Chapter Three

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Application
 Chapter Three:Application

Application

Manipulations of transgenes after incorporation with the help of a cloning vector into
the genome of a host have both practical and fundamental applications in genetic
engineering. In plants, it would be desirable, for example, to remove antibiotic
resistance genes or herbicide resistance genes after they have been used for their
primary purpose, i.e., following selection of transformed cells or after providing
resistance to specific herbicides, respectively. Other applications utilizing transgene
excisions or gene inversions could be useful for controlling developmental processes.
Such manipulations or modifications of transgenes in the plant genome are possible
using certain DNA site-specific recombination systems (Odell and Russell 1994).

3.1 Agricultural Applications

Inserted genes can be classified into several groups based on their use: those that
protect a crop, those that improve the quality of a harvested product, and those that let
the plant perform some new function.

Genes That Protect a Crop

The major use of plant genetic engineering using cloning vector has been to make
crops easier to grow by decreasing the impact of pests. Insect resistance has been
achieved by transforming a crop using a Bt gene. Bt genes were isolated from
Bacillus thuringiensis, a common soil bacterium. They code for proteins that severely
disrupt the digestive system of insects. Thus an insect eating the leaf of a plant
expressing a Bt gene stops eating and dies of starvation. There are many Bt genes,
each of which targets a particular group of insects. Some Bt genes, for example, target
caterpillars. Others target beetles.

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Cloning vectors are also has been used in the battle against weeds. Bacterial genes
allow crops to either degrade herbicides or be resistant to them. The herbicides that
are used are generally very effective, killing most plants. They are considered
environmentally benign, degrading rapidly in the soil and having little impact on
humans or other organisms. Thus a whole field of transgenic crops can be sprayed
with broad-spectrum herbicides, killing all plants except the crops. Corn, soybeans,
canola, and cotton that have been engineered to withstand either insects or herbicides,
or both, are widely planted in some countries, including the United States. In
addition, other crops, including potatoes, tomatoes, tropical fruits, and melons, have
been engineered for resistance to viral diseases.

Genes That Improve Crop Quality

An emerging major use of genetic engineering for crops is use of cloning vector to
alter the quality of the crop. Fresh fruits and vegetables begin to deteriorate
immediately after being harvested. Delaying or preventing this deterioration not only
preserves a produce's flavor, and appearance, but maintains the nutritional value of
the produce. Genes that change the hormonal status of the harvested crops are the
major targets for genetic engineering toward longer shelf-life.

For example, the plant hormone ethylene is associated with accelerated ripening, as
well as leaf and flower deterioration, in fruits that are injured or harvested. Scientists
insert genes that interfere with a plant's ability to synthesize or respond to ethylene,
thereby extending postharvest quality for many fresh products, including tomatoes,
lettuce, and cut flowers. Scientists are also using gene insertion to improve a plant's
nutritional value and color.

Genes That Introduce New Traits

One approach for improving the economic value of crops is to give them traits that
are completely new for that plant. Some crops, including potatoes, tomatoes, and

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bananas, have been engineered with the help of cloning vector with genes from
pathogenic organisms. This is done to make animals, including humans that eat the
crops immune to the diseases caused by the pathogens. The genes code for proteins
that act as antigens to induce immunity. Thus edible parts of plants are engineered to
act as oral vaccines. This approach may be particularly effective for pathogens, such
as those causing diarrhea and other gastrointestinal disorders, that enter the body
through mucous membranes. This is because the "medicine" in the food comes into
direct contact with these membranes and does not have to be absorbed into the blood
stream. Genes have also been engineered into crop plants to direct the plants to
produce enzymes used in the manufacture of paper. Other genes direct crops to
produce small polymers useful in the manufacture of plastics. This general approach
is being termed "plant molecular farming."

Rice is another plant that has been engineered for a new trait. During commercial
processing, a substantial part of the white rice grains are removed, leaving very little
vitamin A. Vitamin A deficiency is a significant health problem in regions dependent
on rice as a dietary staple. Scientists engineered a certain form of rice, known as
"golden rice" because it has a yellow tinge, to express three introduced genes. These
genes let the plant produce the precursor of vitamin A in the portion of the grain that
remains after processing, thereby providing a dietary source of the vitamin.

