REVIEW OF LITERATUREshodhganga.inflibnet.ac.in/bitstream/10603/15106/8... · transferred from Ti...
Transcript of REVIEW OF LITERATUREshodhganga.inflibnet.ac.in/bitstream/10603/15106/8... · transferred from Ti...
REVIEW OF LITERATURE
The discovery of Ti plasmid in Agrobacterium tumefaciens has
given a big thrust to the research on gene transfer in higher
plants. In the last two decades the limitations of conventional
plant breeding have been realized and discussed by plant
biotechnologists and it has been proposed that to overcome the
limitations, gene transfer techniques should be developed.
The biggest obstacle for gene transfer to plant cell was'the non-
availability of a suitable vector. The crown gall diseas~ caused
by Agrobacterium tumefaciens has long been known (Braun, 1958)
but the discovery of Ti plasmid, present in the bacteria was made
only about 10 years ago (Van Larebeke et. al., 1974; Zaenen et.
a I . , 1974) • The potentiality of Ti plasmid was gradually
realised and at present Ti plasmid is considered to be extremely
useful to develop a vector system for gene transfer in higher
plant cells.
AGROBACTERIUM AS A VECTOR SYSTEM
The gram negative soil bacteria, Agrobacterium tumefaciens,
induces neoplastic growth on several dicot plants, called as
crown gall tumor (Smith and Townsend, 1907). Tumor formation is
the result of the transfer (Zaenan et. a I . , 1974) and
integration of a part of Ti plasmid into the plant genome
(Chilton et. al., 1977; Thomashaw et. al., 1980; Willmitzer et.
ale 1980) . Ti plasmids found in all virulent strains of ~
tumefaciens are about 200-250 kb in size and are stably o
maintained in Agrobacterium at temperature below 30 C (Van
5
L~rebeke et.al. 1974). Ti plasmids have two major regions i.e.
'Virulence' region (Vir) and transfer regio~ (T-DNA). These two
regions are involved in conjugative transfer and replication of
the plasmid in Agrobacteria.
T-DNA
The Ti plasmids can be classified on the basis of opine they
synthesize such as nopaline and octopine type Ti plasmids.
Opines are the source of nutrition for Agrobacteria and they are
produced by tumor cell after the integration of T-DNA into plant
genome. 'Nos' locus coding for nopaline synthase and 'ocs' locus
coding for octopine synthase are present on the T-DNA (Guyon et • • a I . , 1980) • Nopaline type plasmid§ like pTiC58 and pTiT37
contain about 23 kb T-DNA (Lemm~rs et.al., 1980) • In some
octopine plasmids (e.g. pTiA6NC, pTiAch5 and pTiB6), the T-region
is divided into two adjacent DNA segments, one is 13 kb (leftT-
DNA) and the other is 7 kb (right T-DNA). These two segments can
b.e transferred" to plant genome either independently or as a
continous stretch (De Beukeleer at.al., 1981) •
. T-DNA functions are expressed in plant cell, several
polyadenylated transcripts of genes residing on T-DNA were
detected (Bevan and Chilton, 1982; Willmitzer et. a I . , 1981,
1982, 1983) .. The position and direction of these transcripts are
well mapped. Using hybridization techniques, it has been found
that octopine and nopaline type T-DNA contAin 9 kb homologous
region, over which are mapped 1,2,4,5,6a,6b of the T-DNA encoded
transcripts (Engler et. ~l., 1981; Willmitzer at.al., 1983) •
Each of these transcripts represent less than 0.0011. of total
6
poly A+ RNA of tumor cells (Klee et.al.,
homologous region represent oncogenic loci
for tumor induction in plant cells.
ONCOGENES
1984) • The 9 kb
("onc") responsible
The studies based on transposon insertion and deletion
functional mutagenesis of T-DNA region have made possible the
analysis of the T-DNA encoded oncogenes. Mutations in gene 1 or
gene 2 induce tumors that produce abundance of shoots, indicating
that these genes encode for functions that suppress shoot
formation
formation
inhibition
(shi) . Mutation in gene 4 results in extensive root
therefore indicating that this gene codes for root
function (roi). The two loci together prevent shoot
and root differentiation and keep the tumor unorganized
(Garfinkel eta ala 1981; Joos et. al., 1983; Inze et. al., 1984).
