Plant Transformation and

Regeneration
Lecture 14
Monday February 13th, 2017
 Plant Transformation:
Critical Components

Transgenic plants are Regeneration

Events Transformation
created from an
Events
overlap of
regeneration and TRANSGENIC
transformation events PLANTS

Plant Explant
‘Plant Regeneration’ is typically a key limiting step
and required to capture stable transformation
events
Agrobacterium tumefaciens
Embryogenesis

Regeneration
Organogenesis

Genome

Plant Cell
 Plant Transformation the Complete Process

TRANSGENIC PLANTS

Regeneration
T-DNA

Acclimatization

Field Testing

Yield/
Cocultivation
mac-cryIAc CaMV35S-GUS CaMV35S-KAN LB
Quality
RB

Gm pDU92.710
Candidate
RB Ubi7-GUS Ubi3-cryIAc mas-KAN LB
Transgenes/Traits
pDU96.3113
Agrobacterium Vector Gm

Binary Plasmid Vector

 Setting up the Agrobacterium-mediated plant transformation

Agrobacterium Co-cultivation
Culture Conditions

+ =

Individual plant cells are

Leaf discs transformed with the
transgenes
Choice of Explants
 Choice of Explants
• Age and development stage of plant
– Shoots
– Apex
– Seedlings
– Germinating seeds
– Flowers
• Type of explant
– Leaves
– Petioles
– Internodal segments
– Roots
– Embryos/seed
• Ratio of wound to explant surface area Leaf discs
– 7mm optimal
• Preconditioning explants
• Agrobacterium Host Strain
– C58 Agrobacterium
– EHA101/105
– Extra copy of virG (pCH32) Culture Conditions
• Cryostorage of strains
– Glycerol 15%
– DMSO
• Plates with antibiotic selection
–
. . . .. . . .
Select for Host chromosome
– Select for Disarmed Ti
...
– Select for Binary backbone . . -80oC
• Overnight culture
– YM
– LB
– 523 medium
• Wash and re-suspend
– 0.5 OD (550nm) Overnight
– 1-5x108 cells/ml
• Incubation Medium (IM)
– MS-medium
– MES buffer 2.5 mM
– pH5.25
– Acetosyringone 100mM
– Betaine/proline 1mM
– Monosaccharides
• Growth in induction medium
– 5hr
– 18-22oC
• Infection
– 10 mins Co-cultivation
– Pat dry on sterile Whatman 1
paper
• Co-cultivation
– 2-5 days
– In dark
– Regeneration medium
– +/- Whatman 1 paper
• Co-cultivation with feeder
cells
– 5-7 days tobacco suspension Individual plant cells are
cells transiently transformed with the
– Whatman1 paper transgenes
Section of an
Leaf Disc
apple leaf disc
infected with
Agrobacterium

Cut Edge

Infecting
Agrobacterium
cells
Moving from transformed cells to transformed plants:
plant tissue culture

• Plant cells are totipotent: any single cell can give rise
to a whole new plant

• Caveat to totipotency: in order to regenerate plants, cells

must be provided with appropriate amounts of plant
hormones, sugars, vitamins, nutrients, etc.

• Transformed cells are not inherently more likely than non-

transformed cells to generate whole plants; to specifically
generate transgenic plants, they must be provided with a
selective advantage
Selectable marker genes and the regeneration
of transgenic plants
Antibiotic resistance
Ubi7 Chitinase Ubi3 Kan
gene; antibiotic will
+ kill plant cells that do
not possess this gene

After co-cultivation, leaf

discs are placed on growth
medium containing antibiotic
• Regeneration Media Regeneration
– Supporting organogenesis
– Stress reduction
• Regeneration Conditions
– In dark
– 4-12 weeks
• Counter-selection
– 500 ug/ml Cef
– Kill Agrobacterium
• Selection
– Selectable marker gene
– Identify transgenic events
– Simulate events
• Record keeping
• Elongation Regeneration
– Transfer to light
– Elongation of shoots
– embryogenesis
• Micropropropagation
– Shoot multiplication
– Secondary embryogenesis
• Plantlet formation
– Rooting of shoots
– Germination of embryos
• Confirmation
– Callus formation in kan
– Rooting in kan
– Marker gene expression
 Regeneration and Transformation of
Carrizo Citrange

