BIOTECHNOLOGY – Genetic Engineering of Plants - J. A.

Thomson

GENETIC ENGINEERING OF PLANTS

J. A. Thomson
Department of Microbiology, University of Cape Town, South Africa

Keywords: Agrobacterium tumefaciens, biolistics, transgenic plants, transformation,

gene silencing

Contents

1. Introduction
2. Transformation of dicotyledonous plants
3. Transformation of monocotyledonous plants
4. Transformation of algae
5. Promoter efficiency and tissue specificity

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6. Targeting genes to organelles

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7. Integration and stability of transgenes
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Acknowledgements
Glossary

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Bibliography
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Biographical Sketch
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Summary
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Genetic engineering can be used to introduce specific traits into plants. It will not
replace conventional breeding but can add to the efficiency of crop improvement. It is
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possible due to the fact that plants are totipotent, enabling regeneration of a new plant
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from an isolated cell. Transformation of dicots is usually carried out using the
bacterium, Agrobacterium tumefaciens. Genes are cloned into plant expression vectors
that carry the right and left border sequences. They are introduced into plants with the
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aid of a disarmed Ti plasmid whose virulence gene products allow the genes to be
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transferred to the plant nucleus where they are integrated into the genome. Monocots are
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usually transformed by a biolistic process, using a “gene gun”. In both cases callus
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tissue is regenerated on media containing an antibiotic or herbicide to select for

transformants. The exception is transformation of the common wall cress, Arabidopsis
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thaliana, where tissue culture is not required. Gene silencing, whereby plants shut off
the expression of multiple copies of a gene, can be a problem when attempting to
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introduce a new trait into a plant. Although the process is not fully understood it can, at
the level of transcription, be due to methylation or ectopic DNA pairing. At the post-
transcriptional level, the transgene RNA could be specifically degraded if tagged by a
small complementary RNA molecule. It is often advantageous for plants to express the
introduced transgenes in specific tissues or under specific conditions. As a result many
genes are cloned downstream of tissue-specific or inducible promoters. High expression
of a transgene may be required under certain circumstances. The 35S promoter of the
cauliflower mosaic virus is commonly used in dicots while the maize ubiquitin promoter
is the monocot promoter of choice. Targetting genes to organelles such as chloroplasts
can also enhance expression. Little is understood of the way in which genes are
integrated into the plant chromosomes. In many cases multiple inserts occur at one

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locus. The field of plant genetic engineering is a fascinating one and will continue to
grow in efficiency and sophistication in the years to come.

1. Introduction

Conventional plant breeding has succeeded in producing a wide variety of commercial

plants and crops with a range of important agronomic traits. It has succeeded in
converting a Mexican grass into maize and Middle East grass into wheat. However, it is
to a large extent a hit-or-miss process, combining large parts of parental genomes in a
rather uncontrolled fashion, although this is currently being improved due to the modern
technique of marker assisted breeding. Genetic engineering, on the other hand, allows
scientists to transfer very specific genes into plants, resulting in the introduction of one
or more defined traits into a particular genetic background. This process is called
transformation and the genes involved are expressed to form a protein responsible for
the particular trait. The traits involved include herbicide and drought tolerance, and

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resistance to viral, bacterial and fungal pathogens as well as to herbivorous insects. The

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added advantage is that the transferred gene(s), or transgene(s), can come from any
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organism as long as its expression is compatible with its new host.

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Figure 1: Plant transformation. Plant material is transformed with DNA carrying a

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selectable marker and the gene of interest. Callus tissue develops on selective media.
Shoots develop on the addition of cytokinins. Withdrawal of cytokinins promotes root
development.

