SET: 1
1. What do you understand by micropropagation? What are the different pathways of morphogenesis in vitro?
2. Can you expect double haploid production during anther culture in vitro. How you can differentiate between haploid and diploid plants during the growth until maturity.
3. What are secondary metabolites. What is the biosynthetic origin of secondary metabolites, why is it synthesized and how it is regulated in plants.
4. Describe in detail Agrobacterium mediated gene transfer in Plants.
5. Write in detail how herbicide resistant plants can be produced for Triazines and Phosphinothricine.
6. What do you understand by abiotic stress, discuss various abiotic stresses and their effect on plants.
7. What do you understand by molecular farming, explain with few examples.
8. What is metabolic engineering. Discuss about the strategies for the manipulation of secondary metabolic pathways.
SET: 2
1. What are the minimum facilities needed for the development of a plant tissue culture set up.
2. What are somaclonal variations, discuss their application with suitable examples.
3. How important are the secondary metabolites for the plant itself and for biotechnological applications.
4. Give an account of gene transfer methods in plants for the production of transgenic plants.
5. Describe about the strategies employed for genetically engineered plants for herbicide resistance.
6. Discuss the global status of abiotic stress resistant transgenic crops.
7. Discuss how plants can be used as bioreactors for the production of foriegn proteins.
8. Give an example of the first metabolic engineering of secondary metabolism in plants and discuss in detail.
SET: 3
1. What do you mean by surface sterilization of explants. Discuss the procedure for establishment of in vitro culture.
2. What are haploids? Discuss their significance in plant improvement.
3. What do you understand by plant cell immobilization and what are its advantages for secondary metabolite production.
4. Give a detailed note on plant transformation vectors.
5. Describe how insect resistant transgenic plants can be developed using protease inhibitor and alpha-amylase inhibitor.
6. What is oxidative stress, discuss how superoxide dismutase gene helps to produce transgenic plants resistant to oxidative stress.
7. Describe about the methods for the expression of foreign proteins in transgenic plants.
8. Discuss how overproduction of Petunia chalcone isomerase in tomato results in fruit with increased levels of flavanols.
SET: 4
1. What plant growth regulators would you recommend for callus induction in vitro. What explants would you recommend to obtain embryogenic callus in vivo. Explain in detailwith its applications.
2. Discuss the process of cryopreservation in detail and write about its implications in the germplasm conservation on plants.
3. Describe with some examples where in vitro cell and tissue culture systems are exploited commercially for the production of secondary metabolites.
4. Give a detailed outline of plant transformation technology and describe different methods.
5. Give your opinion about the transgenic plants extensively used by farmers for cultivation in India.
6. There is great success for transgenic plant production for biotic stress, however there is a lacuna which needs to be unplugged for abiotic stress, justify with example.
7. Discuss with suitable examples production of pharmaceutically important pesticides in plants.
8. Give an account of the genetic engineering of plants for production of Vitamin E.
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PLANT BIOTECHNOLOGY Question Papers (Regular, 2008)
Monday, December 22, 2008
METABOLIC PATHWAYS (Unit 8 for JNTU, PBT)
Thursday, December 18, 2008
Metabolic pathways are series of consecutive enzymatic reactions that produce specific products. Their reactants, intermediates, and products are referred to as metabolites. Since any organism utilizes many metabolites, it has many metabolic pathways which are interconnected.
Metabolic pathways can be divided into two categories : those invoving degradation (catabolism) and those involving biosynthesis (anabolism).
In catabolic pathways complex metabolites are exorgonically, broke doen to simple products.
Cataboism converts a large number of diverse substances (carbohydrates, lipids and protiens) to common intermediates.
These intermediates are then metabolized in a central oxidative pathway that terminates in a few end products.
Carbohydates, lipids and protiens are degraded to a common intermediate, acetyl- CoA, this is followed by oxidation of acetyl group to CO2 and H2O by a series of reactions.
Anabolism carries out the opposite process
Relatively few metabolites serve as starting materials for several varied bio-synthetic products.
Four important characteristics of metabolic pathways are:
1) Metabolic pathways are irreversible
2) Every metabolic pathway has a first commited step
3) All metbolic pathways are regulated
4) metabolic pathways in eukaryotic cells occur in specific cellular locations
METABOLIC PATHWAYS ARE IRREVERSABLE :
They are highly exorgonic (have large –ve free energy changes) so that their reactions go to completion. This characteristic provides the pathway with direction. Consequentially if two metabolites are metabolically interconvertible the pathway from the first to the second must differ from the second back to the first.
The reason for this difference is that if the route from the first metabolite to the second is exergonic, free energy must be sulloied in order to bring it back uphill. This requires a different pathway for atleast some of the reaction steps.
The existance of independent interconversion routes is an important property of metabolic pathways because it allows independent control of the rates of the two processes.
If metabolite 2 is required by the cell it is necessary to turn off the pathway from 2 to 1 while turning on the pathway from 1 to 2.
EVERY METABOLIC PATHWAY HAS A FIRST COMMITTED STEP :
Although metabolic pathways are irreversable, most of their component reaction function clise to equilibrium. Early in each pathway however, there is generally an irreversible (exergonic) reaction that commits the intermediate it produced to continue down the pathway.
ALL METABOLIC PATHWAYS ARE REGULATED :
In order to extert control on the flux of metabolites through a metabolic pathway it is necessary to regulate its rate limiting step.
Most metabolic pathways are therefore controlled by regulating the enzymes that catalyse their first committed steps. This is the most efficient way to exert control because it prevents the unnecessary synthesis of metabolites further along the pathway when they are not required.
METABOLIC PATHWAYS IN EUKARYOTIC CELLS OCCUR IN SPECIFIC CELLULAR LOCATION :
The synthesis of metabolites in specific membrane bounded subcellular compartments makes their transport between these compartments a vital component of eukaryotic metabolism.
Biological membranes are selectively permeable to metabolites because of the presence on membranes of specific transport proteins
PHENYLALANINE – SHIKONIC ACID PATHWAY (unit 8 for JNTU, PBT)
The biochemical pathways leading to the production of valuable plant secondary metabolites are being studied with a virus to engineer increased production and to reduce cost of production. Over expression of the gene that encodes for the first enzyme (generally the rate limiting enzyme) in a pathway results in higher levels of the desired end product. This has been successfully done in the enhancement of taxol production from transformed tissue cultures of Taxus species with taxidine synthase gene.
The biosynthetic pathways for the production of plant phenolics are relatively well worked out.
Plant phenolics are a large variety of secondary metabolites that contain a phenol group, a hydroxyl functional group on an aromatic ring as shown below.
Most plant phenolics are synthesized through shikonic acid pathway and phenyl propanoid pathway.
The shikonic acid pathway converts simple carbohydrate precursors derived from glycolysis and the pentose phosphate pathway to the aromatic amino acids, which act as the precursors for the synthesis of phenolics as briefly given in the following
(CLICK ON THE IMAGE TO ENLARGE)
To enhance phenylalanine synthesis it is essential to knockout any feedback inhibition of the pathway from chorismate to phenylalanine.
The next step was to remove competing pathways i.e the synthesis of tyrosine and tryptophan.
Once all control circuits and competing pathways are removed, attempts were made to increase the carbon flux through the biosynthetic pathway.
Over expressing all the gene in the pathway did not enhance the yield of phenylalanine because the supply of the precursor (E-4P and/or PEP) was rate limiting.
The precursors were increased by cloning transketolase (to enhance E4P levels) and eliminating pyruvate kinase (to enhance PEP levels).
The most abundant class of secondary phenolic compounds in plants are derived from phenylalanine via the elimination of an ammonia molecule to form cinnamic acid.
This reaction is catalyzed by phenylalanine ammonis lyase (PAL). This is perhaps the most studied enzyme in plant secondary metabolites and is situated at a branch point between primary & secondary metabolism, so the reaction that it catalyses is an important regulatory step in the formation of many phenolic compounds.
Reactions subsequent to that catalyzed by PAL lead to the addition of more hydroxyl groups and other substituent’s.
Trans – cinnamic acid, P – coumaric acid and other derivatives are simple phenolic compounds called phenylpropanoids, that form the important building blocks of the more complex phenolic compounds.
The phenyl propanoid pathway is clearly explained in the below diagram :
(CLICK ON THE IMAGE TO ENLARGE)
The enzyme PAL is the first committed step for phenyl propanoid pathway.
This enzyme has been over expressed in tobacco plants and the transgenic plants showed resistance to pathogens like viruses, bacteria, fungi and grazing insect larvae. While suppression of PAL reduced resistance
FLOWER COLOUR (unit 8 for JNTU, PBT)
‘Novel flower colours’ is a target of biotechnology. Anthocyanins are major flower pigments in higher plants.
Metabolic engineering provides a powerful tool to generate novel flower colors with out changing the other desired characteristics of a cultivar.