Shoot Organogenesis Rooting of New Shoots Acclimatization

Chromogenic Analysis of
Regeneration of Transgenic
Transgenic Activity (GUS)
Shoots (85%)
Carrizo Seedling Epicotyl Regeneration and Transformation System

Shoot Proliferation

+ve GUS assay

Shoot Formation
Rooted Shoots

Plants in Soil
 Transformation of Grapevine

Immature Anthers Embryogenic Callus Kanamycin Selection Germination

GREEN HOUSE TESTING

PCR Validation

Aguero, Uratsu and Meredith

Acclimatization
Summary Points:

Plant tissue culture may be employed for many useful applications such as meristem
culture for pathogen elimination, rapid multiplication of genetically identical plants through
micropropagation, embryo rescue, somatic hybridization, ploidy manipulation and
modification through somaclonal variation.

Leaf-disc Transformation: Is a procedure to obtain transgenic plants. Leaf explants are

infected with Agrobacterium and then the leaf explants are subjected to genetic selection
using antibiotic resistant genes and allowed to undergo organogensis to produce
transgenic plants. Transgenic plants arise only when the cells that are being selected due
to their new genetic background are also the ones being regenerated.

All functional transformation systems consist of three critical components: Useful genes
(selection and new traits), a method for gene transfer and a regeneration method
capable of the selection of single, transformed cells and inducing them to become
complete plants.

Transformation forms a useful bridge between more conventional methods of plant

improvement such as breeding and newer technologies based in genomics.
 Source Materials
Aguero, C.B., C.P. Meredith and A.M. Dandekar. 2006. Genetic transformation of Vitis vinifera L. cvs.
‘Thompson Seedless’ and ‘Chardonnay’ with the pear PGIP and GFP encoding genes. Vitis 45(1): 1-8.
Aguilar, J., J. Zupan, T.A. Cameron and P.C. Zambryski. 2010. Agrobacterium type IV secretion system and
its substrates form helical arrays around the circumference of virulence-induced cells. PNAS 107: 3758-
3763.
Dandekar, A.M. and H.J. Fisk. 2004. Plant Transfromation: Agrobacterium-Mediated Plant Transformation. In
Transgenic Plants: Methods and Protocols. Ed: L. Pena. Pub in Methods in Molecular Biology series by
Humana Press. New Jersey, USA. pp. 35-46.
Dandekar, A.M., G. Teo, S.L. Uratsu and D. Tricoli. 2006. Apple (Malus x domestica). In: Agrobacterium
Protocols. Ed: L. Wang. Pub in Methods in Molecular Biology vol. 344 by Humana Press. New Jersey, USA.
pp. 253-261.
Gelvin, S.B. 2009. Agrobacterium in the genomics age. Plant Physiol. 150: 1665-1676.
Khanna, H.K., J-Y. Paul, R.M. Harding, M.B. Dickman and J.L. Dale. 2007. Inhibition of Agrobacterium-
induced cell death by antiapoptotic gene expression leads to very high transformation efficiency of banana.
MPMI 20: 1048-1054.
Leslie, C.A., S.L. Uratsu, G.H. McGranahan, J. and A.M. Dandekar. 2006. Walnut (Juglans). In:
Agrobacterium Protocols. Ed: L. Wang. Pub in Methods in Molecular Biology vol. 344 by Humana Press.
New Jersey, USA. pp. 297-307.
Vega, J.M., W. Yu, A.R. Kennon, X. Chen and Z.J. Zhang. 2008. Improvement of Agrobacterium-mediated
transformation of Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep. 27: 297-305.
Yuan, Y-C., E. Haudecoeur, D. Faure, K.F. Kerr and E.W. Nester. 2008. Comparative transcriptome analysis
of Agrobacterium tumefaciens in response to plant signal salicylic acids, indole-3-acetic acid and g-amino
butric acid reveals signaling cross-talk and Agrobacterium-plant co-evolution. Cellul. Microbiol. 10: 2339-
2354.
Ziemienowicz, A. 2010. Plant transgenesis. Meth. Mol Biol. 631: 253-268