Gene 1 (iaaM) codes for enzyme tryptophan 2-mono-oxygenase that
catalyses the conversion of tryptophan into indole acetamide,
which in turn is converted into an active auxin, IAA by the gene
2 (iaaH) encoded enzyme called Indole acetamide hydrolase. The
two loci iaaM and iaaH together present a new pathway of auxin
biosynthesis (Schroder et.al., 1984; Inze et.al., 1984). Gene 4
(iptz)
ca 11 ed
codes for the enzyme involved in cytokinin biosynthesis
isopentenyl
pyrophosphate and
transferase which converts
5'AMP into active cytokinin,
isopentenyl
isopentenyl
adenosine 5 monophosphate (Akiyoshi et.al., 1984; Barry et.al.,
1984) • Garfinkel eLal. (1981) identified a class of mutations
mapp~ng in the right most part of the T-DNA (Octopine type) that
induced unusually large tumors (tml). It can be concluded from
7
the transcriptional analy~es of Willmitzer et. ale (1983)
some of these mutations affect transcripts 6a and 6b.
that
Joos
et.al. (1983) developed various mutant Ti plasmids containing
deletions in 6a and 6b as well as right border. The tumor
morphology induced by this mutant Ti plasmid was not different
from that of wild type but showed attenu~tion of tumorogenesis on
tobacco. In a study of manipulation of hormones endogenously in
transgenic Petunia, iaa M and ipt genes were either put under the
control of 19S promoter or made inducible by hsp70 promoter of
maize .. It was'observed that overproduction of IAA leads to
extreme apical dominance. Similar charcteristics were observed
when chimeric hsp70 liaaM was induced by heat shock. Transgenic
plants containing chimeric ipt gene showed higher level of
greening. The hsp70lipt· containing plants showed these
characteristics even in uninduced conditions (Medford and Klee,
1989, Medford et.al., 1989). Another study concluded that other
oncogenes such as 5 and 6a and 6b do not by themselves influence
plant growth and differentiation or tumor morphology so their
functions remained unclear (Inze et.al., 1984). Some recent
studies on 6b have revealed that it modulates the functions of
iaa and ipt loci (Spanier et.al.,1989; Tinland et.al., 1989;
1990). The oncogenes have also been described as strain specific
(Bonnard et.al.,1989; Huss et.al.,1989; 1990).
T-DNA BORDERS
Ti plasmid is able to carry out the transfer of its T-DNA into
the plant cell by virtue of its virulence genes which are present
in trans with respect to T-DNA. The T-DNA is flanked by 25 bp
8
dir-ec t (imper-fect) r-epeats which act as T-DNA tr-ansfer- signal.
Only these 25 bp dir-ect r-epeats (having two conser-ved domains of
13 and 5 to 7 bp) at the end of T-DNA ar-e r-equir-ed in cis for- its
mobilization to the plant cell because its tr-ansfer- is unaffected
by a deletions of the inter-nal por-tions of native T-DNA or- by
placement of cloned fr-agment car-r-ying only T-DNA bor-der- r-epeats
(Wang et.al., 1984; Per-alta and Ream, 1985). It was' later
demonstr-ated that T-DNA flanked with bor-der-s could be placed even
on separ-ate plasmid or- on chr-omosome without affecting its
tr-ansfer- to plant cell (Hoekema et.al.,1983). Fur-ther- mor-e,
deletion of the fir-st 6 bp or- the last 10 bp of the 25 bp
sequences blocks T-DNA tr-ansfer- (Wang et.al., 1987) • Shaw
et. a I . , ( 1984) r-epor-ted that deletion of the r-egion over-lapping
the left bor-der- 25 bp r-epeat had little effect on Agr-obacter-ium
pathogenicity, wher-eas deletion of the r-ight bor-der- r-egion,
abolished cr-own ga 11 tumor- for-mation. Fur-ther-, if the
or-ientation of the r-ight bor-der- fr-agment is r-eversed, the
efficiency of T-DNA tr-ansfer- is gr-eatly attenuated (Peralta and
Ream, 1985). These r-esults suggested that T-DNA might be
transferr-ed in a r-ight ward to left war-d dir-ection, deter-mined by
the orientation of the bor-der- r-epeats. Recent data suggest that
ther-ear-e sequences adjacent to the 25 bp bor-der- r-epeats that
influence their- efficiency to pr-omote T-DNA tr-ansfer-. A 24 bp
DNA sequence situated· to the right and adjacent (within 60 bp) to
