2018 Solved Old Paper (BOT

Transgenic Plant:-

• Definition:- Stable transformation is achieved when a desired gene is integrated into the genome of a plant, this plant is called transgenic plant.

• These transgenic plants are developed for the following purposes:-

1. Insect Resistance

2. Virus Resistance

3. Seed Protein Quality

4. Gene Silencing

5. Male Sterility

6. Biochemical Production

1. Insect Resistance:-

cry gene transfer:- The cry gene is found in the plasmid of Bacillus thuringiensis (Bt) bacteria. This cry gene makes Crystal protein which is insecticidal.

Ø The cry gene was isolated from the plasmid of Bt and integrated into the cotton genome to form a transgenic plant called Bt-cotton.

Ø This Bt - cotton is resistant to boll worm.

Ø Crystal protein perforates the larva's alimentary canal leading to its death.

2. Virus Resistance:-

cp – gene transfer:- The coat protein is transferred from the TMV virus to the tobacco plant.

Ø The RNA of TMV virus is not come out in the presence of cp-protein. Due to which the tobacco plant becomes resistant to mosaic disease.

Ø Other examples: - Tomato, Alfalfa, Beetroot, Potato

3. Seed Protein Quality:-

SFA8 gene transfer:- In sunflower seeds, the SFA8 gene produces a protein that contains an excess of sulfur-rich amino acids methionine and cysteine. While the proteins present in pea seeds lack methionine and cysteine amino acids.

Ø Therefore the SFA8 gene is transferred to pea seeds.

4. Gene Silencing:-

Slow ripening tomatoes:-

Ø PG (Poly Galacturonase) enzyme digest the pectin. Due to which fruits become soft and fluffy quickly and ripen quickly.

Ø Anti-sense genes are integrated into the genome of tomato plants against genes that code PG enzymes. This inhibits the expression of PG gene and the tomato fruit ripens late.

Ø Antisense gene made against PG gene inhibits its expression, which causes tomato to ripen late and can be easily exported.

5. Male Sterility:-

Producing male sterility:-

Ø Flavonoid are essential for maturation of pollens. If we stop its formation then male sterility can be created in the plant.

Ø The CHS (Chalcone Synthase) enzyme is required for flavonoid synthesis.

Ø Anti-sense genes are integrated into the plant's genome against genes that code CHS.

Ø Absence of a flavonoid causes pollen to become inactive. The plant becomes male sterile.

6. Biochemical Production:-

• PHB (Poly Hydroxy Butyrate):- It is prepared from Acetyl CoA. From this bio-degradable plastic is made.

• The transgenic plant is developed by isolating the phb - B and phb - C genes from the Alcaligenes eutrophus bacterium and transfer to Arabidopsis thaliana. Now PHB is obtained from this plant.

Golden Rice:-

• The genes that form β - carotene are integrated into the embryo of rice within the genome to prepare golden rice.

• 2 genes are isolated from Daffodils plant and 1 gene from Erwinia uredovora bacterium.

• β - carotene is yellow in color, which makes rice yellow.

• β - carotene is the precurssor of vitamin A.

PCR: Its principles and uses:-

• PCR:- The process of multiplication of DNA segments using DNA polymerase and DNA primers is called PCR.

• Discovery:- The PCR technique was discovered by Kary Mullis in 1985.

• Thermocyclers are used to achieve different temperatures.

• PCR has three main steps -

i. Denaturation:- When dsDNA is heated, both its chains separate at a temperature of 90°C.

ii. Primer Annealing:- Primers are attached at the 5 'end of single chains at a temperature of 55°C.

iii. Polymerization:- DNA polymerase enzyme polymerize the primers at 70°C temperature.

• Heat stable DNA polymerase:-

Ø Normal DNA polymerase is heat sensitive. It is destroyed due to its deformation at 90°C temperature. Therefore, we cannot use normal DNA polymerase in PCR.

Ø Instead, we use heat stable DNA polymerase in PCR which can tolerate high temperature and does not deform.

Uses of PCR:-

i. The amplification of gene fragments as fast alternative of cloning.

ii. The modification of DNA fragments.

iii. The sensitive detection of pathogenic microorganisms, if desired followed by an accurate genotyping.

iv. DNA analysis of arachaeological specimens.

v. The detection of mutations relevant for inherited diseases, malignant transformation or tissue typing.

vi. The analysis of genetic markers for forensic applications, for paternity testing and for the mapping of hereditary traits.

vii. The species-specific amplification of DNA segments between interspersed-repeat elements.

viii. The study of gene expression.

