Studies on haploid embryogenesis and its application in plant breeding

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Binucleate pollen grains of tobacco were fractionated by percoll density gradient centrifugation and cultured in 0.4 M mannitol solution (starvation treatment).After therr days in culture the pollen grains were transferred to a modified MS medium containing mannitol, galactose, glutamine and ABA. About 80% of the pollen grains divided and about 40% of the dividing pollen grains developed into seedlings through haploid embryogenesis after transfer to an appropriate medium. On the other hand, when binucleate pollen grains obtained after Percoll density gradient centrifugation were cultured in basal medium with glutamine, most pollen grains underwent normal development of pollen.By using these culture methods, we examined biochemical changes which occurred specifically during pollen dedifferentiation. Qualitative and quatitative changes in amino acids and nucleotides were observed during the dedifferentiation, but the changes were also observed during maturation of pollen grains. Changes in the rates of protein, RNA and DNA synthesis and in patterns of de nove protein synthesis were similar in both dedifferentiation and maturation pathways of pollen grains.Patterns in protein phosphorylation were examined by using 32P inorganic phosphate. Four proteins (a-d) were phosphorylated specifically during pollen dedifferentiation. On the other hand, 5 other proteins (e-i) were phosphorylated spedifically during pollen maturation. We examined several factors affecting pollen dedifferentiation and protein phosphorylation such as glutamine, cytokinin, EDTA and developmental stage of pollen grains. Phosphorylation of proteins a - d was directly correlated with pollen dedifferentiation. Proteins a - d are localized in particular fractions such as mitochondria and chloroplasts. Now efforts are being made to elucidate role(s) of the proteins a - d in pollen dedifferentiation.