.
Moreover, strains with induced octopine in tumor cells are able t o utilize
octopine as a source of N for bacterial growth; strains which incude nopaline
tumors can utilize nopaline as a source of N. Genetic experiments (Bomhoff et
al., 1976) have examined the plasmid-linked nature of octopine or nopaline
utilization and systhesis. The plasmid-linked genes have an immense advantage
since cells containing this plasmid may be selected and characterized.
The crucial question of whether or not the plasmid is integrated into the host
cell DNA has not been answered. Neither has it been established that the plasmid
per se is capable of causing neoplastic growth of plant cells. Since cultured
crown gall cells are growth-regulator independent, and since other biochemical
properties are associated with the presumptive tumor-inducing plasmids, it
should be possible, using protoplasts, to establish unequivocally whether the
isolated plasmid can induce the tumorous state. We have begun research with
this specific object in mind.
2. Requirements of the Molecular Vector
There are other requirements which we believe essential to utilize such DNA
vectors for gene transfer in plants. First, it will be necessary to multiply the
hybrid molecules in some convenient organism and at present the bacterium
Escherichia coli is most suitable. Therefore the replication sequence that enables
the hybrid molecule to multiply in plants must be combined with a replication
sequence (i.e., of a bacterial plasmid) permitting multiplication in bacteria. Thus
the hybrid molecule could multiply in plants or bacteria. If efficient infection
and multiplication of the vectors previously discussed can be achieved in plant
cells, as has been obtained for tobacco mosaic virus, then indeed plant cells may
provide a convenient milieu in which to adequately multiply the hybrid molecule.
The hybrid DNA must aIso carry genetic information that is expressed in both
plant and bacterial cells in order to select those few cells which contain the
hybrid DNA, As mentioned earlier (Section VII, B), tobacco cells are sensitive to
similar concentrations of kanamycin (but not carbenicillin, tetracycline, or
SOMATIC CELL GENETICS AND PLANT IMPROVEMENT
69
neomycin) which inhibit protein synthesis in bacteria. No doubt other drugs or
antimetabolites will be found that equally inhibit growth of plant and bacterial
cells. The A . tumefaciens plasmids have the gene for octopine or nopaline
utilization which can be selected for, at least in bacteria. If the induction of
growth regulator autotrophy in plant protoplasts can be associated with infection by purified plasmid, then this provides another double selection system.
If the hybrid DNA molecule can be taken up, multiplied, and expressed in
plant cells, the ultimate object is to integrate i t into the plant genome. As
pointed out earlier (Section VI, B) kanamycin resistance appears t o have been
stabilized in tobacco cells following transformation by bacterial plasmid DNA.
The concomitant use of yirradiation may have led to integration brought about
by breakage and reunion. However, this evidence is only circumstantial.
3. Insertion Sequences and Transposons
It has generally been believed that integration of DNA into some specific
genome can be achieved only as a consequence of recombination between
segments of DNA having extensive nucleotide sequence homology or by breakage and reunion. Recently a new class of genetic elements has been characterized
in bacteria in which recombination only occurs at the termini of these elements
(see review by Cohen, 1976). The nucleotide sequences at these termini are
called insertion sequences and they have a defined length of 800-1400 base
pairs. There are several different insertion sequences. When such a sequence
inserts into a gene the function of that gene is abolished and indeed any other
genes distal to the point of insertion relative to the promoter for that operon.
When the sequence is excised from a gene the function of that gene, and any
other genes in the same operon which were affected, is restored. Any DNA
sequence can be contained between two insertion sequences and these termini
are arranged so that one is an inverted repeat of the other. Such complexes are
known as transposons because they can move from one hereditary element t o
another, e.g., from plasmid to plasmid, plasmid to chromosome, or plasmid to
bacteriophage, conferring on the receptor element the genetic function contained between the insertion sequences. These transposable genetic elements are
probably responsible for the immense genetic diversity of bacterial plasmids, and
indeed for the evolution of prokaryote genetic systems. Moreover, the transposable controlling elements that regulate phenotypic expression in maize (McClintock, 1956; Fincham and Sastry, 1974) are consequentially similar to
transposons in that they transpose to different chromosomal locations and in so
doing influence a variety of genetically controlled functions.