Plant transformation is possible due to the fact that plants are totipotent, enabling
regeneration of a new plant from an isolated cell. Thus if a gene is transferred to a plant
genome in a cell the regenerated plant will contain that gene in every cell. In practice
the gene(s) of interest are introduced together with a selectable marker such as
resistance to a herbicide or antibiotic to which the plant is sensitive. Cells and
regenerating plants are grown in gel-like media containing that herbicide or antibiotic
and only plants expressing the genes for resistance will grow. The hormone auxin is
used to initiate and maintain callus. Once cells have been transformed, cytokinin
hormones are incorporated into the medium to allow shoot development. Withdrawal of
cytokinin promotes root growth. Once plants are fully developed they are taken out of
the media and planted in soil for “hardening off”. The process is shown

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diagrammatically in Figure 1.

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2. Transformation of dicotyledonous plants

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Dicotyledonous plants are those which develop from two cotyledons in the seed. They
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can be recognized by the branching veins in their leaves. Dicots of commercial value
include many horticultural plants such as petunias, and crops such as tobacco, tomatoes,
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cotton, soybean and potatoes. Petunias have been engineered to produce a range of
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attractive flower colors and patterns. Tobacco, due to its ease of transformation, initially
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became the workhorse of plant genetic engineering, but more recently the common wall
or thale cress, Arabidopsis thaliana, has become very popular. It has the advantage of
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not requiring tissue culture during its transformation. Tomatoes have been transformed
to delay their ripening, cotton to insect resistance and herbicide tolerance, soybeans to
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improved oil quality and herbicide tolerance, and potatoes to resist viruses.
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2.1 Transformation using Agrobacterium tumefaciens

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Agrobacterium tumefaciens is a plant pathogenic soil bacterium. It makes (tumefacient

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= swollen: Latin = tumefacere) a tumor on plants it infects and as these are often on the
crown region where the stem meets the roots, the disease is called Crown Gall.
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Scientists were amazed to discover that the bacteria transfer part of their DNA to the
plant nucleus where it becomes integrated into the plant genetic material. The
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transferred DNA, or T-DNA, is part of a large tumor-inducing (Ti) plasmid. The T-

DNA carries an onc (oncogenic) region, which, by coding for the production of plant
growth hormones, results in the proliferation of plant cells forming a tumor or gall. It
also codes for the production of unusual derivatives of arginine, such as nopaline or
octopine, which the bacteria can use as growth substances. This bacterial-plant
interaction is known as genetic colonization. It was not long after this discovery that
scientists realized that the introduction of a foreign gene into the T-DNA would enable
its transfer to the plant cell nucleus. This led to the development of plant transformation
using a disarmed, onc-, version of the Ti plasmid that could transfer DNA into plants
without causing the production of a tumor.

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BIOTECHNOLOGY – Genetic Engineering of Plants - J. A. Thomson

The Ti plasmid is very large, in the order of 200 kb, and therefore unwieldy to work
with in vitro. It was soon discovered that all that is required for a gene to be introduced
into a plant are the 25-bp repeat sequences at the borders of the onc region, known as
the left and right borders (LB and RB), and the virulence genes (vir) of the Ti plasmid.
It was possible, therefore, to separate these in a system of binary vectors. Genetic
manipulation is done in Escherichia coli on a small plasmid carrying a multiple cloning
site (MCS) downstream of a plant promoter, and a gene coding for resistance to a
herbicide or antibiotic that is toxic to the plant of interest, situated between the LB and
RB. This plasmid is then transformed into a strain of A. tumefaciens carrying a disarmed
Ti plasmid, which essentially consists only of the vir region and an origin of replication
(Figure 2).

