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Ganeshan Sivanandhan,Sol hee Bae,Chae min Sung,Su Ryun Choi,Geung-Joo Lee,Yong Pyo Lim 한국원예학회 2021 한국원예학회 학술발표요지 Vol.2021 No.10
Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and a lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transient transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. Unfortunately, there is no standardized method for Chinese cabbage. Hence, in this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were optimally isolated following a 4-h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) Macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl₂, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 10<SUP>6</SUP> protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with 40 μg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study will be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.
Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell
Ganeshan Sivanandhan,Chinnathambi Arunachalam,Venkatachalam Vasudevan,Gnanajothi Kapildev,Ali Alharbi Sulaiman,Natesan Selvaraj,Andy Ganapathi,임용표 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.2
Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day precultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD600, infection time of 6 min, AS concentration of 150 lM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.