Virus resistance
Many plant viruses are transmitted by insects that feed by sucking plant sap. In 1986
it was reported that when the coat protein gene for the RNA virus that causes tobacco
mosaic was introduced into the host plant its expression interfered with the replication
and systemic spread of the virus, making the plant resistant (Abel et al.1986). This
phenomenon has also been observed in other plant viruses and their hosts (Turner et
al.1987). Naturally occurring virus resistance is uncommon, and the discovery
showed much promise. The first commercial application of this discovery is the use
of viral coat protein genes of cucumber mosaic, zucchini yellow, and watermelon
mosaic viruses in squash (Tricoli et al.1992). The engineered squash contains viral
coat proteins that account for <0.1% of the total protein of the squash fruit. This

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compares favorably with the somewhat greater virus content of commonly consumed
fruit from plants that are naturally infected with virus. Other forms of virus resistance
include viral genes that, when expressed in plants interfere with replication of the
viral genome or impede virus movement from cell to cell (Golemboski et al.1990;
Cooper et al.1994). The pokeweed plant produces several ribosomal inhibitor
proteins. One of these expressed in transgenic plants protects them against a broad
range of viruses (Lodge et al.1993).

Resistance to pathogenic fungi

Many fungal pathogens have cell walls made of chitin. Novel transgenic plants that
express bean or Serratia marcescens chitinase genes have been reported to show
resistance (Benhamou et al.1993). Four genes for resistance to different plant
pathogens were recently cloned from higher plants with the help of cloning vector
(Moffat, A.S .1994). Their structural similarity-all had regions of DNA with repeat
lysine encoding motifs-suggests that they have a role in signaling biochemical
defense responses. An earlier example was of a corn gene that appears to encode the
structure of a membrane receptor protein to which a fungal pathotoxin binds (Briggs
1992). General methods for recovering and redeploying such genes will help to
reduce our dependence on pesticides.

3.2 Food Application

Controlled ripening
The first food product from this recombinant DNA technology, genetically engineered
chymosin (rennin), is widely used in Europe and the United States for cheese making.
The first genetically engineered food plant to be introduced in the United States, the

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Flavr Savr tomato (Calgene, Davis, CA), was released early in 1994. These tomatoes
incorporate a Ti vector that carries an antisense form of the tomato gene that encodes
Polygalacturonase (PG). Produced during fruit ripening, this enzyme breaks down
pectin, the cement that holds the cells of the fruit tissue together, and is responsible
for fruit softening. The PG antisense gene reduces the mRNA transcript formed and
hence the amount of PG. The result is a fruit with a longer shelf life at full ripeness.
Phytase production
Genetic engineering promises improvements in the nutritional value of livestock feed.
Phytate (myoinositol hexaphosphate) is the principal storage form of phosphorus in
plant
seeds. Inorganic phosphate is released from phytate by an endogenous enzyme,
phytase, at seed germination. However, when plant seeds are fed to monogastric
animals, such as pigs and poultry, these animals cannot use phytate and thus cannot
access the phosphorus. Phytase,prepared from Aspergillus niger, can replace the
phosphate supplement (Beers et al.1992). A gene from As. niger that encodes the
enzyme was used to transform tobacco with an Agrobacterium vector (Pen et
al.1993). Phytase-expressing plants were normal in growth and development and their
seeds were enriched in phytase. Seeds from transgenic tobacco were tested in feeding
trials with male broiler chicks. This supplemented diet was compared with diets
supplemented with and without inorganic phosphate and As. niger phytase. Growth in
animals fed diets containing milled transgenic phytase-expressing seeds was
significantly greater than that in animals fed control seeds or diets without
supplementation. The production of transgenic oilseed rape and soybean that
constitutively overexpress phytase in their seeds is now under way. Other work has
shown that phytase added to soil increases the availability of phosphorus to plant
roots (Findenegg et al.1993). These findings suggest that the expression of the
enzyme in the roots of transgenic plants may have a similar effect.
Reducing phytate in soybean and other vegetable products may play a role in human
nutrition. Phytate is an effective chelator of divalent cations, such as iron and zinc.
Consuming protein sources that contain relatively high concentrations of phytate

