EBB1, an AP2/ERF Transcription Factor, Promotes Transgenic Shoot Development in PopulusJoseph Ree3/15/12

OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements

OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements

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

Agrobacterium is a natural plant genetic engineer

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

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

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

Summary of Regeneration

Callus on stem explant

Shoot beginning to form

Clearly defined shoot

Rooted plant

Selection

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

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

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

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

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

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

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

OverviewBackgroundMethodsDiscussionConclusionAcknowledgements

Inserted Genes

GA20ox7 or EBB1 p409St409

SpNOS NPTII (Kanamycin)

tNOS

pNOS NPTII (Kanamycin) tNOS

Empty Vector

Tree Genotypes

Model poplar hybrid Populus tremula x P. alba (clone INRA 717-1B4)

• Recalcitrant Populus trichocarpa (N-1).(Sequenced Genome)

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

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

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

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

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

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

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

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

OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements

Genes had no beneficial impact on callus formation (717)

•Overall, no beneficial impact of either tested gene

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%

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%

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

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

Genes improved the number of shoots per explant (717)

•EBB1 increased shoots per explant by 2.14 for leaf and 1.52 for stem

Genes had distinct differences on transgenic shoot recovery (717)

•EBB1 proportion of transgenic shoots increased by 50.8% in leaf•GA20ox7 heavily promoted escapes

OverviewBackgroundMethodsResultsDiscussionConclusionAcknowledgements

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

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

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

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

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

OverviewBackgroundMethodsDiscussionConclusionAcknowledgements

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

AcknowledgementsSteve StraussCathleen MaLiz EtheringtonStudents

◦Faye Sanders◦Heather Hilligas

Wanda Crannell