Genetic manipulation of nitrogen fixation:-

> The subunits of nitrogenase from different nitrogen fixing microorganisms can be mixed to produce

functional system

> Genes can be manipulated to improve the fixation of dinitrogen.

> The microorganisms are modified in the host so that they are unable to assimilate the fixed nitrogen until the function of the nitrogenase is over and release NH4+ directly to the plants.

> The nitrogen fixation can be regulated by leguminous plants by:-

i. Reducing the number of root nodule formation

ii. Regulating the carbon flow to the microorganisms.

> The well known nitrogen fixing bacteria K. pneumoniae resembles closely to non-nitrogen fixing bacterium E.coli. The genes of these two species could be transferred and expressed in either of the organisms.

- Nif- mutants of K. pneumoniae that are deficient in fixing nitrogen are located between genes for histidine biosynthesis (his) and shikimik acid uptake (shi A).

- The his and nif regions can be actively transferred from a strain of K. pneumoniae to an E.coli

strain which require histidine.

- E.coli cells that do not require histidine anymore have acquired the ability to fix nitrogen.

- The conjugative plasmid pRDI that picked up the nif and his genes was selected and transferred to other bacterial genome.

- The gene, nifL, that serves as the repressor of nitrogen fixation can be deleted thus allowing constitutive expression of nif promoter mediated by nifA and ntrC (nitrogen regulator) gene products.

- The nitrogenase activity has been observed in E.coli that carries the nif plasmid pRDI which was very

much similar to that of K. pneumoniae strain.

> Agrobacterium tumefaciens, an obligate aerobe not resembling Klebsiella did not result in nitrogen fixing recombinants upon transfer of pRDI to itself.

> Mutants of Azotobacter vinelandii that lack either of the components of nitrogenase regained the

nitrogenase activity when pRDI was transferred to them.

> Nitrogenase can be protected from the inhibitory action of oxygen by a protein leghaemoglobin. The genes encoding these proteins can be isolated and transferred to other nitrogen fixing systems so as to

protect the nitrogenase from oxygen activity.

> Nitrogenase activity is correlated with the hydrogenase activity that evolve hydrogen hence it

requires more energy. The energy can be saved if the evolved hydrogen is further reduced to water

releasing electrons. Many nitrogen fixing bacteria possess ‘uptake hydrogenases’ which consists of two subunits HupS and HupL and it is advantageous to introduce it together with the nif genes into hosts that do not possess uptake hydrogenase system.

Western Blotting:-

Principle:-

> Western blot is performed by using polypropylene gel electrophoresis. SDS-PAGE allows protein samples to be separated and transferred to a solid support, such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane.

> The solid support can absorb the protein and keep its biological activity unchanged. The transferred solid support membrane is called a blot and is treated with a protein solution to block the hydrophobic binding site on the membrane.

> The membrane is treated with the antibody (primary antibody) of the target proteins.

> Only the proteins to be studied can specifically bind to the primary antibody to form an antigen-antibody complex.

> After the primary antibody is washed and removed, only the position of the target protein binds to the primary antibody.

> The primary antibody-treated membranes are treated with a labeled secondary antibody after washing.

> After treatment, the labeled secondary antibody that binds to the primary antibody forms an antibody complex that can indicate the location of the primary antibody, both the location of the protein being studied.

Procedure:- There are six steps involved in western blot -

i. Sample preparation:-

> Proteins can be extracted from different samples, such as tissues or cells.

> Since tissue samples display a higher degree of structure, the tissues are first broken down by the mechanical invention, such as homogenizer or sonication.

> Protease and phosphatase inhibitors are commonly used to prevent the digestion of the sample at cold temperatures.

> After protein extraction, it is important to detect the concentration of proteins, which permits the mass of proteins loaded into each well. And a spectrophotometer is often used for proteins concentration.

ii. Gel electrophoresis:-

> The most commonly used gel is polyacrylamide gels (PAG) and buffers loaded with sodium dodecyl sulfate (SDS).

> Western blot uses two types of agarose gel:

Stacking gel:- that is used for concentrate all proteins in one band

Separating gel:- that allows for separating proteins according to their molecular weight.

> Smaller proteins migrate faster in SDS-PAGE when a voltage is applied.

> PAGE can separate proteins ranging from 5 to 2,000 kDa according to the uniform pore size which is controlled by the Different concentration of PAG.

> Typically separating gels are made in 5%, 8%, 10%, 12% or 15%.

