Plant Cell, Tissue, and Organ Culture

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Plant Cell, Tissue, and Organ Culture HORT
515 Plant Genetic Transformation

• DNA Delivery
• Screening and Selecting for Transgene
  Expression
• Plant Regeneration
• Identification of Transgenic Plants
• Genetic Transformation of Plants - transferring
  into cells (protoplasts) foreign DNA
• Integration of the DNA into a plant genome
  (nuclear or cytoplasmic)
• Expression of the foreign DNA in planta and
  mitotic and meiotic inheritance of the encoded
  genetic material

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1. DNA Delivery
A. DNA Delivery Methods Agrobacterium
tumefaciens mediated Ti plasmid mediates
Agrobacterium infection and transformation Manip
ulation of Agrobacterium for plant
transformation Process of Agrobacterium
transformation Direct DNA uptake Micro-projec
tile bombardment Polyethylene glycol or
electroporation mediated DNA uptake into
protoplasts B. Cell, Tissue and Organ Targets
for Transformation Morphogenetically
competent cells In planta transformation
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• DNA is constructed into vectors, typically
  bacterial plasmids, and these are the DNA
  delivery vehicles
• Vectors contain plant expression cassettes,
• enhancer/promoter (controls transcription)
• transcribed DNA (encodes mRNA)

Plant Expression Cassette

Coding

Terminator/
Leader 5-UTR
Enhancer/Promoter


sequence

AAA/3-UTR


Multiple expression cassette (e.g. selectable
marker, reporter and target genes) are
constructed into a plasmid vector
Plasmid Vector w/Expression Cassettes
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Agrobacterium mediated transformation -
Agrobacterium tumefaciens (crown gall) or
Agrobacterium rhizogenes (hairy root), gram
negative bacteria that are phytopathogens Bacteri
a infect plant cells, and transfer DNA is
integrated into the nuclear genome, replicated
and inherited Three genetic components that are
relevant to plant transformation T-DNA and
Virulence (Vir) regions located on the Ti
tumor- inducing (Ti) plasmid (200
kb) Chromosomal virulence (Chv) genes that are
responsible for chemotaxis and attachment of the
bacteria to the plant cell wall, example
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Agrobacterium Infection and T-DNA Transfer into
Plant Cells
Tzfira and Citovsky (2002) Trends Cell Biol
12121-129
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• Ti plasmid mediates Agrobacterium infection and
  transformation
• Ti plasmid contains the virulence region in
  which the Vir genes are located and Vir gene
  products
• sense plant based signal molecules
• facilitate bacterial infection
• excision of the transfer DNA (T-DNA) from the Ti
  plasmid
• transport of the T-DNA from the bacterial cell
  to the plant cell
• import of T-DNA into the nucleus
• integration of T-DNA into the host genome

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Ti plasmid mediates Agrobacterium infection and
transformation Seven necessary loci VirA,
VirB, VirC, VirD, Vir E, VirG and Vir
H Signaling VirA (receptor) and VirG
(transcription factor), VirA sense phenolic (or
sugar) products of wounded plant cells, transmits
a phospho-relay signal to facilitate release of
VirG from the membrane to activate other Vir
genes Processing of T-DNA VirD1 and VirD2
are endonucleases that recognize the 25 bp
repeats on the left and right T-DNA borders,
VirD2 attaches to the 5-end of the T-DNA strand,
and VirE protein packages the DNA. Processing
facilitates transport and genome
integration Transport of T strand complex into
plant cells transport through the bacterial
membrane via a type IV secretion system composed
of VirB and VirD4 proteins, example
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Agrobacterium Infection and T-DNA Transfer into
Plant Cells
Tzfira and Citovsky (2002) Trends Cell Biol
12121-129

• Agrobacterium recognition and attachment to host
  cell
• Sensing of plant signals by VirA-VirG two
  component receptor
• VirG mediates signaling and activation of Vir
  genes
• Production of mobile T-DNA (VirD1 and VirD2),
  complex with Vir proteins (VirD2 and VirE2
• 5. Transport of T-DNA w/Vir proteins through
  VirB-VirD4 type IV transporter