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Figure 2: Binary vectors. ApR, ampicillin resistance for selection in E. coli; SmR,
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streptomycin resistance for selection in A. tumefaciens; ori E.c, origin of replication for
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E. coli; ori A.t, origin of replication for A. tumefaciens; RB, right border; NPTII,
kanamycin resistance for selection in plant cells; MCS, multiple cloning site; LB, left
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border
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This strain of A. tumefaciens is then used to transform plants. The earliest species to be
transformed was tobacco, Nicotiana tabacum, which rapidly became the model dicot
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plant. However, more recently the workhorse has changed to Arabidopsis thaliana
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which has a very small genome of 120 Megabases and is easier to transform. In order to
transform tobacco, and most other dicots, leaf disks are cut and placed in a Petri dish
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containing a liquid medium. The A. tumefaciens strain is placed on the surface of the
disks and co-cultivation carried out for 2-3 days. The cutting of the leaf disks results in
the plant producing wound-response compounds, such as acetosyringone, which induces
the virulence genes. The leaf disks are then transferred to selection media containing the
herbicide or antibiotic of choice. This is often kanamycin as many binary vectors carry
the neomycin phosphotransferase gene (NPTII) which codes for kanamycin resistance.
Transformation occurs along the cut edges of the disks, resulting in the formation of
callus tissue which carries the DNA between the LB and RB integrated at random into
the plant genome. The callus tissue is then transferred to regeneration medium also
containing kanamycin, which only allows transgenic plants, expressing kanamycin
resistance, to develop. The whole process takes about three to four months.

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During the regeneration process care must be taken to inhibit the growth of
Agrobacterium as false positive results could be due to the expression of the T-DNA-
carrying genes in the bacteria rather than in the plant. This is often found despite the
fact that the genes are expressed from eukaryotic promoters. Antibiotics such as
carbenicillin or cefotaxime can be used to eliminate the bacteria but they are not always
sufficient. Another strategy is to introduce into the T-DNA a GUS gene, coding for ß-
glucuronidase, which carries a plant intron. The enzyme is very easy to detect
histochemically and fluorometrically and will only be correctly spliced if it is expressed
within the plant and not in A. tumefaciens.

Arabidopsis transformation is very simple and does not require tissue culture. This is
advantageous because during the tissue culture process somatic mutations can occur
which may adversely affect the plant of being very simple and not requiring any tissue
culture. To transform Arabidopsis young flowering plants are inverted into a suspension
of A. tumefaciens cells under a vacuum. This causes the bacteria to infiltrate into the

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flowers and transfer the T-DNA into the DNA of the developing seeds, which are

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collected and germinated on the selected antibiotic. Only transgenic seeds will
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germinate and although the frequency of transformation is only about 1%, Arabidopsis

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produces such copious amounts of seed that transgenic plants are readily obtained.

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(Figure 3).
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Figure 3: Top row: untransformed Arabidopsis plants. Bottom row: transformed

Arabidopsis plants stained blue due to expression of the β-glucuronidase gene

2.1.1 The role of A. tumefaciens chromosomally encoded proteins and the Vir
proteins

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At least two loci on the Agrobacterium chromosome are required for the initial stage of
binding the bacterium to the plant cell. The products of the genes chvA and chvB
(chromosomal virulence) are responsible for the synthesis of a polysaccharide on the
bacterial cell surface. ChvB is a membrane protein, which catalyzes the conversion of
UDP-glucose into cyclic glucans, which are transported into the periplasm by ChvA.

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Figure 4: VirA (sensor), a membrane-spanning protein in A. tumefaciens, reacts to the

presence of acetosyringone, produced by wounded plant tissue. It autophosphorylates
itself and then passes the phosphate group onto VirG. VirG in this activated state binds
to the vir boxes in the promoters of the vir genes, inducing their expression. VirD1
and D2 nick the T- DNA in the RB and unwind the double helix. DNA synthesis lays
down a complementary strand in the Ti plasmid. VirD2 becomes covalently attached to

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the leading edge of the T- DNA, guiding it to the plant nucleus, and VirE2 coats the
single-stranded DNA, protecting it from nucleases

VirA and VirG make up a two-component signal transduction system. VirA is a

periplasmic membrane protein, which senses the presence of wound-induced phenolic
compounds, such as acetosyringone. VirA interacts with VirG, activating it by
phosphorylation. In this state VirG acts as a transcriptional activator of the vir genes by
binding to specific sequences, vir boxes, in their promoters. These Vir proteins have a
number of functions. VirD1 and VirD2 nick the T-DNA within the RB, and VirD2
becomes covalently attached to the 5’ end of the single-stranded T-DNA. It contains
nuclear localizing signals and will guide the DNA into the plant nucleus. When the nick
is made it provides a priming end for the synthesis of a DNA single strand. This
displaces the old strand, laying down a complementary strand in the Ti plasmid. The old
strand is the one transferred. VirE2 proteins coat the single-stranded T-DNA, preventing
it from degradation. VirB proteins form a pore in the bacterial membrane through which

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the coated T-DNA is transported (Figure 4).