> When we choose the appropriate percentage of the separating gel, we should consider the size of the target proteins.

> The smaller the known weight of proteins is, the higher percentage of gels should be used.

iii. Proteins transfer:-

> After separating proteins by gel electrophoresis, proteins are moved from within the gel onto a solid support membrane to make the proteins accessible to antibody detection.

> The main method for transferring proteins is called electroblotting, which uses an electric field oriented perpendicular to the surface of the gel, to pull proteins out of the gel and move into the membrane.

> It can be done semi-dry or wet conditions, while wet conditions are usually more reliable as it is less likely dry out the gel.

> The membrane is placed between the gel surface and filter. The transfer sandwich is created as follows: a fiber pad (sponge), filter papers, the gel, a membrane, filter papers, a fiber pad (sponge).

iv. Blocking:-

> Blocking is an important step in the western blot to prevent antibodies from binding to the membrane non-specifically.

> The most commonly used typical blockers are BSA and non-fat dry milk.

> When the membrane is placed in the dilute solution of proteins, the proteins attach to all places in the membrane where the target proteins have not attached. In this way, the “noise” in the final product of the western blot can be reduced and result in clearer results.

v. Antibody incubation:-

> After blocking, the primary antibody binds to target protein when the primary antibody is incubated with the membrane.

> The choice of a primary antibody depends on the antigen to be detected.

> Washing the membrane with the antibody-buffer solution is helpful for minimizing background and removes unbound antibodies.

> After rinsing the membrane, the membrane is exposed to the specific enzyme conjugated secondary antibody.

> When performing secondary antibody incubation, the labeled secondary antibody can bind to the primary antibody which has reacted with target proteins.

> Based on the species of the primary antibody, we can choose the appropriate secondary antibody.

vi. Protein detection and visualization:-

> A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance. It enables us to know the densitometry and location of the targets protein. And the size approximations are taken by comparing the proteins bands to the marker.

> There are several detection systems are available for protein visualization, such as colorimetric detection, chemiluminescent detection, radioactive detection, and fluorescent detection.

> The electrochemiluminescence (ECL) system is the most common detection method.

Gene concepts and molecular biology of gene:-

Modern Concept of Gene:-

> A gene can be described as a polynucleotide chain, which is a segment of DNA.

> It is a functional unit controlling a particular trait such as eye colour.

> Beadle and Tatum concluded by various experiments that gene is a segment of DNA that codes for one enzyme. They proposed one gene-one enzyme hypothesis.

> But as some genes code for proteins that are not enzymes, the definition of gene was changed to one gene-one protein hypothesis.

Protein Hypothesis:-

> Since proteins are polypeptide chains of amino acids translated by mRNA, gene was defined as one gene-one polypeptide relationship.

Some proteins have two or more different kinds of polypeptide chains, each with a different amino acid sequence. They are products of different genes.

> For example, haemoglobin has two kinds of chains a andβ chains, which differ in amino acid sequence and length. They are encoded by different genes.

> Thus, gene is defined as one gene-one polypeptide relationship.

Structural and Regulatory Genes:-

> Even the one gene-one polypeptide definition is not complete as it does not include gene which codes for rRNA and tRNA. Only mRNA is translated into proteins. Therefore genes which code for polypeptides and RNAs are called structural genes.

> In addition to structural genes, DNA also contains some sequences that have only regulatory function. These regulatory genes constitute signals, which “turn on” and “turn off” the transcription of structural genes and perform various other regulatory functions.

> In this way the definition of gene includes structural genes as well as regulatory genes.

> Benzer coined terms for the gene, they are Cistron which is the unit of function, Recon which is the unit of recombination and Muton which is the unit of mutation.

Molecular Definition of a Gene:-

> According to Lodish and others, gene is defined as the entire nucleic acid sequence that is necessary for the synthesis of a functional gene product, which may be polypeptide or any type of RNA.

> In addition to structural genes (coding genes) it also includes all the control sequences and non-coding introns.

> Most prokaryotic genes transcribe polycistronic mRNA and most eukaryotic genes transcribe monocistronic mRNA.

Number of Genes on a Single Chromosome:-

> Total number of genes on a single chromosome is different in different organisms.

> Bacteriophage virus R17 consists of only three genes, SV40 consists of 5-10 genes.

> E. coli bacteria have more than 3000 genes on single 1 mm long chromosome.

Size of a Gene:-

> In E. coli there are more than four million pairs of nucleotides (4638858 base pairs). It has been estimated that there are about 3000 genes in E. coli.