A major target has been the development of blue pigmented flowers in species in which the development of blue color does not naturally occur, such as rose (or) carnation.
Delphidin is the anthocyanin that normally leads to blue pigmentation.
A research group in Australia identified and cloned a gene from petunia hybrid encoding a flavanoid 3’5’–hydroxylase (F3’5’ – H), which is required for the biosynthesis of delphidin.
This provides information necessary or producing transgenic plants with blue flowers.
Transgenic violet carnations have been produced by the introduction of F 3’5’H gene and its high suspension in the petals
Modification of intensity of flower color has been achieved.
White and pink flower varieties have been obtained by introducing sense and antisense chalcone synthase (CHS) transgenes into petunia. Chrysanthemum, rose, carnation, etc.
Flower color has been changed by the introduction of pathway.
Specific transcriptional activators control the genes in anthocyanin biosynthesis.
Expression of maize transcriptional factor “R” (Lc allele) under the control of the CaMV 35S promoter elevated the amount of anthocyanin in transgenic Arabidopsis and tobacco.
Expression of Lc gene in petunia resulted in purple coloration due to accumulation of anthocyanin.
This approach may allow novel color patterns to be introduced into ornamental species by the controlled expression of anthocyanin and flavanoids.
Flower color novelty in particular the generation of unknown blue & yellow flowering cultivars, is one of the major driving force in ornamental Plant breeding.
In a combination of classical & molecular methods, metabolic engineering provides powerful tools to generate novel flower colors with out losing other desirable characteristics of a pre-existing cultivar.
With this view point, production of anthocyanins has also been manipulated in several plants this resulting in making flowers with novel phenotypes such as pink petunia commonly known as “surfinia” were obtained through antisense suppression of chalcone synthase (CHS) gene.
Similar attempts have also been made to generate yellow/cream or deep purple dianthus (commonly known as moondust or roomshadow) and magenta, pale yellow and dark pink coloured petunia.
DISEASE RESISTANCE
A large number of plant defense response genes encoding antimicrobial proteins have been cloned. Most of these are transcriptionally activated in response to infection or exposure to microbial elicitor macromolecules.
1. PATHOGENESIS – RELATED (PR) PROTEINS :
These are low molecular weight proteins which accumulate to significant levels in infected plant tissues. The ability of hydrolytic enzymes e.g. chitinases & 1 – 3 beta – D Gluconase, to break chitin & glucan in the cell walls of fungal pathogen has been exploited to develop crops resistance to pathogen.
Chitinase gene of soil bacterium serratia marcescens was introduced into tobacco. The tobacco is resistant to Alternaria longipes, causing brown spot disease.
A chitinase gene of bean (Phaseolus vulgaris) under the control of promoter of CaMV 35S has been introduced into tobacco. The transgenic tobacco plants are resistant to the fungal pathogen Rhizoctonia solani, causing damping off of seedling.
The 1 – 3 beta – D Gluconase together with chitinase gene showed fungal resistance in tobacco (fungal disease caused by Cercospora nicotinae) & tomato (fungal disease caused by Fusarium oxysporum lycopersci).
2. ANTI MICROBIAL PROTEINS :
Plants and other organisms may contain antimicrobial proteins that are not necessarily associated with induced defence response, but the presence of these proteins exhibit resistance to pathogen.
Barely α – thionin gene when transferred to tobacco showed resistance against pseudomonas syringae pv tobacco
Antimicrobial proteins of non plant origin include lytic peptides & lysozymes.
Lytic peptides form pores in bacterial membrane (e.g. attacin, ucropin etc.)
Ucropins been expressed in transgenic potato and tobacco & attacins in apple plants.
A ucropin gene when transferred to tobacco showed resistance against Pseudomonas syringae & bacterial pathogen.
Expression of the bacterial gene bacteriopsin (bo) encoding a proton pump in transgenic tobacco resulted in complete resistance to P.syringae pv tobacco.
Bacteriophage T4 lysozyme gene transferred to potato showed resistance to Erwinia carotovora, subspecies atrosepta.
When human lysozyme was introduced into tobacco, transgenic tobacco showed enhanced resistance against Erisiphe uehoracearum & P.syringe.
When bovine lysozyme gene was introduced into tobacco produced bovine lysozyme that destroys the cell wall of invading pathogenic bacteria.
As the plant cells destroy bacteria, the plants can resist the bacterial pathogens.
The fungal endo – α – 1,4 – D polygalacturonases are partly responsible for the dissolution of the plant cell wall.
The cloning of a bean PGIP (Poly Galacturonase Inhibiting Protein) results in over expression of the inhibitor in transgenic plants & there by fungal disease development in plants is inhibited.
3. ENGINEERING TOXIN INSENSITIVITY :
Enzymes inactivating the toxins produced by bacterial and fungal pathogens are used to engineer disease resistance in plants.
The bacterial haloblight pathogen of bean, Pseudomonas phaseolica, produces a peptide toxin, phaseolotoxin, which causes the chlorotic halos.
Phaseolotoxin inhibits the enzyme ornithine trans carbamylase (OC).
Bacteria have been selected which contain a phaseolotoxin – insensitive OC and the gene encoding this enzyme, has been transferred to tobacco, where its expression prevents the disease symptoms.
Transgenic sugarcane expressing an albicidin detoxifying gene (alb) from pantoea dispera (bacterium) provides control against leaf & cold disease (caused by Xanthomonas albilineans) under the control of maize ubiquitin promoter.
4. PHYTOALEXINS :
Phytoalexins are secondary metabolites synthesized by plants and contribute to disease resistance.
During infection, stored phytoalexins (usually present in special cells or in a conjugated inactive) are mobilized, while genes are induced for the synthesis of more phytoelixins.
Resveratol is one of the commonest synthesized.
The key enzyme in resveratol synthesis is resveratol synthase (often known as stilbene (phytoalexins) synthase or STS)
STS gene of peanut transferred to tobacco proved its fungitoxic action (againt botrytis cinerea).
5. MANIPULATION OD DISEASE RESISTANCE GENES :
A gene from maize, which confers resistance to Cochlibolus carbonum has been cloned by transposoon tagging.
This gene encodes that inactivates the fungal toxin.
The gene that confers resistance to a pathogen in a resistant crop variety is transferred to a susceptible variety.
VIRUS RESISTANCE
Viral infection reduces photosynthesis rate & growth region of crop plants, thereby lowering crop yields.
Viral infection does not occur in plants already infected with certain viruses.
This principle is called “cross protection”.
Cross protection is the ability of one virus to prevent or inhibit the effect of a second challenge virus.
If a susceptible strain of a crop is inoculated with a mild strain of a virus, then the susceptible crop strain develops resistance against more virulent virus strain.
This principle is used by genetic engineers to develop transgenic virus resistant plants.
The transgenic plants produced viral coat proteins or viral nucleiprotein (or) satellite RNA’s in their cells.
Therefore viral infection does not take place.
Crop varieties raised by conventional breeding method are resistant to only one virus and sometimes the resistance may be reverted. Transgenic technology tries to overcome this problem.
1) TRANSGENIC PLANTS WITH VIRAL COAT PROTEIN
Genes for viral coat proteins have been introduced into plants to make them produce the viral proteins.
Transgenic plants having such viral coat proteins are resistant to viral infection.
e.g. TMV resistant tobacco & tomato.
TMV is a single stranded RNA virus & the RNA is enclosed in a protein coat.
Powell – Abel et al (1986) first demonstrated that transgenic tobacco expressing tobacco mosaic virus (TMV) coat protein showed resistance.
Coat protein mediated resistance is correlated with the inhibition of virus replication at the initial point of infection.
This resistance takes the form of reduced numbers of infection sites on inoculated leaves, suggesting that an initial step in the virus life cycle has been disrupted.
It has been demonstrated that TMV cross protection may result from the coat protein of the protecting virus preventing uncoating of the challenge virus RNA.
Powell – Abel et al (1986) made cDNA of the TMV coat protein & introduced it into tobacco plant through disarmed Ti plasmid of Agrobaterium.
The transgenic tobacco plants produced TMV coat protein in their cells & were resistant to viral infection.
2) TRANSGENIC PLANTS WITH VIRAL NUCLEOPROTEIN
RNA of some plant viruses is found associated with certain proteins. These are necessary for replication replicase & a protein that stabilizes its shape.
Several of these nonstructural replicase proteins have been found to provide a high degree of resistance to virus infected when expressed in transgenic plants.
The cDNA of nucleoprotein is constructed & fused with a 35S promoter of CaMV & introduced into plants through disarmed Ti Plasmid of Agrobacteriumn.
Transgenic tobacco, tomato, lettuce, groundnut, pepper etc have been developed to resist viruses by introducing nucleoproteins gene of tobacco rattle virus (TRV) & tobacco spotted wilt virus (TSwV).