the r-ight copies of the native 25 bp bor-der r-epeats of TL and TR
T-DNA elements of the octopine Ti plasmid, called over-dr-ive, is
essential for efficient tr-ansfer of constr-ucts carrying only
synthetic 25 bp r-epeats. In case of octopine type T-ONA, ver-y
9
poor transfer of T-DNA occurred in the absence of overdrive
sequence and this sequence alone was active in the absence of its
neighboring sequences. Overdrive acts like an enhancer, it can
stimulate T-ONA transfer when placed in either orientation, on
either side and at variable distance from synthetic borders
(Peralta et.al., 1986; Van Haaren et.al., 1987). No satisfactory
model to explain the activity of borders and overdrive· has
emerged, on the basis of present data available. Other important
informations reported on overdrive are highly dissimilar
sequences immediately surrounding octopine or nopaline borders
are. In addition, there are no sequences adjacent to Nop right
border with good homology to the octopine 24 bp overdrive
seq'-:lence (Wang et.al., 1987) • Thus any potential overdrive
sequence in nopaline Ti plasmid must be either very different , '
f~om those in octopine Ti plasmid or further away from the
border. The existing hypothesis to explain the enigma 01
overdrive in nopaline is that, the nopaline vir proteins may
inherently be more active than their octopine counterparts.
Support for this hypothesis has been provided by the experiment
where the presence of overdrive is not required for efficient T-
DNA transfer from the octopine Ti plasmid if the concentration of
vir specific products is elevated by increasing the copy number
of ONA sequences overlapping the vir region (Zambryski, 1988).
10
VIRULENCE REGION
During ihe infection of plant with A. tumefaciens and before the
development of tumor a complex set of reactions take place as a
result of which T-DNA
cell. The abil i ty
is
of
transferred from Ti plasmid to the plant
the bacteria to do so is defined as
'virulence' •
chromosomal
The virulent bacteria contain two sets of genes
virulence (chv) genes (Douglas et.al., 1985) and Ti
plasmid virulence (vir) genes (Stachel and Nester, 1986). While
chv are constitutively expressed, vir gene expression is induced
by plant signal molecules such as acetosyringone and dC-OH
acetosyringone (Stachel et.al., 1~85). The chv A and chv B loci
are essential for virulence and specify the binding of
Agrobacteriumto plant cells (Douglas et.al. 1985). The chv A
and chv B are located on a 15.5 kb segment of Agrobacterium
chromosome. The 8.5 kb chv B codes for a membrane protein of
approx 235 kd that acts as an intermediate in the synthesis of
cyclic -1,2 glucan and chv A may code for a transport function
(Zorreguieta . et.al. ,1988). Another locus of chromosomal
virulence called as psc A, is approx 3 kb and is. required for the
synthesis of the major neutral and acidic extracellular
polysaccharides (Thomashaw et.~l., 1987). All three loci (chv A,
chv Band psc A) have dramatic effects on the surface composition
of bacterial cells, but it is not known exactly how their
products enhance attachment to plant cells.
Ti PLAMID VIRULENCE (Vir) GENES
Wounded plant cell s are susceptible to infection by
Agrobacterium. Earlier it was thought that wounding is important
11
since it removes physical barrier (cell wall) for the
penetration. However, wounded but metabolically active cells
have been shown to excrete low molecular weight signal molecules
recognized
The signal
by the Agrobacterium to induce vir gene expression.
molecules were purified from the culture media of
tobacco cells and identified as acetosyringone (AS) and hydroxy
et • a I . , acetosyringone (HO-AS) (Stachel et.al., 1985; Stachel
1986) • AS and HO-AS resemble products of phenylpropanoid
the major pathway to produce plant secondary metabolism,
metabolites lignin and flavonoids which are important to plant
under injury. AS can act as a chemo attractant for Agrobacterium
in vitro, suggesting that its presence at plant wound sites in
nature may serve a chemotactic role (Ashby et.al., 1987).
