not by me.. for the student of cdbt

1. Marker and Reporter genes used for
plant transformation
Deepak Raj Pant
Central Department of Botany
Tribhuvan University, Kirtipur
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2. Genetic Manipulation of plants
Techniques of introducing DNA into plant cells: 10 hrs
Marker and reporter genes used for plant transformation, Model
plants and their Role in genetic manipulation, Indirect transformation:
Genetic transformation of plant tissues with the use of Agrobacterium,
Ti-plasmid and mechanism of T-DNA transfer (different protein
involved and their rolr, vir region and other genes involved), Ti plasmid
derived plant vector systems; binary and cointegrative vectors
transformation process, regeneration of the transformed lines, Plant
Viruses as biological vectors. Direct gene transfer methods in plants
(Microprojectile bombardment, Electroporation; polyethylene glycol
(PEG)- mediated gene transformation, Silica carbom fibres whiskers).
Transformation of protoplasts with naked DNA
Genetic modified plant and their Application 5 hrs
Genetic engineering for plant improvement: Development of Pest
resistance, herbicide resistance, resistance against viruses, improving
stress tolerance, Protoplast fusion and its implication, Importance of
GM plants
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3. Genetic Manipulation of plants
• Manipulation of genome of an
organism
• Can be done by traditional method
(breeding) or by using the techniques of
molecular cloning and genetic
transformation (i.e., by genetic
engineering)
• Genetic engineering is used for genetic
manipulation due to its advantages
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4. Genetic Manipulation of plants
Genetic engineering is advantageous
because
• Changes can be manipulated in lab and
introduced into target organisms/tissues/
cells without affecting other genes
• It overcomes the requirements for sexual
compatibility
• It enables us to study the gene function by
inactivation of gene, altered expression of
gene, ectopic expression of genes, altering
the proteins, etc
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5. Genetic Manipulation of plants
What we need?
• A fully functional gene with all its regulatory
sequences
• A competent target (Embryogenic
competence; competence for transgene uptake)
• Proven method of delivery (biological,
chemical, or physical)
• Method of verifying the progress of
transformation and selection of transformants
(i.e., reporter and selectable marker genes)
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6. Selectable markers
• The selectable markers are the genes
which enable the selection/recovery of
transformed cells/tissues from
untransformed tissues in the growth media
containing selecting agent (usually an
antibiotic or a herbicide)
• These genes provide selective advantage
for the transformants (which occur at very
low frequency) during the course of
selection, i.e., the growth of cells/tissues in
media containing the toxic substance
(selecting agent)
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7. Selectable markers
• The selectable markers usually encode for
a protein that detoxifies the compound used
for selection of transformed cells
• Some of the selectable markers also
involve the genes that are mutated and
produce target proteins that are insensitive
to toxic substances (usually antibiotics or
herbicides) used for selection
• Some encode for proteins that convert a
non-metabolite into a metabolite
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8. Antibiotics Resistance as Selectable markers
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Selectable marker gene(s) Resistance against Uses
Neomycin phosphotranferase II
(npt II)
G418 sulphate, Kanamycin,
Neomycin, etc
Dicots/ monocots
Hygromycin phosphotransferase
(hpt or aphIV)
Hygromycin B Dicots/monocots
Aminoglycoside -3
adenyltransferase (aad A) or
streptomycin resistance gene (spt)
Streptomycin/
Spectinomycin
dicots
Gentamicin 3-N- acetyl transferase
(gat)
Gentamicin Dicots
Dihydrofolate reductase (dfr) Methotrexate Dicots
Unknown gene (ble) of Tn5
transposon
Bleomycin

9. Herbicide Resistance as Selectable markers
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Selectable marker gene(s) Resistance against Uses
EPSPS/ gox from Achromobacter
sp.
Glyphosate/ Roundup Dicots/monocots
Bialaphos (Basta) resistance (bar)
or phosphinothricin acectyl
transferase (pat)
Glufosinate/ PPT/Basta/ Dicots/monocots
Chlorosulfuron resistance (csr) Sulfonylurea
(chlorosulfuron)
Dicots/monocots
Bromoxynil nitrilase (bxn) Bromoxynil (Buxtril) Dicots/monocots
Mutant of Acetolactate synthase
(als)
Imidazolinones
(Imazethapyr)
Dicots
Unknown monoxygenases Auxins like 2,4-D, 2,4,5-T Dicots