3) TRANSGENIC PLANTS WITH VIRAL SAT RNA
RNA of certain plant viruses contain some non – coding sequences called satellite RNA’s or SAT RNA.
SAT RNA’s are a class of small (approx 300 nucleotides), single stranded RNA molecule that are dependent upon a helper virus for replication & virion packaging to cause infection elsewhere.
SAT RNA depends on virus for its replication & transmission, even though its unrelated to viral genome.
A number of SAT RNA’s have been shown to modulate the replication & symptom of diseases of their helper viruses.
Satellite RNA’s that attenuate symptoms can be potentially used to reduce the disease severity of the helper virus.
Hence its use in transgenics to confer resistance in crops finds an important place.
Trin et al (1987) Trin & Causui (1991) demonstrated that the deliberate inoculation of a mild strain of CMV with a symptom attenuating satellite RNA successfully protected tobacco, pepper, tomato & cucumber plants from a virulent strain of CMV & reduced yield losses. This strategy is limited to those virus systems in which attenuating satellite RNA’s are found.
Gehrlach et al (1987) introduced a cDNA of a satellite RNA of tobacco ringspot virus (TRV), into tobacco through disarmed Ti plasmid.
The transgenic tobacco is resistant to tobacco ringspot virus.
It produces viral SAT RNA’s that attenuate the disease symptoms.
4) TRANSGENIC PLANTS WITH ANTISENSE RNA :
A strategy for the control of plant viruses is the transgenic expression of antisense segments of viral RNA’s.
“Anti – sense RNA“ is RNA complementary to mRNA.
Normal mRNA of a gene is said to be a “sense” because it carries the codons that are “read” during translation to produce specified sequence of amino acids.
The complement of mRNA “sense” strand will not contain a sequence of codons that can be translated to produce a functional protein.
Thus this complementary strand is antisense RNA.
The principle of this strategy is to bind viral RNA with complementary RNA sequences Expressed by the plant.
Potentially prevent accessibility accessibility of the viral RNA for replication or gene expression.
Thus, antisense constructs should be used to block the initial steps important in the establishment of viral infection.
This strategy was used to develop viruses resistant tobacco.
e.g. CuMV (cucumber mosaic virus)
In CuMV, viral protein is encoded by RNA3.
DNA that produce antisense RNA for RNA3 was linked with a 35S promoter & inserted into a disarmed Ti plasmid.
THE rDNA was introduced into tobacco calli, through A. tumifaciens.
Tobacco regenerated from the calli are resistant to CaMV.
INSECT RESISTANCE
Several microbes & plants produce proteins toxic to specific insects, pests that feed on plants.
One goal of plant genetic engineering is to transfer the genes encoding these protein toxins to agronomically important plants with the hope that expression of the toxin genes in these plants will provide biological control of some serious insect pests.
Currently insect pests are controlled by the use of broad spectrum chemical insecticides.
However there is reason for concern about the potential damage to ecosystems & pollution of ground water that might result from the wide spread use of these chemicals on agricultural crops.
The best known example of the use of natural gene products to control plants pests or the insects are toxin of Bacillus thuringiensis.
1. B.thuringiensis produces endotoxin called (toxin of Bacillus thuringiensis) Bt toxin.
Each of the B.thuringiensis toxin genes encodes a large protein that aggregates to form protein crystals in spores & the protein crystals are highly toxic to certain insects.
Some of the insects that are killed by the protein are plant pests of major economic importance.
Different subspecies of B.thuringiensis produce toxins that kill different insects.
For example, the toxin produced by B.thuringiensis subspecies (krustak), kills lepidopteran larva such as the tobacco hornworm.
The gene that encodes this toxin has been isolated & shown to synthesize a functional toxin in E.coli . the gene is put under the control of a 35s promoter of CaMV & introduced into plants through disarmed plasmid.
Transgenic tomato plants were regenerated & shown to express the gene.
The toxicity of the gene – product synthesized in the transgenic plants was tested by allowing tobacco hornworm larvae to feed on the transgenic plants died within a few days.
Larvae applied to control plants remained healthy & eventually consumed the entire plant.
Transgenic tomatoes with Bt gene are resistant to tobacco hornworm (Manduca sexta) tomato fruit worm,(Heliothis zea) & tomato pinworm (Keferia lycopersicella ).
The Monsanto company (
The transgenic cotton is called killer cotton.
It is resistant to attack by tobacco budworm (Heliothis virescens), cotton bollworm, pink bullworm, beatarmy worms.
2. TRANSGENIC PLANTS WITH Bt TOXIN GENE & SERINE PROTEASE INHIBITOR GENE.
Bt toxin produced in transgenic plants is not effective against a broad range of insect pests, as it is present in low concentration. for enhancing the insecticidal activity a serine protease inhibitor gene (SPI Gene) is fused with a Bt toxin gene & CaMV promoter.
The DNA construct is introduced through disarmed Ti plasmid.
The transgenic plant produce Bt toxin as well as serine protease inhibitor, pesticidal activity of such transgenic plant is twenty times greater than that of transgenic plant having Bt toxin alone.
NOTE : plants contain peptides acting as protease inactivating protein (PIPts). The different proteinases are serine, cysteine, aspartic acid & metallo proteinases. They catalyze the release of aminoacids from dietary protein thereby providing the nutrients crucial for normal growth & development of insects. The proteinase inhibitor deprive the insect of nutrients by interfering with digestive enzymes of the insect.
3. TRANSGENIC PLANTS WITH COWPEA TRYPSIN INHIBITOR
Cow pea is resistant to many storage insects in grainaries. It has a protein called cowpea trypsin inhibitor (CPTI)
CPTI found in cowpea is the most active inhibitor upto date.
This inhibitor gene produces antimetabolite substances that provide protection against major storage pest Brichid beetle (Callosobrichus maculatus).
The gene is also harmful to various Lepidoptera insects (Heliothis verescens), spodopteran insects (Manduca sexta), colepteron insects (callosobruchus, anthonomus grandis), orthopteran insects (Locusta migratoria), but is not harmful to mammals.
The gene coding for CPTI was transferred to tobaccp through a binary vector system made from Ti plasmid. The transgenic tobacco has been resistant to many insect pests including tobacco budworm.
It produced 2 microgram of CPTI per mg, which is not toxic to man & animals.
4. TRANSGENIC PLANTS WITH Nicotiana alata PROTEINASE :
Nicotiana alata (an ornamental plant) has insecticidal property because of presence of a proteinase inhibitor called Nicotiana alata proteinase inhibitor. (NAPI).
It is encoded by NAPI gene.
Transgenic tobacco, clovers & peal have been developed by introducing cDNA of NAPI genes.
TRANSGENICS IN CROP IMPROVEMENT
Plants containing introduced DNA are known as transgenic plants or genetically engineered plants. They acquire a new trait from introduced DNA.
The acquired trait is heritable. This principle is called “gain of function”.
Some introduced DNA blocks the expression of certain original genes of the plants. This is called “cassey function”.
The development of transgenic plants is the result of integrated application of r – DNA technology, gene transfer methods & tissue culture techniques.
Transgenic plants in food, fibre, vegetable & fruit crops have led to improved crop yields.
Genes are transferred from entirely irrelevant organisms (notably bacteria & viruses) to crops producing new useful traits in crops.
Genetic transfer facilitates introduction of only desirable genes from donor without cotransfer of any undesirable genes from donor species, which normally occurs by conventional breeding techniques.
The list of plant species that can be transformed by vector mediated (Agrobacterium) and vector – less methods has been growing continuously.
At present transfer capability has been extended to more than 120 species in atleast 35 families.
Monocot species were earlier considered outside the host range of Agrobacterium tumifaciens, which led to the development of direct DNA introduction or vector–less methods for transformation.
Thus most success in development of transgenic plants in monocots have been due to vectorless systems.
However, recently Agrobacterium mediated transfer has been reported in monocot species including the most important food crops like rice (1994), maize (1996) & wheat (1997).
Genetic transformation has led to the possibility of transforming crops for enhanced resistance to insects and diseasescaused by fungal & bacterial pathogens & viruses. Herbicide resistance in crop plants has led to effective use of herbicides to control weeds.
HERBICIDE RESISTANT PLANTS :
Plants that can tolerate herbicides are called herbicide resistant plants.
Herbicide resistance of crop varieties help us in the effective use of herbicides to control weeds.
So transgenic crop plants with herbicide resistance have been developed by adopting genetic engineering.
GLYPHOSATE RESISTANT PLANTS :
Glyphosate is the active ingradient of many broad spectrum herbicides such as :
- a) Glyphos
- b) Round up
- c) Tumble weed
It enters the plant cells and inhibits the enzyme 5 enol pyruvyl shikimate 5 phosphate synthetase [EPSP Synthetase]
This enzyme is present in chloroplast.