The induction of vir gene expression was shown to be at the level
of transcription (Janssens et.al., 1986). The vir genes required
for T-DNA transfer are located in trans with the border sequences
in 40 kb Vir region (Stachel and Nester, 1986). Gen~tic analysis
of Ti plasmids have shown that Vir region encodes at least six
separate complementation groups - vir A,B,C,D,E,G. The induction
and regulation of vir genes expression is coupled with two types
of processes, extracellular recognition and intracellular
response. These two processes are mediated by the products of
virA and virGo Mutation in virA severely attenuates and that in
virG totally abolishes the induction of other vir loci (Stachel
and Zambryski, 1986). virAl virG coupled function shares analogy
with other pairs of bacterial proteins which act as sensor
regulator of gene expression in response to environmental stimuli
12
ego env Z / Omp R, nt~ B /nt~ C, pho R /pho B genes of ~ coli
~espond to
concent~ations
changes in osmola~ity, ni t~ogen and
~espectively (Ronson et.al., 1987).
phosphate
The fi~st
gene in each pai~ codes fo~ a memb~ane p~otein that di~ectly
senses the envi~onment. The second gene acts as an activato~ of
t~ansc~iption of othe~ genes. The t~ansfe~ of info~mation f~om
senso~ to ~egulato~ could be via phospho~ylation and
dephospho~ylation, which
(Ninfa and Magasanik,
is best unde~stood fo~ nt~
1986) . By analogy, vi~A
B /nt~ C
most likely
functions as a chemo~ecepto~ which senses the p~esence of AS and
t~ansfe~s the info~mation to the inside of bacte~ia by
modification of vi~G, which is as yet not unde~stood.
·Like its homologous counte~pa~ts, vi~A has a t~ansmemb~ane domain
and cell f~actionation expe~iments have localized vi~A on to
inne~ memb~ane (Le~oux et.al., 1987). How vi~G activates othe~
vi~ genes is not yet unde~stood. vi~A is constitutively
exp~essed, whereas vi~G ~egulation is ve~y complex. The~e a~e
two distinct vi~G messages which diffe~ at thei~ 5'- te~mini. One
.is constitutive message which is p~esent du~ing vegetative and
induced conditions. The othe~ one is induced message, p~oduced
only du~ing induction and is found to be 50 bp longe~ at its 5'
te~mini. The induced message is p~esent at 10-fold highe~ levels
than the constitutive message (Stachel and Zamb~yski, 1986) •
Vi~G exp~ession is fu~the~ complicated by the fact that its
induction is ~egulated at two diffe~ent leve15. One level is
independent of the p~esence of a wild type copy of vi~G, since
vi~G mutants show a significant ( 25%) level of plant induced
vi~G t~ansc~iption. The second level is dependent on intact vi~
13
G. Thus virG positively autoregulates its own expression (Stachel
and Zambryski, 1986). Further, virG is produced at high level
which is an unusual property for an activator. One explanation
could be that the virG product is not very efficient molecule
and is required in high turnover. This hypothesis is supported
by the discovery of super-virulent Aqrobacterium A 281, which has
been found to produce higher levels of virG product (Jin et.al.,
1987).
In the vir region, virA and virG are the only monocistronic loci,
coding for 70 kd and 30 kd respectively. Vir C is 2 kb and codes
for two proteins 26 kd and 23 kb (Yanofsky and Nester, 1986).
The 2 kb virE region predicts two polypeptides 7 kd and 60.5 kd
(Winans et.al., 1987). The virD region predicts four
polypeptides of 16 kd, 47 kd, 21 kd and 75 kd (Jayaswal et.al.,
1987; Porter et.al., 1987). Vir B is the largest locus of about
9.5 kb and the nucleotide sp.quence of virB predicts 11
23.5 polypeptides 25.9 kd, 12 kd, 11.7 kd, 21.6 kd, 65.7 kd,
kd, 31.7 kd, 5.7 kd, 26.1 kd, 72.7 kd, 38 kd. (Ward
1988). However, virB data of Ward et.al (1988) does not
with genetic studies using AS-induced Aqrobacterium
et.al,
agree
which
identified 33 kd, 80 kd, 25 kd by N-terminal half of the locus
(Engstrom et.al., 1987). The most abundant Vir proteins produced
in AS-induced Agrobacterium are virE and virB polypeptides. VirE
encodp.s a single stranded (ss) DNA binding proteins which could
stoichiometrically cover T-DNA molecules (Christie et.al., 1988;
Citovsky et.al., 1988; Das, 1988). While the function of virB
products is not known, their ~ssociation with cell envelop as
studied in cell fractionation experiments suggest that they are
14
transmembrane proteins and might be playing some role in
directing T-DNA transfer which occur at the bacteria cell
surface. Their high level of production tits well in this theory
(Engstrom et.al., 1987). The virD locus of pTiA6 was sequenced
and computer analysis indicated five possible ORFs. However only
two polypeptides of mol. wt. 16 and 56 kds, the product of vir 01
and virD2 were detected in ~ coli. Vir 0 products exhibited
double stranded (ds) T-DNA border specific endonuclease activity.
