EUKARYOTIC DNA PACKAGING

 DNA is the genetic material. It is a large biomolecule. Eukaryotic DNA is present in the nucleus. It has millions of base pairs. The total length of DNA is 1.8 m in a cell. Such a large molecule is to be packed in the nucleus of an 8 micrometer in size. 

Metaphase DNA is packed into chromosomes that are visible through the light microscope. This packaging occurs approximately 10,000 times. The original thickness of the DNA strand is the two-nanometer.  The thread of DNA is packed into a 1400 nanometer thread which is the breadth of the chromosome. This packaging requires multiple types of proteins that are associated with DNA molecules.

 The first level of Packaging of DNA: Nucleosome 

 The 11-nanometer structure is known as a nucleosome.  This structure requires a special type of protein those are known as histone proteins. 

There are five types of histone proteins which are named H1, H2A, H2B, H3, and H4. The histone proteins are positively charged due to their composition of amino acids. The amino acids present in histone proteins are lysine and arginine, these give histone proteins a positive charge.

 Eight histone proteins form the core of the nucleosome. The core of the nucleosome is octamer which has two subunits each of H2A, H2B, H3, and H4. 

146 base pair of DNA coil around the histone octamer, H1 histone then clip both the ends of DNA to the histone octamer. This further takes 20 base pairs of DNA.

 

The DNA between the two histone octamer is called linker DNA this reduces the length of DNA from 1m to 14 cm which reduces the size of DNA to 1/7. The Packaging of DNA into nucleosomes, that results in the formation of a string of beads. This is visible under the electron microscope. 

30 nm fiber

 Even after this level of packaging DNA is too large to be packed into the nucleus of the cell for this the histone octamer is packed into a solenoid or zig-zag structure of the 30-nanometer fiber.

 In the solenoid structure, the linker DNA is present in the center. It is a compact structure.

Zig-zag 

In this folding, the 30 nm fiber is formed by the zig-zag arrangement of nucleosomes. Here the linker DNA is outside the center. 

CHROMATIN

 This 30-nanometer fiber is then packed into 300nm and the 300 nm fiber coil around proteins to form 700 nm fiber. This is called chromatin.

 The 700-nanometer fiber is then packed into a chromosome that has 1400 nanometers this thickness appears in the metaphase chromosome.

SIGNIFICANCE OF DNA PACKAGING 

Interphase DNA is diffusely distributed and in form of chromatin. This is in an irregular shape.

This allows a huge amount of DNA to be packed in the small nucleus of a cell.

DNA packaging in chromosomes allows the distribution of genetic material equally to the daughter nuclei during cell division.

The numerical and structural study is possible of the metaphase chromosome.

Karyotype and banding pattern study is done on metaphase Chromosomes, this helps in identifying any genetic disorder in feotus. This study is not on unpacked DNA.

BIOREMEDIATION

 BIOREMEDIATION is curing the polluted soil, air, or water using living organisms. It is eradicating the pollutants. It is a practical application of biotechnology in ecology. 

The basis of strategies used for bioremediation, it is basically two types: 

The in situ bioremediation

The ex-situ bioremediation

The in situ bioremediation

It is eradicating pollutants at the site of pollution. In situ bioremediation is require different strategies such as

Bioventing

Biosparging

Percolation

Air sparging

Pump and treat

Bio-slurping

Bioremediation eradicates pollutants from the environment. Microorganisms are used for this purpose. The microorganisms convert pollutants into CO2 and H2O or biomass. The pollutants are degraded to non-harmful molecules. The degradation of pollutants requires oxygen. Some ions facilitate the metabolism of pollutants. Supplying these nutrients to the site of the pollution is called bio stimulating. Improving the strain of microorganisms for better metabolism of pollutants is done with the help of Biotechnology.

Bioventing

It is bioremediating the underground water. Microorganisms present at the site can eradicate the pollutants from the underground water. But their action is insufficient. To effectively eradicate the impurities oxygen is required by the microorganisms. Oxygen is supplied to the polluted site by creating a vacuum.  A well is dug on the site of action. The vacuum is created by using a pump and fans. Air passes through the vent and fastens the aerobic metabolism of the pollutants.