Glyphosate is the competitive inhibitor for EPSP Synthetase.
It inhibits the synthesis of aromatic amino acids from phospho enol pyruvate. Therefore the plants die when glyphosate is added.
It blocks the biosynthesis of tryptophan & phenylalanine. It does not effect man and animals.
The cell line of petunia resistant to glyphosate over produced the EPSP Synthetase.
The mRNA of EPSP Synthesizing gene was isolated from the cell line & cDNA was made from that mRNA, CaMV (cauliflower mosaic virus) promoter was linked to the 5 end of cDNA & cDNA in construct was introduced into Agrobacterium through disarmed Ti Plasmid.
The Agrobacterium were allowed to inject leaf discs of petunia sensitive to glyphosate. The transgenic petunias are resistant to glyphosate by over producing the enzyme EPSP Synthetase.
The EPSP Synthesizing genes of petunia are also transferred to tomato, tobacco, maize, cotton etc.
The soil bacterium Salmonella typhimurium & E.coli are resistant to glyphosate. They produce glyphosate tolerant EPSP Synthetase.
They produce EPSP Synthetase which is normal in activity but poor in affinity to glyphosate.
This enzyme is coded by a gene called “aro A gene”.
The aro A gene transferred to many plants through disarmed Ti plasmids for producing glyphosate resistance in them.
Glyphosate tolerant petunia, potato, tobacco, tomato, cotton etc have been made by this method.
SULPHONYL UREA RESISTANT PLANTS :
Sulphonyl urea tolerant plants have been developed by cDNA technology.
Sulphonyl urea inhibit acetolactate synthetase (ALS) which synthesizes branched chain aminoacids.
Arabidopsis ALS Gene produces ALS which does the usual function but has poor affinity to sulphonyl urea.
The ALS Gene of Arabidopsis is transferred to tobacco through disarmed Ti Plasmid. The transgenic tobacco is 100 times more resistant to sulphonyl urea.
ATRAZENE :
It inhibits QB Protein, of photosystem II in chloroplast. Hence in Atrazene sensitive plants photosynthesis declines & finally the plants die.
A mutant amaranthus hybridus has Atrazene tolerant QB protein & hence the plant show Atrazene resistance.
The gene for QB protein in Amaranthus hybridus was transferred to tobacco & some other plants. The transgenic plants are resistant to Atrazene.
The gene in Maize for glutathione – s – transferase can detoxifythe atrazine & thereby tolerate the Atrazene.
PPT is the active ingredient of the commercial herbicide “Basta”.
The enzyme phosphinothricin acetyl transferase in streptomyces hygroscopicus can detoxify the PPT, it is encoded by “bar” gene.
The bar gene of streptomyces hygroscopicus was transferred to tobacco, wheat, maize, soyabeen etc.
The transgenic plants were resistant to PPT.
BROMOXYNIL TOLERANCE :
Here a gene called bxn enables Klebsiella pneumoniae to resist bromoxynil.
The bxn gene codes for the enzyme Nitrilase that can detoxify the Bromoxinyl residues.
ABIOTIC STRESS
Adverse environmental conditions such as droughts, extreme temperatures and salinity impose stress on plants and effect plant growth and yield. These abiotic stress cause cell death and senescence.
The soil water potential is a combination of the effects of surface area of soil particles and small soil pores that absorb water (matrix potential) the effects of dissolved substances (solute/osmotic potential) and the atmospheric pressure effects(pressure potential).
Water potential = matrix potential + solute potential + pressure potential
ΨW = ΨM + ΨS + ΨP)
In non salty well drained soil the matrix potential is almost equal to the water potential
When solubility is high the contribution of solute potential (Ψ)S to water potential(ΨW) is high.
Soil capacity of a soil is the soil moisture % held in the soil at a water potential(ΨW) of -1/3 barr, which is the condition of newly wetted soil after gravitational water have drained from macro-pores.
The permanent wilting potential is the soil moisture at -15 barr
Plant available water (water available to plants) is the water in soils held between -1/3 barr and -15 barr.
Some plant species can extract water held at water potential of -60 barrs or lower but water held at potential less than -15 barrs is absorbed only slowly and in small quantities.
Crop plants wilt if only -15 barrs water is present because water loss through transpiration is faster and greater than that amount absorbed by roots at these low moisture potentials.
Most soluble salts in soils are composed of cations of sodium, calcium and magnesium and anions of chlorine, sulphate and carbonate ions.
Although toxicity due to high concentration of sodium and chlorine and other ions can occur, salts usually effect plants growth because of the osmotic effect.
High salt concentration lowers the solute potential there by lowering the water potential it more difficult for plant roots to extract water from soil
During a drying period salts in soil solutions may be so concentrated so as to cause water to move from root to soil (exosmosis) there by killing the plant.
Barley & cotton have considerable salt tolerance
Several abiotic stresses such as drought, salinity and extreme temperature have a common consequence of causing cellular water deficit.
The response of plants to water deficit is the synthesis and accumulation of low molecular weight compounds called as osmoprotectants.
These osmoprotectants within the cells lower the osmotic potential and help maintain turgor
These solutes include inorganic ions, organic ions, soluble carbohydrates including polyols (sugar, alchohols) , aminoacid (proline) and ammonium compounds such as glycine betaine.
Several plant genes that encode key enzymes of osmolyte biosyntheic pathways including sugars, alchols, glycine betaine and proline have been cloned.
Strong correlations have been reported between proline accumulation and tolerance to draught and salinity stresses.
Gamma pyrroline – 5 – carboxylate synthetase (P5 CS) is the limiting enzyme in the synthesis of proline.
Transgenic tobacco plants were generated with P5 CS enzyme & they showed enhanced plant growth under salt & draught stresses.
The ability to synthesize & accumulate glycine betaine is synthesized by the two step oxidation of choline via the intermediate betainealdehyde the reaction beung catalysed by choline monoxygenase (CMO) and betaine aldehyde dehydrogenase (BADH)
Transgenic plants of tobacco, arabidopsis and rice with BADH have shown increased salt resistance.
Polyols like mannitol contribute to drought tolerance in plants.
Polyols are derivatives of sugars.
An E.coli gene MtPD (mannitol – 1 posphate dehydrogenase) evolved in the biosynthesis of mannitol was introduced into tobacco through disarmed Ti plasmid.
The transgenic tobacco plants producing mannitol were tolerant to draught & salt stresses.
Fructose and poly fructose molecules that are produced by many plants and bacteria and play a role in adaptation to osmotic stress due to their high soluble nature.
Fructosyl transferase gene of Bacillus subtilis was transferred to potato and the transgenic potato showed tolerance to drought stresses.
Trehalose a non – reducing disaccharide of glucose is known as a trehalose has been shown to stabilize protein and membrane lipids
Transgenic tobacco plants with the trehalose – 6 – phosphate synthetase gene (TPS 1) from E.coli accumulated trehalose and exhibited improved drought tolerance.
Heat shock protein (stress response proteins) synthesized in increased amounts in response to elevated temperature or other stressful treatments and which usually helps the cells to survive the stress.
Osmotic stress induces the accumulation of a set of low molecular weight proteins such as late embryogenesis abundant protein (LEA).
A barley LEA gene HVA, when transferred to rice resulted in rice tolerance to drought and salinity.
High temperature stress – tolerant transgenic Arabidopsis plant were produced by increased production of heat shock proteins transcription factor (Ab HSP) by a CaMV 35S promoter.
In conditions of drought and salt stress there is over production of over active oxygen species in plant cells.
Hence a plants ability to be scavenge these toxic oxygen species is considered to be critical for abiotic stress tolerance.
Among the enzymatic antioxidants, super oxide dismutase (SOD) catalyses the dismutation of superoxide to catalase(CAT) and peroxidase breakdown hydrogen peroxide to water
An effective detoxification mechanism against hydrogen peroxide is the ascorbate glutathione cycle, where ascorbate peroxidase (AP) , monodehydro ascorbate reductase and glutathione reductase (GR) work together
Genes pr cDNA’s encoding SOD, CAT, AP & GR have been isolated from several crops and transferred to other crops to confer draught & salt resistance. SOD from rice and AP from pea were transferred to tobacco to confer tolerance to salt draught and chilling stresses.
Oxygen deprivation is detrimental to most species of higher plants.
This can occur during periods of flooding and in drained soils.
Several toxic oxidant products are frequently formed.
Genes encoding proteins that increase intracellular oxygen concentration and/or its transport hence been isolated.
Expression of nonsymbiotic haemoglobin from barley (Hb) and bacteria vitriscella iteroacia (VHB) in maize and tobacco resulted in faster seed germination & higher plant growth rates.
Arabidopsis is a small green plant. It contains acyl glycerol phosphate (AGP) which makes the plant tolerant to prolonged chilling.
AGP is made from glycerol diphosphate by the enzyme glycerolposphate acyl transferase (GPAT).