10. Selectable Markers from AA synthesis pathway
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Selectable marker gene(s) Resistance against
Dihydropicolinate synthase (dhps) from
bacteria
S-aminoethyl L-cysteine (AEC), a toxic
analogue of L-Lysine
Aspartate Kinase (AK) from bacteria Lys and Thr (These normally inhibit AK
and cause starvation for methionine)
Tryptophan Decarboxylase (TD) from
Catharanthrus roseus
4-methyl tryptophan (a toxic analogue
of tryptophan)
Other selectable Markers
Selectable marker gene(s) Resistance against
Phosphomannose isomerase (pmi) from E.
coli
Mannose; can utilise mannose as substrate;
convert mannose 6-p to fructose 6-P
Xylose isomerase (xyl A) from Streptomyces
rubiginosus
Xylose; conversion of D-Xylose to D-xylulose
Ipt, BADH, rol A , etc can also be used Cytokinin autotrophic growth,
osmoprotectant, hairy roots

11. 11
Negative Selectable markers
Selectable marker gene(s) Killed by
Nitrate reductase gene (nia) Chlorate on medium containing NH4
+
Cytosine deaminase (codA) 5-fluorocytosine, a non toxic compound, due to
its conversion into 5-fluorouracil (toxic)
• Negative selectable markers are not much useful in
routine transformation
• These can be used in transposon tagging to select
the non-mutant populations

12. Reporter genes
• The reporter genes encode for the
products (scorable markers) that can be
easily scored/quantified in transformed
tissues by simple assays
• These reporter genes also help
detect/screen the transformation events
during gene transfer experiments
• These markers encode for products that
are themselves fluorescent/ coloured or
give fluorescence/ colour reactions upon
treatment with other substrates
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13. Reporter genes
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Reporter
gene
Enzyme/potein Substrate Assay
gfp gfp none Fluorescence observation
Gus or
uidA*
Β-Glucuronidase X- Gluc Histochemical,
photometric, fluorometric
Lux/ luc Bacteria/ Firefly
Luciferase
D- Luciferin, ATP Fluorometric
phoA Alkaline phosphatase Phosphate Fluorescece measurement;
histochemical
cat Chloramphenicol
acetyl transferase
14C-
chloramphnicol,
Acetyl CoA
Acetyl chloramphenicol
detection by
chromatography/
autoradiography
lacZ Β-Galactosidase IPTG and X-Gal Colour formation (blue/
white)
Npt II Neomycin phospho-
transferase
Kanamycin and 32P-
ATP
Detection of radioactivity
in tissues (in situ)

14. Green Fluorescent protein (gfp)
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• From Jellyfish Aquoria victoria,
glows in blue light 395 nm giving
green fluorescence (510 nm)
• Can be detected in vivo (non
destructively) by using fluorescence
microscope
• Different variants like EGFP, Red
GFP, EYFP, etc available
• Does not require any substrate, can
be detected directly

15. Gus gene (uid A)
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• From Escherichia coli
• Codes for the β glucuronidase which
breaks x-gluc (5 bromo-4 chloro-3 indol β D
glucuronide) into blue colour; can be used
for histochemical analysis of gene
expression
• Converts 4MUG (4- methylumbelliferyl-β-
D glucuronide) into a fluorescent
compound 4MU (Methyl Umbelliferone);
can be used for quantification by
fluorescent measurement
• Can also be quantified spectro-
photometrically by using p-nitrophenyl
galactoside as substrate
Gus expression in young
leaves

16. Gus gene (uid A)
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17. Luciferase gene luc or lux
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• From firefly (luc) or bacteria (lux)
• Converts luciferin into oxyluciferin in the presence of
oxygen and ATP
• Oxyluciferin is highly unstable, singlet excited
compound which emits fluorescence upon relaxation to
ground state

18. Alkaline phosphatase pho A
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• From Bacteria E. coli
• Cleaves compounds containing phosphate group like o-
nitrophenly phosphate, x-phos (5 bromo-4 chloro-3
indolyl phosphate) or 4MUP (4-methyl umbelliferyl
phosphate)
• Can be used for colorimetric or histochemical detection
(with o-nitrophenyl phosphate or X-phos) or by
fluorescence measurement (with 4-MUP)
• Forms yellow nitrophenol with o-nitrophenyl
phosphate
• Gives dark blue coloured precipitate with X-phos in the
same manner as does β-glucuronidase with x-gluc

19. • Chawla H.S. 2009. Introduction to Plant Tissue
Culture. Third Edition. Oxford and IBH
• Guerineau, F. 1996. Tools for expressing foreign
genes in plants. In: Jones, H. (ed), Plant gene transfer
and expression protocols. Humana press. Pp 1-32
• Gupta, P.K. 2009. Biotechnology and Genomics.
Rastogi Publications, pp 442-444
• Slater, A., Scott, N.W. and Fowler, M.R. 2008. Plant
Biotechnology, the genetic manipulation of plants.
Oxford University Press.pp 86-91
References
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