Deletion analysis demonstrated that this activity is encoded
within the 5' proximal 1.7 kb portion that carries ORF1 and ORF2.
Neither of the ORF showed endonuclease activity independently
(Jayaswal et.al.,1987 ). Another study demonstrated that after
cleavage of T-DNA border by vir 0 products both ds, nicked T-DNA
molecules and ss T-DNA (T strands) were present. It was
determined by deletion analysis that vir 02 product is tightly
associated with and probably covalently attached to the 5' end of
the T strands (Young and Nester, 1988). In the Agrobacterium
culture induced by AS , about 5 additional proteins, designated
as virulence related proteins (VRPs) have been detected (Engstrom
et~ al., 1987). The expression of VRP genes is under the control
of vir A Ivir G. One VRP of mol. wt. 45 kd, is encoded by pin F
(plant inducible locus F) which maps immediately adjacent to vir
A (Stachel and Nester, 1986). Another VRP of 27 kd is not yet
mapped on Ti plasmid, while the other VRPs are most likely
chromosomaly encoded. pin F locus has been shown to be non
essential, other VRPs may directly function in T-DNA transfer by
providing some regulatory component (such as sigma factor for RNA
polymerase) or a component of transfer system (such as membrane
15
protein) (Zambryskiet.al., 1989) •
T-DNA TRANSFER INTERMEDIATE MOLECULES
Hypothetically, there exist two possibilities, (1) Transfer by
cleavage at border sites resulting in the loss of T-DNA from Ti
plasmid, (2 ) Site specific cleavage and copy of the T strand
without losing T-DNA.
The initial studies, however, suggested that the T-DNA
intermediate might be a ds circular molecule. This work was
designed to select only circular T-DNA molecules. The T-circles
were rescued in ~ coli with total DNA prepared from vir induced
Agrobacterium (Koukolikova-Nicola et.al., 1987; Tim~erman et.al.,
1988) • Initially, the theory of T-circles seemed to be an
attractive one, but taking into account the frequency
circles production which is calculated to be between 3 X -3
(Timmerman et.al., 1988) and 10 (Machida et.al., 1986),
of T-3
10
one
cannot strongly support it. the transfer intermediate production
cannot be a rare event occurring at such low frequency. The
second possibility is ds linear molecule. This structure was
speculated on the basis of the data that. detected ds breaks at
the borders of Ti plasmids (Veluthambi et.al., 1987).
However, any intermediate molecule derived as the result of T-DNA
loss, seems to be an unlikely possibility because the T-DNA
transfer process would not evolve to be suicidal. The analysis of
Agrobacterium DNA after Vir induction has shown that the major
population of T-DNA intermediate is that of linear ss copy of T-
DNA region designated as the T-strand (Stachel et.al., 1986;
Stachel et.al., 1987; Veluthambi et.al., 1987).
16
T-STRAND AS THE TRANSFER INTERMEDIATE
Total DNA from Agrobacterium induced by AS was isolated and
electrophoresed and blotted on the membrane filter. Fine
techniques of DNA analysis demonstrated the presence of a linear
ss DNA copy of the T-DNA region (Stachel et.al., 1986). This
molecule called as T strand, is produced at about one copy per
bacterium and corresponds to the bottom strand of the nopaline T-
DNA. Based on the discovery of T-strand and functions of Vir
loci, following model is being considered to explain the
mechanism of T-DNA transfer T-strand may be packaged into a viral
like particle coated with Vir E2 protein but the. fact that T
strands are not produced in abundant quantities and that T-strand
transfer requires close physical contact between Agrobacterium
and the plant cell suggests that the process can not be. analogous
to viral infection. The T-DNA transfer process may be similar to
conjugation (Lichtenstein and Fuller, 1987; Ward and Barnes,
1988), wherein close contact is important. The borders nicks
are analogous to nicks at the origin of conjugal transfer. The
T-strand is analogous to linear ss donor DNA. AS-induction
might result in a T-strand synthesis analogous to replacement
strand synthesis. A very strong experimental support comes from
the study wherein ori T from conjugative ~ coli was used as a
substitute for the T-DNA borders (Buchanan-Wollaston et.al.,
1987).