The GPAT gene of arabidopsis along with CaMv 35S promoter and transferred to tobacco through disarmed Ti plasmid
The transgenic tobacco accumulates AGP in the cells and tolerates chilling to allow for its cultivation in cold area.
Nishizaura et al in 1996 introduced a desaturase gene from cyanocobacterium into tobacco. This resulted in increased chilling tolerance in tobacco ssedling.
Expression of fish (winter flounder fish) antifreeze protien (AfP) in transgenic tobacco plants inhibit the information of ice crystals in cells and there by confer tolerance to freezing stress.
MOLECULAR FARMING
Certain compounds of commercial interest that were earlier produced only from exotic sources of plants, animals & microbial origin are now produced in domesticated crops by using genetic engineering techniques.
Transgenic plants can act as living bioreactors for low cost of production of pharmaceuticals & chemicals.
This is know as molecular farming. It has been possible to produce vaccines, industrial enzymes, polypeptides,fatty acids, carbohydrates and biodegradable plastics through molecular farming.
APPLICATION OF PLANT BIOTECHNOLOGY FOR PRODUCTION OF QUALITY OILS
Fats and Oils differ only in that fats are solids and oils are liquid at room temperature.
Fats & Oils that occur in plants and animals consist of mixtures if triacylglycerol s (also referred to as triglycerides).
These non-polar, water-insoluble substances are fatty acid tri-esters of glycerol
Their fatty acid composition vary with the organism that produce them.
Plant oils are usually richer in unsaturated fatty acids than are animal fats.
Fatty acids are carboxilic acids with long chain hydrocarbons side groups.
Unsaturated fatty acids contain one or more double bonds between carbon atoms. In saturated fatty acids all of the carbon atoms are bonded to the max. number of hydrogen atoms.
Some biological fatty acids
Saturated fatty acids
Lauric acid 12:0
Palmitic acid 16:0
Stearic acid 18:0
Arachdonic acid 20:0
Unsaturated fatty acids
Palmitoic acid 16:1
Oleic acid 18:1
Linoleic acid 18:2
α – linolenic acid 18:3
γ- linolenic acid 18:3
Arachidonic acid 20:4
Plant species use different polymers generally oil & starch, to store energy required for seedling growth. The oil may be present in seeds (e.g. ground nut, mustard, rapeseed, sunflower & soya bean which contain upto 50% of the seed dry weight) or fruits (e. g. olive, ovocado and oil palm fruits which contain high level of oils)
Vegetable oil is a major commodity for both food & industrial needs.
The fatty acid composition of the oil used for food or industrial applications differ.
Oil quality(fatty acid composition) & yield can be improved by genetic engineering to suit our needs. An example for such kind of genetic modification is the high lauric acid ( a fatty acid with 12 carbon atoms ) rape seed (canola) oil.
The wild type rape seed contain 60% oleic acid with other fatty acids.
The genetically engineered rapeseed plant variety (laurical) produced lauric acid a 12 carbon fatty acid used to make soaps and detergents.
The researchers introduced only one gene coding for a thioesterase enzyme from the California bay tree which contain high levels of lauric acid.
The shut off fatty acid synthesis after 12 carbons rather than allowing the acid to grow to 18 carbon length (oleic acid) normal for the plant while having little effect on the productivity.
There was a dramatic change in the spectrum of fatty acids in the oil i.e. a change from 60% oleic acid to 60% lauric acid. The genetically engineered oil is already in commercial production.
Mono unsaturated fatty acid level in plants is desirable so that the net value of oils is enhanced. The transgenic soyabean with modified oil composition from about 25% oleic acid to over 85% has been developed and this improved oil has important food and non-food uses.
Rape seed is poor in stearate & stearic acid. An antisense DNA was constructed for the critical enzyme and introduced into embryos of rape sedd.
The transgenic rape seed oil with high level of stearate was produced and is used in the manufacture of margarine (a butter like solid oil)
A ∆15 desaturase gene was introduced into rape seed. The transgenic plant produced oil rich in α – linolenic acid.
The gene for the biosynthesis of oil in cocoa was transferred to soya through disarmed Ti plasmid. The transgenic soya bean produced cocoa oil that can be used for chocolate making.
γ- linolenic acid (GLA) is the first intermediate in the bioconversion of linoleneic acid to arachidonic acid.
GLA is important in alleviating hyper cholesteromia and many caronary heart diseases.
It is not produced in oil seed crops.
This conversion is catalysed by by ∆6 desaturase, and the gene encoding this enzyme has been cloned from the cyanobacterium synechocystis.
Expression of this gene in transgenic tobacco resulted in the production of GLA.
In 1998, this desaturation gene has been cloned from filamentous fungi, moteirella alpina and transferred to canola.
The hydroxy fatty acid vicinoleic acid has industrial applications in the manufacture of nylon paints, varnishes, resins, lubricants, and cosmetics.
Transgenic arabidopsis expressing castor oleate hydroxylase gene accumulated ricinoleic acid upto 17% of the total lipid fraction
Somerville & coworkers of michigan state university have cloned the arabidopsis gene which codes for an enzyme that catalyses the synthesis of polyunsaturated fatty acids
Dietary poly unsatured fatty acuds have a role in lowering bloof cholesterol and are needed for human growth.
Since then the eight different desaturase genes from arabidopsis that control the polymerisation of plant oils have been isolated.
These genes have been inserted into plants such as soyabean, canola & flax that typically make more saturated oils
This resulted in the production of nutritionally improved oils in the crops
Thus it is possible to tailor plant oils to specific nutritional needs.
EDIBLE VACCINES
THEORY BEHIND EDIBLE VACCINES
One of the major challenges of biotechnology is to reduce clinical innovations to economically viable practices. Plant derived edible vaccines were first conceived and are continuing to be developed with this prime directive in mind: merging innovations in medical science and plant biology for the creation of efficacious and affordable pharmaceuticals. Since the emergence of the original idea about 10 years ago, it was embraced by a growing number of laboratories in academia and industry.
Despite notable success, traditional vaccine technology has its limitations. Almost all vaccines now commercially available consist of either inactivated or attenuated strains of pathogens which are almost always delivered by injection (the oral polio vaccine is an exception). In contrast, many of the currant vaccine development efforts focus on subunit vaccines, and these are being considered for either mucosal or parental delivery.
A subunit vaccine refers to a pathogen derived protein (or even just an immunogenic domain of a protein, i.e., an “epitope”) that cannot cause disease but can elicit a protective immune response against the pathogen. Very often the subunit vaccine candidate is a recombinant protein made in transgenic production–hosts (such as cultured yeast cells), then purified, and injected into vaccines to immunize against a specific disease. Subunit vaccines are generally considered safer to produce (eliminating the need to culture pathogenic organisms) and more importantly, to use.
However, immunization by injection (parental delivery) rarely results in specific protective immune responses at the mucosal surfaces of the respiratory, gastrointestinal and genito -urinal tracts. Mucosal immune responses represent a first line of defense against most pathogens. In contrast, mucosally targeted vaccines achieve stimulation of both the systemic as well as the mucosal immune networks. In addition, mucosal vaccines delivered orally increase safety and compliance by eliminating the need for needles. While subunit vaccines are effective, they currently depend on capital-intensive fermentation-based technology and a “ cold chain ” (refrigeration) for delivery. Both of these factors create constraints in use in the developing world, where vaccines are needed the most. Combining a cost effective production system with a safe and efficacious delivery system, plant edible vaccines, provide a compelling new opportunity.
THEORY PUT TO CLINICAL TRIALS
In 1992 the expression of Hepatitis-B surface antigen (HbsAg) in tobacco plants was described. To prove that plant derived HbsAg can stimulate mucosal immune responses via the oral route, potato tubers were used as an expression system and were optimized to increase accumulation of the protein in the plant tubers. The resulting plant material proved superior to the yeast-derived antigen in both priming and boosting of immune responses to oral immunogen in mice. In parallel with evaluation of plant derived HbsAg, Mason and Arntzen explored plant expression of other vaccine candidates including the ‘labile toxin B subunit (LT-B) of entertotoxigenic Escherichia coli (ETEC) and the capsid protein of Norwalk virus(NVCP). The plant derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals.
Success in mouse experiments provided motivation for conducting Phase І / ІІ human clinical trials to test the safety and immunogenicity of plant produced LT-B, NVCP and HBsAg. In the three cases tested, humans who consumed raw potato tubers containing tens of microgram amounts of the antigens developed specific serum and more importantly mucosal immune responses. Significantly the three antigens in these studies come from three very different pathogens. Taken together these results provide the basis for wider-scale clinical trials with these antigens.
Although mucosal and systemic antibody titers were evaluated in vaccines who received the plant-based oral vaccines, we do not yet have evidence of protection against pathogen challenge. Ethical considerations usually preclude (prevent) clinical trials from directly assaying protection except in few cases. In contrast working with veterinary vaccines provides researchers an opportunity to asses the degree of immune protection more directly.
