2024 Plant Biotechnology Post Doctoral Oral Presentation Competition

First Place

Agrobacterium T-DNA Genes as Tools to Promote Regeneration of Transgenic Woody Plants

Barriers to transformation continue to hamper application of recombinant DNA-based biotechnologies in most crops, including for gene editing. There has been a great deal of excitement about the use of developmental regulator (“DEV”) genes as parts of transformation systems in recent years, with striking improvements in monocot transformation as a result, though dicot systems have also seen improvements. We have been studying the extent to which a variety of DEV genes can aid the regeneration of transgenic tissues from clonally propagated forest trees, with a focus on poplar and eucalypts.  Generally, the results have been disappointing; the genes have variously helped or inhibited transformation, and effects vary widely with gene source, expression, and plant genotype. We have obtained better and more genotype-independent results with genes from Agrobacterium. Using a set of 6 genes from the T-DNA of a little used “shooty” strain first discovered by researchers at INRA in France in the 1990s, we developed a co-transformation (“altruistic”) system where these genes promote the recovery and rate of regeneration of transgenic poplars. This method was more efficient (2.3x) at regenerating transgenic shoots on a per explant level and reduced time to shoot production by six weeks relative to the conventional approach. Resulting shoots did not integrate the Agro genes and were phenotypically normal. Deletion testing revealed that the hormone biosynthesis genes alone were insufficient to induce altruistic shoot production in poplar, and introduction of premature stop codons in each gene identified 6B as the major factor required for non-cell autonomous shoot proliferation. Our results suggest that sets of “DEV” genes from Agrobacterium can be powerful tools, and when used altruistically avoid integration of DEV genes into the genome. Further exploration of the many still uncharacterized Agrobacterium strains and T-DNA genes is likely to be fruitful for discovery of new and useful DEV genes. 

Greg S. Goralogia, Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR. Abstract presentation P-1001

Second Place

Seed Plastids: A Novel Platform for Recombinant Protein Expression

Recombinant proteins expressed in chloroplasts should be immediately processed because the leaves are perishable. Here, we report that recombinant proteins can be expressed in seed plastids where they are well protected. We produced three reporter proteins in tobacco seed plastids: GFP, the green fluorescence protein; mScarlet, a variant of red fluorescent protein, and GUS, or β-glucuronidase. First, we introduced the reporter genes in the plastid genome with the strong rRNA operon promoter and an engineered binding site of the PPR10 RNA binding protein in the 5’-UTR. Seed-specific expression in plastids was ensured by transcription of the plastid-targeted PPR10^GG RNA binding protein gene from the napin A promoter in the nucleus. For seed expression the nucleus of transplastomic GFP lines was transformed with the PnapA:PPR10^GG gene, and independent transgenic lines were screened for seed-specific accumulation of reporter proteins. GFP accumulation was detected only in two (#4, #5) out of 17 independently transformed lines. The active PnapA:PPR10^GG #4 allele was then introduced into the transplastomic mScarlet and GUS backgrounds. GFP, mScarlet, and GUS accumulated at 780, 770, and 26 µg/kg^-1 in the seed. Accumulation of recombinant proteins was correlated with PPR10^GG accumulation. Combination of the seed-specific nuclear PPR10^GG gene with a cognate RNA binding site in the 5’UTR of plastid mRNAs enabled the accumulation of recombinant proteins in seed plastids. Seed plastids are a suitable compartment for metabolic engineering without interfering with plant development, and for the accumulation of non-glycosylated proteins, making seed plastids a useful source of vaccine antigens in animal feed.

Malihe Mirzaee, Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854-8020. Abstract presentation P-1003

Third Place

Leaf Transformation of Different Maize (Zea mays L.) Genotypes and Elite Inbred Lines

Maize is one of the most important model crops for genetics and biotechnology research. It is used as livestock feed as well as for human consumption. Increasing populations and climate change require maize improvement by the introgression of desirable traits into agriculturally important genotypes. Genetically modified maize development is extremely challenging due to genotype-related recalcitrance to transformation. Transformation of immature embryos is the most widely used method for maize. It is a very labor-intensive method that requires a high level of expertise and efficient greenhouse facilities are needed for growing maize year-round. However, leaf transformation of maize has been recently reported. Unlike maize embryo transformation, leaf transformation uses young seedling leaves, which are more readily available than embryos. The approach utilizes ectopic expression of growth-stimulating morphogenic genes during the early stages of leaf transformation. Our key interest is to study the response of different maize genotypes and helper plasmids. For this study, we used a previously published leaf transformation method, which utilizes a construct containing a maize-optimized Baby Boom (Bbm) and Wuschel2 (Wus2) developmental gene combination (Wus2/Bbm). The construct also includes a green fluorescent protein (GFP) gene for assessing transformation progress. Overall in this presentation we would like to showcase the response of different genotypes and a publicly available helper plasmid to maize leaf transformation.

Ritesh Kumar, Boyce Thompson Institute, Ithaca, NY 14853. Abstract presentation P-1002