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.

Devising an Innovative, Genotype-free Transformation Protocol for Recalcitrant Indica Rice Genotypes: Samba Mahsuri and Sundarbans’ Salt-tolerant Indica Rice Cultivars White Getu and Hamilton to Establish an Ingenious Platform for CRISPR/Cas9-mediated Genome Editing

BackgroundPlant genetic transformation involves in vitro callus induction & regeneration strategies that are quintessential for introduction of novel agronomical traits employing CRISPR/Cas9-based genome editing. However, lack of effective regeneration and transformation techniques for indica rice cultivars pose as the foremost hurdle towards genetic improvement in rice crop. We devised an astounding road-map to genotype-independent and efficacious in vitro callus induction, transformation and shoot regeneration protocol that emerges as an optimal therapy, universally adaptable to invariably any rice cultivar, in order to establish an ingenious CRISPR/Cas9-based genome editing platform in this crop.ResultsWe developed a genotype-independent regeneration and transformation protocol employing mature seed-derived calli for indica rice (one mega variety- Samba Mahsuri and two salt tolerant wild genotypes- White Getu & Hamilton) genotypes to introduce important agronomical traits via CRISPR/Cas9-based genome editing system. MS- and N6-salt based media reinforced with 2,4-D (2.5 mg/L); dicamba (1.5 mg/L); TDZ (0.1 mg/L), proline (1000 mg/L), and glutamine (2.5 mg/L) exhibited highest percentage (95-98%) of embryogenic calli initiation and development. Employing this novel protocol, we achieved unparalleled regeneration efficiencies within untransformed calli (90-94%) and transformed calli (81-86%) in these recalcitrant indica genotypes and significantly enhanced number of shoots (18-20) on MS medium containing BAP (1.5 mg/L), NAA (0.5 mg/L), TDZ (1.0 mg/L), zeatin (0.2 mg/L) and proline (500 mg/L). We successfully transformed rice calli with pCAMBIA1300-based marker- free NICTK-1_pCRISPR-Cas9 vector harbouring the cassette of plant codon optimized Cas9 via biolistic approach that exhibited notably enhanced transformation efficiencies (67-69%). The integration of Cas9 gene into rice genome was validated by PCR, Southern blotting and Sanger sequencing analyses. The transgenic lines were phenotypically indistinguishable from the wild type as no significant differences in phenotypic performances were revealed between transgenic and wild type lines. ConclusionWe devised a promising, time-efficient, universally adaptable, optimal hormonal-media therapy for triggering enhanced embryogenic callus formation, regeneration and transformation efficiencies, across recalcitrant indica rice genotypes.

Xerotolerance: A New Property in Exiguobacterium Genus

The highly xerotolerant bacterium classified as Exiguobacterium sp. Helios isolated from a solar panel in Spain showed a close relationship to Exiguobacterium sibiricum 255–15 isolated from Siberian permafrost. Xerotolerance has not been previously described as a characteristic of the extremely diverse Exiguobacterium genus, but both strains Helios and 255–15 showed higher xerotolerance than that described in the reference xerotolerant model strain Deinococcus radiodurans. Significant changes observed in the cell morphology after their desiccation suggests that the structure of cellular surface plays an important role in xerotolerance. Apart from its remarkable resistance to desiccation, Exiguobacterium sp. Helios strain shows several polyextremophilic characteristics that make it a promising chassis for biotechnological applications. Exiguobacterium sp. Helios cells produce nanoparticles of selenium in the presence of selenite linked to its resistance mechanism. Using the Lactobacillus plasmid pRCR12 that harbors a cherry marker, we have developed a transformation protocol for Exiguobacterium sp. Helios strain, being the first time that a bacterium of Exiguobacterium genus has been genetically modified. The comparison of Exiguobacterium sp. Helios and E. sibiricum 255–15 genomes revealed several interesting similarities and differences. Both strains contain a complete set of competence-related DNA transformation genes, suggesting that they might have natural competence, and an incomplete set of genes involved in sporulation; moreover, these strains not produce spores, suggesting that these genes might be involved in xerotolerance.

