Studies on haploid embryogenesis and its application in plant breeding

采用percoll密度梯度离心分离烟草双核花粉粒，在0.4 M甘露醇溶液（饥饿处理）中培养。培养3天后，将花粉粒转移到含有甘露醇、半乳糖、谷氨酰胺和ABA的改良MS培养基上。约80%的花粉粒分裂，约40%的分裂花粉粒在转移到合适的培养基后通过单倍体胚发生发育成幼苗。另一方面，经Percoll密度梯度离心获得的双核花粉粒在谷氨酰胺的基础培养基中培养时，大部分花粉粒发育正常。通过这些培养方法，我们研究了花粉去分化过程中发生的生化变化。氨基酸和核苷酸在去分化过程中发生了定性和定量的变化，但在花粉粒成熟过程中也发生了变化。在花粉去分化和成熟途径中，蛋白质、RNA和DNA合成速率以及de - nove蛋白质合成模式的变化是相似的。用32P无机磷酸盐检测蛋白磷酸化模式。4个蛋白（a-d）在花粉去分化过程中被特异性磷酸化。另外5个蛋白（e-i）在花粉成熟过程中被特异性磷酸化。研究了谷氨酰胺、细胞分裂素、EDTA和花粉发育阶段等影响花粉去分化和蛋白磷酸化的因素。a - d蛋白磷酸化与花粉去分化直接相关。蛋白质a - d定位于特定的部分，如线粒体和叶绿体。目前正在努力阐明a - d蛋白在花粉去分化中的作用。

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.