Biosparging/ Air sparging

This is just the opposite of bioventing. In sparging air is pumped through pipes. This aerobic facilitates metabolism. 

Pump and treat

It is pumping the groundwater and then treating it.

Bio-slurping

It is vacuum enhanced dewatering technique for hydrocarbon-contaminated sites. Water is sucked through a vacuum pipe and treated.

Bioslurping is the combination of both bioventing and free product recovery. Here the pollutants belong to two different categories. It is used to treat petroleum and other hydrocarbons impurities in groundwater.

Ex-situ bioremediation

Ex-situ bioremediation is treating the polluted soil or water away from the site of pollution. It involves the transportation of soil/water at the site of treatment. The special typeS of plants are established for the treatment of polluted soil or water.

Ex-situ bioremediation includes land farming, compost piles, and irrigation, piles.

Land farming

It is the method of treatment for contaminated soil. It utilizes volatilizing and treatment of the soil using microorganisms. Contaminated soil is extracted and spread on an open farm. The volatile pollutants are vaporized and other substances are treated with the help of microorganisms. 

Compost piles and irrigation 

It is remediating the polluted soil by adding manure or increasing air circulation by adding wood filings. Water holding capacity is increased in the contaminated soil. Irrigation of contaminated soil increases the microbial bioremediation of contaminated soil.

Biopiles 

It may be combining phytoremediation and microbial bioremediation. The contaminated soil is treated by this method. Biopiles involve engineering too. It involves irrigation, aeration, and adding manures to the contaminated soil. Biopiles are made on a farm of non-contaminated soil. The two soils are separated with the help of thick polyethylene. Aeration is done with the help of pipes. Microbes are mixed in the soil. Alginate beads are added for bioremediation. Plants are used to treat the uppermost layer.

 

Thuringiensis toxin as a natural pesticide

 Thuringiensis toxin is produced by Bacillus thuringiensis. The toxins are called cry proteins. These are endotoxins. The bacterium is present in the soil. Thuringiensis toxin (cry protein) has been used for protecting crops against insect pests.  

Tissues sprayed with this toxin kill the insect pests. This toxin is an effective biocontrol against insect pests. Through genetic engineering, the Cry gene has been inserted into plants. The transformed plants produce their own thuringiensis toxin.

Using the chemical Pesticides

 The pesticides have always been erratic in their performance as;

 Their concentration is not equally distributed on the plant tissues that are sprayed with the toxin

They may not be eaten by insects. 

Sprayed the toxin may be washed away by the rain, so the endotoxin performs irregularly.

The use of toxic pesticides pollutes the soil. 

It may be dissolved in water that pollutes the water as well.

MECHANISM OF ACTION OF CRY PROTEIN

When sprayed on the crops it has been found that Bacillus thuringiensis produce crystalline protein. This is known as cry protein this endotoxin when entered into an insect is metabolized by insect Gut enzymes which convert the inactive cry protein into active cry protein and this protein breakdown the cells of the insect gut and causes paralysis of insect gut muscles in this way this toxin kills the insect pest. 

MOLECULAR MECHANISM OF ACTION

The crystalline protein is insoluble and inactive.

It is converted into soluble protein by proteases of the insect gut.

The soluble cry AC protein bind to cell membrane protein cadherin.

This can kill the gut cells by two processes.

MOLECULAR MECHANISM OF cry1AC PROTEIN

They form pores in the cell membrane. As binding of cry oligomer to the cadherin protein activates binding it to GPI anchor protein, this causes pore formation in the cell.

The cell dies due to leakage.

Another method is a cascade of events. This initiates programmed cell death.

PLASMID FOR Bt GENE

 Gene for cry protein has been isolated from the bacterium this gene is under the constitutive promoter of cauliflower mosaic virus CaMV and G7 Terminator. The gene has been inserted into the plasmid and an expression vector has been created. These genes are also associated with an antibiotic-resistant marker gene. 

cry1AC plasmid (simplified)

This plasmid was inserted into Agrobacterium tumefaciens. This bacterium is selected for its transformation. The bacterium is then utilized to transfer genes into the crop of interest, in this case, it is cotton.

