2. Introduction
► Transgenic plants: - Plants whose genetic material has been modified by insertion of a
foreign gene to produce the product of our interest.
► Transgenic plants are an attractive and cost-effective alternative to microbial systems for
the production of biomolecules.
► Products produced by these types of plants includes bioactive peptides, antigens for
vaccine, antibodies, nutritional supplements, enzymes and biodegradable plastics.
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3. Need of Transgenic Plants
► Proteins produced by prokaryotic and mammalian cells have limitations in terms of
suitability, purification and post translational modification.
► The basic objective of using plant as a bioreactor is the production industrially important
bioactive biomolecules like carbohydrates, lipids and plant metabolites.
► Their major need is to produce heterologous proteins.
► A low-cost and easy-to-scale-up solution in agriculture has a variety of advantages,
including the absence of animal contamination.
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6. Optimization of Gene Expression
1) At transcription and translation levels:
► Promoter region: contains site for RNA Pol binding thus has control over transcription.
► Initiation codon: the sequences around the start site are the most conserved sequences so using
them as a gene construction, increases the protein production upto several folds.
► 5’ UTR sequences: leader sequences, role in enhancing the translation efficiency of enzymes.
► 3’ UTR sequences: These sequences are mainly responsible for polyadenylation and thus
stability of mRNA. So can be artificially edited leading to overproduction of mRNA.
► Coding sequences: by altering the genes coding for the desired protein.
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7. Optimization of gene expression
2) Post translational modifications:
► Mainly include glycosylation, phosphorylation, polyadenylation, methylation, etc.
► Despite their ability of glycosylation plants do add certain non-desirable residues like plant
specific fucose and xylose. So transgenic plants have been developed to produce humanized
proteins.
3) Subcellular sorting:
► This type of modification is directly limiting the protein production to certain specific
organelles (to reduce the difficulties in product recovery).
► Organelles like endoplasmic reticulum, storage vacuoles and oil bodies are included.
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9. Strategies to achieve Uniform Transgene Expression
► There is a lot of diversity in transgene expression within populations of transgenic plants
transformed with the same transgene.
► Hinders proper evaluation and are undesirable when high-throughput transgene screening is
intended.
► Several strategies have been explored to Minimize variation and stabilize transgene expression
1. Generation of single-copy transformants
2. Artificial chromosomes: targeted delivery eliminates the possible entry of transgene into the
inactive region of the genome
3. Mini-chromosome construction: all the transgenes would reside on a mini-chromosome, not
linked to any endogenous genes; thus linkage drag can be avoided
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10. Downstream Purification Strategies
► The common steps for the recovery are fractionation of plant tissue, extraction of r-protein and
purification.
► Unwanted molecules of the plants must be eliminated during r-protein purification.
► Several techniques have been developed that purifies the product without cell disruption and
prevents the release of phenolic chemicals and other pollutants from plant cells.
► Protein bodies, which are insoluble oil bodies and are light in weight (can be separated by
centrifugation) can be targeted for the desired protein.
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11. Types of Bioreactors
► Seed based bioreactor Platform: - Plant seeds have high rates of protein synthesis and
accumulate a huge amount of proteins during seed development, which makes them an
ideal habitat for storing recombinant proteins.
► Seed protein storage vacuole as bioreactor: - In seed bioreactors, protein storage vacuoles
(PSVs) are the main compartments for storing recombinant proteins. Soluble proteins
contain distinct targeting cues thus travel through particular transport channels to reach
the seed PSV and its sub compartment.
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12. Types of Bioreactors
► Seed oil body as bioreactor: - Seed oil body (OB) is a desirable bioreactor because it can
store a significant number of macromolecules. In plant seeds, OBs are surrounded by a
high-density protein called oleosin.
► Chloroplast as a bioreactor: - Chloroplasts in plants and algae have been regarded as an
ideal organelle for producing recombinant proteins.
► The hairy root system: - The secretion system of hairy root also called Rhizosecretion
formed by Agrobacterium rhizogenes, offers a simplified method for the isolation of
recombinant proteins using
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14. Nutritional Components
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► The majority of the nutrients required in the human diet may be found in plants.
Thus transgenic plants have been developed to improve the nutritional quality.
► Carbohydrates: Plants are capable of producing polymeric carbohydrates (such
as starches and fructans) as well as individual sugars (e.g. Sucrose and Fructose).
Fructans are an important ingredients in foods for a healthy colon. Eg: Fructan
accumulation by Beta vulgaris.
15. Nutritional Components
⮚ Lipids: - Different categories of fatty acids have been modified like
Saturated fatty acids: to modify fatty acids the degree of desaturation needs to be inhibited.
Medium chain fatty acids: they are required for synthesizing medium chain triglycerides
for detergent production.
⮚ Vitamins: - In addition to completing standard photosynthesis duties, the plastids of higher
plants generate a variety of nutritionally valuable chemicals such as carotene (Vit A),
tocopherols (Vit E) and ascorbate (Vit C).
⮚ Micronutrients: Improvement in the level of iron and zinc.
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16. Advantages
► Plant – bioreactors as cost-effective alternatives
► Plant bioreactor in Transient expression
► Plant – bioreactors in Nuclear and Plastid stable transformation
► Examples of plant bioreactors :-Human lysosomal enzyme can be produced in plant
bioreactors. The earliest plant produced pharmaceutical product is the human growth hormone
produced in transgenic tobacco. Several plant types like cereals, legumes, etc. have been
exploited for production of recombinant molecules like antibodies and subunit vaccine proteins
and human biopharmaceuticals.
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17. Limitations
► It cannot control dosage and dose consistency of orally delivered plant-based
recombination proteins.
► Only a few species are known to be transformed and not all .
► Production of the desired biomolecules may be affected by climatic factors like sunlight.
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