This document discusses in planta transgenic approaches for crop improvement using molecular techniques. It provides information on various physical, chemical and biological plant transformation methods including microinjection, biolistics, electroporation, and Agrobacterium-mediated transformation. The document then focuses on Agrobacterium tumefaciens and its Ti plasmid, describing the mechanism of T-DNA transfer to plant cells. It discusses binary vector systems and various in planta transformation protocols like floral dip and vacuum infiltration methods. Finally, it presents a case study demonstrating the successful use of floral dip transformation to produce transgenic rice plants.

1. COURSE (PP-606) – MOLECULAR APPROACHES FOR
IMPROVING PHYSIOLOGICAL TRAITS
TOPIC - INPLANTA TRANSGENIC APPROACH FOR
CROP IMPROVEMENT
COURSE INSTRUCTOR –
DR. R K PANDA
DEPT. OF PLANT PHYSIOLOGY
CA, OUAT, BBSR
PRESENTED BY –
JYOTI PRAKASH SAHOO
01ABT/PHD/17
DEPT. OF AGRIL. BIOTECH.
CA, OUAT, BBSR
DEPARTMENT OF PLANT PHYSIOLOGY
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2. Physical Chemical Biological
• Microinjection
• Biolistics - gene gun
• Particle bombardment
•Electroporation
•Silica/carbon fibers
•PEG
•Calcium phosphate
•Artificial lipids
•Proteins
• A. tumefaciens
• A. rhizogenes
In planta
• Meristem transformation
• Floral dip method
• Pollen transformation
Plant Transformation Methods
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Microinjection Biolistics - gene gun Electroporation

3.  Efficient, quick and tissue culture independent system for crop plants
improvement useful for those plants that lack tissue culture and regeneration
system.
 Two most common Agrobacterium mediated in-planta methods such as floral dip
and vacuum infiltration have been successfully used by many researchers in both
dicot and monocot plants.
 Main advantages of in-planta transformation are to produce large number of
transgenic plants and accumulation of high concentration of total soluble
protein in short time.
Inplanta transgenic approach
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Types of Tissues to be transformed
 Callus transformation
 Immature embryo transformation
 Pollen transformation
 Shoot apex transformation
 In planta transformation
 Floral transformation
 Seed transformation

4. auxA auxB cyt ocs
LB RB
LB, RB – left and right borders (direct repeat)
auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine
Agrobacterium tumefaciens (Ti-Plasmid)
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Crown galls caused by
A. tumefaciens
 Having 4 chromosomes; ~ 5500 genes and Ti Plasmid
~ 250kbp

5. Vir (virulent) genes
 virA - transports AS into bacterium, activates virG
 virG - promotes transcription of other vir genes
 virD2 - endonuclease/integrase that cuts T-DNA at the borders
 virE2 - form channels in artificial membranes
 virB - operon of 11 proteins, gets T-DNA through bacterial membranes
 virD2 & virE2 - gets T-DNA to the nucleus of plant cell
Vir genes Functions
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Activated by
Acetosyringone (AS)
(a flavonoid) released by
wounded plant cells

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Mechanism of transfer of T-DNA
Phosphorylated Vir A protein
Vir G protein dimerises
Expression Vir operon
Topoisomerase and endonuclease Vir D1 activates Vir D2
Act as primer for
DNA synthesis
Exonuclease activity signal sequence, drives it into the
nucleus of plant cell

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T-DNA enters plant cell as a single stranded structure immediately
converted into double stranded form
Vir E2 (nuclear localization sequence) responsible for transfer of T
DNA into plant cell nucleus
For integration 23-79 base pair deletion takes place at the integration
or target Site
Activation of auxins, cytokinins and opines genes
uncontrolled growth in the form of tumor
Integration of T-DNA in the plant genome

8. Binary vector system
Strategy:
1. Move T-DNA onto a separate, small plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin resistance) gene in T- DNA.
4. Vir genes are retained on a separate plasmid.
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.
A plasmid
containing vir
region but no T-
DNA, therefore no
T-DNA transfer
takes place in
plant genome
Another plasmid
containing T-
DNA with Right
border (RB) and
Left border (LB)
but no vir genes.
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9. 9
Co- integrated vector system