> The minimum size of a gene that encodes a protein can be directly estimated, Each amino acid of a polypeptide chain is encoded by a sequence of three consecutive nucleotides in a single strand of DNA. Therefore by measuring the size of the polypeptide chain, the size of a gene can be directly measured.

> The average polypeptide chain has about 450 amino acids, which are encoded by 1350 nucleotides. Therefore, in E. coli the number of genes will be around 3000 (4000000/1350 = 3000).

> Human genome contains about 30000 genes.

> Simple round worm C. elegans has about 20000 genes.

> A single copy of chromsome is composed of more than 3 billion base pairs.

> Coding regions of these genes take up only 3% of the genome.

Fine Structure of a Gene:-

> A gene is present only in one strand of DNA, which is a double stranded helix.

> A gene consists of several different regions.

> The main region is the coding sequence which carries information regarding amino acid sequence of polypeptides.

> The region on the left side of coding sequence (upstream or minus region) and on the right side (downstream or plus region) consists of fairly fixed regulatory sequences.

> Regulatory sequences consist of promoters which are different in prokaryotes and eukaryotes.

Types of Genes:-

i. Simple Genes:-

> Simple genes have a coding sequence of bases in one DNA strand.

> Upstream the coding region, the promoter is present.

> Downstream, the termination region is present.

ii. Split Genes (Interrupted genes):-

> In most of eukaryotes, many non-coding sequences are present between coding sequences.

> The coding sequences of DNA of the genes are called exons.

> In between exons are present non-coding sequences called introns.

> Exons alternate with introns.

> Normally introns do not possess any genetic information and are not translated.

> Splicing:-

- The mRNA transcribed from this DNA is called precursor mRNA (pre-mRNA) and contains exons as well as introns.

- The introns are removed by excision and discarded. This process is known as splicing.

- The remaining segments or exons are joined together to form the mature mRNA which takes part in translation.

- The mature mRNA is much smaller than the pre-mRNA for example α -globin has two introns, ovalbumin has seven introns and α-collagen has 52 introns.

iii. Overlapping Genes:-

> Most genes consist of DNA sequences that code for one protein.

> But there are some sequences that code for more than one protein.

> Fredrick Sanger discovered this phenomenon in bacteriophage φ x 174.

> Overlapping genes are common in many viruses.

> Here the small length of viral DNA is exploited for synthesizing different proteins.

> This is achieved in different ways.

> In some cases, one gene generates two proteins by having different starting points. Similarly, the same gene generates two proteins by terminating the expression at different points.

> In other cases, a sequence of DNA makes no distinction between exons and introns. This sequence of DNA, which uses only exons for expression, also uses adjoining introns at other times for expression.

> The differential splicing of a single stretch of mRNA leads to overlapping and therefore different proteins. In this way, multiple proteins can be generated from a single stretch of DNA.

iv. Jumping Genes or Transposons:-

> Earlier it was thought that genes are static and have definite and fixed locus.

> However, recently it has been discovered that segments of DNA can jump to new locations in the same or different chromosome.

> First of all it was discovered by Barbara Mc Clintock in Indian maize corn. It has cobs with kernels of different colours. The light coloured kernels were caused by segments of DNA that move into genes coding for pigmented kernels, thereby inactivating pigmented kernels.

> These mobile genes are called transposable elements or transposons.

> They can jump within the genome, thus affecting the gene expression.

> Transposable elements are components of moderately repetitive class of DNA.

> A transposon has well defined ends. It consists of a long central portion. On either end each transposon has specific sequence of bases which are inverted repeats or palindromes on opposite strands. These terminal repeats help in identifying transposons.

> The site where a transposon is inserted is called target site or recipient site.

> Transposable elements can lead to change in the expression of genes.

>They can also cause mutations.

> In bacteria, they are present on plasmids.

v. Variable Genes:- Certain polypeptides are coded not by one gene but they are coded by more than one gene present on the same or different chromosomes.

Open Reading Frame:-

> A gene is a segment of genome which is transcribed into RNA.

> If the RNA is a transcript of a protein coding gene then it is called messenger RNA or mRNA. This is translated into protein.

> If the RNA is non-coding as ribosomal RNA (rRNA) or transfer RNA (tRNA) it is not translated.

> The part of the protein coding gene which is translated into protein is called open reading frame. It has triplet nucleotide codons.

> Open reading frame starts with an initiation codon and ends with a termination codon.

> The region of DNA before a gene is called up-ream region denoted with a minus (-) sign while region after the gene is called downstream denoted with a plus (+) sign.