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Ti plasmid mediates Agrobacterium infection and
transformation Nuclear localization nuclear
localization signals on VirD2 and VirE2 and other
sites on the proteins facilitate interaction with
numerous host proteins including chaperones,
phosphatases, nuclear pore complex proteins, etc.
Integration host proteins facilitate genome
integration, example
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Agrobacterium Infection and T-DNA Transfer into
Plant Cells
Tzfira and Citovsky (2002) Trends Cell Biol
12121-129

• Formation of mature T-complex, interaction with
  plant proteins
• T-complex nuclear import, plant targeting
  proteins
• Transport to the host chromosome, promiscuous
  recombination
• and integration, single strand gap repair

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Ti plasmid mediates Agrobacterium infection and
transformation Determinants of Agrobacterium
host range Host plant inhibitors of
Agrobacterium sensing interfere with
chemotaxis, phenolic signal recognition,
attachment Host plant proteins that facilitate
or inhibit Agrobacterium recognition, T strand
transport into the plant cell and nucleus or
integration VirF is an Agrobacterium host
range factor- VirF is secreted through the type
IV secretion system where it interacts with host
proteins and thought to facilitate T-complex
integration
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Ti plasmid mediates Agrobacterium infection and
transformation T-DNA contains genes that
encode enzymes in the plant cytokinin/auxin
biosynthetic pathways (tumorization) and opines
(carbon/nitrogen sources), illustration
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Ti Plasmids
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Manipulation of Agrobacterium for plant
transformation - Separate T-DNA and disarmed Ti
plasmids (minus T-DNA) are harbored in
Agrobacterium (binary system, binary vector)
T-DNA plasmid (5 to 20 kb) consists of right
and left borders - 25 bp inverted repeat
fragments that flank (left and right borders) the
expression cassettes and are recognized by Vir
gene products for transfer into the plant
cell T-DNA plasmid (binary) is an E. coli
compatible plasmid and molecular genetic
engineering utilizes E. coli as the host
organism, semi-multicopy plasmid, rec-, selection
markers, etc. The plasmid is transformed into
Agrobacterium by triparental mating or by direct
uptake, electroporation or freezing and thawing,
example
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Manipulation of Agrobacterium for plant
transformation
Binary vector (Bin)
Binary
'Disarmed' Ti
Ti
Bin
LBA4404
KnR Selection
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• Process of Agrobacterium transformation infect
  wounded plant tissues in vitro and induce plant
  regeneration
• Leaf disks or suitable explants are
  co-cultivated, transformed cells/tissues are
  isolated based on screening for expression of
  reporter or marker genes, and plants are
  regenerated from the cells
• T-DNA insertions are random within the genome
  and occur on average of 1.5 times/transformation
  event
• However, inserts often include T-DNA concatamers
  or are results of promiscuous recombination,
  incomplete insertions, illustrations

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Agrobacterium-mediated Transformation by
Co-cultivation w/Leaf Disks
Co-cultivation
Incubation
Plant Regeneration
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Plant expression cassettes two basic types of
plant expression cassettes Transgene open
reading frame (ORF) expression drive by an
ectopic promoter Often constitutive but may be
conditional Translation fusions Promoter
expression typically driving expression of a
reporter gene for monitoring, illustrations
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Example of Transgene Vector PBI121 (modified)
Transgene
LB
CaMV 35S promoter
NOS-ter
RB
NPT II(Kanr)
NOS-pro
NOS-ter
Transgene open reading frame expression cassette
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Example of Reporter Gene Vector pBI101
Promoter
TGA
ATG
LB
ß-glucuronidase
NOS-ter
RB
NPT II(Kanr)
NOS-pro
NOS-ter
Promoter (reporter gene translational fusion)
expression cassette
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1. DNA Delivery