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Little is known about the integration mechanism within the nucleus. The integrated

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DNA has a well-defined right junction with the plant DNA, retaining 1 – 2 bp of the

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RB. The left junction is variable, with the junction being at the LB or at one of a series
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of sites ~100 bp within the T-DNA.
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This is because the LB is nicked with less precision, often resulting in the T-DNA
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extending further into the Ti plasmid. Sometimes multiple tandem copies of T-DNA are
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integrated at a single site.

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2.2 Other transformation methods

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For many years the only alternative to A. tumefaciens-mediated transformation was the
direct uptake of naked DNA by plant protoplasts, achieved by electroporation or
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mediated by polyethylene glycol (PEG). This depends on the ability of plants to

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regenerate from protoplasts, which varies considerably between species. Early

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experiments focused on Solanaceae species which regenerate readily. Success has

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subsequently been obtained with Arabidopsis, rice, maize and forage grasses. Important
parameters for successful PEG-mediated protoplast transformation include ion
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concentrations, the presence of inert carrier DNA, the molecular weight and
concentration of PEG and the physical configuration of the nucleic acid. Linearized
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double-stranded plasmid DNA molecules are expressed and integrated most efficiently.

Now the most widely used alternative to transformation by A. tumefaciens is biolistics

(see 3.1). Other techniques, which have been reported but not extensively used, include
pollen co-cultivation, microinjection of somatic embryos and liposome fusion with
protoplasts.

3. Transformation of monocotyledonous plants

Monocotyledonous plants develop from a single cotyledon in the seed. They can be
recognized by the parallel veins in their leaves. Most of the world’s crops belong to the

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monocots, including maize, wheat, rye and sorghum. Transformation of monocots with
A. tumefaciens was not possible until relatively recently, and then only with certain
plant varieties and bacterial strains. The exact reasons for the recalcitrance to this
method are not fully understood. Initially it was thought that the triggering of the
virulence process was impaired by the fact that monocots do not produce wound
exudates such as acetosyringone. However, external addition of this compound did not
facilitate the process. Resistance to A. tumefaciens is likely to be due to a number of
factors, probably including integration into the chromosome, a process that is not well
understood.

3.1 Biolistic transformation

In 1987 Klein et al. described a procedure in which high velocity microprojectiles were
used to deliver nucleic acids into living cells. In their experiments they demonstrated
transient expression of RNA or DNA in epidermal cells of onion, Allium cepa. This

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technique of particle bombardment, or biolistics, is the most versatile and effective way

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of creating many transgenic plant species, including elite lines. Genes can be introduced
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into mitochondria and chloroplasts, present in many copies in a cell, as alternatives to

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delivery into the nucleus. This has the advantage of increasing the number of copies of

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the transgene. As these organelles are maternally inherited and therefore not transmitted
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by pollen, there is minimal chance of the transgene being transferred by cross-
pollination, to plants in the field.
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Figure 5: (A) A transgenic maize plant resistant to the herbicide bialaphos together with
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a non- transgenic plant. Both were sprayed with bialaphos. (B) The Biolistic® Accell®
gene gun
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To transform plants biolistically, DNA is coated onto suitable metal particles, usually
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gold or tungsten, which are chemically inert. These are blasted into plant tissue using a
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biolistic device driven by a gas, usually helium. A series of baffles or mesh screens is
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used to reduce cell death. The type of plant tissue used can vary between different plant
species, the most common being immature embryos, embryogenic callus and meristems.
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There are a number of instruments available for use in biolistics, based on various
accelerating mechanisms. The most widely used is the one currently marketed by Bio-
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Rad, Inc (Biolistics®) but the Accell® device, based on a particle inflow mechanism,
has been very useful in developing variety-independent gene transfer methods for
recalcitrant monocots (Figure 5).