These insertion sequences may provide the mechanism for integrating the hybrid
molecules into the plant genome. Such a mechanism would mitigate the restraint of
the substantial need for sequence homology to effect integration. In the case of the
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W. R. SCOWCROFT
association between the plasmid of A. tumefaciens and crown gall, Schell et al.
(1977) propose as a working model that insertion sequences are involved in the
mechanism of transformation of plant cells to the neoplastic state. If this
proposition is substantiated, then molecular genetic engineering in plant improvement is a very real possibility.
Provided that a molecular vector can be developed for transferring foreign
genetic information into plants, the remaining problem is to decide what genes,
or gene-controlled functions, might usefully be incorporated into a plant breeder’s population. A related problem is the identification of the nucleotide sequence for the particular gene in the hybrid molecule, because for the transfer of
specific genes it is essential that the hybrid containing the appropriate nucleotide
sequence be obtained in quantity. If a bacterium is to be used to multiply the
vector, and since eukaryote genes are poorly expressed by prokaryotic protein
synthesizing systems, it is probably essential that purified mRNA appropriate for
that nucleotide sequence be used as a probe (Kedes et al., 1975; Grunstein and
Hogness, 1975). Since such mRNAs are not readily available for plant genes, t h s
is unlikely to be of much value. Alternatively a “shotgun” approach can be used
where a large number of hybrid molecules, containing random pieces of DNA
from the donor genome, are made and inserted into plant cells. The specific
hybrid molecule containing the desirable nucleotide sequence is selected by
virtue of a property that the gene confers on the recipient plant cell. The specific
hybrid selected in plant cells, from among a random set, could then be multiplied in an appropriate bacterium. Even the integration of a vector, containing
random pieces of DNA from another plant genome, into a plant species of
choice would markedly increase the quantity, and hopefully the quality, of
genetic variability on which plant breeders could practice selection.
D. PLANT IMPROVEMENT AND DESIRABLE GENES FOR
MANIPULATION
In this review, and where experimental information is available, I have presented examples where the asexual transfer of genetic information may be of
value to plant improvement (Section IV). These examples have included aspects
of disease resistance, tolerance to stress conditions such as salinity or flooding,
and the possibility of selecting for amino acid overproducing mutants. Other
physiological characters may be subject to genetic modification in cellular
systems. For example, freezing injury in plants has long been thought to be due
to dehydration injury (Burke et al., 1976), and Towill and Mazur (1976) have
recently demonstrated a close correspondence between freezing injury and
osmotic injury to cultured plant cells. This would certainly be amenable to cell
culture and protoplast studies where resistance to osmotic shrinkage could be
selected. The consideration of other possibilities, such as specific gene(s) modifi-
SOMATIC CELL GENETICS AND PLANT IMPROVEMENT
71
cation which affect yield, would require the expertise of plant breeders, physiologists, biochemists, and geneticists which is beyond the scope of this review.
However, one of the commonly advertised aims of cell culture and genetic
engineering is to confer on nonlegume crop plants the ability t o fix atmospheric
nitrogen.
1. The Special Case of Nitrogen Fixation
If nonlegumes could be developed w h c h were able to meet even part of their
nitrogen requirement directly from biological nitrogen fixation, the benefits
from reduced fertilizer use might be enormous. In the best known agricultural
nitrogen fixing system, the legume-rhizobia symbiosis, it has been estimated
that grain legumes fix as much nitrogen (about 40 X lo6 tons/annum) as is
currently provided by the application of chemical nitrogen fertilizers (Hardy and
Havelka, 1975). In addition it is estimated that by the turn of the century the
demand for nitrogenous fertilizers will rise to 200 X l o 6 tons/annum. There is
an obvious need for an alternative.