17
Ti BASED PLANT GENE TRANSFER VECTORS
Attempts have been made to modify Ti plasmid into a useful gene
transfer vector. To monitor gene transfers, one marker genes
which have been routinely used in animal systems were cloned in
T-DNA. Since direct cloning in Ti plasmid is not possible, gene
is first inserted into T-DNA by site specific insertion. The
resulting vectors is then recombined with wild type Ti plasmid.
This procedure depends on a double crossover event for insertion
of marker gene (Matzke and Chilton, 1981). Further improvements
were done in construction of vectors. Among the first of such
improved vectors is cointegrate vectors, which are the results of
a single- cross over event between T-DNA sequences and an ~ coli
plasmid. (Herrera-Estrella et.al., 1983). This method would
still result in oncogenic Ti vectors which will induce tumorous
growth. Therefore, a new type of vectors were constructed which
were non oncogenic i.e. deleted in most of the T-DNA borne genes.
The efficient non-oncogenic vectors consisted of pBR322 sequences
flanked with T-DNA borders. Such vectors could be used for
single step recombination for introducing foreign gene in T-DNA.
One such vector is termed as pGV3850 (Zambryski et.al., 1983).
The chimeric genes of nos or ocs promoter and npt2 coding region
were constructed and introduced into non-oncogenic Ti vectors.
Successful transformation followed by regeneration of whole
plant expressing foreign marker gene were achieved (De Block
et.al., 1984; Horsch et.al., 1985). Some useful marker genes
constructed, thereafter, are - neomycin phosphotransferase II
I I ) (Reiss et.al., 1984); hygromycin phosphotransferase (npt
(hpt) gene from ~ coli (Waldron et.al., 1985), phosphinotricin
18
acetyl transferase (PAT) gene from streptomyces (Thompson et.al.,
1987), a mutated mouse dihydrofolate reductase (dhfr) conferring
versatile resistance to methotrexate (Eichholtz et.al., 1987) ,
marker gene for gene regulation studies iSfl-glucoronidase (GUS)
from ~ coli (Jefferson et.al., 1987), luciferase gene (lux A
and lux B) from Vibrio photimus (.Ow et.al., 1986) . Another
modification carried out in Ti vectors was a contribution of
Monsanto Co., USA. The new vector was called as split-end-vector
(SEV) that carried the two T-DNA borders on separate plasmids.
Only the left border incJuding3kb TL-DNA is present on the
avirulent plasmid TiB6S3SE. It is complemented by pMON200 which
provides right border including nos gene, a polylinker containing
multiple cloning site, a marker gene (nos/npt2), bacterial
selectable marker gene and a reg~on of homology to the TL-DNA of
pTiB6S3SE. Since pMON200 alone cannot replicate in
Agrobacterium, cointegrates carrying engineered T-DNA can be
selected for by bacterial resistance marker (Fraley et.al.,
1985) • Another strategy of vector construction was used to
develop binary vectors. In an Agrobacterium, reside two types of
plasmids (1) a modified Ti plasmid providing vir functions in
trans (2 ) a broad host range plasmid carrying a gene construct
of interest, mobilization and replication functions of RK2 and a
bacterial selectable market gene. All these sequences combined
in a plant gene vector cassette can be maintained, replicated and
mobilized back & forth between ~ coli and
(Hoekema, et. a 1., 1983).
19
Agrobacterium
PLANT TRANSFORMATION
Initially, wounding and infection was the m06t common method of
developing tumor. When only non modified Ti plasmids were
available, regeneration could not be achieved. Wounding was done
either on whole plant or explants. transformants were selected
for phytohormone autotrophy.