3.1.1 Gene silencing

Biolistic transformation is a useful transformation technique for plants not susceptible to

transformation by A.tumefaciens. However there are negative features associated with
this system such that multiple inserts are often obtained and there may also be genomic
rearrangements. While naively this might be considered to be advantageous, in practice
it is not so as the plant often responds by shutting off the expression of the transgene

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through a process referred to as gene silencing. Gene silencing is a natural defense

pathway against viruses and transposons. Referred to as RNA interference in animals,
post transcriptional (PTGS) in plants and quelling in fungi, the process of silencing is
sequence specific and can be at the level of transcription, transcriptional gene silencing
(TGS) or post transcriptional (PTGS). DNA methylation is involved in both TGS and
PTGS. Transcriptional cis-inactivation is due to methylation when the transgene, in
one or more copies, integrates in or next to hypermethlyated sequences. TGS involves
the inhibition of transcription. However, silencing can also occur when transgenes
integrate at hypomethylated regions. This can be due to differences between the DNA
of the transgene and that of surrounding sequences, for instance when DNA from a
dicot is transformed into a monocot, leading to specific methylation and silencing of
foreign DNA. Methylation usually involves CG or CNG bases. Transcriptional trans-
inactivation can also occur, probably by some sort of DNA-DNA pairing. This is in
some cases correlated with inverted repeats, suggesting ectopic pairing between
transgene copies or between transgenes and homologous host genes. This could lead to

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the synthesis of aberrant RNA that triggers specific degradation of all homologous RNA

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in the cytoplasm. LS

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PTGS is associated with high transgene copy number and strength of the promoter. In

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PTGS, transgenes are transcribed at apparently normal rates in the nucleus but there is a
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strong reduction in the cytoplasm of steady-state mRNA levels. This is because when
the transgene mRNA reaches a threshold level, the silencing mechanism is triggered and
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results in degradation of all homologous RNA in the cytoplasm. However, not all
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PTGS is consistent with the threshold model. Weakly or even untranscribed transgenes
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can also trigger silencing of homologous host genes. One explanation that has been
proposed is that transgene RNA could be degraded if tagged by specific small
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complementary RNA molecules. A plant RNA dependent polymerase, using the

transgene RNA as a template, could synthesize these. They can act with mRNA
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forming duplexes that act as targets for double stranded-RNAses. Viruses themselves
are also able to trigger their own silencing on entry into a plant even in the absence of a
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transgene.
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Plant viruses have however developed mechanisms to overcome being silenced by

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plants by expressing inhibitors that interfere with the PTGS pathway. These inhibitors
are mainly proteins that interfere with one or more steps in the silencing pathway.
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Examples include the Cucumber mosaic virus 2b protein, p25 protein in Potexviruses,
C2 gene in Tomato leaf curl virus and p19 protein from Tobamoviruses.
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With a better understanding of gene silencing, however, design of vectors that minimize
transgene silencing is being used to ensure that the transgene is not silenced upon entry
into the plant cell. In addition, gene silencing can be harnessed to advantage to study
gene function, for genetic improvement of crop plants and development of virus
resistance.