From an examination of known systems for biological nitrogen fixation it is
clear that the critical process of reduction of nitrogen to ammonia by the
enzyme nitrogenase is restricted to the prokaryotes. There is n o unequivocal
example of a eukaryote which synthesizes this enzyme (see Postgate, 1974;
Dilworth, 1974). Among the prokaryotes the ability to fix atmospheric nitrogen
is fairly widespread, particularly among those classified as primitive. These
microorganisms inhabit the rhizosphere and phyllosphere of plants and are
found in the water of rice paddies. The contribution of free-living nitrogen-fixing
nicroorganisms t o the nitrogen nutrition of plants is poorly understood and
probably minimal. Nonpathogenic associations of nitrogen-fixation microorganisms with lower and higher eukaryotes are also widespread in nature, and
include the blue-green algal associations with fungi (lichens) and the aquatic fern
Azolla (see appropriate chapters in Quispel, 1974). In addition more than a
hundred species of nodulated nonlegume plants are known to fix nitrogen; the
microsymbiont is almost certainly an actinomycete (Bond, cited in Quispel,
1974). Recently, von Bulow and Dobereiner (1975) have described the natural
occurrence of a nitrogen-fixing association between the bacterium Spirillum
Zipoferum and roots of monocots. Apart from pasture grass species, this association has been found in roots of maize and sorghum. The extent to which this
association provides fixed nitrogen for the host plant is still a matter for
conjecture. Clearly then eukaryote plants, during the course of their evolution,
have taken advantage of biological nitrogen fixation by entering into associations
with microorganisms.
Because of its agricultural importance the best understood nitrogen-fixing
association is that of the legume-rhizobia symbiosis, where the contribution to
the nitrogen nutrition of the host, and t o the nitrogen status of agricultural soils
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W. R. SCOWCROFT
is considerable. Recent evidence from our, and other, laboratories has shed
additional light on the relative roles of the bacterium and plant in legume
symbiosis which indicates that legume symbiosis is not as rigid as previously
believed. It was largely accepted that the legume host provided essential genetic
information for the synthesis of nitrogenase in legume nodules (Ddworth and
Parker, 1969). However, it is now known that at least some species of Rhizobium can fix nitrogen independently of the host (Pagan et al., 1975; McComb et
al., 1975; Kurz and LaRue, 1975). Moreover, these rhizobia can also fix nitrogen
when cultured with nonlegume plant cells such as tobacco (Scowcroft and
Gibson, 1975), wheat, rape, bromegrass (Child, 1975), carrot, and rice (Kurz and
LaRue, 1975). It seems therefore that the cellular environment of nonlegumes
does not inhibit the process of nitrogen fixation. Studies on the regulation of
nitrogen fixation in free-living rhizobia (Scowcroft et al,, 1976; Bergersen and
Turner, 1976) indicate that nitrogenase synthesis is not directly regulated by the
adenylylation/deadenylylation of glutamine synthetase as has been found for the
anaerobic nitrogen fixer Klebsiella pneumoniae (Shanmugam and Valentine,
1975).
A further index of the potential flexibility of nitrogen fixation by rhizobia is
apparent in its symbiosis with a nonlegume. Trinick (1973) showed that nodules
formed on the nonlegume Trema cannabina were due to a slow-growingstrain of
Rhizobium. In these nodules, leghemoglobin, which is normally required in
legume nodules to regulate the oxygen flux, was not found (Coventry et af.,
1976). It is possible that in these Trema nodules an oxypolyphenol oxidase may
act as an alternative 02-carrier, to maintain the nodule O2 flux required to
support N2 fixation. Alternatively, the rhizobia forming these nodules may have
undergone evolutionary adaptation to enable the nodule bacteria to survive and
fix N2 without an O2 stabilizing system. This could also account for the fact
that only some strains of rhizobia will fix nitrogen under free-living conditions
(Pagan et al., 1975). Trinick and Galbraith (1976) have further shown that
nodule development in T r e m is more rudimentary than that found in the
legumes. They suggest that nitrogen fixation occurs in the extensive infection
threadlike structures found in Trema roots as well as in the bacteria-filled
host cells.
Extensive studies have characterized the genetic regulation of nitrogen fmation in several bacteria, particularly Klebsiella pneumoniae (Brill, 1975). The
nif' gene(s) of Klebsiella can be mobilized and transferred to other bacteria
using bacterial plasmids. When such information is transferred to the closely
related species, Escherichia coli, the nif 'gene is expressed and can be integrated
into the E. coli chromosome (Cannon et al., 1974). Using a wide host range
plasmid, the Klebsiella nif' gene has also been transferred to nifAzotobacter
vinelandii cells (Cannon and Postgate, 1976), and to Rhizobium meliloti and
Agrobacterium tumefaciens (Dixon et al., 1976). It was only in the first
species that the Klebsiella nif' gene was expressed. In the latter two there