> Many genes are split between exons and introns. The introns are removed by splicing to produce a functional RNA before translation.

Secondary Metabolites:- These are organic compounds produced by any lifeform, e.g. bacteria, fungi, animals, or plants, which are not directly involved in the metabolism for normal growth, development, or reproduction of the organism.

Classification:-

1. Terpenes:-

> These are the largest single class of compounds found in essential oils.

> They are responsible for the aromas, flavors, and colours.

> These are polymer of isoprene molecules. Hence also called as isoprenoids. Each isoprene molecule contains five carbon atoms with double bonds.

Isoprene

> Examples:- Geraniol (Monoterpene), Cafestol (Diterpene), Lycopene (Tetraterpene), Gutta-percha (Polyterpene)

2. Phenolics:-

> Most abundant secondary metabolites of plants.

> They have potent antioxidant properties.

> They are polyphenols which are ubiquitous means found in all plant organs.

> Examples:-

i. Lignin (Structural polymer)

ii. Flavonoids and Carotenoids (Attractants)

iii. Flavonoids (UV screens)

iv. Salicylic acid and Flavonoids (Signal compounds)

v. Tannins and Phytoalexins (Defense response chemicals)

vi. Coumarins

3. Alkaloids:-

> These are N - containing cyclic organic compound.

> The term ‘alkaloid’ was coined by W. Meibner, a German pharmacist, meaning ‘alkali like’. Latter it was demonstrated that the alkalinity was due to the presence of a basic nitrogen atom.

> Morphine was the first alkaloid which was isolated by F.W. Serturner in 1806 from the latex of the opium poppy.

Types of Alkaloids:-

a. Based upon type of nitrogen and the biochemical origin:-

i. True alkaloids:- They are derived from amino acids and have nitrogen in a heterocyclic ring. Eg.- Cocaine, Morphine, Quinine etc.

ii. Proto alkaloids:- They are derived from amino acids and do not have nitrogen in a heterocyclic ring. Eg.- Yohimbine, Mescaline, Hordenine etc.

iii. Pseudo alkaloids:- They are not derived from amino acids but have nitrogen in a heterocyclic ring. Eg.- Capsaicin, Caffeine, Ephedrine etc.

b. Based upon ring structure:-

i. Tropane alkaloids:- They are abundantly found in the Solanaceae family. They are derived from ornithine and acetoacetate. Eg.- Hyoscyamine, Cocaine, Atropine, Scopolamine

ii. Pyrrolizidine alkaloids:- They occur in the plants from Asteraceae and Fabaceae family. Eg.- Senecionine

iii. Piperidine alkaloids:- Presence of odor is the common feature. Eg.- Trigonelline, Coniine, Arecoline, Lobeline, Pelletierine, Nicotine, Anabasine, Piperine and Ricinine.

iv. Quinolines alkaloids:- These alkaloids are obtained exclusively from the bark of the Cinchona plant. Eg.- Cinchonine, Cinchonidine, Quinine, and Quinidine

v. Isoquinoline alkaloids:- These groups of alkaloids have huge types of medicinal properties like antiviral, antifungal, anticancer, antioxidant, antispasmodic, and an enzyme inhibitor. Eg.- Morphine, Codeine, Papaverine, Narcotine, Hydrastine, Berberine, Emetine, Cephaëline, Tubocurarine, Corydaline.

vi. Indole alkaloids:- This is the largest and most interesting alkaloid group derived from tryptophan. Polyhalogenation is a common feature of these alkaloids. Eg.- Ergometrine, Ergotamine, Physostigmine, Ajmaline, Serpentine, Reserpine, Yohimbine, Aspidospermine, Vincablastine, Strychnine, Bruceine.

vii. Steroidal alkaloids:- 1,2-Cyclopentane phenanthrene ring system is the characteristic of this type of alkaloids. Eg.- Solanine, Veratrum, Funtamine, Conessine.

viii. Imidazole alkaloids:- The imidazole ring structure is the characteristic of this type of alkaloids. Eg.- Pilocarpine.

ix. Purine alkaloids:- They contain purine ring. Purine is the nitrogenous base. Eg.- Caffeine, Theobromine, Theophylline.

x. Pyrrolidine alkaloids:- Eg.- Hygrine, Stachydrine, Ficine, Brevicolline.

Stratagies to increase production of secondary metabolites in tissue culture:-

1. Origin of plant tissue:- The genetics of the plants have a high influence on the production of secondary metabolites. For example, high fielding cultivars produce high nicotine content compared to low fielding cultivars.