3.2 Agrobacterium transformation

Multiple inserts during Agrobacterium transformation are less frequent than with
biolistics. Hence, if a plant can be transformed by this method it is usually the preferred

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technique. Although the host range of A. tumefaciens is broad, encompassing a large

number of species of dicotyledonous plants and some monocots and Gymnosperms, a
number of important monocot crops are recalcitrant to the method. These include maize,
rice, wheat and other cereals. There has thus been considerable emphasis on the
improvement of A. tumefaciens strains to transform these recalcitrant species. The
greatest success to date has been with rice. Hiei and colleagues produced transformants
with an efficiency similar to that of transformation in dicotyledons but using a ‘super-
binary’ vector. Optimization of the conditions of tissue culture and of the condition of
co-cultivation was of critical importance and the choice of tissues as starting material
was one of the most important factors. Callus cultures initiated from scutella were
excellent materials for transformation whereas immature embryos and shoot apices were
not. The DNA was randomly integrated into the rice genome and rearrangements, such
as the deletion of part of the T-DNA, was occasionally observed. However this is also
the case in Agrobacterium-mediated transformation of dicotyledons. More recently
successful transformation of maize by Agrobacterium has been achieved (ref).

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4. Transformation of algae
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Although this area of investigation is in its infancy there are reasons to be optimistic

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about the use of recombinant viruses as vectors of foreign DNA. There is growing
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interest in farming with marine microalgae in particular, and genetic modification could
be used to develop strains, which can produce a variety of proteins, polysaccharides and
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high value secondary chemicals. The large brown algae, commonly known as kelps, can
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measure up to 50 m in length and weigh hundreds of kg. They are the basis of
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economically important industries worldwide. Viruses, or more often electron

microscopic observations of viral like particles, have been described in a number of
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brown algae. Henry and Meints have isolated a strain of marine brown algae, which
harbors a stable virus infection. The viral DNA appears to be stably integrated into the
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genome of the host and may have potential as a vehicle for the introduction of foreign
genes into algae.
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5. Promoter efficiency and tissue specificity

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The 35S promoter of cauliflower mosaic virus (35S) was one of the earliest used in the
transformation of dicots. It is still widely used but has been improved by duplicating a
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250-bp up-stream enhancer. An A/T-rich positive regulatory region upstream of the

translation start site of the pea plastocyanin gene can also enhance it. This enhancer acts
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as a quantitative element in all transgenic plant tissues.

In monocots the promoter of choice is from the maize ubiquitin gene, which consists of
the promoter, the 5’ untranslated exon and the first intron. Splicing out of the intron
appears to increase gene expression by possibly increasing the stability of the mRNA or
increasing the level of mature cytoplasmic RNA.

It is often advantageous to develop a transgenic plant in which the gene of interest is

only expressed in certain tissues or under certain conditions. This could minimize the
genetic load on a plant by ensuring the gene is only expressed in the targeted tissues or,
for example, only after pathogen infection. Thus, for example, nematode resistance

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should be targeted to the roots, delayed fruit ripening to the fruit or stalk borer
resistance to the stalk. This could also assist safety and regulatory aspects if, for
example, nematode resistance is not expressed in edible fruits.

A recent example comes from work by Rao et al. who introduced the snowdrop lectin
into transgenic rice to confer resistance to rice brown planthopper. These insects feed by
phloem abstraction and cause ‘hopper burn’ as well as transmitting an important virus.
The snowdrop lectin has been shown to be toxic to the insects. The lectin gene was
driven by a phloem-specific promoter from rice and was expressed only in the phloem.
Transgenic plants were shown to have increased resistance to the brown planthopper,
which showed decreased survival, lower fecundity and retarded development.

A tissue-specific promoter has also been used in rice. Rice (Oryza sativa L.), the major
food staple for more than two billion people, contains neither β-carotene (provitamin A)
nor precursors thereof in its kernel endosperm. To improve the nutritional value of rice,
Burkhardt et al. introduced the genes for β-carotene synthesis into rice behind an

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endosperm-specific promoter and showed accumulation of the provitamin in the
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kernels.

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A useful way of detecting whether a particular promoter is able to target the desired
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tissue is to fuse it to a reporter gene that is readily visualized in a manner that is not
destructive to the plant. One such gene is the luciferase gene, lux, which originates from
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the bacterium, Vibrio harveyi or from the firefly (Photinus pyralis). Light emission can
be monitored visually, photographically or electronically. It is rather pretty observing
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plants that glow in the dark!

