PLANT REGENERATION AND TRANSFORMATION OF MESOPHYLL PROTOPLASTS OF NICOTIANA GLUTINOSA L.
Liu Chun-ming and Xu Zhi-hong (Shanghai Institute of Plant Physiology,Academia Sinica)
In the present report we first suc-ceeded in plant regeneration from mesophyll protoplasts of Nicotiana glutinosa.Low tem-perature (10℃) pretreatment to the plant material significantly increased the stability and division frequency of protoplasts isolated subsequently.The enzyme incubation mixture consisted of 1% Onozuka R-10,0.2% Mace-rozyme R-10 and 0.5M mannitol at pH 5.6.Protoplasts divided to form cell colonies in NT medium with the division frequency of 22% at 7 days.Somatic embryogenesis were induced from protoplast-derived calli subcul-tured on MS medium containing 2,4-D at the concentration of 0.5-1.5 mg/1 for 40-50 days with the highest frequency being 45% at 1.0 mg/1 2,4-D.Plantlets regenera- ted when the embryoids were transferred to hormone-free medium. Cyto-histological observation showed that embryoids were initiated from the cells in surface or peripheral areas of calli and cell clumps.The starch changes in the cells of embryoids and neighbour tissue during em-bryogenesis were observed,and its possible function was discussed. Using the method of co-cultivation,N.glutinosa protoplast-derived cells were trans-formed by Agrobactenum tumefaciens strain with reconstructed Ti plasmid 3851,and clo-ned transformants were obtained on MSo medium.Presence of nopaline demonstrated that T-DNA genes were expressed in the transformed tissues.