10. Common In Planta Transformation Protocol
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Leaf disc/

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Leaf-disc transformation method
(regeneration with tissue culture)

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Floral dip method
Simple submersion of plant into bacterium suspension
– No vacuum is needed
– Conducted with plants grown until just flowering
– Progeny seeds are harvested and germinated using selective antibiotic
Plant leaf disks are placed in a suspension of bacteria and vacuum
pulled
– Air is release like a sponge being squeezed
– Vacuum is released and solution floods tissue
– Plant disk is cultured
Vacuum infiltration method
(does not require tissue culture)

13. Selectable Markers
• Antibiotic resistance
• Herbicide resistance
• Positive selection genes - that allow use of some necessary media component.
– NPTII - kanamycin (antibiotic)
– Hpt - hygromycin
Novel Selection Genes
• Luciferase - gene from fireflies – substrate
• Green Fluorescent Protein - from jellyfish - under lights
and filter the transgenic plants - GFP
• GUS - glucuronidase gene will convert added substrate
to blue color.
(X-glcA (X-gluc or X-glc or X-glcU) - substrate for GUS)
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Case Study

15. Plant materials and growth conditions
Agrobacterium-mediated transformation
 Agrobacterium strains - AGL1 or EHA105
 Vector - pCAMBIA1304 carrying a gusA gene
 Culture medium - Luria Bertini (LB) broth supplemented with
kanamycin (50 mg/L) and rifampicin (40 mg/L)
 Murashige and Skoog (MS) suspension medium (pH - 5.7)
Rice (Oryza sativa L.) variety RD41 grown in pots with natural light until
flowering in a greenhouse.
The inflorescences at the growth stage (the beginning of panicle
emergence and tip of inflorescence emerged from sheath) (Lancashire et
al, 1991) were used for transformation.
Materials and Methods
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pCAMBIA1304

16. Screening for transgenic rice by PCR analysis
Genomic DNA was extracted from T0 transgenic rice leaves using
(CTAB) method
Transgenic lines were verified using specific primers designed from the
gusA sequence of pCAMBIA1304 binary vector
PCR reaction - using pair of specific primers
gusA-F1 and gusA-R1 to amplify the 989 bp fragment
gusA-F2 and and gusA-R2 to amplify the 361 bp fragment
Histochemical glucuronidase (GUS) assay
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17. Evaluation of a simple and effective inoculation medium for
floral-dip transformation of rice
 The addition of 200 μmol/L acetosyringone (AS) gave the
transformation efficiency no different from the standard
medium (AS free)
AS could enhance the transformation efficiency of both
monocot and dicot crops
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Results

18. Potential of A. tumefaciens strain EHA105 for anther
transformation via floral-dip method
The gusA expression analysis demonstrated that the anther
transformation efficiency of A. tumefaciens strain EHA105 was slightly
higher than strain AGL1
This suggests that the virulence of Agrobacterium strains AGL1
and EHA105 is different among plant species even those plants are
in the same group (monocot or dicot). 18

19. Transgenic rice production from floral-dip transformation
PCR amplification of a 989 bp
expected product for positive
transformed plants using a pair of
primers gusA-F1 and gusA-R1
PCR amplification of a 361 bp
expected product for positive
transformed plants using a pair of
primers gusA-F2 and gusA-R2
gusA expression analysis of 20
individual T0 plants from the
PCR-positive bulk tests
Nos. 2, 4, 7 and 13
1 kb 1 kb
 Floral-dip transformation could give transgenic rice with at least 1.4% (4 transgenic plants
from 286 tested T0 lines) transformation efficiency.
gusA transgene in
this line was
silenced.
no GUS-stained leaf
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The bulk tests Nos. 2, 4 and 7 carried one
GUS-stained leaf each

20.  In-planta transformation is a tissue culture independent, quick and efficient
direct transformation system that produced large number of plants in very short
time.
 Floral dip and vacuum infiltration methods are the two main in-planta
transformation methods that were successfully used to transformed gene of
interest in cereal crops, vegetables, oil seeds crops and many other plants.
Results of the case study indicated that floral-dip transformation is a potential
tool for production of the transgenic rice, which can be used for molecular
breeding via genetic engineering in the future.
Summary
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References

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