2. Culture Media:- The composition of the culture medium influences both, biomass production and the synthesis of secondary metabolites. So, ideal conditions are required to be maintained to keep a balance between these two.

3. Growth Regulators:- They have a significant effect on the production of secondary metabolites, as they can either be synergistic or antagonists in their actions. For example, the addition of gibberellic acid to the Taxus cuspidate’s medium increases the taxol production.

4. Carbon source in the medium:- Sucrose is one the most widely used carbon source in the culture media. However, there are also some others, which include glucose, fructose, and galactose.

5. Temperature:- A higher temperature is reported to enhance the production of secondary metabolites.

6. pH:- A perfect pH between 5-6 is required to be maintained for the production of secondary metabolites.

7. Light intensity:- Apart from stimulating the production of secondary metabolites, light intensity also has a role in determining the amount of the production.

8. High-cell Density culture:- A high-density culture increases the production of secondary metabolites. The case is reported in Japonic cells where they have grown up to 759 \ L of cell mass to increase productivity.

9. Elicitation:- Elicitors are compounds of biological origin involved in plant microbe interaction. Elicitors are considered as mediator compounds which induce secondary metabolites formation in cells cultures. Varieties of elicitors have been used for production of secondary metabolites. Elicitation improves the efficiency of Sec. Product accumulation in plant cell culture by:

i. Minimizing up on time

ii. Avoiding change of media

iii. Induction of enzymes involved in biosynthetic pathway

iv. Inducing excretion of metabolites into the medium

Some of the biotic elicitor- induced products

10. Precursor feeding:- Under in vitro circumstances, often the precursors of metabolite die and can’t carry out the reaction. This can be overcome by supplying enough precursors to cells, which can be done by two methods:

a. Add precussors:- By adding precursors in the medium so that their availability is not limited.

b. Prevent resistant precursor analogs:- By preventing resistant precursor analogs and increasing the intracellular levels of precursors for the metabolite production. Eg.-

i. Production of shikonin increased three-fold after the addition of L-Phenylalanine to the medium in Lithospermum species.

ii. Datura sp. cell suspension cultures are supplemented with hydroquinone, in traces, the arbutin synthesis increase considerably.

Antisense - RNA:- It is single-stranded RNA molecule that can bind and inhibit specific mRNA translation to protein.

Principle:-

Sense RNA:-

> messenger RNA (mRNA) is single-stranded.

> Its sequence of nucleotides is called "sense" because it results in a gene product (protein).

> Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.

Antisense RNA:-

> RNA can form duplexes just as DNA does.

> All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand.

> The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense.

Example:-

5´ C A U G 3´ mRNA

3´ G U A C 5´ Antisense RNA

Antisense technology:-

> When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked.

> This may occur because the ribosome cannot gain access to the nucleotides in the mRNA or because the duplex RNA is quickly degraded by ribonucleases in the cell.

> With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.

Applications of Antisense technology:-

1. Treatment of viral infection:-

> Antisense oligonucleotides are selective and highly specific and binds with targeted viral mRNA and down regulate the expression of viral proteins, thus inhibit the virus replication.

> Thus this antisense technology can be used to design therapeutic compounds targeted to specific mRNA to treat various viral diseases.

> Vitravene (fomivirsen):- It is the first antiviral Antisense drugs used to treat cytomegalovirus retinitis. Other potential targets of antisense drugs include human papillomavirus, HIV, hepatitis-B virus, influenza A virus, and herpes simplex virus (HSV).

2. Treatment of cancer:-

> As antisense drugs are less toxic than conventional drugs, many antisense oligonucleotides are currently investigated to treat various cancers in humans or for the deactivation of oncogenes.

> For the first time an antisense oligonucleotide in combination with cisplatin was approved to treat bladder cancer.

3. Gene therapy:-

> Antisense oligonucleotides are being used in genetic research to treat various genetic disorders.

> In genetic disorders, some point mutations resulting in the formation of a defective mRNA and then defective protein.

> Antisense oligonucleotides blocks the formation of defective mRNA and then defective proteins.

> β-thalessemia:- A genetic blood disorder can be completely treated using antisense technology in which O-alkyl oligonucleotides or morpholino-oligonucleotides have been used.

4. Medical applications:-

> Antisense technology is used to investigate protein function in the living brain to study central nervous system (CNS) proteins such as transmembrane receptors, ion channels, transporters, G proteins, and growth factors.