The development of non-oncogenic engineered vectors with
selection markers made it possible to cocultivate Agrobacterium
with protoplasts or cell suspension. This method proved more
efficient since it was possible to get mora, number of
regenerated transformed plants (Wullems et.al., 1981 and Marton •
et • a 1 • , 1979) The method was further improved by incorporation
of feeder layer culture system (Fraley et.al., 1984). However,
the above mentioned methods relied on preparation of protoplasts
which remains a problem for several plant species. Therefore,
the method of leaf disc cocultivation with Agrobacterium hAS been
widely practised. The simplicity and efficiency of leaf-disc
transformation method has made it most popular method so far
(Horsch et.al., 1985; Lloyd et.al., 1986; Mc Cormick et.al.,
1986). Similarly cocultivation of other explants such as stem and
calli have proved to be equally successful. For fast
regeneration and lowering of transformed plant (for genetic
analysis), the approach of cocultivation with epidermal segments
of flowering branch has been very useful (Trinh et.al., 1987) .
The very similar methods have been useful for the transformation
of explants with ~ rhizogenes, which induces hairy roots
formation.
20
STRUCTURE AND INHERITANCE OF FOREIGN GENES
Using protoplast and Agrobacterium cocultivation method, it was
.possible to isolate clones of T-DNA transformed cells. It was
observed that phenotypic expression of T-DNA genes was often
aberrant. These clones were shown to contain short T-DNAs
(Marton et.al., 1979; Ooms et.al., 1982). Tumors deficient in
iaa genes produce abnormal shoots (teratoma). They form roots on
ipt inactivation. They lack nopaline or agrocinopine when nos or
acs are defective. It was demonstrated that such phenotypic
variations are caused by loss of T-DNA or methylation. The
physical mapping data suggest that early in the ·transformation
cycle~ of Agrobacterium, a replication step of a pre-sele~ted T-
DNA occurs before integration into the plant genome (Van
Lijsebettens at.al., 1986).
Several studies have is usually
integrated into nuclear a Mendelian
manner. The integration distributed
allover the genome (Ambros et.al., 1986). Recently, it has been
demonstrated in plants that foreign gene can be targeted on to a
specific site where it integrates as a result of homologous
recombination. In most of the cases of random insertions, foreign
DNA sequences are integrated at a single locus or as a cluster of
tandem copies which behave as single dominant Mendelian trait.
However, multiple insertions into two or more different sites on
different chromosomes were also observed. Cotransformation with
two strains
segregation
1984) .
having distinct genes has demonstrated the
of traits in the F1 generation (De Block et.al. ,
575- '7: ~g ShSI
• 21 ,.lJ
-r~':l ...
Peerbolte et.al. (1987) reported the occurrence of somaclonal
variation in 3 years old cultures of crown gall tissues. The
change in the morphology of tumor line, apparently, resulted from
a considerable rearrangement of DNA sequences accompanied by
deletions and possibly amplifications. Methylation of T-DNA in
crown gall tumors was studied in several lines. In all tumor
lines, atleast one T-DNA copy was unmethylated (Gelvin et.al.,
1983) . Phenotypic variation in a tumor line induced by anti
auxin treatment were cloned and stable variants were selected.
The molecular analysis revealed that the basis of variation is
suppression of
( Amas,ino
found to
et.al.,
have
T-DNA oncogenes as a result of DNA methylation
1984) • Similarly, 'nos' gene expression was
been switched off as a result of cytosine
methylation (Hepburn et.al., 1983). The new trait developed as
a result of methylation could be reversed by the treatment with
5-azacytidine. The hybridization analysis of plant genome
carried out before and after integration of T-DNA using T-
DNA/plant DNA junctions as the probes revealed that several types
of rearrangements resulted from integration of T-DNA such as
formation of direct repeats of target plant sequences, deletion
and insertion events at the junctions. The study suggests that
T-DNA insertion is a multiple step process of recombination
accompanied by local replicative and repair activities mediated
by host cell enzymes (Gheysen et.al., 1987).
22
BIOCHEMICAL BASIS OF CELL PROLIFERATION/DIFFERENTIATION
The biochemical and molecular events governing cell proliferation
or differentiation are not quite explored. One basic reason of
the lack of knowledge in this field could be the non-availability
of an ideal plant system, that can be manipulated for
proliferation and differentiation in defined conditions. Tobacco
tissue culture is a model system where shoot differentiation is
well controlled by cytokinin. Howevwer, in many plant systems
hormones fail to induce differentiation. Crown gall tumor is
an ideal system to study cell proliferation. However, almost no
report on biochemical studies on crown gall tumor exists .except
for the ones describing hormonal analysis.