SECOND GENERATION EDIBLE VACCINES
Multi component vaccines that provide protection against several pathogens are very desirable. An elegant approach to achieve this goal, based on epitope fusion to both subunits of the cholera toxin (CT), was demonstrated by Yu and Langridge. CT provides a scaffold for presentation of protective epitopes of rotavirus and ETEC, acts as a vaccine candidate by its own right and as a mucosal adjuvant devoid of toxicity. The trivalent edible vaccine elicited significant humoral responses, as well as immune memory B cells and T-helper cell responses, important hallmarks of successful immunization. Commonly, foreign proteins in plants accumulate to relatively low levels (0.01% to 2% of total soluble protein). In the clinical trials described above, 100gm of raw potato tubers expressing LT-B of ETEC in three doses had to be consumed in order to overcome digestive losses of the antigen and to elicit a significant immune response. Less immunogenic proteins would require even larger doses to be effective. Even with more palatable alternatives to potatoes (e.g. bananas), these accumulation levels may limit the practicality of edible vaccines.
Two solutions to overcome this limitation are being explored. First, techniques to enhance antigen accumulation in plant tissues are being explored. These include, optimization of the coding sequence of bacterial or viral genes for expression as plant nuclear genes, and defining the sub cellular compartment in which to accumulate the product for optimal for optimal quality and quantity. Several laboratories are also developing alternative expression systems to improve accumulation. For example the expression in plastids is advocated by some. Other systems involve plant viruses for expression of foreign genes or coat-protiens fusions and even viral assisted expression in transgenic plants.
The second approach is to enhance the immunogenicity of the orally delivered antigens by using mucosal adjuvants. One such approach is making use of bacterial enterotoxins such as CT or LT (e.g. Yu and Langridge, 2001), mammalian and viral immune-modulators as well as plant derived secondary metabolites.At door step of the 21st century, the fear of a surge in naturally occurring epidemics is heightened by the threat of bio-terrorism. This new reality makes disease prevention through vaccination a necessity in our ever more interconnected world. Any tools we can master and all tools we can afford will have to be employed. Technical problems and skeptics aside, edible vaccines have passed the major hurdles of an emerging vaccine technology.
APPLICATION OF PLANT BIOTECHNOLOGY FOR INDUSTRIAL ENZYMES
Currently most infustrial enzymes are produced from microbes by large scale fermentation.
An alternative approach to the production of such enzymes would be to express and recover these enzymes from transgenic plant.
The production of industrial enzymes in transgenic alfalfa has certain advantages.
Alfalfa is widely grown perennial crop capable of three or more harvests in year.
It is beneficial environmentally since it does not require annual tilling and planting and needs fewer application of fertilizers and pesticides than conventional row crops.
Furthermore technology for extracting protien from alfaalfa while leaving avaluable residue for animal feed is well developed. Some enzymes and their applications are listed below:
a) α – amylase : Textiles, starch syrups, laundry and dish washing, detergents, paper designing, fermentation of ethanol, animalfeeds.
b) Cellulase : Dish washing detergents, animal feeds, textiles, bioenergy production.
c) Invertase : Manufacture of syrup from cane and beet sugar.
d) Lactase : Eliminates lactose from dairy food
e) Pectinase : fruit processing
f) Acid protienases : baking (improves dough handling)
g) Alkaline proteases : detergent, leather & fur.
h) Pepsin : cheese production
i) Subtilisin : chiral resolution of chemical compounds (or) pharmaceuticals
j) Lysozymes : anti bacterial
k) Isomerase : conversion of glucose to high fructose corn syrup
l) Alchohol dehydrogenase : chiral synthesis of chemicals
m) Peroxidase : laundry & wood pulp bleaches
n) Acetolactate decarboxilase : brewing industry
o) Histidine : cosmetics
p) Xylanase : paper industry
Manganese dependent lignin peroxidase from the fungus phanerochaete chrysosporium has been expressed in transgenic alfalfa plants.
This enzyme has potential for large scale industrial usage for lignin degradation and as a bleaching agent in bio-pulping processes (paper industry).
Expressing of this enzyme IN alfalfa may also have the potential for increasing the fibre degestability of forages for ruminants
α – amylase from Bacillus licheniformis has been expressed in alfalfa.
It was found to be a robust enzyme that is active over a wide pH and temperature range.
Transgenic alfalfa expressing the animal feed enzyme phytase (from Aspergillus niger) and two different cellulases have been field tested.
Phytase was expressed at levels upto 2% total soluble protein in some transgenic lines.
This makes it an economically viable alternative to microbially produced phytase.
The production of animal feed supplements in transgenic alfalfa is particularly appealing since the expressed juice or leaf meal preperation can be added to animal rations with no intervening extraction (or) purification neccesary.
They contain proteins and xanthophyll, a yellow pigment used to enhance the colour of poultry, making it more attractive to cusumers.
It has been recently dicovered that the cellulase enzyme can replace the pumice stones used in textile industry to produce stone wash denim. This will help to counter the damage that pumice stone can cause to fabric.
The cellulase enzyme can also be used as a biopolishing agentas it removes the fuzz from the surface of the cellulose fibres.
Proteases and hydrolases are used in laundry detergents and starch processing respectively.
Genetic manipulation can generate simple molecules from known chemical structures to ore active compounds.
For instance, sweetness of cornsyrup can be increased by chemical transformation using the glucose isomerase enzyme.
These developments may have very wide applications in pharmaceutical, food and agricultural areas.
Transgenic cotton plants with insect resistance have been produced by transferring a gene from the bacteria Bacillus thuringiensis. With this success, scientists are now trying to develop transgenic coloured cotton, which could replace the bleaching and dying process.
The manipulation of enzyme structure for having maximum catalytic activity and resistance to reaction environment is called enzyme engineering.
Many enzymes are sensitive to heat. Some enzymes catalyse reversible reactions. Some enzymes are sensitive to pH fluctuations.
The reaction kinetics are very poor while using such enzymes.
Genetic engineers have manipulated the genes coding for the enzymes in such a way as to have desired features.
More number of disulfide bonds in enzymes, increases the thermostability as well as resistance against extreme pH changes.
Nucleotides coding for cystine are introduced into the gene coding for the enzyme, by site directed mutagenesis.
The modified gene produces enzymes with some more cystine residues and establishes some additional disufide bonds.
The engineered enzymes are more stable at high temperature and extreme pH changes.
As the major portion of the enzymes remain the same, there is no change in the catalytic activity.
Another reason for poor stability towards high temperature is the presence of more asparagine in the enzyme molecule.
One or two asparagine residues are replaced by threonine or isoleucine by site ddirected mutagenesis in the gene coding for that enzyme.
The resulting enzymes are more stable towards heat
PLANTIBODIES
INTRODUCTION
The term "plantibodies" was created to describe the products of plants that have been genetically engineered to express antibodies and antibody fragments in planta, with many applications. (Antibodies were first produced in tobacco plants approximately twelve years ago, Smith, 1996). Plants are being used in this technology as antibody factories, using their endomembrane and secretory systems to produce large amounts of clinically viable proteins which can later be purified from the plant tissue. The purified antibodies have many possible applications, for example, as diagnostic tools, in immunochromatography or in medical therapy (Jaeger et. al., 2000) . This site will focus on the use of plantibdodies in MEDICAL THERAPY and IMMUNOMODULATION in plants.It has been shown that plants can produce fully functional antibodies, capable of binding and aggregating Streptococci bacteria. Both IgA and IgG have been successfully assembled and purified in tobacco plants. (Stabila et. al., 1994)A lot of research has been carried out in this field as molecular biology techniques are improving, the production of antibodies by plants for therapeutic purposes and plant pathogen resistance will be possible in the near future with much research currently in clinical trials. Inducing the production of specific antibodies in plants has many commercially viable applications not only to the pharmaceutical industry but also the plant breeder. Plantibodies reduce the risk of human disease contamination found with cell fermentation procedures & hence has the potential to forgo expensive screening for viruses and bacterial toxins
This is a completely novel technology as plants are being engineered to express proteins that are naturally, exclusively expressed in animal species. Antibodies were previously produced in mice and purified with a view to using the antibodies to treat disease. However, when administered to patients, they gave severe immune responses. Humanised monoclonal antibodies were then created with very similar gene sequence to those genes coding for human antibodies. These are created from murine immunoglobulins, the constant domains and framework regions of the variable domain are altered to give high sequence homology to human antibodies. This process was not only laborious, but also expensive and the mouse antibodies were very difficult to purify. The production of antibody fragments by plants is not only cheaper, but more efficient. Since plants do not produce antibodies naturally, the purification process is much simpler and plants are capable of producing unlimited amounts of the protein. It has been shown that plants are capable of correctly assembling and folding recombinant gene products, even full-length heavy and light chains into complete antibodies. (Ma and Hein, 1995)
HOW ARE ANTIBODIES EXPRESSED IN PLANTS?
