Research on the genetic transformation system of Canna×generalis

【Objective】This study aimed to establish an Agrobacterium-mediated genetic transformation system for Canna×generalis， providing a reference for further study of gene function study and transgenic breeding of Canna×generalis.【Method】Using the embryogenic callus of the Canna×generalis Bailey. ‘Mohong’ as the recipient material， the calli were inoculated into MS medium containing different concentrations of kanamycin （Kan） （0， 20， 40， 60， 80， and 100 mg/L） and cefotaxime （Cef） （0， 100， 300， 500， and 600 mg/L）， and they were then cultured to screen the optimal concentrations of antibiotics and antimicrobial agents. The orthogonal experiments were conducted to investigate the effects of 16 combinations， including pre-culture duration （0， 1， 2， and 3 d）， microbial liquid concentration （OD_{600 nm}） （0.3， 0.4， 0.5， and 0.6）， infection duration （8， 10， 12， and 15 min）， co-culture duration （1， 2， 3， and 4 d） and acetosyringone （AS） concentration （0， 100， 150， and 200 μmol/L）， on the genetic transformation efficiency of Canna×generalis. The optimal conditions for genetic transformation were identified， and eight positive Canna plants were randomly selected for PCR detection.【Result】The Canna×generalis calli were relatively sensitive to Kan and Cef： when the Kan concentration increased to 80 mg/L， the survival rate of calli after 30 d of culture was 15.00%， and most of them turned brown and died alongside little proliferation， indicating the optimal Kan concentration for culturing Canna×generalis calli was 80 mg/L； at a Cef concentration of 300 mg/L， the survival rate of calli after 30 d of culture was 26.67%， and the calli exhibited vigorous growth and proliferation， with some calli capable of budding， indicating the optimal Cef concentration for culturing Canna×generalis calli was 300 mg/L. Orthogonal experiment analysis revealed that， under the conditions of pre-culture for 1 d， OD_600nm of 0.3， AS concentration of 100 μmol/L， 12 min of infection， and co-culture for 4 d， the highest positive transformation rates for calli （38.00%） and buds （12.16%） were achieved， which were significantly higher than those of other combinations except for Combination 4 and Combination 8 （P<0.05）. Bacterial liquid concentration and pre-culture duration were the primary factors affecting the transformation rates of positive callus and positive bud， respectively. Among the eight positive Canna plants， six exhibited distinct target bands at 843 bp， confirming successful expression of the exogenous eGFP gene in these six positive Canna plants.【Conclusion】An Agrobacterium-mediated genetic transformation system for Canna×generalis is successfully established， and the optimal conditions for the genetic transformation are pre-culture for 1 d， OD₆₀₀=0.3， AS concentration of 100 μmol/L， infection for 12 min， and co-culture for 4 d.