> Antisense oligonucleotides can be used to inhibit expression of a particular enzyme. For eg. Inhibition of acetyl cholinesterase enzyme is the molecular target for the treatment of diseases like Alzheimer’s disease.

> In inflammatory diseases, antisense oligonucleotides is used to demonstrate the relative importance of various signaling components at the molecular level.

> A novel respirable antisense drug called RASONS is used to treat various respiratory diseases including asthma, influenza, bronchitis, pulmonary fibrosis, pneumonia, and lung cancer.

> Antisense oligonucleotide can be used target specific membrane component that influences the pathophysiological mechanisms in renal and cardiovascular disorders.

5. Flavr Savr tomatoes:-

> Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they reach the customer.

> Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.

> The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe.

Molecular farming:- It describes the production of recombinant proteins and other secondary metabolites in plants. This technology depends on a genetic transformation of plants.

The strategies of plant transformation:- Molecular farming depending on the production of transgenic plants. It has been operated by two general methods:

1. Stable or permanent expression systems:-

a. Stable nuclear transformation:-

> It refers to the integration of foreign genes into the nuclear genome of plants, which results in the change of genetic structures and consequent expression of transgenes after integration with the host genomes.

> The largest amount of recombinant proteins has been produced by one of the most common methods of stable nuclear transformation.

> A method exploited for aggregating proteins in dried beans of maize culminates in a long-term storage in the beans at the room temperature without decomposition of proteins.

> In addition, it has a considerable potential for producing crops like cereals that actually grow everywhere.

> Limitations:-

i. A long production cycle for some crops.

ii. Potential collisions of some crops with natural species or food products.

b. Stable plastid transformation:-

> Numerous advantages:-

i. Preventing transgene escape through amphimixis, because plastids are inherited through the maternal tissue in the majority of species.

ii. Absence of chloroplasts in pollen and consequent improbability of their transfer, which reduces environmental concerns.

> The transformed transgenic plants with homoplasmic chloroplasts (all chloroplasts carry transgenes) were selected after several generations of plant regeneration from bombarded leaf explants.

> Selection was conducted on a medium containing spectinomycin or combined with streptomycin.

> The researchers have already extracted a human pharmaceutical protein, more than 3% to 6%, from the total soluble proteins in the chloroplasts of tobacco.

> Recently, Oey reported a very high level (70% of an entire soluble protein) for a protein antibiotic with the chloroplast system, which, till today, has been the highest concentration of recombinant proteins.

> Functional limitation:-

i. Chloroplast transformation only in tobacco is practically possible, but unfortunately this plant is inedible and full of poisonous alkaloids.

ii. Long lasting storage in refrigerators will bring about changes in protein stability.

c. Plant cell suspension culture:-

> This method involves the removal of cell walls and gene transfer to the obtained protoplasts and suspension culture.

> The purification system and its downstream processing are cheaper and easier.

> In addition, the use of suspension culture can decrease heterogeneity in proteins and sugar (N-glycans) regarding the uniformity of the type and size of cells.

> Furthermore, as a fast system there is no need for the production of transgenic plants.

> Some samples of plant suspension cultures can be used for producing biomedicines, including:-

i. Vaccines of Newcastle disease virus of chicks

ii. Recombinant human glucocerebrosidase for treating diabetes.

> Though this method is cheaper, safer, and easier in comparison to the other methods of genetic manipulation, cell suspension has not yet been suggested as an optimal production choice of production in plant systems.

> Limitation:- It is believed that the ultimate products and their usability are constrained by reducing the level of recombinant proteins during the stationary phase because of the enhanced proteolytic activity.

2. Temporary or transient expression systems:-

a. Agrobacterium transformation method:-

> Infiltration of recombinant agrobacterium suspension into tobacco leaf tissue is achieved without stable gene transfer, which facilitates the transfer of T-DNA to a very high percentage of cells, where the transgenes are expressed at a high level without a stable transfer of genes.

> Presently, this method has been very efficient for the production of clinical biomedicines with a fast expansion.

b. Viral infection methods:- The viral infection method depends on the capability of plant viruses, such as tobacco mosaic virus and X potato virus, which functions as a vector to convey foreign genes into plant genomes without fusing with the genome of that plant.

c. Magnifection system:-

> Expression systems based on viral vectors and agrobacterium methods suffer from some constraints for the co-expression of two or more polypeptides required for the production of hetero-oligomeric proteins.

> Thus, a new transient expression system known as MagnICON technology has been developed by Icon Genetics Company.

