Microspore culture in Nicotiana with special reference to its possible application in Hevea brasiliensis.

Immature pollen of Nicotiana tabacum cv Wisconsin 38 and cv Burley, isolated at stages from the late uninucleate to early bicellular, was successfully cultured to give embryoids and green plantlets. This is the first report of successful culture of tobacco pollen isolated direct from the plant. The method represents a substantial advance on existing methods which relied on both prior treatment of the excised buds and a short period of anther culture, to induce the pollen into embryogenesis, The pollen was isolated and pretreated for about 7 days in an appropriate medium. For bicellular pollen, from which the largest number of embryoids was obtained, this medium was simply water; for uninucleate pollen, which required a higher osmotic pressure for survival, it was a solution of inorganic salts. After the pretreatment, nutrient medium with sucrose was added, upon which embryoids and plantlets formed. Not more than 6% of the pollen grains developed into microscopic embryoids. Still fewer survived to become plantlets. Attempts to induce more pollen grains into embryogenesis by means of growth substances and other empirical modifications of the pretreatment medium were unsuccessful. Plantlet yields were increased by increasing nutrient availability after the pretreatment.The low fraction of pollen grains induced into embryogenesis was consistent with findings of previous workers which suggested that embryogenesis is predetermined by events that precede culture. Isolation of the pollen into the complete nutrient medium instead of into water was deleterious to embryogenesis. This contrasts markedly with the situation in anther culture in which high yields of pollen plantlets can be obtained by supplying the complete medium at inoculation. The deleterious effect of sucrose and other organic compounds on embryogenesis in the case of isolated pollen suggests that in anther culture the anther tissues act as a screening agent preventing access of the deleterious compounds to the pollen during the initial induction period. Attempts to culture isolated pollen of N. sylvestris were less successful. A few microscopic embryoids were obtained, but none developed into plantlets. Isolated pollen of N. sylvestris may have more stringent nutritional and osmotic requirements than that of N. tabacum. Changes in the anatomy of N. sylvestris anthers during culture were investigated. They could not be correlated with the potential for embryogenesis. Details of anther and pollen development were, studied in Hevea brasiliensis as a preliminary to anther and pollen culture studies. Anthers or staminal columns bearing many anthers were cultured, but multicellular embryoids were not observed in any of the treatment tested. Time did not permit for the findings on the culture of isolated tobacco pollen to be tested in any detail in Hevea.