Plant Transformation:

Selectable Marker &

Reporter Genes
F. Chidzwondo
Room 173
Department of Biotechnology and Biochemistry
Expected Outcomes
1. Distinguish between selectable marker genes
& reporter genes
2. Discuss the use of each type of gene
Modern Plant Biotechnology
• Based on delivery, integration & expression of defined
foreign genes into plant cells which can then be grown in
vitro to regenerate plants
• Permits access to an unlimited gene pool through transfer
of desirable genes between any 2 species of interest,
irrespective of their evolutionary or taxonomic relationships
• Recovery of transgenic plants requires:
1. Effective gene transfer techniques
2. Appropriate use of signals for the regulation of transgene
expression
3. Vectors for transgene amplification & transfer
 Identification of Transformants
• A marker gene = gene used to determine if a nucleic acid sequence
has been successfully inserted into an organism's DNA
• Ideal marker gene should be:
• Absent from the cells used in the study or easily distinguishable from the
native form of the gene
• Assayed conveniently
• Have a broad linear detection range
• To identify the transgenic clones among large populations of
untransformed cells:
• Selectable marker genes or visible reporter genes (screenable/scorable
marker genes) are co-transformed with the gene of interest
• Both allow one to distinguish between transformants & non-transformants but
in addition, reporter genes allow one to measure the level of expression of the
transformed gene
What is a Selectable Marker Gene?
• A selectable marker gene facilitates selection of putative
transformants on a medium containing a selection
agent, such as:
• Antibiotic (nptll, hpt, acc3, aadA)
• Herbicide (bat; pat)
• Antimetabolite (dhfr)
• Selectable markers are typically genes for antibiotic resistance,
which give the transformed cell the ability to live in the presence
of an antibiotic, eg:
• Beta-lactamase confers ampicillin resistance to bacterial hosts
• Neo gene from Tn5 confers resistance to kanamycin in bacteria
geneticin in eukaryotic cells
Examples of Selectable Marker Genes
1. Neomycin phosphotransferase
2. Chloramphenicol acetyltransferase
3. Hygromycin phosphotransferase
4. Bromoxynil nitrilase
Neomycin Phosphotransferase, NPTII
• The most widely used selectable marker for plant
transformations

• Enzyme is encoded by the nptll (or neo) gene, derived from the
transposon Tn5, & inactivates aminoglycoside antibiotics such
as kanamycin, neomycin & geneticin by phosphorylation

• Kanamycin is mostly used as the selective agent, normally in

concentrations ranging from 50 to 500 mg/l

• Geneticin is generally more toxic than kanamycin

What is a Reporter Gene?
• Reporter genes allow for the identification of transformed cells
without the need for selective media, as they encode proteins
that are more readily apparent

• Reporter genes eg cat, lacZ, uidA, luc, gcfP allow one to

distinguish transformed from non-transformed plants AND to
study regulation of different cellular processes such as:
• Gene expression
• Protein localization &
• Intracellular protein traffic
What is a Reporter Gene?
• Such genes are called reporters because the
characteristics they confer on organisms expressing
them are easily identified & measured
• Also called screenable markers

• By employing vital markers such as GFP, these

characteristics can be observed in situ, without the need
of destroying plant cells
• GFP = Green fluorescent protein, a marker for screening
Examples of Reporter Genes
1. Beta glururonidase (GUS) uidA protein from
Escherichia coli– needs substrate X-gluc for blue
colour
2. Luciferase proteins from bacteria & firefly - yield light
when substrate luciferin is present
3. Green fluorescent protein (GFP) from jellyfish - an
example of an autofluorescent protein that changes
colour when excited by certain wavelengths of light
Reporter Genes for Plant Transformation
Selectable Markers vs Scorable Markers
Selectable Markers
• Typically used to recover
transgenic plant cells from
non-transgenic cells
• Antibiotic resistance markers
& herbicide resistance
markers are most common
Scorable Markers
(Reporter Genes)
• Can help visualize transient
expression
• Can help visualize if tissue is
stably transgenic
• Useful for cellular &
ecological studies
Beta Glururonidase (GUS)
• GUS provides for a histochemical stain
• Sprayed with herbicide or grown on antibiotic
• Transformed plants survive
• Efficiencies now as high as 5%
• Direct selection
• Removal possible but extremely difficult
GUS-Positive Plants and Cells
Firefly Luciferase Produced in Tobacco
35S:GFP Canola
Green and other Colour Fluorescent Proteins

• Why use other colours?

ER Targeting
• Signal peptide directs GFP to endoplasmic reticulum,
ER for secretion
• But HDEL tag sequesters assembled GFP in ER
• Protected more accumulation

• ER retention dramatically improves orange fluorescent protein,

OFP brightness (monomers)
Red Foliage as an Output
• Arabidopsis MYB transcription factor PAP1 regulates the
expression of anthocyanin biosynthesis genes: overexpression
of PAP1 results in a red-plant phenotype
 Production of Transgenic Plants
Without Marker Genes
• Marker gene could be toxic and/or allergenic upon ingestion.
• Moreover genes that confer antibiotic resistance could be
transferred to bacteria of intestinal flora
• Efforts being made to do without marker genes