• DNA Delivery Methods
• Agrobacterium tumefaciens mediated
• Direct DNA uptake
• Micro-projectile bombardment
• Polyethylene glycol or electroporation mediated
  DNA uptake into
• protoplasts

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Direct DNA Uptake - DNA is delivered into cells
but transport to the nucleus and genome
integration are not facilitated Developed
because monocots (cereals) were thought to be
outside of the host range of plants that can be
infected by Agrobacterium
Microprojectile bombardment - gold or tungsten
particles (1 to 2 ?m in diameter) are coated with
DNA and delivered to cells as high velocity
microprojectiles Plant cells remain viable,
undergo mitosis and then plants are
regenerated Numerous factors affect the
delivery process, e.g. size, number and velocity
of the microparticles, DNA type and amount, type
and physiological state of target cells, etc,
illustrations
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Biolistic PDS-1000 Particle Bombardment System
Figure 7. Biolistic PDS-1000 particle bombardment
system.
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DNA coated particles
Macrocarrier holder
mylar
Macrocarrier
Stopping screen
Particle Bombardment System
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Microparticle Delivery to Cells
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Sorghum Inflorescence Explants for Particle
Bombardment and Culture onto Induction Medium
Note damage caused by particle bombardment,
injury can be reduced by pre-culture/osmotic
stabilization
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GUS Activity in Sorghum Inflorescence Explants
after Particle Bombardment, and Somatic
Embryogenesis
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Direct DNA Uptake - DNA is delivered into cells
but transport to the nucleus and genome
integration are not facilitated
Microprojectile bombardment Polyethylene
glycol (PEG) or electroporation mediated uptake
into protoplasts - DNA uptake is facilitated by
PEG or electric impulse (electroporation) Cell
wall must be re-synthesized and then plants
regenerated, illustration
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Transient gene expression assays
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Agrobacterium tumefaciens strains have been
identified that can infect monocots Rice
transformation is achieved by Agrobacterium
tumefaciens transformation, Garg et al. (2002)
Proc Natl Acad Sci 9915898-15903 used
LBA4404 Maize transformation and the
transformation of other cereals are achieved by
Agrobacterium transformation, but the strains are
trade secrets
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1. DNA Delivery

• DNA Delivery Methods
• Cell, Tissue and Organ Targets for Transformation
• Morphogenetically competent cells
• In planta

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Cell, Tissue and Organ Targets for Transformation
- each transformation event is a cellular based
process, so cells, individual or within tissues
or organs, are targets for DNA delivery and
transformation

• Primary regenerated plants are heterozygotes
  (hemizygotes, transgene locus and null)
• DNA delivery to cells that are morphogenetically
  competent cells - individual cells are
  transformed within an explant and plants are
  regenerated from these cells in vitro,
  illustration

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DNA Delivery to Competent Cells and Regeneration
of Transgenic Plants
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Cell, Tissue and Organ Targets for Transformation

• DNA delivery to cells that are morphogenetically
  competent cells
• In planta transformation

• Arabidopsis inflorescence transformation
  pre-anthesis inflorescences (maximize number of
  receptive flowers by pruning plants), likely
  targets are microspores/ megaspores, T1 plants
  are heterozygotes
• Agrobacterium solution should contain surfactant
  and sucrose
• Keep inflorescences moist for at least 24 hours
  as this facilitates transformation because
  Agrobacterium is motile, illustration
• Shoot meristem/apex transformation - DNA is
  delivered to cells in the shoot apical meristem
  and there is lineage of the transformation event
  to germ cells

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In Planta Transformation of Arabidopsis thaliana
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Agrobacterium-mediated in planta Transformation
of Arabidopsis
A
A'
C
B

• and A. different methods of transforming
  Arabidopsis
• B. plants covered with Saran wrap for incubation
  period
• C. plants shortly after transformation with dead
  leaves.

