INSERTION OF MICROSCOPIC OBJECTS THROUGH PLANT-CELL WALLS USING LASERMICROSURGERY

A detailed protocol is presented for precisely inserting microscopic o bjects into the periplasmic region of plant callus cells using laser m icrosurgery. Ginkgo biloba and Agrobacterium rhizogenes were used as t he model system for developing the optical tweezers and scalpel techni ques using a single laser. We achieved better than 95% survival after plasmolyzing G. biloba cells, ablating a 2-4-mu m hole through the cel l wall using a pulsed UV laser beam, trapping and translating bacteria into the periplasmic region using a pulsed infrared laser beam, and t hen deplasmolyzing the cells. Insertion of bacteria is also described. A thermal model for temperature changes of trapped bacteria is includ ed. Comparisons with other methods, such as a reverse-pressure gradien t technique, are discussed and additional experiments on plants using laser microsurgery are suggested. (C) 1998 John Wiley & Sons, Inc.