Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

A Novel Method for High-efficiently Obtaining the Transgenic Lines by combining a convenient, rapid and efficient Visual Screening method and an efficient genetic transformation System in B. napus

Background Brassica napus is an important oilseed crop which offer a considerable amount for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few such aforementioned systems available for efficient application in B. napus .Results Based on the well-established genetic transformation system in Brassica napus , five vectors companied with red fluorescence protein from Discosoma sp. ( DsRed ) were constructed and transformed via agrobacterium -mediated hypocotyl transformation. The average of 59.1% tissues were marked with red fluorescence by visual screening method in tissue culture medium, and 96.1% of which were amplified with the exogenous genes in eight different rapeseed varieties on average. In addition, the final transgenic-positive efficiency of the rooted plantlets was up to 90.7% from the red fluorescence marked tissues, which was much higher than that of the previous reports. Besides, DsRed also could be applicable to the seedlings, including of seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the high-efficiency genetic transformation system combining with the transgenic-positive visual screening method presented here will help us to efficiently and conveniently obtain transgenic-positive rapeseed plantlets.Conclusion A novel method was developed for rapidly, conveniently and high-efficiently obtaining the transgenic lines, which would help to obtain the higher proportion of transgenic positive regenerated plantlets, thereby avoiding the long period plant regeneration. This will benefit the gene functional study especially in high throughput molecular biology research.

Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven

Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘White Heaven’, respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino–green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.