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Another useful marker is the green fluorescent protein of the jellyfish, Aequorea
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victoria. In order to visualize the fluorescence in green tissues the mutant form of the
gene with improved emission must be used and driven by a very strong promoter.
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6. Targeting genes to organelles

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As mentioned above, targeting genes to chloroplasts and mitochondria can be

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beneficial. Recently, Kota and colleagues developed transgenic tobacco plants

expressing a Bacillus thuringiensis toxin in their chloroplasts. Strains of B. thuringiensis
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(Bt) produce protein toxins specific to a variety of herbivorous insect pests. By

expressing the gene in chloroplasts, which are present in cells in high numbers, the
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authors achieved toxin levels of between 2% and 3% of total soluble protein. This was
so high that even Bt-resistant insects were killed when they were fed leaves from the
transgenic plants. The added advantage is that the genes will be maternally inherited and
therefore not spread by pollen, since there is concern that cross-pollination could spread
transgenes in an uncontrolled fashion. This would be a particular problem if herbicide
resistance were spread to potentially weedy relatives.

7. Integration and stability of transgenes

One of the first attributes of a transgenic plant to be determined is whether the

transgenes are stably integrated, inherited in a Mendelian fashion and stably transferred

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to subsequent generations. This is usually the case when a few genes are integrated in
one copy at one site. In many transgenic plants one or a few genes are transferred into a
plant’s genome along with the selectable marker. This has resulted in the production of
transgenic crops expressing herbicide tolerance, resistance to viruses, fungi and
bacteria, as well as to insect pests. Improved agricultural characteristics have also been
achieved by overexpression of specific genes in a metabolic pathway or by inhibiting
gene expression using antisense sequences. However, many agronomic characteristics
are polygenic, requiring the engineering of multiple genes. This necessitates the
integration of many transgenes, which must be stably inherited and expressed. Recently
Chen and co-workers succeeded in tranforming rice with up to 11 transgenes. They co-
bombarded the rice embryogenic tissue with 14 different plasmids and found that
integration of the multiple transgenes occurred at either one or two genetic loci.
Inheritance conformed to the 3:1 Mendelian ratio. Coexpression of four marker genes
was followed until the R2 generation and found to be stable.

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Although the mechanism of integration of transgenes into plant DNA is poorly

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understood, the integration of many genes at one or a few loci could not happen by
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chance. They may have been inserted individually at the same locus, or the plasmids

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might have become joined prior to integration. Experiments carried out by Kohli and

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colleagues, also using biolistic transformation of rice, suggest a two-phase integration
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mechanism mediated by the establishment of integration hot spots. In the first stage,
before integration, transforming plasmids appear to be spliced together. The cointegrate
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is then inserted into a site which subsequently acts as a hot spot, facilitating subsequent
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integration of successive transgenic molecules at the same locus.
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A number of studies have been done on the mechanism of integration during A.

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tumefaciens transformation. De Neve and co-workers co-transformed plants with two

Agrobacterium strains each carrying a different T-DNA and found that they were
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frequently integrated at the same locus. Out of 27 Arabidopsis transformants 12

contained the T-DNAs linked to each other in all possible configurations but with a
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preference for those with at least one RB involved. They also propose a model whereby
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separate T-DNAs are ligated prior to integration.

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Hansen and Chilton have used a combination of biolistics and Agrobacterium

transformation to develop transgenic plants carrying only the genes of interest without
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undesired vector sequences. They call this method “agrolistic” transformation. The
virulence proteins VirD1 and VirD2 from A. tumefaciens are required for the excision of
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T-DNA strands, and VirD2, covalently attached to the 5’end of the strand, targets it to
the plant nucleus. They cobombarded a plasmid carrying the virD1 and virD2 genes
with one containing border sequences flanking their genes of interest. Agrolistic inserts
were those which exhibited right junctions with plant DNA that corresponded precisely
to the sequence expected for T-DNA.