Application of polymeric dimethylaminoethyl methacrylate based carriers of plasmid DNA for genetic transformation of Ceratodon purpureus moss

Introduction. Genetic engineering in plants is of great importance for agriculture, biotechnology and medicine, and nanomaterials are widely used for genetic engineering. The aim of present study was to evaluate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based comb-like polymers as gene delivery systems in moss Ceratodon purpureus (Hedw.) Brid. protoplasts and determine the level of phytotoxicity of these polymers. Materials and Methods. In order to confirm the formation of complex of poly-DMAEMA carrier with plasmid DNA pSF3, gel retardation assay was used. The PEG-mediated transformation protocol was adapted to transform the protoplasts of C. purpureus moss with poly-DMAEMA carriers. Light microscopy was used to study a toxicity of polymers for moss protoplasts. The level of the polymers toxicity was estimated as IC50 value. Results and Discussion. The formation of pDNA complex with DMAEMA-based carriers took place at 0.03% concentration of the polymers BGA-21, BGA-22(2ph), BG-24, BG-25, BG-26 or 0.1% concentration of the BGA-22 polymer. Poly-DMAEMA carriers were able to deliver plasmid DNA pSF3 into protoplasts of C. purpureus moss. Three stable transformants of C. purpureus were obtained at using BGA-22 polymer, 2 clones – at using BGA-21 carrier, and 1 clone – at using BGA-22(2ph), BG-24, BG-25, BG-26 polymers. The poly-DMAEMA carriers at the working 0.0025% dose were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-88.4% of viable protoplasts of C. purpureus moss were detected after treatment with studied carriers at 0.0025% dose. A survival ratio of protoplasts reached 66.7-72.9% under the effect of these polymers at 0.025% dose, which is 10 times higher than their working concentration. The IC50 value of poly-DMAEMA carriers was in the range of 0.113-0.164%, that was approximately 10 times higher than that of the PEG-6000 used for gene delivery in plants. Conclusion. Novel synthetic poly-DMAEMA carriers delivered the gene of interest into moss C. purpureus protoplasts and possessed a low phytotoxicity. Thus, these carriers can be useful for gene delivery into plant cells.

Improving Adventitious Shoot Regeneration and Transient Agrobacterium-Mediated Transformation of Apricot (Prunus armeniaca L.) Hypocotyl Sections

The improvement of previously described protocols for the regeneration of shoots from ‘Canino’ mature seed hypocotyl slices has been accomplished. The effects of different factors such as the part of the hypocotyl used, vacuum-infiltration, 2,4-Dichlorophenoxyacetic acid pulse, vacuum-infiltration and sonication on regeneration and transient transformation were analyzed. When the three slices obtained from the hypocotyls were evaluated separately on regeneration medium, the highest percentages of regenerating explants were achieved in the part close to the epicotyl and in the central part. On the other hand, sonication of the explants for 30 s followed by vacuum-infiltration during Agrobacterium infection for 20 min allowed for an increase in the transformation events. The application of these modifications to the procedure increased the regeneration efficiencies, and transient transformation events and may reduce the frequency of failed experiments. An efficient regeneration/transformation protocol could facilitate its use as a biotechnological technique for apricot breeding.

Organogenesis and Sonication-Assisted Agrobacterium-Mediated Transformation of Poplar Roots

Functional genomics along with genetic transformation and plant regeneration are used to identify genes of interest, comprising essential tools to obtain plants with desired characteristics. In this study, we described an organogenesis and a genetic transformation protocol for Populus tremula x Populus alba clone 717-1B4 using roots. In the organogenesis experiments the PGRs zeatin, BA and kinetin at different concentrations were evaluated. The effect of explant age was evaluated and at 30 days “old” roots (6 months old) showed a higher regeneration rate when compared to “young” roots (2 months old). In the genetic transformation experiments kanamycin concentration, the use of sonication (SAAT), co-cultivation period and explant age were evaluated. Sonication positively affected transformation, while co-cultivation time did not interfere. Regarding explant age, no statistical differences were observed. Quantitative real-time PCR (qPCR) data showed that among the six transgenic plants evaluated, two presented two copies and the others only one copy of the gene. In this study, efficient protocols of organogenesis and genetic transformation for poplar roots are presented with a transformation efficiency of 58%.

Two Areas and One Passage: A Simplified Protocol for the Transformation of General Hospitals to Infectious Disease Hospitals Upon the Pandemic Outbreak

Infectious disease hospitals and wards fell far short of demand after the COVID-19 pandemic outbroke. Consequently, it was of urgent importance to transform existing general wards into temporary infectious disease wards for COVID-19 patients. However, due to the limitations of the original structure of public hospitals, general wards could not meet the structural criteria requiring “three areas, two passages, and two hallways” for receiving infectious disease patients. In this article, we present a “Two Areas and One Passage” reconstruction and working protocol, which has been proved to be safe and easy to achieve by medical staff during the epidemic of COVID-19 in 2020. This protocol tremendously helps to save workforce and personal protective equipment compared with standard transformation protocol and thus might be used as an exemplary protocol for the transformation of general hospitals to infectious disease hospitals upon pandemic outbreak. Besides, it also provide a new scope for us to transform the general hospital to military infectious disease hospital if there occur abundant infectious disease suffers in the battlefield.