Antibodies can be expressed in plants as either full length molecules or as smaller fragments. ScFv's (Single chain, Fragment variable) are produced by fusing variable light and variable heavy domains with a flexible linker peptide. This is a highly specific antibody fragment that binds to antigen with high affinity. ScFv's, unlike full length antibodies, can easily be targeted to sub-cellular compartments. This is very useful for the plant breeder when targeting specific pathogen proteins in plants, in order to engineer PATHOGEN RESISTANCE. The production of whole antibodies in planta has more therapuetic potential than fragmented antibodies, however, correct folding and assembly of the chains is more challenging for the plant cell. Fusion of cell signaling molecules e.g. the KDEL signal to the C terminus of the protein increases cytosolic expression. (Conrad and Fiedler, 1998) Each method has its advantages and disadvantages for antibody production. Smaller antibody fragments are both easier to produce and easier for the plant to assemble, however, full length antibodies are much more resistant to proteolysis in the plant. Plants are a favorable way of producing antibodies as they are capable of assembling a complete secretory antibody in one single cell. Mammals require numerous cell types to produce the different components of the antibody i.e. the plasma cells produce the dimerising J chain and epithelial cells synthesize the secretory component of the IgA antibody Firstly, the genes coding for the antibody fragments or full antibody must be engineered into the plant. This can be done using a variety of PLANT TRANSFORMATION METHODS In order to obtain high amounts of antibody production, it has been found that targeting the protein into the extracellular space (i.e. the apolplast) is the most efficient method. Plants, as animals, secrete antibodies after post-translational modification has occured. Proteins that are secreted into the apoplastic space undergo less hydrolysis due to the lack of hydrolytic enzymes in the apoplasm. Signal peptides can be used to target the light and heavy chains to the Endoplasmic Reticulum (ER), chaperones similar to those found in human cells are found here which ensure the antibody undergoes correct folding. Higher accumulation of antibody fragments has been observed in the ER and the apoplast compared to the cytosol
The addition of signal peptides to antibody fragments is obviously an advantage over the use of full antibodies. However, if full antibodies are used, certain domains could be altered to facilitate complement activation upon binding. This would significantly aid the therapeutic properties of the antibody if it were to be used in the treatment of human disease, helping to elicit an immune response. Plant seeds are an attractive organ for the genetic engineer to target protein expression to as they are natural storage organs capable of retaining high protein levels over long periods of time in the absence of degradation. This technique can be carried out by using seed-specific promoters. Plantibodies were first produced in tobacco plants, but it is now being developed in corn which is one of the most common crops grown worldwide. The corn seed kernel is capable of naturally storing plantibodies in a low moisture environment that has a high concentration of protective protease inhibitors. These can then be purified using simple milling techniques which easily separate the high molecular weight antibodies from other components of the kernel
PRODUCTION IN PLANTA
The availability of large amounts of cheaply produced purified antibodies could provide treatment to a wide range of pathogenic organisms. It is possible to raise antibodies to virtually any antigen in the laboratory, however, there are few systems available that facilitate the mass production of monoclonal antibodies. Plants provide an attractive system that could produce large amounts of antibody at a relatively low cost. The production of secretory IgA by plants has numerous therapeutic applications. Lehner et. al. have shown that antibodies produced in plants used to treat Rhesus monkeys for dental caries were devoid of systemic side effects. There have been many experiments carried out to investigate the stability of the antibody in the plant cell. It has been suggested that the difference in glycosylation in plant cells could affect the efficiency of the antibody produced. N-linked glycosylation occurs in all higher eukaryotes, including plants, however, it is thought that plant glycans are smaller, with different terminal sugar residues. It is speculated that the difference in glycosylation could cause lower affinity antigen binding and it may possibly cause the antibody to be immunogenic, raising concerns for systemic applications of plantibodies in humans.(Cabanes-Macheteau et. al., 1999)
PLANT TRANSFORMATION
Plant transformation is the introduction of a foreign piece of DNA, conferring a specific trait, into host plant tissue. The foreign gene (termed the "transgene") is incorporated into the host plant genome and stably inherited through future generations. The correct regulatory sequences are added to the gene of interest i.e. promoters and terminators, then the DNA is transferred to the plant cell culture using an appropriate vector. The gene is attached to a selectable marker which allows selection for the presence of the transgene. Genes conferring resistance to a specific antibiotic are often used to serve this purpose. Once the plant tissue has been transformed, the cells containing the transgene are selected and regeneration back into whole plants is carried out. This is possible as plant cells are totipotent, which means that they contain all the genetic information to control the development of that cell into a potentially fertile plant. Therefore, the gene is contained in every single plant cell, however, where it is switched on is determined by the promoter which is controlling the gene. Plant transformation can be carried out in a number of different ways depending on the species of plant in question. This is discussed in the sections below. Plant transformation was developed as an alternative to conventional breeding methods which are more laborious than this fairly simple (now routine) laboratory procedure.
WHY TRANSFORM PLANTS?
Plants are transformed in order to create transgenic plants that contain genes conferring traits that are desirable to the plant breeder. This includes resistance to specific plant diseases and pests, drought and saline tolerance. It also includes novel uses of plants, such as plantibody production and the production of edible vaccines in plant tissue. Plants can be engineered to express sub-unit vaccines which trigger oral immunity if plant tissues are consumed as food. Vaccines can be created to a huge number of pathogens, such as bacteria that cause diarrhea; responsible for 3 million infant deaths per year in the developing world.(Arntzen, 1998)Conventional breeding methods were seen to be slow and laborious, plant transformation techniques were developed to overcome these problems. The ability to introduce novel genes into plants, often across the species barrier, provides many commercially viable applications to the plant breeder
Two of the major techniques used to transform plants are :
AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION
BIOLISTICS
AGROBACTERIUM-MEDIATED TRANFORMATION
A major method of DNA transfer in plants is Agrobacterium mediated transformation. The natural living soil bacteria, Agrobacterium tumefaciens is capable of infecting a wide range of plant species, causing Crown Gall diseases (see image below). It has natural transformation abilities which can be exploited in plant biotechnology. When A. tumefaciens infects as a cell, it transfers a copy of its T-DNA, which is a small section of DNA carried on its Ti (Tumour Inducing) plasmid. The T-DNA is flanked by two (imperfect) 25 base pair repeats. Any DNA contained within these borders will be transferred to the host cell.(Zupan and Zambriski, 1995)
The T-DNA section on the Ti plasmid can be replaced by the transgene coding for the desirable trait attached to the appropriate regulatory sequences. The recombinant bacteria can then be used to infect both regenerating cell and protoplast cultures. (Protoplasts are wall-less special plant cells)
Marker genes such as those coding for antibiotic resistance are attached to the transgene so that it is possible to select those cells that have been transformed by the bacterium. Cell to plant regeneration is carried out on the selected cells and transgene expression is characterized i.e. it is necessary to check that the gene is expressed at the correct levels in the correct tissues.
Agrobacterium tumefaciens has been used to transform many dicotyledonous plant species with relative ease. However, it is, as yet, not possible to transform monocotyledonous species, which include commercially viable cereal crops such as maize and rice.
The image below shows a crown gall tumor on tomato. Tumors caused by Agrobacterium tumefaciens are often apparent on the plant at ground level
BIOLISTICS
Biolistics (other wise known as Particle Bombardment) involves directly "shooting" a piece of DNA into the recipient plant tissue. This is carried out using a gene gun. Tungsten or gold beads (which are smaller than the plant cells themselves) are coated in the gene of interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. The particles pass through the plant cells, leaving the DNA inside.
This method can be used on both monocotyledonous and dicotyledonous species successfully. It is again a relatively simple laboratory procedure. The transformed tissue is selected using marker genes such as those that code for antibiotic resistance. Whole plants are then regenerated from the totipotent transformed cells in culture, containing a copy of the transgene in every single cell (Nottingham, 1998).