Production of Bt plant

 Transformed plants are selected through marker-assisted methods. These Transformed plants are utilized to reduce modified plants which are known as BT cotton.

CRY PROTEINS

 Cry proteins are classified basically into four classes. Each of these classes is effective against the special class of insects. Cry I is effective against Lepidoptera try to be effective against Lepidoptera and Diptera Cry 3 is effective against Colepetra and Cry 4 is effective against specific Diptera.

Other endotoxins that are produced by Bacillus thuringiensis are- cry which are crystal proteins there is 126 type of Crystal protein that is produced by bacteria as endotoxin cyt proteins that are cytolytic there are 22 classes of this endotoxin, and the toxin is VIP which is vegetative insecticidal protein.

 Bt cotton was first produced in 1987 it got permission to be grown in India only in 2003 Monsanto is a seed company that markets BT cotton seeds. Other crops that have been improved by utilizing the cry gene are Bt brinjal and Bt tobacco.

 Drawbacks of using pest-resistant crop

 The endotoxin produced in young tissue is efficient against the larva of insects. In older tissue effective production of endotoxin has not been achieved yet this endotoxin is effective against certain types of insects only. It has not found its universal application. Using terminator Technology has also made it controversial.

 Cotton happens to be the first crop to receive environmental clearance as a genetically modified crop in Indian agriculture.  Cotton bollworm belongs to Lepidoptera and these are sensitive to cry 1 endotoxin. The cotton bollworm reduces the yield of the cotton crop by up to 50% in India and this endemic effect Rajasthan Haryana and Punjab.

Benefits of using BT cotton

 It helps in managing the bollworm infection without any adverse effect on the environment.

 It reduced the use of pesticides the reduced the cost of production of the crop. It reduced the risk of cotton cropping.  It reduced the adverse effects of utilizing pesticides on soil, it improved the yield of cotton. Bt gene has no adverse effect on human and cattle health. Seeds produced by cotton are used as cattle feed.

  Seeds of Bt cotton can also be used as cattle feed, and they are not harmful to cattle these are easily digested by the cattle. The cry endotoxin is not harmful to cattle. This toxin was not found in cattle after eating reset escaping the gene into the environment is negligible for BT Cotton as the BT gene is inserted into tetraploid cotton with chromosome number 52 and this cotton does not breed with a wild variety of cotton as well cotton does not outbreed with other members of Malvaceae so, there is least of this gene escaping into the environment.

 

 

SOMATIC HYBRIDIZATION

 Somatic hybridization is a fusion of two somatic cells for the production of hybrids. 

The somatic hybrids are intra/inter-specific hybrids. 

Here distant parental cells are fused to obtain somatic hybrids.

 Plant cells have cell walls so it is not possible to fuse intact plant cells. Protoplasts are isolated before somatic hybridization

STEPS INVOLVED IN SOMATIC HYBRIDIZATION

A. Isolated protoplasts of two species.

B. Adhesion of protoplasts

C. Formation of a connection between two protoplasts.

D. Dissolution of the intervening membrane of the two protoplasts.

F. Fusion of the cytoplasm.

E. Formation of heterokaryon.

Different methods of protoplasts fusion

Somatic hybridization involves the isolation of protoplasts, the fusion of protoplasts, isolation of products of somatic hybridization, verification of hybridization, and culture of somatic hybrid.  

Protoplasts can fuse spontaneously, chemical fusion, electrofusion, or through physical methods.

Spontaneous fusion is not favored. Other methods used to produce somatic hybrids are given below.

CHEMICAL FUSION

 Calcium chloride (CaCl₂) is used for chemical fusion. Calcium ions form a bridge between the cells. So, calcium ions are used to fuse somatic cells.

 NaNO₃ is used for the fusion of somatic cells.

Polyethylene glycol

 PEG forms a bridge between the somatic cells.

 In the plant, PEG is a fusogen that forms a bridge between the somatic cells. This ensures the adhesion of protoplast with each other. Once the adhesion is complete, the intervening cell membrane of the protoplast is dissolved. This results in the fusion of cytoplasm. The cell membrane is regularized and the heterokaryon is produced. This heterokaryon then undergoes mixing of the nucleus and results in the formation of somatic hybrids.