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• In planta transformation

• Arabidopsis inflorescence
• Shoot meristem/apex transformation - DNA is
  delivered to cells in the shoot apical meristem
  and there is lineage of the transformation event
  to germ cells, illustrations

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Dicot Shoot Apical Meristem
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Divisions of cells that express the marker
genes. GUS-positive cell lineages in L1 (a and b)
and L2 (c), and anthocyanin-accumulating cell
line in a leaf of wheat (d), 10 days after
bombardment. Arrows mark the only cell of each
lineage that contains a gold particle. Bars
represent 10 ?m.
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2. Screening and Selection for Transgene
Expression

• Reporter - optimization of gene delivery and
  expression
• Selectable marker - identification of transformed
  cells, tissues and organs selection/detoxificatio
  n
• Target gene gene of interest, optimize
  expression

Transient vs Stable Expression Transient - DNA
is transferred to the cell/protoplast, migrates
to the nucleus where it remains largely
extrachromosomal but is transcriptionally active
Stable - one or more intact copies of the
transcriptionally active DNA is integrated into
the genome (current hypothesis is random
insertion), replicated and inherited through
mitosis and meiosis
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• Common reporter genes - used for the analysis of
  transient or stable transgene expression
• ?-glucuronidase (GUS) - encoded by the uidA
  locus of E. coli. Detection is based on
  histochemical (blue) or fluorometric assay
  depending on which ?-glucuronide substrate is
  used.
• Anthocyanin transcriptional regulators -
  transcriptional activators that regulate natural
  anthocyanin biosynthesis, activity is discernible
  as red pigmentation, illustration
• Luciferase (luc or lux depending on whether the
  gene is from firefly or bacteria, respectively) -
  detection requires luciferin, a substrate that
  generates luminescence
• Green Fluorescent Protein (GFP) - luminophore
  gene from jelly fish, excitation at 395 nm and
  emission at 508 nm

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R/C1 and ß-glucuronidase (GUS) Histochemical
Activities
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• Common reporter genes
• ?-glucuronidase (GUS) - encoded by the uidA
  locus of E. coli. Detection is based on
  histochemical (blue) or fluorometric assay
  depending on which ?-glucuronide substrate is
  used.
• Anthocyanin transcriptional regulators -
  transcriptional activators that regulate
  anthocyanin biosynthesis, activity is discernible
  as red pigmentation
• Luciferase (LUC or LUX depending on whether the
  gene is from firefly or bacteria, respectively) -
  detection requires luciferin, a substrate that
  generates luminescence, ?max at 560 nm,
  illustration
• Green Fluorescent Protein (GFP) - luminophore
  gene from jelly fish, excitation at 395 nm and
  emission at 508 nm

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Luciferase Activity
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• Common reporter genes
• ?-glucuronidase (GUS) - encoded by the uidA
  locus of E. coli. Detection is based on
  histochemical (blue) or fluorometric assay
  depending on which ?-glucuronide substrate is
  used.
• Anthocyanin transcriptional regulators -
  transcriptional activators that regulate
  anthocyanin biosynthesis, activity is discernible
  as red pigmentation
• Luciferase (luc or lux depending on whether the
  gene is from firefly or bacteria, respectively) -
  detection requires luciferin, a substrate that
  generates luminescence, illustration
• Green Fluorescent Protein (GFP) - luminophore
  gene from jelly fish, excitation at 395 nm and
  emission at 508 nm