HUMAN APPLICATIONS OF PLANTIBODIES
Plantibodies have many applications. They can be used to protect plants against DISEASE by engineering the plant to express antibodies against a particular plant pathogen. However, this section will describe the use of plantibodies for the treatment, and more importantly, the prevention of human disease.It has been shown that large amounts of complex multimeric immunoglobulin protein can be successfully produced and assembled within the plant. These can be purified using simple affinity chromatography methods. Secretory antibodies survive longer in the body than serum antibodies due to protection from the secretory component present in the mucosa. The secretory IgA antibody protects the mucosal surfaces of the body as a primary defense against pathogen attack. Therefore the production of secretory antibodies in plants has a lot of therapeutic potential.When engineering plants to express antibodies intended for use in medical therapy, the following steps are taken:
Obtain the neutralizing human antibody and determine its sequence
Engineer the genes coding for the antibody into the plant germ line for field scale production
Harvest the crop, mill and purify the antibody product
Formulate the pharmaceutical product
Undergo clinical trials to test for safety and efficacy of product
TREATMENT OF DENTAL CARIES : Plantibodies were first produced in tobacco plants by the biotechnology company EPIcyte in California. However, the first clinical evidence for the effectiveness of plantibodies in the treatment of human disease was obtained at Guys Hospital (Hikmat et.al., 1998). Plantibodies were produced in tobacco against the bacteria Streptococcus mutans. This bacteria is responsible for causing the majority of tooth decay by producing lactic acid which erodes tooth enamel. The research carried out involved painting the purified secretory plantibodies onto teeth for a three week period. It was found that this provided protection from S. mutans for up to four months, with the plantibodies specifically inhibiting bacterial attachment to the tooth surface. It has been shown that it is possible to produce secretory monoclonal antibodies in plants that are capable of preventing bacterial colonization in the human body. The anti Streptococcus mutans plantibody which has been developed not only as a cure, but also a preventative treatment against dental caries. This is an alternative approach to local passive immunization without any systemic side effects. An IgG monoclonal antibody was raised to the cell surface streptococcal antigen I/II. The antibody was repeatedly applied to the teeth of Rhesus monkeys. The monkeys had decreased colonization of the antibody compared to control animals. It is thought that the antibody opsonises the S. mutans facilitating phagocytosis and killing by the gingival traffic of neutrophils.(Lehner et. al.)
TREATMENT OF HERPES: Sexually transmitted disease is one of the most worrying international epidemics of the modern day, therefore, the production of monoclonal antibodies in plants for the treatment of these diseases is promising. . The biotechnology company EPIctye holds the exclusive license for plantibody technology from the Scripps Research Institute in California. The company are currently in pre-clinical trials with a topical gel containing plantibodies against the Herpes Type I and II virus (Zeitlin et. al.). This has already been proved to be effective against the virus when applied to the vagina of mice. It is hoped in the future that a more potent plantibody will be produced that will be used to protect new born babies from transmission of the virus from infected mothers during birth.In October 2000, EPIctye Pharmaceutical Inc. were awarded a grant for Phase I and Phase II trials for the development of antibodies for sexual health products. This grant is to be used for the development of anti-HIV(Human Immunodeficiency Virus) and anti-HSV(Herpes Simplex Virus) plantbodies for the production of a microbicidal lubricant. News from the 2000 International AIDS conference in South Africa concluded that there is a dire need for products to prevent the spread of AIDS (this disease develops as a consequence of infections by the HIV virus.) EPIcyte's plantibody technology provides an attractive way of producing large quantities of monoclonal antibodies in plants for the treatment of this disease . Currently antibody production is too expensive and has limited therapeutic application due to the huge scale-up costs. The production of antibodies in planta could produce unlimited quantities of pharmaceutical grade antibodies at a significantly lower cost than those produced in animal cell culture.
THE FUTURE
The future of monoclonal antibodies is very promising. A lot of this technology is still in clinical trials, but the possibility of producing large amounts of monoclonal antibodies raised to a specific antigen at low cost surely has unlimited therapeutic applications. It is thought that disease prevention is the key application to this technology. Sexually transmitted diseases are becoming increasingly prevalent in todays society, costing the government $12 billion in the USA for treatment. The production of an over-the-counter topical gel derived from plantibodies capable of killing sexually transmitted pathogens may be available in the future. It is also thought that it may be possible to deliver the plantibodies via control release devices such as pessaries. These are not only relatively cheap to produce, they also provided a user-friendly way of delivering treatment to the patient.
PLANT APPLICATIONS OF PLANTIBODIES
Using proteins that belong exclusively to the realm of animals to help plants fight viruses and nematode infections in planta is an important application of plantibody technology. It is possible to modulate phytohormone activity and hence block pathogen infection, however, much research is being carried out in an attempt to achieve high level accumulation of antigen-specific antibodies and antibody fragments. It is possible to engineer specific recombinant antibodies against cellular pathogenic antigens. These are able to block regulatory functions of pathogens or block pathogens directly in specific target plant organs. It is possible to target antibodies to specific cellular compartments and hence block specific secreted antigens. This is termed INTRACELLULAR IMMUNISATION; modulating antigens inside the plant cell.Expressing antibodies in plants can also be used to study the actions of plant hormones such as Abscissic Acid (ABA). This has successfully been carried out by engineering anti-ABA ScFv's targeted directly to the Endoplasmic Reticulum (ER) of leaf cells. This blocks the action of ABA on stomatal functioning facilitating the study of the effects of this hormone on stomata
EXAMPLES
Cytosolic ScFv's were engineered against the Artichoke Mottled Crinkle Virus coat protein. These were found to give decreased viral infection and delayed symptoms.
Anti-Tobacco Mosaic Virus (TMV) transgenic plants containing full-length anti-TMV secreted antibodies were created. These showed decreased TMV necrotic lesions correlated with antibody expression.
Transgenic tobacco plants containing secreted ScFv's against a 25KDalton coat protein from the Beet Necrotic Yellow Vein Virus were created. The presence of the plantibodies gave partial protection against the viral infection and its associated symptoms.
Transgenic tomato plants were engineered to express antibodies against the Tomato Spotted Wilt Virus movement proteins with a view to assessing the levels of antibody mediated resistance to the virus.
PROTECTION AGAINST VIRUS INFECTION IN PLANTS: There are many examples of transgenic plants that express components of the viral genome, conferring resistance to specific viruses. It is thought that by expressing, for example, the viral coat protein genes, interference of viral replication occurs, hence rendering the plant resistant. However, this technique has many risk issues attached to it, due to the presence of viral DNA sequences present in human foodstuffs.It is now possible to express antibodies against viral proteins as an alternative approach. This paper describes the isolation of antibodies against the Artichoke Mottled Crinkle Virus (AMCV) coat protein... termed the F8 antibody. Variable (heavy) and Variable (light) chains were amplified using PCR ( the Polymerase Chain reaction) and their antigen binding affinity was tested using the ELISA staining technique. The ScFv fragments were expressed in the Nicotiana benthamiana plant; a natural host for AMCV. The plants showed reduced AMCV accumulation.This was the first example of a plant phenotype with attenuation of viral infection derived from a constitutively expressed virus-specific antibody
It can be seen that there is an UNLIMITED REPERTOIRE of antibody specificities that can be exploited by the plant breeder.. providing new and novel disease resistances.
ScFv's appear to be crucial as they are functionally stable in the cell cytoplasm and are suitable for immune modulation of selected cytoplasmic antigens
It is unknown exactly how plantibodies work. More information is required on virus-antibody interactions in early infection stages. It has been suggested that antibody binding to Calcium ion binding sites on the coat protein would uncoat the virus
The exact location of the antibody fragment within the cell is crucial, as it appears that antibody Compartmentalization plays a big part in determining protection for the plant.
CONCLUSIONS
Production of antibodies in plants has numerous applications not only to the pharmaceutical industry but also the plant breeder. If clinical trials are successful, plants could provide a "factory system" for the production of huge amounts of monoclonal antibodies for the treatment of disease. They could also be used an internal protection system for plants against their own specific pathogens
Plants can be engineered to express both antibody fragments and full length fragments, it has been shown that plants are capable of correctly assembling and folding complex antibody molecules. Antibody fragments such as ScFv's are often assembled in the plant more efficiently, however, full length antibodies are less prone to proteolysis in the plant cell
Problems have been encountered with regard to differential glycosylation within the plant cell. This is due to the presence of smaller plant glycans (thought to have different terminal sugar residues) used in N-linked glycosylation. When using plantibodies for therapeutic purposes and plant protection, it must be taken into account that differing glycosylation could decrease the antigen-binding affinity of the antibodies produced. It has also been suggested that the presence of foreign glycans could produce an allergenic effect on administration to humans.
There is currently a lot of research being carried out into the applications of plantibodies. In plants, protection against viral and nematode infection are at the primary targets of the technology. In humans, a lot of work has been carried out into treatment of dental caries, caused by the bacteria Streptococcus mutans and the treatment of the Herpes virus.
This technology holds a lot of promise for the future, hopefully contributing to the treatment of many diseases. EPIctye pharmaceuticals are currently developing a topical gel for the treatment of the Herpes Simplex Virus, with a view to using this to treat new born children born to infected mothers. It is hoped that subsequent antibody-based topical contraceptives will be formulated from antibodies produced by plants. The Respiratory Syncytial Virus which causes acute pulmonary infection and Clostridium difficile which is a major cause of diarrhea are also targets for plantibody technology. Eventually, it is hoped that it may be possible to treat the Human Immunodefficiency Virus (HIV) using antibodies derived from plants.