 Electrofusion

 Where the cells are fused under the current of electricity electric current of 0.5-1.5 volt is passed through the suspension medium that contains isolated protoplast. This allows charge separation of isolated protoplast. These protoplasts now behave like a dipole. They line up between the two poles. The charge is disturbed by passing a current of high voltage for a few seconds. The intensity charge is passed between the electrodes, which is 0.125- 1kVcm^-1. The high-intensity charge causes reversible membrane breakdown. This allows the formation of a contact area between the two protoplasts. Fusion of protoplast takes less than 10 minutes in the electric field. The presence of calcium chloride 1mM in the fusion mixture increases fusion frequency protoplast. The density should be 1x10⁴ protoplasts per ml for electro-fusion of protoplasts.

CHARGE SEPARATION OF PROTOPLASTS UNDER ELECTRIC FIELD

 In the microchamber or microdroplet method of protoplast culture, protoplast can be fused in the microchambers. Platinum wires are used as electrodes for electrofusion. Electrofusion is more suitable for mesophyll cells than root or callus protoplasts (Pelletier 1993)

ELECTROFUSION OF PROTOPLASTS IN MICROCHAMBER

 Selection of fusion products

 Morpho-physiological basis of selection

 Fusion product can be selected by studying the morphology of cellular hybrid callus, where the fusion product of Solanum tuberosum to Solanum ceraceifolium where one parent forms a green color callus and the other form brown yellow color callus, and the fusion product forms intermediate callus where green color and purple color cells are present.

 Hybrid vigor

Hybrid vigor where cells of Dianthus chinensis and Dianthus barpatus show hybrid vigor. Hybridized cells of these parents divide and form shoots and roots vigorously.

 

 Markar based selection or complement selection

 

Where metabolic deficiencies of two fusion parents are utilized to select a hybrid. The metabolic deficient parents are eliminated by themselves in the medium that doesn't contain the metabolic component. However, hybrids will survive in this medium.

 The complement selection also utilizes Herbicide resistance, antibiotic resistance, or amino acid analog genes as these can be markers.

 Isolation of heterokaryon

 In the low-density culture where use and products are cultured in the low-density medium so that callus of different colors and morphology are selected individually in this medium.

 Morphologically isolated fusion products can be manually selected through a micropipette, where the two fused protoplasts have a different color to that of their parent. This process was first determined by Hoffmann in 1978-1979. 

Fluorescent isolation of heterokaryons 

Dual fluorescence labeling system where protoplast are labeled by Green pigment the Fluorescein diacetate (1 to 20mgl^-1 ) which emit green color and the other set of protoplast are labeled with red color using Rhodamine isothiocyanate (10 to 20mgl^-1). This labeling is achieved by adding enzymes and the fluorescent product to the culture mixture. Manual isolation of product was done through Pasteur pipette.

 The dual-labeled products can also be isolated using FACS where cells are sorted on the basis of the wavelength they emit. Due to their staining, different wavelengths are emitted by the cells. Thus the cells are selected into the different containers by FACS.

 Verification of hybrid product

 It is done to study morphology where flower color or expression of leaf variegation can be used to determine the hybridization of the fusion product. 

Cytological analysis

  Where the number of chromosomes is estimated for hybrid cells. The chromosomes number could be multiple of the number of chromosomes that were present in parents.

 Isozyme analysis

 Here the isozymes that are present in two different parents that reflect different band patterns are studied through molecular isolation. Isozymes studied for hybrid verification are Phosphatases, esterases, peroxidases, and phosphoglucomutase.

DNA analysis

 Restriction enzyme polymorphism studied for hybrid product establish the hybrid ability or DNA fingerprinting can be used for hybrid products.

 The fate of the genome of hybrid

 The fate of the genome depends upon the number and type of cells fused.

Genome segregation occurs during cell division, after fusion genome segregation during regeneration of the plant.

 Inter-parent recombination of plastid-genome occurs rarely so plastids are selectively eliminated in hybrid products.