Acknowledgments

The author thanks Nikki Campbell for artwork and Dionne Miles for critical reading of
the first edition manuscript. She thanks Betty Owor for rewriting the section on gene
silencing for the second edition.

©Encyclopedia of Life Support Systems (EOLSS)

BIOTECHNOLOGY – Genetic Engineering of Plants - J. A. Thomson

Glossary

Biolistics: the process whereby DNA is introduced into an organism using

a particle gun
Callus: a mass of relatively unspecialized tissue used in plant tissue
culture as the starting material for the propagation of plant
clones
Dicotyledonous plant which develops from two cotyledons in the seed.
plant:
Ectopic: occurring in an unusual place or in an unusual form or manner
Epigenetic: describing mitotically and/or meiotically heritable traits that are
not due to an organism’s DNA
Eukaryote: any organism whose cells contain a nucleus bounded by a
nuclear membrane
Explant: a piece of living tissue taken from its normal situation to a
culture medium

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Monocotyledonous LS plant which develops from a single cotyledon in the seed
plant:

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mRNA: messenger RNA transcribed from DNA in the nucleus, exported
into the cytoplasm and translated into protein

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Oncogene: gene involved in the formation of cancerous tissue
Organelle: any discrete structure in an individual cell of a multicellular
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organism that is adapted and/or specialized for the performance

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of one or more vital functions
Prokaryote: any organism in which the genomic DNA is not enclosed by a
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nuclear membrane within the cell

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Promoter: the region of a gene where RNA polymerase binds to initiate

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transcription
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Protoplast: a spherical, osmotically sensitive plant cell without its cell wall
but retaining an intact cell membrane
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RB, LB: right border and left border

Reporter gene: a gene used to disclose the function of potential regulatory DNA
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sequences upstream of the reporter gene

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Selectable marker: gene coding for resistance to an antibiotic or herbicide which

can be used to select for transformed tissue or plants
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Silencing: the process whereby an organism shuts down the expression of a

gene
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T-DNA: the region of the Ti plasmid that is transferred to plants

Ti plasmid: the tumor inducing plasmid of Agrobacterium tumefaciens
Totipotent: having the potential to develop in any of the directions
inherently possible, given its particular genetic constitution, and
thus to form a new organism or to regenerate any part of an
organism
Transcription: the synthesis of RNA on a template of DNA
Transformation: the transfer of genetic information by means of ‘naked’ foreign
DNA from one organism to another
Transgenic plant: plant carrying a foreign gene
Translation: the process by which a particular sequence of bases in

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BIOTECHNOLOGY – Genetic Engineering of Plants - J. A. Thomson

messenger RNA (mRNA) determines a sequence of amino acids

in a protein
Bibliography

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patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of

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BIOTECHNOLOGY – Genetic Engineering of Plants - J. A. Thomson

Ow, D.W., K.V. Wood, M. DeLuca, J.R. De Wet, D.R. Helinski, and S.H. Howell. (1986) Transient and
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Vaucheret, H., C. Béclin, T. Elmayan, F. Feuer bach, C. Godon, J-B. Morel, P. Mourrain, J-C. Palauqui,
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[This is one of the early papers on gene silencing in plants.]

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Biographical Sketch
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Jennifer Thomson has a BSc in Zoology from the University of Cape Town, an MA in Genetics from
Cambridge and a PhD in Microbiology from Rhodes University. She was a post-doctoral fellow at
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Harvard Medical School. She was a lecturer, senior lecturer and Associate Professor at the University of
the Witwatersrand, Director of the CSIR Laboratory for Molecular and Cell Biology before becoming
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Professor and Head of the Department of Microbiology at the University of Cape Town. She is currently
Professor in the Department of Molecular and Cell Biology at UCT. Her main research interest is in the
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development of maize resistant to viruses and tolerant to drought.

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