A detailed protocol is presented for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery. Ginkgo biloba and Agrobacterium rhizogenes were used as the model system for developing the optical tweezers and scalpel techniques using a single laser. We achieved better than 95% survival after plasmolyzing G. biloba cells, ablating a 2-4-mu m hole through the cell wall using a pulsed UV laser beam, trapping and translating bacteria into the periplasmic region using a pulsed infrared laser beam, and then deplasmolyzing the cells. Insertion of bacteria is also described. A thermal model for temperature changes of trapped bacteria is included. Comparisons with other methods, such as a reverse-pressure gradient technique, are discussed and additional experiments on plants using laser microsurgery are suggested.