Recombinant DNA Technology and Plant Breeding

Study Notes

Plant breeding, a practice used since early human civilization, aims to improve agricultural traits like yield and disease resistance.
Classical breeding involves controlled pollination or interbreeding to create new crop varieties with desirable traits. This method often uses selective mating or hybridization, along with in vitro techniques like tissue culture, embryo rescue, and protoplast fusion.
A drawback of classical breeding is its slow generation time. Genetic engineering offers a faster way to accelerate breeding.
Modern plant breeding, also known as molecular breeding, uses applied genetics and molecular biology techniques to modify genes directly, introducing a desired trait into plants.

Genetic engineering utilizes molecular techniques to modify target plant traits, resulting in transgenic plants or genetically modified organisms (GMOs).
Classical breeding, relying on natural processes, focuses on mating organisms with desirable traits for enhanced genetic diversity and elimination of undesirable traits.

Recombinant DNA technology, also known as genetic engineering, involves a set of techniques for manipulating genes/DNA.
This technology has wide applications in fields like medicine, forensics, pharmaceuticals, and improving livestock/crops.
Naturally, DNA recombination occurs through meiosis I (crossing over), fertilization, or mutations/transformations.
Recombinant DNA technology is used to modify and recombine DNA sequences for creating new products like proteins, and altering organisms (plants/animals) with desirable traits.

Key tools include target DNA (gene of interest), restriction enzymes (to cut DNA into fragments), DNA cloning vectors (e.g., plasmids or bacteriophages to carry target genes into host cells), a host cell (bacteria), and modifying enzymes (e.g., DNA ligase and Taq polymerase).

Restriction enzymes are naturally occurring bacterial enzymes that function as a defense mechanism against bacteriophages(viruses).
These enzymes cut DNA at specific DNA sequences called restriction sites.
Restriction sites are palindromic sequences (same sequence in both DNA strands when read in opposite direction).
Enzymes can cut DNA in a staggered manner (sticky ends) or a straight cut (blunt ends).

Cloning vectors are used to carry foreign DNA into a host cell via insertion.
Bacteria plasmids (small, circular DNA molecules) are a prominent example of cloning vectors.
Vectors include bacteriophages, cosmids and YACs.

Gene cloning involves four key steps:
- Isolate the target gene.
- Insert the target gene into a vector (gene carrier).
- Introduce the recombinant vector to a host cell.
- Amplify the target gene within the host cell and select for clones.

Two main methods: cutting the gene from a complete chromosome using restriction enzymes or producing a complementary DNA (cDNA).
Examples include cloning the insulin gene by extracting human DNA and mixing with specific restriction enzymes.

Insert the target gene into a cloning vector, which carries the gene into a host cell.

Recombinant plasmids (vectors containing the target gene) are introduced to host cells.
Methods like transformation utilize calcium chloride to increase cell permeability for plasmid uptake.

Transformed cells grow in a medium that only allows cells with the plasmid (e.g., ampicillin resistance).
Screening methods like blue-white screening identify the bacterial colonies with the target gene inserted in vector.

PCR is a molecular biology technique used to create multiple copies of a specific DNA sequence.
Use Taq polymerase to amplify (increase DNA copies) short segments of DNA by repeating the same step: Denaturation, Annealing, Extension.

Widely applied in agriculture, medicine, pharmaceuticals, forensics, and basic research.
Examples include production of useful proteins (like insulin) in microorganisms and gene therapy for human diseases.