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EBB1, an AP2/ERF Transcription Factor, Promotes Transgenic Shoot Development in PopulusJoseph Ree3/15/12
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OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements
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OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements
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Introduction to transformationTransformation is the transfer of
genes across kingdomsAllows technological advances
that would be otherwise impossible◦Human insulin from bacteria◦Crop resistances to disease
Multiple methods for transformation
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Agrobacterium is a natural plant genetic engineer
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Basics of using Agrobacterium for transformation Plasmids carrying the transgenes are inserted into
disarmed Agrobacterium tumefaciens Explants are harvested tissues
◦ Leaf disks◦ Stem segments
Explants are submerged into Agrobacterium induction media
Leaf disk
Stem segmentFlask of Agrobacterium
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Regeneration: growing whole plants from a single cellPlant cells are able to dedifferentiate
and divide to become a new plantMedia containing sugar, water, and
hormones promote regenerationCell growth, division, and
differentiation is based on gene expression
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Antibiotics in plant tissue culturelimit growth to engineered cells
•A non-transformed regenerated shoot is an “escape”•Selectable markers limit growth of non-transgenic cells
-Antibiotic resistance
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Summary of Regeneration
Callus on stem explant
Shoot beginning to form
Clearly defined shoot
Rooted plant
Selection
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Recalcitrance to transformation
Not all plants are practical can be transformed effectively
Natural barriers prevent the use of certain species and cultivars◦ Poor gene uptake◦ T-DNA silencing◦ Poor regeneration
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Past Efforts to Improve Transformation and Regeneration
Increased Agrobacterium virulence◦Virulence factors
Tissue culture environment modification◦Media to facilitate regeneration
Modifying hosts◦Histone and other modification
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Populus is economically and scientifically valuableMany poplar species are
cultivated for wood pulp and paper◦Potential as a biomass crop
Model tree species◦Rapid growth◦Easily propagated◦Genome and gene expression
sequences
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EBB1’s discovery and observations
EBB1 discovered using activation tagging◦ Resumed growth faster
EBB1 encodes a transcription factor that alters gene expression
Distinct phenotype◦ Shoot proliferation◦ Poor rooting ability
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EBB1 potentially improves regenerationIncreased number of shoots per
explant in leaf disk explants were observed in past studies
Similar AP2/ERF genes ESR1 and WIND1 also observed to improve regeneration of tissue in Arabidopsis
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GA20ox7 as a potential regeneration gene
GA20ox7 encodes a highly active enzyme in the later steps of gibberellic acid (GA) production
Increased numbers of transgenic GA20ox7 events were recovered compared to control
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Gibberellic acid’s effect on plant growthGibberellic acid (GA) is a
hormonal regulator that affects many plant processes
Heavily tested for use in agriculture◦Increased wheat and rice harvest◦Improved tree growth
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OverviewBackgroundMethodsDiscussionConclusionAcknowledgements
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Inserted Genes
GA20ox7 or EBB1 p409St409
SpNOS NPTII (Kanamycin)
tNOS
pNOS NPTII (Kanamycin) tNOS
Empty Vector
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Tree Genotypes
Model poplar hybrid Populus tremula x P. alba (clone INRA 717-1B4)
• Recalcitrant Populus trichocarpa (N-1).(Sequenced Genome)
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Experimental designThree transformation experiments
per genotype◦717 experiments began in September
2010◦N-1 experiments began in December
2010five one-month-old trees were
harvested for each gene per experiment
Between 3 to 7 Petri dishes per explant type
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Explant Population (717)
Experiment EBB1 GA20ox7 Empty Vector
One 149/172 104/141 114/114
Two 144/126 129/122 145/133
Three 167/148 169/98 140/134
Combined 460/446 400/361 399/381
# Leaf explants / # Stem explants
Stem:1,188Leaf:1,259
Total:2,447 explants
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Explant Population (N-1)
Experiment EBB1 GA20ox7 Empty Vector
One 98/119 57/143 67/35
Two 100/95 118/111 138/100
Three 137/156 153/126 124/98
Combined 335/370 328/380 329/233
# Leaf explants / # Stem explants
Stem:983Leaf:992
Total:1,975 explants
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Callus Formation
Two days of Agrobacterium growth on explants◦ Infection of plant cells
