EBB1, an AP2/ERF Transcription Factor, Promotes Transgenic Shoot Development in Populus Joseph Ree 3/15/12

Overview Background Methods Results Discussion Conclusion Acknowledgements

Overview Background Methods Results Discussion Conclusion Acknowledgements

Introduction to transformation Transformation is the transfer of genes across kingdoms Allows 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 segment Flask of Agrobacterium

Regeneration: growing whole plants from a single cell Plant cells are able to dedifferentiate and divide to become a new plant Media containing sugar, water, and hormones promote regeneration Cell growth, division, and differentiation is based on gene expression

Antibiotics in plant tissue culture limit 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 valuable Many 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 regeneration Increased 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 growth Gibberellic 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

Overview Background Methods Discussion Conclusion Acknowledgements

Inserted Genes GA20ox7 or EBB1 p409S t409S pNOS 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 design Three transformation experiments per genotype ◦ 717 experiments began in September 2010 ◦ N-1 experiments began in December 2010 five one-month-old trees were harvested for each gene per experiment Between 3 to 7 Petri dishes per explant type

Explant Population (717) ExperimentEBB1GA20ox7Empty Vector One149/172104/141114/114 Two144/126129/122145/133 Three167/148169/98140/134 Combined 460/446400/361399/381 # Leaf explants / # Stem explants Stem:1,188 Leaf:1,259 Total:2,447 explants

Explant Population (N-1) ExperimentEBB1GA20ox7Empty Vector One98/11957/14367/35 Two100/95118/111138/100 Three137/156153/126124/98 Combined 335/370328/380329/233 # Leaf explants / # Stem explants Stem:983 Leaf: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 Formation All explants placed on media with cytokinin to promote growth of shoots Shoot growth was determined at 30 days after placed on media containing cytokinin Explants with clearly defined shoots were counted ◦ Yes/no

Shoots per Explant Explants were transferred to shoot elongation media ◦ Lesser cytokinin concentration ◦ Number of shoots per explant counted after 30 days Evaluated using a known 10mm mark on a scalpel

PCR Verification Two shoot samples per explant were randomly selected for PCR analysis ExperimentEBB1GA20ox7Empty Vector One138/7758/3761/62 Two72/6944/2361/104 Three26/8169/4588/35 Combined236/227171/105210/201 # Leaf samples/ # Stem samples EBB1, GA20ox7, kanamycin Stem:533 Leaf:983 Total:1,516 PCR samples

Statistical approach All 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

Overview Background Methods Results Discussion Conclusion Acknowledgements

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) AA A A B A 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 ExperimentConstruct EBB1GA20ox7Empty Vector One80.57%48.94%49.12% Two82.11%63.11%44.36% Three16.22%66.33%64.18% Pooled Average59.60%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 stage No further data could be taken on N-1 regeneration ◦ no shoots regenerated even after several months Highly 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

Overview Background Methods Results Discussion Conclusion Acknowledgements

Summary of Trends EBB1 Explant Callus (N-1) Callus (717) Shoot (717) Shoots/ Explant (717) Transgenic /Escape (717) Leaf Stem GA20ox7 Explant 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 regeneration EBB1 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 plants Unfavorable 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 design Increased number of repetitions Reconsider genes ◦ Would other genes be more effective? Using the same genes ◦ p409S promoter constitutively expressed ◦ Tailor promoter to target meristem formation

Overview Background Methods Discussion Conclusion Acknowledgements

Conclusions Regeneration 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

Acknowledgements Steve Strauss Cathleen Ma Liz Etherington Students ◦ Faye Sanders ◦ Heather Hilligas Wanda Crannell