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, Barbara Reed, Terry Hu, and Sergei Krasnyanski, were impressed with all of the contestants’ knowledge and preparation. They recognized Lauren Erland from the University of Guelph with the 3rd place award, Jameson Coopman from the University of Florida with the 2nd place award, and Andika Gunadi from Ohio State with the 1st 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 Jeffrey Beringer
First Place Award
Soybean (Glycine max) Promoter Characterization Through CRISPRi
Gene expression in all organisms is largely regulated by the promoter, which is located upstream of the gene coding region. The promoter contains a variety of regulatory elements that are recognized by DNA-binding proteins, leading to spatio-temporal regulation of gene expression. Promoters and promoter regulatory elements have typically been studied by evaluating the effects of either promoter truncation or regulatory element modulation on gene expression. In this study, CRISPR interference (CRISPRi) was evaluated as an additional tool for promoter characterization using a constitutive soybean (Glycine max) promoter (GmScreamM8). Through the use of a deactivated Cas9 protein (dCas9), promoter sequence-specific guide RNAs were designed to bind to the targeted promoter sequence, thereby competitively displacing any DNA-binding proteins required for transcription. DNA constructs, containing the native promoter fused with the green fluorescent protein (gfp) gene, as well as dCas9 and target-specific guide RNA cassettes were generated and co-introduced into lima bean (Phaseolus lunatus) cotyledon tissue using particle bombardment. The 6 different guide RNA targets consisted of 4 distinct sequences within the promoter, along with a sequence in the GFP coding sequence, and a sequence in the plasmid backbone. Transient GFP expression of 6 biological replicates per target was monitored for 66-140 hours post-bombardment using a semi-automated image capture platform. Based on image analysis, use of one of the guide RNAs to a target in the promoter region led to a clear reduction in gene expression while many of the guide RNAs were not effective in reducing promoter activity. Among the GFP-expressing cells in the lima bean cotyledons, the epidermal cells showed different expression patterns from the parenchyma cells, which seemed to be less responsive to CRISPRi. These findings represent the first documented promoter characterization using CRISPRi in plants.
Andika Gunadi, Department of Horticulture and Crop Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH. In Vitro Cellular and Developmental Biology, 53:S30, 2017
Second Place Award
Effect of Desiccation Stress and Subsequent Recovery on the In Vitro Growth of the Epiphytic Orchid Dendrophylax lindenii
The Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe, is an endangered leafless epiphytic orchid that is anecdotally considered to be difficult to grow and manage under greenhouse conditions. Successful growth supposedly requires still air and high humidity, but this may not be the case. Recent in vitro, greenhouse, and field observations suggest that the species may exhibit desiccation tolerance. To determine this, the desiccation tolerance and recoverability of D. lindenii was tested. Individual Ghost Orchid plants were randomly placed into sterile, filter-capped baby food jars within air tight chambers maintained at 10% relative humidity using a supersaturated potassium hydroxide salt solution for 1, 2, 3, or 4 weeks. At the end of the desiccation period, the orchids were recovered in vitro on P723 Orchid Sowing Medium + 30 g/L Banana Powder for 4 weeks. Data on root weight, number, and length, as well as number actively growing root tips were collected. Water potential values were collected for each desiccation exposure time. Preliminary results indicate that the Ghost Orchid exhibits high tolerance to desiccating conditions. After 4 weeks at 10% relative humidity, plant survival was 80%. Desiccating conditions overall led to a decrease in viable root number per plant. Of the surviving plants subjected to 4 weeks of desiccation, 63% remained alive after the recovery period and the number of viable roots increased demonstrating a resumption of active growth. Understanding the desiccation tolerance of D. lindenii to long periods of extreme water stress could have positive applications to enhance both conservation and horticulture production of this species. In horticulture, successful propagation could be increased through drier growth and maintenance conditions, and in conservation this desiccation tolerance could facilitate the direct field establishment of plants from culture, avoiding the time and expense of Stage IV greenhouse acclimatization.
Jameson Coopman, Environmental Horticulture Department, University of Florida, Gainesville FL. In Vitro Cellular and Developmental Biology, 53:S30, 2017
Third Place Award
A New Balancing Act: Melatonin and Serotonin as Mediators of Plant Morphogenesis
Melatonin (Mel) and serotonin (5HT) are indoleamines first identified as neurotransmitters in vertebrates; they have now been found to be ubiquitously present across all forms of life. Though Mel and 5HT possess important roles in plant growth and development, their roles in morphogenesis are still poorly defined. We hypothesize that Mel and 5HT function as a novel class of plant growth regulators (PGRs). To investigate this, we used a dual approach: phytochemical analysis and in vitro culture experiments. First, a simple and efficient method for the phytochemical analysis of Mel, 5HT and several established classes of PGRs, was developed for in vitro grown plants. Second, we examined the morphogenetic effects of Mel and 5HT in several plant culture systems including breadfruit (Artocarpus altilis) and St. John’s wort (Hypericum perforatum: SJW). In breadfruit, though Mel had a minimal effect on growth, 5HT appeared to act as both an anti-browning agent and to possess cytokinin-like effects in culture. Particularly, it was found that 5HT (100 µM) could replace kinetin supplementation, in multiplication medium. Our lab possesses unique lines of SJW, a model for the study of Mel and 5HT, with high (L4) and low (L112) endogenous Mel levels, in comparison to wild-type plants. Neither root, nor shoot explants of the three lines showed a significant difference in growth. Grown on media supplemented with Mel, 5HT or their precursors (5, 10 or 30 µM), both root and shoot cultures showed a dose-dependent morphogenetic response, particularly with respect to shoot and root initiation. High levels (10 – 30 µM) of these compounds induced a generally inhibitory effect, while low concentrations (5-10 µM) showed improved growth and regeneration. Additionally, L4 showed inhibition at lower levels than did L112, supporting the dose dependent nature of Mel and 5HT, a defining characteristic of PGRs. Together this research presents a) a platform for the investigation of Mel and 5HT in morphogenesis, and b) suggests Mel and 5HT should be classified as a novel class of PGRs.
Lauren Erland, University of Guelph, 50 Stone Road W, Guelph, ON N1G 2W1, Canada. In Vitro Cellular and Developmental Biology, 53:S13-14, 2017