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results in decreased mineral bioavailability, and consuming vegetable proteins with

low concentration of phytate promotes mineral absorption

3.3 In Health Sector

Vaccines and antibodies play a key role in healthcare. However, the cost of
production and maintaining a chain for vaccine distribution has so far hampered
realizing their full potential. Expression of antigens as vaccines, and of antibodies
against antigens of pathogens in transgenic plants is a convenient and inexpensive
source for these immunotherapeutic molecules. Various antigens and antibodies have
already been expressed successfully in plants with the help of cloning vector and
have been shown to retain their native functional forms. Edible plant vaccine against
diarrhoea, expressed in potato, and antibody against dental caries, expressed in
tobacco, is already in pre-clinical human trials. Attempts are being made to express
many proteins of immunotherapeutic use at high levels in plants and to use them as
bio-reactors of the modern era.

Transgenic plants for immunotherapy

Since 1984, when transformation of tobacco – the first plant to be transformed with a
foreign gene using cloning vector – was reported(De Block et al.1984), various
foreign proteins including serum albumin, human α -interferon, human
erythropoietin, and murine IgG and IgA immunoglobulins have been successfully
expressed in plants (Cramer et al.1996). In recent years, several attempts have been
made to produce various antigens and antibodies in plants (Mason et al.1995; Ma et
al.1995). Antigens or antibodies expressed in plants can be administered orally as any
edible part of the plant, or by parenteral route (such as intramuscular or intravenous
injection) after isolation and purification from the plant tissue. The edible part of the
plant to be used as a vaccine is fed raw to experimental animals or humans to prevent
possible denaturation during cooking, and avoid cumbersome purification protocols.

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Vaccines

While plant system may have the capability of producing any vaccine in large
amounts and in a less expensive manner, purification of the product may require the
use of existing or even more cumbersome procedures. Attention therefore has been
paid to mainly those antigens that stimulate mucosal immune system to produce
secretory IgA (S-IgA) at mucosal surfaces, such as gut and respiratory epithelia. In
general, a mucosal response is achieved more effectively by oral instead of parenteral
delivery of the antigen. Thus, an antigen produced in the edible part of a plant can
serve as a vaccine against several infectious agents which invade epithelial
membranes. These include bacteria and viruses transmitted via contaminated food or
water, and resulting in diseases like diarrhoea and whooping cough. The first report
of the production of edible vaccine (a surface protein from Streptococcus) in tobacco,
at 0.02% of total leaf protein level, appeared in 1990 in the form of a patent
application published under the International Patent Cooperation,Treaty(Mason et
al.1995). Subsequently, a number of attempts were made to express various antigens
in plants (Table 3.1). Since acute watery diarrhoea is caused by enteroxigenic
Escherichia coli and Vibrio cholerae that colonize the small intestine and produce one
or more

Protein Plant
Hepatitis B surface antigen Tobacco
Rabies virus glycoprotein Tomato
Norwalk virus capsid protein Tobacco
E.coli heat labile enterotoxin B sununit potato
Cholera toxin B subunit potato,Tobacco
Mouse glutamate decarboxylase potato
VPI protein of foot and mouth disease virus Arabidopsis
Insulin potato
Glycoprotein of swine transmissible gastroenteritis Arabidopsis
coronavirus

Table 3.1: Antigens produced in transgenic plants

enterotoxin, an attempt was made towards the production of edible vaccine by

expressing heat-labile enterotoxin (LT-B) in toabacco and potato(Haq et al.1995).

