Research Advances in the Development of Transgenic and Gene Edited Products in Sri Lanka Dr. Pradeepa C.G. Bandaranayake Director, Agricultural Biotechnology Centre Faculty of Agriculture University of Peradeniya
teosinte Early cultivated maize Crop biotechnology has a long history Tomato ancestor (5 g) Beefsteak (1000 g!) Modern maize History of crop improvement By trial and error for almost 9900 years By scientific principles of breeding for last 100 years By chemical induced mutation for last 75 years By rdna technology last 20-25 years By gene editing less than 10 years
Regulatory framework in Sri Lanka - not ready yet! No transgenic plant moved beyond planthouse No gene edited plant - moved beyond tissueculture lab While waiting for the regulatory framework Methods are being optimized New protocols are developed Some scientists are waiting Some are moving away More research investments on Marker assisted breeding Genomics Transcriptomics Wild relatives!
Source: Sanwen Huang/ Chinese Academy of Agricultural Sciences
Department of Agriculture Sri Lanka Encourages Mutation Breeding Several Programs on Major Crops Vegetables Fruits Rice
Double Strand DNA Breaks Naturally Crossing over during meiosis In somatic cells in response to irradiation, UV-light, chemicals
What if we could decide and direct double strand breaks? What do we need? Ability to recognize a specific DNA sequence Ability to cleave DNA a nuclease CRISPER/Cas9 RNA recognizes DNA Need a way to get following components into plant cells Cas9 protein Guide RNA(s) Donor template DNA Regenerate a plant from that cell
Methods for gene editing Transgenic methods Ex: Using plant transformation constructs/vectors encoding both Cas9 gene and Guide RNA(s) Eliminate the transgenes from the edited plants Non-transgenic methods Insert Cas9 protein and Guide RNA(s) into the cells Regenerate a whole organism from the edited cell The proteins and RNAs are naturally destroyed Available methods Particle bombardment Protoplasts Viral mediated Many advantages of using non-transgenic methods
Particle Bombardment Particle bombardment of Cas9 protein, guide RNA(s) and donor template followed by regeneration of whole plants
Protoplast Isolation, Transfection and Regeneration Seed germination 18 o C 2 weeks old seedlings Protoplast Isolation Microscopy and assay Growth at 24 o C Incubation at 18 o C
Methods optimized for soybean
Methods are optimized for tomato and chili Vector Control Gene silencing-ribosomal16_7
Methods optimized for Duckweeds
Exploring the possibility of utilizing genes from wild relatives The genus Oryza has 24 species only two cultivated Rest - 22 species are wild - o5 in Sri Lanka One endemic to Sri Lanka - Oryza rhizomatis has branched rhizome with thick root system perennial drought resistant, pest resistance
Non Food Crop No wild relatives in Sri Lanka Gerbera hybrida Dream!!!
Selected DFR and F3 5H Full length genes were PCR amplified Cloned into the pdonr221 and then to the pk7wg2.0 Agrobacterium mediated transformation Vitis vinifera Clitoria ternatea
What is next? Some regulatory genes MYB transcription factors Gene editing promoter
Developed a tissueculture protocol Biolistic transformation Regeneration Phenotype GUS Assay