Transformation of Tobacco cpDNA with FusionE7GGG/GUSGene and Homologous Recombination Mediated Elimination of the Marker Gene

Gene introduction approaches in chloroplast transformation and its applications

“…This step ensures the metal particle used is free from contaminants or other foreign substances to prevent the DNA construct from being affected throughout the process. The DNA construct will be introduced to the metal particles [ 5 , 6 , 8 , 9 , 23 , 63 ], where the DNA construct will coat the metal particle by using the precipitation principle [ 5 ] and the metal particles will be attracted to the DNA construct which is a negatively charged compound [ 9 ]. This step requires the use of calcium chloride and isopropyl for the formation of the DNA-tungsten / gold complex.…”

“…The microcarrier is loaded into the chamber, where it is located around 7 inches away from the rupture disk [ 5 , 9 , 38 ]. After the macro-carrier is loaded into the biolistic chamber, the petri dish containing the desired chloroplast to be transformed is placed at the bottom of the biolistic chamber and the pressure within the chamber is reduced to the desired level [ 5 , 8 , 9 , 38 ]. The pressure applied varies based on the type of cells used and the distance between the rupture disk and the Petri dish [ 5 , 8 , 29 ].…”

“…Commonly, two methods will be used for the bombardment of the complex, one of which involves charged electricity and the other involves pressurized gas [ 9 , 23 , 38 ]. It is common to use helium gas, where pressurized helium gas is applied on the macro-carrier to transfer the complex into the chloroplast at high speed [ 5 , 8 , 9 , 23 , 29 , 38 , 39 ]. After the bombardment, the DNA construct is incorporated into the desired cells followed by 2 weeks of incubation for the healing of the cells to take place and further process is carried out once the callus formation is observed [ 5 ].…”