 The nuclear chromosome may be selectively eliminated from the hybrid products, so this may result in the formation of hybrids and result in a novel combination of the plastid-mitochondrial genomes. 

 USES OF SOMATIC HYBRIDIZATION

Tomato hybrids have been developed that are resistant to TMV and spotted wilt virus. 

Environment tolerance and stress-tolerant plants can be developed through hybridization.

  The high-yielding plants can be developed through symmetric hybridization.

  

 

SYNTHETIC SEEDS

 

 Somatic embryogenesis results in the production of somatic embryos. These embryos can be propagated through clonal propagation or these embryos can be converted into synthetic seeds, synseeds, or artificial seeds.

 Synthetic Seeds are produced by encapsulating the somatic embryos in a protective layer.

 The synthetic seeds should have a coating layer that should be mildly protective.

This layer should protect the seeds from diseases and should be easily grown.

 It must contain nutrients growth hormones

 It is easy to handle and the seed must be protected from rough, handling.

 They can be grown using farm machinery.

TYPES OF SYNTHETIC SEEDS

 There are two types of synthetic seeds

The desiccated synthetic seed and hydrated synthetic seed

 Desiccated synthetic seeds

These were produced by Kitto and Janic in 1982 they used polyoxyethylene for encapsulating the somatic embryos.

Polyoxyethylene is non-toxic to somatic embryos and does not allow the growth of microorganisms. 

Somatic embryos were mixed with a 5% weight by volume solution of polyoxy Ethylene such that the final concentration becomes 2.5 percent of polyoxy.

This suspension was dispensed in 0.2 ml drops they were spread on a Teflon sheet the drops dry in 5 hours into wafers. 

For culture these Wafers were dissolved in a suspension medium, as a result, the survival of the somatic embryo was low.

 Hydrated synthetic seeds

 A number of methods have been started for the production of hydrated synthetic seed somatic embryos mixed into a 2 percent w/v  solution of sodium alginate and dropped using a plastic pipette in a 100 millimolar solution of calcium nitrate.

 Ion exchange results in the production of calcium alginate on the coats of seeds. Surface complexing is complete within 30 minutes the size of the capsule can be controlled by the size of the pipette.

Calcium alginate capsules are difficult to handle as they are sticky and are dried immediately once exposed to the atmosphere as they lose water.

 So, calcium alginate encapsulated somatic embryos are coated with Elvax 4260 (ethylene Vinyl Acetate acrylic acid terpolymer). 

SYNTHETIC SEED PRODUCTION

Hydrated synthetic seeds

 Large scale production of synthetic seed as described by Onishi et al. 1994 and Sakamoto et al. 1995.

PROMOTION OF EMBRYO DEVELOPMENT

 Promotion of embryo development by culturing somatic embryos in a suspension medium of high osmolarity with 10% Mannitol under 16 hours of light 300 Lx illumination.  It increased the size of somatic Andreas from 1-3 mm to 2 -8 mm.

DEHYDRATION OF EMBRYOS

 Dehydration of embryos to reduce their water content from 95 to 99% to 80 to 90% to keep them for 7 days on multiple layers of filter paper with 16 hours of photoperiod and 14μm²/s irradiance.

POST DEHYDRATION CULTURE OF SOMATIC EMBRYOS

Post dehydration culture of the somatic embryo on SH medium containing 2% Sorbitol, 0.01 milligram per liter BAP 0.0 1 mg/l GA3, and air enriched with oxygen in 16-hour photoperiod and 20ºC temperature for 14 days so that, somatic embryos acquired autotrophic nature.

 The storage of sugar and nutrients in the seeds is done with 3% sucrose. Microscopic capsules are formed with a mixture of 8% Elevax 4260, bee wax, and 0.1% Topsin M which is the fungicide.

 This microcapsule release sucrose slowly over a period of 3 to 21 days at 20ºC and at 4ºC, but it is not released. 

The gel is made self-breaking for its efficient germination. This involves rinsing the seeds thoroughly under tap water, followed by immersion in 200 mM solution of KNO₃ for 60min and finally desalting done by rinsing under tap water for 40 minutes. 

Encapsulating somatic embryos in calcium alginate has lowered the cost of artificial seed production.

