GM Food - Benefits:
1. High yield without pest.
2. 70% reduction of pesticide usage.
3. Reduce soil pollution problem.
4. Conserve microbial population in soil.
Risks - believed to:
1. Affect liver, kidney function and cancer.
2. Hormonal imbalance and physical disorder.
3. Anaphylactic shock (sudden hypersensitive reaction) and allergies.
4. Adverse effect in immune system because of bacterial protein.
5. Loss of viability of seeds show in terminator seed technology of GM crops.

Properties of Vectors:
1. Vectors are able to replicate autonomously to produce multiple copies of them along with their DNA insert in the host cell.
2. It should be small in size and of low molecular weight, less than 10 Kb (kilo base pair) in size so that entry/transfer into host cell is easy.
3. Vector must contain an origin of replication so that it can independently replicate within the host.
4. It should contain a suitable marker such as antibiotic resistance, to permit its detection in transformed host cell.
5. Vector should have unique target sites for integration with DNA insert and should have the ability to integrate with DNA insert it carries into the genome of the host cell. Most of the commonly used cloning vectors have more than one restriction site. These are Multiple Cloning Site (MCS) or polylinker. Presence of MCS facilitates the use of restriction enzyme of choice.

A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites.
Types
(i) Exonucleases - which remove nucleotides one at a time from the end of a DNA molecule. e.g. Bal 31, Exonuclease III.
(ii) Endonucleases - which break the internal phosphodiester bonds within a DNA molecule. e.g. Hind II, EcoRI, PvulI, BamHI, TaqI.
(iii) The restriction enzymes are called as molecular scissors
(iv) They are used in recombinant DNA technology as they recognize & cut DNA within a specific sequence.

(i) ECORI is from Escherichia ( E ) coli (co), strain RY 13 (R) & first endonuclease (I) to be discovered.
(ii) It contains 2 different antibiotics resistance genes & recognition site for several restriction enzymes.
(iii) This sequence is referred to as a restriction site & is generally -palindromic which means that the sequence in both DNA strands at this site read same in 5' - 3' direction & in-the 3'-5' direction.

(iv) The exact kind of cleavage produced by a restriction enzyme is important in the design of a gene cloning experiment.
(v) Some cleave both strands of DNA through the centre resulting in blunt or flush end.
(vi) These are known as symmetric cuts.
(vii) Some enzymes cut in a way producing protruding & recessed ends known as sticky or cohesive end.
(viii) These are known as staggered or asymmetric cuts.

Polyethylene Glycol (PEG) and Dextran Sulphate.

Restriction endonuclease. e.g: Hind II, EcoR I, Pvul, Bam H I, Taq I.

Bioremediation:
It is defined as the use of microorganisms or plants to clean up environmental pollution.
Some examples of bioremediation technologies are :
(i) Phytoremediation - use of plants to bring about remediation of environmental pollutants.
(ii) Mycoremediation - use of fungi to bring about remediation of environmental pollutants.
(iii) Bioventing is the process that increases the oxygen or air flow to accelerate the degradation of environmental pollutants.
(iv) Bioleaching is the use of microorganisms in solution to recover metal pollutants from contaminated sites
(v) Bioaugmentation is the addition of selected microbes to speed up degradation process.
(vi) Composting is the process by which the solid waste is composted by the use of microbes into manure which acts as a nutrient for plant growth.
(vii) Rhizofiltration is the uptake of metals or degradation of organic compounds by rhizosphere microorganisms
(viii) Rhizostimulation is the stimulation of plant growth by the rhizosphere by providing better growth condition or reduction in toxic materials

Direct or Vector less Gene Transfer
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant without the help of a vector, The following are some of the common methods of direct gene transfer in plants.
(i) Chemical mediated gene transfer: Certain chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.
(ii) Microinjection: The DNA is directly injected into the nucleus using fine tipped glass needle or micro pipette to transform plant cells. The protoplasts are immobilized on a solid support (agarose on a microscopic slide) or held with a holding pipette under section.
(iii) Electroporation Methods of Gene Transfer: A pulse of high voltage is applied to protoplasts, cells or tissues which makes transient pores in the plasma membrane through which uptake of foreign DNA occurs.

(iv) Liposome mediated method of Gene transfer: Liposomes the artificial phospholipid vesicles are useful in gene transfer. The gene or DNA is transferred from liposome into vacuole of plant cells. It is carried out by encapsulating DNA into the vacuole. This technique is advantageous because the liposome protects the introduced DNA from being damaged by the acidic pH and protease enzymes present in the vacuole. Liposome and tonoplast of  vacuole fusion resulted in gene transfer. This process is called lipofection.

(v) Biolistics: The foreign DNA is coated onto the surface of minute gold or tungsten particles (1-3 \(\mu\)m) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/ micro projectile gun/shotgun) Then the bombarded cells or tissues are cultured on selected medium to regenerate plants from the transformed cells.