Agrobacterium is washed off and placed on callus-inducing media◦ Placed in dark for 20 days
Callus growth evaluated based on new growth◦ Yes/No
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Shoot FormationAll explants placed on media with
cytokinin to promote growth of shootsShoot growth was determined at 30
days after placed on media containing cytokinin
Explants with clearly defined shoots were counted◦ Yes/no
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Shoots per ExplantExplants were transferred to shoot
elongation media◦ Lesser cytokinin concentration◦ Number of shoots per explant counted after 30
daysEvaluated using a known 10mm mark on a
scalpel
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PCR VerificationTwo shoot samples per explant
were randomly selected for PCR analysis
Experiment EBB1 GA20ox7 Empty Vector
One 138/77 58/37 61/62
Two 72/69 44/23 61/104
Three 26/81 69/45 88/35
Combined 236/227 171/105 210/201
# Leaf samples/ # Stem samples
EBB1, GA20ox7, kanamycin
Stem:533Leaf:983Total:1,516 PCR samples
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Statistical approachAll data was compiled by
genotype, explant type, construct, and pooled over all experiments
Two-way ANOVA tests were used to compare genes
Each individual explant or shoot sample assumed to be independent◦Explants were not fully randomized
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OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements
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Genes had no beneficial impact on callus formation (717)
•Overall, no beneficial impact of either tested gene
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Genes promoted callus formation on stem explants (N-1)
A A
A A BA
•Callus production was increased by both genes in stem explants• EBB1: 116%• GA20ox7: 76.8%
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Shoot formation outlier removed (717)
•EBB1 third experiment was an outlier, and thus removed
Shoot Formation on 717 Leaf Explants
Experiment Construct
EBB1 GA20ox7 Empty Vector
One 80.57% 48.94% 49.12%
Two 82.11% 63.11% 44.36%
Three 16.22% 66.33% 64.18%
Pooled Average 59.60% 58.50% 52.80%
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EBB1 had a beneficial impact after outlier was removed (717)
•EBB1 (Adjusted) shoot formation increase by 54% in leaf•EBB1 had no effect on shoot production in stem
•GA20ox7 had detrimental effect in stem explants
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N-1 regeneration halted at callus stageNo further data could be taken on
N-1 regeneration ◦no shoots regenerated even after
several monthsHighly recalcitrant, thus
transformation is sporadic
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Genes improved the number of shoots per explant (717)
•EBB1 increased shoots per explant by 2.14 for leaf and 1.52 for stem
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Genes had distinct differences on transgenic shoot recovery (717)
•EBB1 proportion of transgenic shoots increased by 50.8% in leaf•GA20ox7 heavily promoted escapes
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OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements
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Summary of TrendsEBB1
ExplantCallus(N-1)
Callus (717)
Shoot(717)
Shoots/Explant(717)
Transgenic /Escape(717)
Leaf
Stem
GA20ox7Explant
Callus(N-1)
Callus (717)
Shoot(717)
Shoots/Explant(717)
Transgenic /Escape(717)
Leaf
Stem
Greater regeneration
No difference
Lesser regeneration
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EBB1 has a beneficial effect on regenerationEBB1 promoted more shoots with
improved proportion of transgenic shoots in 717
Noticeable improvement in both a model and recalcitrant phenotype◦Advantage given to transgenic cells◦Outcompete escapes for media
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Using EBB1 to improve regeneration of transgenic plantsUnfavorable phenotype
◦Makes EBB1 useful during regeneration only
System to use and then remove EBB1◦Insert ZFN or recombinase with EBB1◦Inducible promoter triggers
ZFN/recombinase synthesis◦EBB1 ceases to function
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GA20ox7 for promoting calli
GA20ox7 increases GA concentrations to non-transgenic cells◦GA can diffuse out of cells◦could promote growth of escapes
Problematic for model 717◦Reduced recovery of transgenic shoots
Potentially useful for recalcitrant genotypes◦Promotes calli growth◦ Increased regeneration with more escapes
still beneficial for recalcitrant
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Recommendations for experimental designIncreased number of repetitionsReconsider genes
◦Would other genes be more effective?
Using the same genes◦p409S promoter constitutively
expressed◦Tailor promoter to target meristem
formation
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OverviewBackgroundMethodsDiscussionConclusionAcknowledgements
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ConclusionsRegeneration genes
outperformed control at certain stages during regeneration
EBB1 potentially useful in both in model and recalcitrant genotypes
GA20ox7 could be useful in promoting callus growth
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AcknowledgementsSteve StraussCathleen MaLiz EtheringtonStudents
◦Faye Sanders◦Heather Hilligas
Wanda Crannell