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Green Fluorescent Protein (GFP) Activity
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AtSIZ1 Is Localized to Nuclear Bodies
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Selectable marker genes - inactivate toxic
chemicals, e.g. antibiotics or herbicides.
Selection is based on survival of plant cells,
tissues and organs.
Antibiotics Kanamcyin or Geneticin (G418) -
resistance gene/enzyme - nptII (E. coli)/neomycin
phosphotransferase Hygromycin - resistance
gene/enzyme - hpt (E coli)/hygromycin
phosphotransferase Herbicides Phosphinothrici
n (PPT), bialaphos, glufosinate, basta, or Ignite
- resistance gene/enzyme - bar (Streptomyces
hygroscopcus)/phosphinothricin acetyltransferase,
also pat, example
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Common Selective Agents and Selectable Markeres
Used for Plant Transformation
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Target transgenes Majority of GM crops (corn,
cotton, soybeans) are herbicide or insect
resistant Virus resistance Pharmaceuticals
and nutriceuticals (biofortification) Stress
tolerances
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GM Crops Worldwide
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3. Plant Regeneration
Morphogenesis - Plant regeneration from
derivative transformed single cells occurs
through morphogenesis, organogenesis or
embryogenesis DNA delivery must target cells that
are morphogenically competent. Processes of
morphogenesis are either Adventitious
organogenesis - formation of shoots and roots
that are derived from single cell origin Somatic
embryogenesis - embryos derived from cells other
than germ cells, often immature embryos,
illustration In planta -
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Adventitious Shoot Formation and Somatic
Embryogenesis for Regeneration of Transgenic
Plants
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3. Plant Regeneration
Morphogenesis In planta Arabidopsis in planta
transformation circumvents the problem of
morphogenesis by targeting gametophytes,
illustration Shoot apex transformation or
transformation of multicellular structures will
result in formation of chimeras that can be
eliminated, albeit not easily, during mitosis or
meiosis (via germ cells)
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Generating a T-DNA Tagged Population (Floral
Transformation)
Herbicide Selection (T1)
Pool Amplification (T2)
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3. Plant Regeneration
Morphogenesis In planta Arabidopsis in planta
transformation circumvents the problem of
morphogenesis by targeting gametophytes Shoot
apex transformation or transformation of
multicellular structures will result in formation
of chimeras that can be eliminated, albeit not
easily, during mitosis or meiosis (via germ cells)
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• Identification of Transgenic Plants

• Molecular detection of the transgene
• Transgene expression
• Inheritance of the transgene

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• Identification of Transgenic Plants

Molecular detection of the transgene DNA blot
analysis - Southern blot analysis with transgene
probe, illustration Polymerase chain
reaction amplification - primers for detection
(amplification) of transgene sequences
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Southern Blot of Transgenic Sorghum Plants Probed
with a bar cDNA Insert
Different restriction patterns indicating
independent transgenic events
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• Identification of Transgenic Plants

Molecular detection of the transgene DNA
blot analysis - Southern blot analysis with
transgene probe, ion Polymerase chain
reaction amplification - primers for detection
(amplification) of transgene sequences,
illustration
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(No Transcript)
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• Identification of Transgenic Plants

Transgene Expression Reporter/marker gene
activity - same as reporter gene assays,
illustration Transcript or protein levels -
northern (RNA) or western (protein)
blots Enzymatic assay - e.g. ppt assay
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Stress Regulated Expression of HVA1GFP Chimeric
Gene
Control
Cold
Salt
Dark
ABA
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• Identification of Transgenic Plants

Transgene Expression Reporter/marker gene
activity - same as reporter gene assays
Transcript or protein levels - northern (RNA)
or western (protein) blots, illustration Enzyma
tic assay - e.g. ppt assay
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RAG1 Transcript Level in a Transgenic Plant
35SRAG1 rag1-1
rag1-1
RAG1
HSP70
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• Identification of Transgenic Plants

Transgene Expression Reporter/marker gene
activity - same as reporter gene
assays Transcript or protein levels - northern
(RNA) or western (protein) blots Enzymatic
assay - e.g. ppt assay, illustration
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Phosphinothricin Acetyltransferase Activity in
Transgenic Sorghum Plants
14C-acetylCo-A
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Inheritance of the Transgene - functional
insertion at a single locus is phenotypically
equivalent to a dominant allele, i.e. will be
inherited according to Mendelian principles

• Restriction fragments - DNA polymorphisms of the
  transgene, if Arabidopsis, then it is possible to
  define the allele at the locus
• Phenotype - e.g. Ignite inheritance in progeny,
  illustration