POLYAMINES - In higher plants, Put is prQduced-'b~~ two pathw~ys
(1) L-Ornithine is converted to Put and the reaction is catalyzed
by ornithine decarboxylase (DOC) (2) arginine decarboxy~ase
(ADC) catalyzes the conversion of arginine to Put through the
intermediate agmatin. ADC has been reported to have a role in
cell elongation response to light (Kaur-Sawhney and Galston,
1979) and osmotic shock (Flores & Galston, 1984). DOC has been
reported to be affected by various stimuli such as hormones drugs
and growth factors (Bagni eta al., 1983). Polyamines are known
to play an important role in cell proliferation and
differentiation. Several studies have reported the alteration in
the individual and cumulative levels of polyamines in
proliferative and differentiating culture .. The precise function
of polyamines is not known. They may be involved in signal
transduction mechanism (See Smith, 1985; Evans and Malmberg,
23
1989). It was reported that exposure to red light increased the
level of putrescine, agmatin and spermidine in the bud of pea
seedlings whereas in internodes a decrease was observed (Goren
et.al., 1982). Another study on carrot reports that inhibition
of ADC with specific inhibitors and therefore, decrease in Put
level had no effect on cell number but increased fresh weight by
cell expansion, spermine titer increased on ADC inhibition
(Fallon .and Phillips, 1988).
ROLE OF GLYOXALASE-I IN CELL PROLIFERATION - Glyoxalase enzyme is
found in all living organism investigated. It catalyzes the
conversion of methylglyoxal (MG) to D-lactic acid. The enzyme
system consists of two units, glyoxalase-I and glyoxalase~II
having reduced glutathione as a cofactor. MG is synthesized from
dihydroxyacetone phosphate catalyzed by MG synthase or by
aminoacetone catalyzed by amino oxidase. MG is highly toxic
substance and its removal is necessary for a healthy cell.
Glyoxalase-I has been purified and studied extensively in animal
systems, and microorganisms but only a few studies have been
carried out in plant systems (Thornalley, 1990). In plant
systems, glyoxalase-I was first reported in pea seedlings, the
enzyme activity was found to be higher in meristematic
In Datura, glyoxalase-I activity increased with the increase in
DNA and protein synthesis (Ramaswamy et.al., 1983; 1984).
Glyoxalase-I activity was correlated with increase in fresh
weight and cell proliferation in Amaranthus (Das et.al., 1987).
24
PHOSPHOINOSITIDE CYCLE (PI) The membrane lipids,
phosphoinisitides, have been shown to undergo hydrolysis in
response to external stimuli (Berridge and Irvine, 1984). The
turnover of myo-inositol containing phospholipids has been
studied and proposed to have a very important role in signal
transduction (Berridge, 1987). The agonist mediated stimulation
of PI kinases results in formation of phosphotidylinosito 4,5
bisphosphate (PIP ), which is hydrolyzed by phospholipase C into 2
diacylglycerol (DAG) and inositol triphosphate (IP), both of 3
which are considered as second messengers to initiate a cascade
of signal transduction IP causes release of intracellular Ca++ 3
from ER and DG stimulates protein kinase C. PI cycle
intermediates have been determined in Samanea saman pulvini
(Morse et.al., 1987). It was also determined that PI turnover
increased on light stimulation in Samanea saman pulvini (Morse
et.al., 1987). In a recent report PI turnover has been
correlated with plant cell differentiation caused by amino acids
and polyamines inhibitors (Sethi et.al., 1990).
Thus, biochemical basis of cell proliferation and differentiation
in normal plants have been studied by a few groups but, almost no
report exists on transformed plant tissue. Tobacco tumor raised
by infection with nopaline strains gives rise to t@ratoma, shooty
tumor. We consider tumor and teratoma to be an ideal system to
study the biochemical basis of cell proliferation and
differentiation. Recently the biochemical and molecular basis
of plant cell differentiation has been studied and reviewed by
Sethi and Guha-Mukherjee (1990).
25