Tissue Culture

Totipotent

Totipotent

Capacity to divide and differentiate into a whole plant

Somatic embryogenesis

Process of forming plant embryos from non reproductive cells

Organogenesis

Roots and shoots are formed from a single cell or callus

Somatic hybridization

Allows manipulation of cellular genome by protoplast fusion

Allows breeding of plant species that could not happen otherwise

Symmetric somatic hybrids

combination of nuclear and cytoplasmic genetic info of both parents

Ploidy level increases

Asymmetric somatic hybrids

Lose some cytoplasmic or nuclear DNA

Donor parent and recipient

Cybrids (Cytoplasmic hybrids)

Harbors only one parental nuclear genome and either cytoplasmic genome of other or both parents

Somatic Hybridization Advantages

Species bred which are sexually incompatible

Cytoplasmic transfer in short duration

Mitochondria can be combined with chloroplast of other species

PCR reactions need

DNA Template

Primers

dNTPs

DNA polymerase or Taq Polymerase

Denaturation Step

94-98 degrees C

Disrupts H bonds and yields single strands of DNA

Annealing Step

50-65 degrees C

Allows annealing of primers to strands

Stable H bonds form

DNA polymerase binds to primer template and begins synthesis of complementary strands of DNA

Extension Step

72 degrees C

DNA polymerase synthesizes a new DNA strand complementary to template. dNTPS help to do this

Cloning DNA into plasmid vector

Restriction endonuclease cleave target

Phosphatase vector

Ligate target into vector

Transform into host cells

Transformation Frequency

Ratio of transformed cells to total number of treated cells

Transformation efficiency

Number of transformed cells produced by 1 ug of plasmid DNA into a given volume of competent cells

Methods for Analysis of Gene Expression

PCR

RNA sequencing

Microarry

Northern blotting

Regulation of gene expression depends on

Promoter and terminator sequences

Strength of ribosome binding site

Number of copies of cloned gene

efficiency of translation in host

Nucleotides Contain

A phosphate group

A sugar (deoxyribose)

A nitrogen base (A T C or G)

Purine

Adenine and Guanine

Pyrimidine

Cytosine and Thymine

A-T Bond type

Two H-bonds

C-G

3 H-bonds

DNA replication

Unzips and 2 strands act as templates for 2 new complementary strands

DNA polymerase + enzymes required

5’ to 3’

DNA gyrase

Unwinds DNA coil in replication

DNA Helicase

Splits H bond between bases

DNA Vs. RNA

DNA has a deoxyribose (H)

RNA has a ribose (OH)

Types of RNA

mRNA (messenger RNA) for transcription

rRNA (ribosomal RNA)

tRNA (transfer RNA) for translation

Number of types of amino acids

20

How many stop codons

3

Degenerate codon

more than one codon for many amino acids

Protein Synthesis (Translation)

Auxins

Stimulate root growth

Cytokinin

Stimulates shoot growth

Explant

mother plant where tissue was extracted from

Cell/tissue plasticity

Ability to initiate cell division from any tissue of the plant.

ability to regenerate organs or undergo developmental pathway in response to external stimuli

Competent cells

Bacterial cells that can take up foreign DNA through the transformation process

Replicon

DNA originated from a single replication origin

Transcription

RNA polymerase binds to promoter region and unzips the DNA strands

The polymerase adds complementary base pairs (which will be the mRNA) as it moves across the template strand. Polymerase will also zip DNA back up as it goes

The polymerase eventually releases when it reaches the terminator sequence

DNA replication

Helicase enzyme unzips DNA and 2 strands act as template for synthesis of two new complementary strands

Primase binds DNA fragments

DNA polymerase binds to primer and copies each strand (+ other enzymes needed)

Lagging strand has Okazaki fragments

Leading strand complementary base pair

Nucleotides always added 3’

Translation

Small ribosomal subunit binds to mRNA.

tRNA binds to start codon (AUG)

Next tRNA enters ribosome through the A site and binds to the methionine in start codon.

First tRNA leaves ribosome as the previous tRNA moves to the P site

Stops when stop codon is reached