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Transgene Inheritance in Sorghum
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T-DNA Or Transposon Tagging For Mutant Population
Generation
T-DNA or transposons produce insertional
mutations, these mutations typically disrupt gene
function, however, T-DNA vectors can be
constructed when inserted into certain regions of
genome (gene) can activate expression After a
gain-or-loss of function mutant is identified,
then the affect gene is isolated easily because
it is "tagged" either by the T-DNA or transposon.
T-DNA tagging loss-or-gain of function, T-DNA
insertion disrupts gene function or activates
gene expression, illustration Transposon DNA
tagging Transposon Tagging - illustrated is the
Ac/DS system Ds is a non- autonomous element
requiring Ac (transposase) for transposition
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(No Transcript)
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Hyper-osmotically Responsive Stress Mutants
2nd screening
No stress
Cold
ABA
ABA
Stress
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(No Transcript)
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TAIL-PCR Nucleotide Sequence of a T-DNA Tagged
Mutant Encodes an a-Importin
CTNTGTTCGTTCCTTTANACGACTCACTATAGGGCGAATTGGGTACCGGG
CCCCCCCTCGAGGTCNACGGTATCG ATAAGCTTGATTTTGACCATCATA
CTCATTGCTGATCCATGTAGATTTCCCGGACATGAAGCCATGTAGATAGA
A GTCCACCTACTGATAATGTGATAAAATCTGGAGTTGTGCCACGTTTTG
TAGAGTTTCTTAAGAAGGATGATAACC CTAAGCTTCAGGTTAGACTTAA
AGTTCCCATCTTTGATGATTAGTGTAATCTTTGGTTCTGAATTCTTTGAT
CTT TTTTATAGTTTGAGGCTGCTTGGGCTTTAACAAACATTGCTTCTGG
CGCATCTGAGCATACCAAGGTAGTGATTG ATCACGGTGTTGTGCCACTC
TTTGTTCAGCTTCTTGCTTCCCCTGATGATGATGTCCGTGAGCAGGTATA
GTGCT TCTTCTTACTCGCTAATCGAATTCCTGCAGCCCGGGGGATCCAC
TAGTTCTAGAGCGGCCGCCACCCGCGGTGGA GCTTCAGCTTTTGTTCCC
TTTAGTGAGGGGTAATTTCGAGCTTGGCGTAATCATGGCATAGCTGTTTC
CTGTGGA AATTGTATNCGCTCACAATTNCACACACATACGAGCCGGAAG
CATAAAGTGTAAAGCCTGGGGTGCCTAATGAGT GAGCTAACTACATTAA
TGCGTTGCCTCCTGCCCGNTTTCAATCGGGAANC
white - cloning vector, yellow - left border
(primer sequence), a-importin
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Activator (Ac) and Dissociation (Ds) Transposon
System
Transposon tagging of mutant loci Increasing the
number of transgene insertion sites
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Transposition of the Ds (dissociation)
transposable element in maize (Zea mays) and
Arabidopsis. In (a), a maize kernel has purple
sectors caused by excision of Ds from the R gene,
which is required for anthocyanin pigmentation.
In (b), an Arabidopsis seedling, germinated on
medium containing the antibiotic streptomycin,
has green sectors caused by excision of Ds from a
transgene conferring streptomycin resistance5.
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RD29aLUC Expression is Hyper-responsive to
Cold, ABA or NaCl in cpl1 and cpl3 Plants
Control
Cold ( 0 C, 48 h)
(x 106)
A
B
C24
cpl3
C24
cpl3
C24
cpl1
6
cpl3
Counts/seedling
4
2
0
cpl1
cpl1
Cold
ABA
NaCl
Control
cpl3
cpl3
C24
C24
cpl3
cpl3
cpl3
cpl1
C24
cpl1
cpl3
C24
cpl1
C24
cpl1
C24
RD29ALUC RNA blot
Low exposure
cpl1
cpl1
ABA (100 ?M, 4 h)
NaCl (300 mM, 4 h)