This year’s In Vitro Biology meeting featured an oral presentation competition for Plant Biotechnology Students. Presenters were evaluated on experimental design, data analysis, proper interpretation of the results, originality of the study, technical difficulty, and presentation skills. Our expert panel of judges, Randall P. Niedz, Ajith Anand and Carlos Manuel Hernandez, were impressed with all of the contestants knowledge and preparation. They recognized Stephen Jinga (University of Illinois Urbana-Champaign) with the 1st place award, Mst Shamira Sultana (The University of Tennessee) with the 2nd place award, and Chi L. Nguyen (University of Florida) with the 3rd place award. The winners were presented with a certificate and a cash award at the meeting. We encourage all plant biotechnology students to consider this opportunity to develop their presentation skills at future meetings.
Submitted by Veena Veena
Multiplex Genome Editing in the Illinois Long Term Selection Experiment
The Illinois Long Term Selection Experiment (ILTSE) is a unique maize germplasm resource for studies of genome evolution and genetic variants that contribute to phenotypic traits. Moreover, some of the ILTSE genotypes create highly regenerable embryogenic type I callus, which enables transformation and genome editing approaches to characterize gene function. Here, we describe results from site-specific mutagenesis by CRISPR-Cas9 in Illinois Low Protein (ILP). We have initially targeted two genes of interest: Lemon White 1 (Lw1) and L-Asparaginase (ASNase). The Lw1 experiments were performed as a proof of concept to generate albino plants easily detectable in a population of regenerated plants, while ASNase experiments attempt to validate the contribution of this gene to the phenotypic variation for grain protein concentration observed in the ILTSE. For each gene, we designed multiple guide RNAs and tested their function using an in vitro Cas9 cleavage assay. Plasmids were created where Cas9 and ribonuclease Csy4 were controlled by the maize ubiquitin 1 promoter and an array of four guide RNAs separated by Csy4 spacers were expressed as a single transcript driven by the strong viral Cestrum (CmYLCV) promoter. This strategy allows us to minimize plasmid size and for multiplexed guideRNAs. Vectors were delivered to embryogenic calli using biolistics in the absence of morphogenic regulators, and transgenic events selected. Multiple mutant albino plants indicative of biallelic mutations were recovered from the Lemon White 1 experiment in both the ILP and H99 control genotypes at ~2% efficiency. DNA sequencing demonstrated the creation of various types of mutant alleles. Screening of ASNase events is ongoing with a population of over 100 regenerated plants. Our experiments demonstrate the first CRISPR-Cas9-Csy4 multiplexed editing in maize, which serves as a platform for continuing experiments in studying kernel composition and nitrogen use in the ILTSE.
Stephen J. Jinga, University of Illinois Urbana-Champaign, Urbana, IL. In Vitro Cellular and Developmental Biology, 55:S35, 2019
Development and Validation of a Novel Soybean (Glycine max (L.) Merr.) Cell Suspension Culture for High-throughput Promoter Screening
Transient reporter gene assays can be valuable to rapidly estimate expression characteristics of heterologous promoters. The challenge for maximizing the value of such screens is to combine relevant cells or tissues with methods that can be scaled for high throughput screening, especially for crop- rather than model species. We describe herein a novel leaf-derived soybean cell suspension culture (LDSC) that is amenable for a low-cost method for protoplast isolation, which was used for screening endogenous promoters. LDSC-derived protoplasts were validated against known promoter expression profiles from tissue-derived protoplasts (leaves, stems and immature cotyledons). LDSCs can reliably produce 2.82 ± 0.94×108 protoplasts/g fresh culture mass with a transfection efficiency of 30.65 ± 2.70% at 48 hours after incubation. Transfected LDSC-derived protoplasts harboring promoter-reporter gene DNA expression levels were highly relevant to that of leaf- and stem-derived protoplasts (correlation coefficient of 0.99 and 0.96, respectfully) harboring the same constructs. LDSC expression was also significantly correlated to endogenous promoter-gene expression in leaf tissues as measured by qRT-PCR analysis (correlation coefficient of 0.80). In summary, a reliable leaf-derived soybean cell suspension culture was developed that enables low-cost, facile protoplast isolation and transformation, a necessary requirement for automation of heterologous promoter screening for use in soybean biotechnology.
Mst Shamira Sultana, University of Tennessee, Knoxville, TN. In Vitro Cellular and Developmental Biology, 55:S35, 2019
Development of Variegated Lettuce Using CRISPR/Cas9 Technology
The need for gene edited plants to combat issues of growing population rate, extreme weather, and reduced agricultural land availability is more evident than ever as growers are struggling to adapt to the changing environment. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has emerged as a powerful approach for precision breeding to create plants with desirable traits. However, gene editing efficiency of CRISPR/Cas9 in plant relies on the efficacy of generating transgenic plant or tissues with high expression of Cas9 endonuclease. Here, we have constructed a CRISPR-Cas9 vector containing a fused NPT-GFP for high-efficiency gene-editing in annual flowering plants. The high expression of GFP during plant regeneration allowed us to minimize the positional effect on T-DNA expression and preferentially select transgenic seedlings with high expression of Cas9. With this construct, we have targeted the variegation gene LsVar2 in lettuce. Forty independent lines were generated, 27 of which showed strong GFP signals. Six of 27 T0 transgenic lines with GFP signals exhibited variegated leaves. Albino seedlings were observed in segregated progenies of these variegated lines, and sequencing results revealed that homozygous mutations created by Cas9 editing occurred to all these albino seedlings. By contrast, heterozygous mutations were only identified in the plants with variegated leaves and significantly reduced chlorophyll content, and these plants were able to grow and mature normally. Therefore, in addition to confirming the efficiency of the CRISPR-Cas9 vector, our data also provide a proof-of-concept for targeting VAR2 gene in ornamental plants where variegation phenotype is highly favorable. In conclusion, the unique designing of our CRISPR/Cas9 construct allow us to improve gene-editing efficiency and efficiently screen non-TDNA mutants through detecting GFP signals during plant regeneration and progeny segregation.
Chi Nguyen, University of Florida, Apopka, FL. In Vitro Cellular and Developmental Biology, 55:S34-35, 2019