To support the Society’s vision to encourage education and scientific informational exchange and recognize outstanding post docs, the Plant Biotechnology Section held a Plant Biotechnology Post-Doctoral Oral Presentation Competition on Monday June 7th, 2021. A panel of judges evaluated the presentations using the following criteria: experimental design, data analysis, proper interpretation of the results, originality of the study, technical difficulty, appearance and ability of the post-doctoral candidate to present their research. The judges were Viktoriya Coneva (CTC Genomics), Tejinder Mall (Inari Agriculture), and Massimo Bosacchi (KWS). Andika Gunadi from Boyce Thompson Institute received the first place for his talk “Illuminating the Functional Components of Pyrenoid-based Carbon-concentrating Mechanism in Hornworts (Anthocerophyta) using Fluorescent Fusion Protein and Inducible Systems”. The second place went to Ayman Eid from the University of Florida for his talk titled “Turning Up the Heat on Editing of the Highly Polyploid Sugarcane Genome”; and the third place went to Kriaa Dorsaf from Tunisian Bank of Gene for her talk on “In Vitro Culture of Arbutus unedo L.: Micropropagation and Callus induction”.
Submitted by Carlos M. Hernandez-Garcia
Illuminating the Functional Components of Pyrenoid-based Carbon-concentrating Mechanism in Hornworts (Anthocerophyta) Using Fluorescent Fusion Protein and Inducible Systems
Photosynthesis in many species of hornworts (a lineage of bryophytes) and algae is enhanced through the pyrenoid: a specialized CO2 concentrating compartment inside the chloroplast with aggregated RuBisCo. Although unique among land plants and relevant for agricultural crop improvement, pyrenoid-based carbon-concentrating mechanism (CCM) in hornworts is greatly understudied compared to C4 and CAM that are present in some vascular plants. To elucidate the genetic basis of hornwort CCM, we compared the protein sequence and transcriptome profiles of the model hornwort Anthoceros agrestis (having constitutive pyrenoid) and the model algae Chlamydomonas reinhardtii (having inducible pyrenoid), and identified more than six putative hornwort CCM-related genes. Next, to facilitate functional studies and to rapidly determine the protein localization of these genes, we established a particle bombardment-mediated transient expression assay using hornwort elongation factor-1A promoter regulating both a RuBisCo small subunit coding sequence fused with mScarlet (RbcS::mScarlet), and a low-CO2 inducible protein homolog coding sequence fused with mVenus (LCIB::mVenus). Within 7d post bombardment, >500 transiently expressing A. agrestis gametophyte cells were observed per bombarded plate, with RbcS::mScarlet fluorescence localized consistently within the pyrenoid, while LCIB::mVenus fluorescence consistently enveloped the pyrenoid and outlined the chloroplast membrane. Growth medium conditions for A. agrestis tissues were successfully optimized to reduce the time and increase the efficiency of recovering stably transformed fusion protein overexpression lines. Additionally, we recently discovered that pyrenoid formation in A. fusiformis, a close relative of A. agrestis, is inducible in vitro under low CO2 conditions. Efforts are underway using both A. agrestis and A. fusiformis to illuminate the functions of other hornwort CCM-related genes, paving the way towards engineering pyrenoids as an alternative strategy to boost photosynthesis in other land plants.
Andika Gunadi, Boyce Thompson Institute, Ithaca, NY. In Vitro Cellular and Developmental Biology, 57:S35-36 2021
Turning Up the Heat on Editing of the Highly Polyploid Sugarcane Genome
Gene editing is a very promising technology for the improvement of polyploid crops like sugarcane (2n=10–13x=100–130). However, the high level of genetic redundancy in polyploids requires co-editing of multiple alleles to create loss of function phenotypes. Different variants of the RNA guided nuclease Cas9 (e.g. NgCas9) display increased range of target sites. The relaxed PAM-site requirement of NgCas9 can be useful for co-editing of multiple alleles. The objectives of this work are to explore if co-editing of multiple target copies/alleles can be elevated by: 1. Heat treatment of tissue cultures after the delivery of editing reagents; 2. Compare the co-editing efficiencies of NgCas9 and Cas9 and 3. Compare the co-editing efficiencies with monoscistronic or polycistronic expression of 2 sgRNAs. This evaluation was accelerated by targeting magnesium chelatase (MgCh) a key gene in chlorophyll biosynthesis. Knock out mutations of multiple copies/alleles of MgCh result in chlorophyll depletion which can be scored during regeneration of plantlets from tissue culture. Plant expression constructs carrying two sgRNAs in monocistronic or polycistronic configuration, nptII selectable marker and Cas9 or NgCas9 expression cassettes were generated with a combination of conventional cloning and Golden Gate assembly. Both single guides RNAs (sgRNAs) used in the study were validated with an In vitro assay. Each recombinant DNA construct was transferred to embryogenic sugarcane calli via biolistic gene transfer. The regenerated plantlets were analyzed phenotypically for chlorophyll depletion. PCR amplicons from events with chlorophyll depletion were analyzed for restriction site loss by gel electrophoresis, followed by Sanger sequencing and next generation sequencing. We will present data comparing gene editing efficiencies of CRISPR/Cas9 or CRISPR/NgCas9, the effect of heat treatment and incubation at standard temperature and the response to two monoscistronic or polycistronic expressed sgRNAs. The resulting protocol allowed efficient multi-allelic editing in sugarcane.
Ayman Eid, Agronomy Department, University of Florida and DOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL. In Vitro Cellular and Developmental Biology, 57:S35, 2021
In Vitro Culture of Arbutus unedo L.: Micropropagation and Callus Induction
Arbutus unedo L. is a species of strawberry tree, widely represented in the Mediterranean region and western europe. In addition to its use as an ornemental tree, for alcoholic drinks, jams and marmalades it knows an increasing interest because of its chemical and pharmaceutical uses. Micropropagation can be a multiplication technique that can helps to meet the growing demand for this species. For this purpuse we develop an In Vitro propagation protocol in order to encrease multiplication rates of Arbutus unedo L. Nodal explants were cultured on MS (Murashige and Skoog, 1962) media supplemented with different hormonal combinations. The medium wich induce the best axillary budding rate was MS medium (Murashige and Skoog, 1962) supplemented with 5 µl/l Zeatin and 1 µl/l NAA with a percentage of 88.88%. Rising the concentration of Zeatin to 10 µl/l or the concentration of NAA to 2 µl/l seems to reduce axillary budding rate. The emergence of axillary buds was observed from the third week of culture.
Dorsaf Kriaa, Tunisian Bank of Gene. Boulevard du Leader Yesser Arafet, Tunis, TUNISIA. In Vitro Cellular and Developmental Biology, 57:S35, 2021