> This method includes removing coat proteins (responsible for systemic movement) of non-competitive virus stains and systemic delivery of the derived viral vectors to all of the plants using agrobacterium as the medium of primary infection.

> This method not only optimizes the infection but also significantly increases proliferation, and finally results in the co-expression of several polypeptides and the rise of functional protein production more than 100 times.

The advantages of utilizing transgenic plants as bioreactors:- Comparison of different expression systems reveals the advantages of plants in comparison with other expression systems as follows:

i. The healthiness of derived products (plants cannot be the host of human pathogens and bacterial toxins).

ii. Capability of post-translational processing (respecting the features of eukaryotic cells).

iii. The possibility of using breeding methods and sexual crosses to obtain active recombinant multi-chain proteins (therefore, there is the possibility of producing antibodies without application of a double transformation).

iv. Reducing the costs of production (plants can produce biological materials by the use of carbon dioxide, solar energy, and inorganic materials. Moreover, the scale of production can be manipulated regarding scalability).

v. Reducing the costs of storage and transportation of recombinant proteins (when they are produced in dry textures like grains).

vi. Removing the purification step (when the plant tissues containing recombinant protein are edible).

Biotechnology and Society:- Biotechnology is the application of biological organisms, cells, enzymes, and genetic engineering to develop products and technologies that improve human life. It has transformed agriculture, medicine, industry, environmental management, and food production. While biotechnology offers enormous benefits, it also raises important ethical, social, environmental, and legal concerns.

Applications:-

1. Improvement in Agriculture:-

> Development of high-yielding crop varieties.

> Production of genetically modified (GM) crops resistant to pests, diseases, and herbicides.

> Improvement in nutritional quality (e.g., Golden Rice enriched with Vitamin A).

> Development of drought- and salinity-tolerant crops.

> Reduced dependence on chemical pesticides through Bt crops.

2. Healthcare and Medicine:-

> Production of recombinant insulin, vaccines, antibiotics, and therapeutic proteins.

> Gene therapy for treating inherited diseases.

> Early diagnosis using PCR, ELISA, and DNA fingerprinting.

> Development of personalized medicine based on genetic information.

> Stem cell therapy for regenerative medicine.

3. Environmental Protection:-

> Bioremediation for cleaning polluted soil and water.

> Waste management using microorganisms.

> Biofertilizers and biopesticides reduce environmental pollution.

> Biofuels such as ethanol and biodiesel provide renewable energy.

> Conservation of endangered species through tissue culture and cryopreservation.

4. Industrial Applications:-

> Production of enzymes for food, textile, leather, and detergent industries.

> Fermentation technology for producing alcohol, organic acids, vitamins, and antibiotics.

> Manufacture of biodegradable plastics to reduce plastic pollution.

Social Benefits:-

> Increased food security due to higher agricultural productivity.

> Better healthcare with affordable medicines and vaccines.

> Employment generation in biotechnology industries and research institutions.

> Improvement in quality of life through better nutrition and disease control.

> Economic growth through biotechnology-based industries.

Ethical and Social Concerns:-

> Safety of genetically modified organisms (GMOs).

> Possible effects on biodiversity and ecological balance.

> Ethical issues related to cloning, stem cell research, and gene editing.

> Patent rights on genes, seeds, and living organisms.

> Unequal access to biotechnology between developed and developing countries.

> Public concern regarding food safety and long-term health effects.

Biosafety and Bioethics:- To ensure safe use of biotechnology, governments have established biosafety regulations and ethical guidelines.

> Risk assessment before releasing GM crops.

> Monitoring environmental and health impacts.

> Protection of biodiversity.

> Respect for human rights and informed consent in medical biotechnology.

> Prevention of misuse of biotechnology for harmful purposes.

Government Initiatives:- Many countries, including India, promote biotechnology through:

> Department of Biotechnology (DBT)

> Biotechnology Industry Research Assistance Council (BIRAC)

> National Biotechnology Development Strategy

> Promotion of biotech startups and research institutions

> Biosafety guidelines under the Environment (Protection) Act

Challenges:-

> High research and development costs.

> Public acceptance of GM products.

> Ethical controversies.

> Regulatory hurdles.

> Intellectual property rights issues.

> Lack of awareness in rural communities.

Future Prospects:- Advances in CRISPR gene editing, synthetic biology, artificial intelligence, personalized medicine, and climate-resilient crops will further strengthen biotechnology's role in society. Responsible innovation, strict biosafety measures, and public awareness will ensure that biotechnology benefits humanity while minimizing risks.