SOMATIC EMBRYOGENESIS

 Somatic embryogenesis This is the formation of embryos from somatic cells in vitro. It was first observed in suspension cultures (Stewart) of carrots.

 Somatic embryos are formed directly or indirectly in tissue culture. Somatic embryogenesis depends on totipotency and polarity. Totipotency is the ability to produce all types of body cells. Polarity is the condition where cells become polar, i.e. some cells are determined to form shoot and other cells are determined to form root. 

SOMATIC EMBRYOGENESIS: IN VITRO PRODUCTION OF SOMATIC EMBRYOS

Explants are root tissue in the case of carrot, mesophyll cells can also form somatic embryo callus cultured in vitro may also result in the formation of somatic embryogenesis. The scutellum is used for the production of the somatic embryo in the case of rice. Monocots are less viable for the formation of somatic embryos. In dicots variety of tissues are used for the formation of somatic embryos.

 Somatic embryogenesis can be divided into 3 steps first is Induction, the second is embryogenesis, and the third is the maturation of induction.

INDUCTION

 Induction requires the culture of the explant in auxin rich medium.  Embryonic induction is actually the determination of cells to produce embryos. Embryonic induction can occur in auxin free medium. These PEMs are formed in auxin free medium if the cell can synthesize its own auxin.

FORMATION OF SOMATIC EMBRYOS

The induced cells form pre-embryonic masses. These pre-embryonic masses are then cultured in an auxin-free medium so that the cell can form somatic embryos.

 In the auxin-free medium, the pre-determined cells divide and form somatic embryos. These cells undergo globular, heart shape, and torpedo stages of embryonic development. Delaying the culture of callus in auxin free medium removes the induction of cells. The cells will now be used for the formation of plantlets through organogenesis.

 Maturation of embryos

It is the development of a complete embryo through somatic embryogenesis in a culture medium.  The torpedo stage or cotyledonary stage is achieved in somatic embryogenesis.

These embryos are used for Artificial seeds production, or they may be allowed to develop into complete plantlets in a culture medium.

Types of embryos

Embryos can be zygotic embryos or non-zygotic embryos. 

Zygotic embryogenesis occurs naturally after the fertilization of gametes. Where the zygotic cell is formed which is a totipotent cell, it became polar, it develops into an embryo. Parthenogenesis is non-zygotic embryogenesis using female gametes.

 Somatic embryogenesis on the other hand involves the formation of embryos from a somatic cell. Somatic embryogenesis can be direct or indirect. Direct somatic embryogenesis occurs directly from the cultured tissue.

Indirect somatic embryogenesis involved the formation of embryos after the formation of the callus.

 Non-zygotic embryogenesis involved the formation of embryos from non-zygotic cells or any of the body cells except the zygote.

 Non-zygotic embryogenesis can occur through parthenogenesis where unfertilized eggs develop into embryos. 

Polyembryony which is observed in citrus plants, where suspenser cells can develop into multiple embryos, this is also non-zygotic embryogenesis.

 Embryogenesis can also occur through androgenesis where microspores develop into embryos.

  Stages of development of embryos

Somatic embryogenesis involved all stages of development that are taken up by zygotic embryogenesis, it involves a globular, heart shape, torpedo stages, and cotyledonary stages of embryogenesis. From torpedo stages/cotyledonary stage of embryogenesis, can be utilized to form plants. SEs do not involve the formation of cotyledons that are formed in the case of zygotic embryogenesis.

 Limitation of somatic embryogenesis

 Somatic embryogenesis can be formed only in the case of some specific plant.

 Only specific tissues of a plant respond to the formation of somatic embryos.

 Cotyledons are not formed in somatic embryos, so not a regular seed is formed in somatic embryogenesis. Somatic embryos are very fragile. It is impossible to store them as we can store seeds.

 The uses of somatic embryogenesis

 It utilizes suspension culture well somatic embryos produced in suspension culture.

 The plants can be produced through somatic embryogenesis.

 Distant species can be hybridized and somatic embryos can be produced that are otherwise fail to fertilize.

 Artificial seeds are produced through somatic embryogenesis. This process can be scaled up to produce synthetic seeds.