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