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Fig 3.1: Strategies for expression of antigen in plant

One of the alternative strategies of producing a plant-based vaccine is to infect the

plants with recombinant viruses carrying the desired antigen that is fused to viral coat
protein. The infected plants have been reported to produce the desired fusion protein
in large amounts in a short time. The technique involved either placing the gene
downstream a subgenomic promoter, or fusing the gene with capsid protein that coats
the virus (Fig 3. 1). The latter strategy is perhaps the strategy of choice since fusion
proteins in particulate form are highly immunogenic. It should, however, be kept in
view that recombinant viruses need to be highly purified for parenteral administration
or partially purified for oral administration. Modelska et al. (Modelska et al.1997)

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have shown that immunization of mice intraperitoneally or orally by gastric

incubation or by feeding of plants infected with the recombinant alfalfa mosaic virus
(AIMV) carrying rabies peptide CPDrg 24 mounted local as well as systemic immune
response. Oral administration could stimulate both serum IgG as well as IgA
synthesis. After immunization, 40% of the mice were protected against the challenge
with a lethal dose of the virus.

Protein Plant Carrier

Influenza antigen Tobacco TMV
Murine zona pellucida antigen Tobacco TMV
Rabis antigen Spinach AIMV
HIV-1 antigen Tobacco AIMV
Mink enteritis virus antigen Black eyed bean CPMV
colon cancer antigen Tobacco TMV

Table 3.2: Transient production of antigens in plants after infection with plant viruses
expressing a recombinant gene.

Expression and assembly of antibodies in plants

Transgenic plants are also being looked upon as a source for producing large-scale
antibodies which can serve the purpose of passive immunization by direct application,
in addition to providing a tool for drug targeting or interactive inactivation of
undesirable molecules(Hiatt et al.1993; Ma. Et al.1995). Gene technology has
provided great impetus to the utility of antibodies, since antibody genes can be altered
to order. Thus not only genes coding for both the light and heavy chains have been
expressed, but modified genes capable of expressing only Fab fragments (assembled
light chains and shortened heavy chains) or scFV (single peptide chains where
variable domains of heavy and light chains are covalently linked by a short flexible
peptide) have also been expressed in bacteria and mammalian cells(During et al.1990;
Benvenuto wt al.1991; Owen et al.1992; Tavladoraki et al.1993; Zeitlin et al.1998)
(Table 3.3).

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Transgenic plants not only provide the means to express antibodies but also enable
the glycosylation and entry into secretory pathway which allow assembly of complete
antibodies and Fab fragments. Variable fragments (Fv) can be produced in cytosol,
directed to different compartments and fused with proteins such as protein A and
phosphatase to improve the detection and purification of single chain Fv (scFv). In
plants, antibody production (1–5% of total plant protein) has been achieved by

Antibody Antigen Plant

IgG(k) Transition stage analog Tobacco
IgM(λ) NP(4-hydroxy-nitrophenyl) Tobacco
acetyl hapten
Single domain(dAb) Substance P Tobacco
Single chain Fv Phytochrome Tobacco
Single chain Fv Artichoke motiled crinkle Tobacco
Fab;IgG(k) Human creatin kinase Arabidopsis
IgG(k) Fungal cutinase Tobacco
IgG(k) and SIgG/A hybrid S. mutans adhesin Tobacco
Single chain Fv Abscisic acid Tobacco
Single chain Fv Nematode antigen Tobacco
Single chain Fv β-glucuronidase Tobacco
β-1,4 endoglucanase
Single chain antibody Atrazine,Paraquat Tobacco
fragment
IgG Glycoprotein B of herpes Soybiean
simplex virus

Table 3.3: Antibodies and antibody fragments produced in transgenic plant

cross-pollination of individually transformed plants expressing light or heavy chains.

Other approaches involve double transformation, or transformation by constructs
having genes for both light and heavy chains on the same vector. Despite the fact that
production of antibodies in plants takes longer, the low cost of production and
capability of increasing production simply by increased propagation make plant
antibodies an attractive proposition.

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Although the first human clinical trials for plant-based vaccine (Tacket et al.1998)
and antibody(Ma et al.1994) with the help of cloning vector have been performed
recently, many challenges including maximization of expression levels, stabilization
during post-harvest storage, remain to be met. Edible vaccines can be improved for
their oral immunogenecity by the use of appropriate adjuvant which could be used
either as a fusion to the candidate gene or as an independent gene. Concern about
immune tolerance and allergy to plant-based vaccines has been expressed and needs
to be addressed suitably. Antigens produced by diarrhoea and whooping cough
causing organisms are promising candidates. It is also desirable that the concept of
edible vaccine should first be tested in animals.

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