The Establishment Of Regeneration System And MRNA Differential Display During Somatic Embryogenesis In TM-1(Gossypium Hirsutum L.)

The somatic embryogenesis system of cotton affords an important role for genetic improvement and bioengineering. So far, the regeneration system of TM-1 don't been built becaues of extreme difficulty for induction of somatic embryogenesis. A vast of results indicated that the induction of somatic embryogenesis became a bottleneck the process of the regeneration system. To-date, quite a number of reports had been mainly focused on factors of environment and cytology during the embryogenesis of plant. So the studies of gene expression and molecular regulation during embryogenesis were the foundation for optimizing and consummating plant regeneration farther.The objectives were to build the somatic embryogenesis regeneration system of an upland cotton (Gossypium hirsutum L.) variety, TM-1, from its hypocotyls by optimizing the conditions and hormones combinations. Furthermore we used cDNA-AFLP to analyze gene expression profiles during somatic embryogenesis and to isolate stage-specific genes in hypocotyls, calluses and embryogenic calluses of TM-1. The main experimental results were presented as follows:1. The Establishment of regeneration system of TM-1 (Gossypium hirsutum L.)Calluses were induced in media containing the optional combination of 2,4-D .01mg/L, IAA 0.01mg/L, KT 0.01mg/L, 3% sucrose and agar 6g/L by 7 day hypocotyls of sterilized plant. After 35 days, the calluses were yellow and loose.The calluses subcultured on MS medium of different hormone combination until the embryogenic calluses initiated. But the induction rate of embryogenic calluses was very low. Somatic embryogenesis was facilitated through increasing the active carbon 2g in the medium while active carbon also can decrease the brown phenomenon.The plantlets were obtained on MS medium containing VB5+3% sucrose++0.1mg/L IAA+0.1mg/L 6BA. The survival rate of regeneration plant transferring reached 90%.2. mRNA differential display during somatic embryogenesis in TM-1We used cDNA-AFLP to analyze gene expression profiles during somatic embryogenesis of hypocotyls, callus and somatic embryo in TM-1.One hundred and eighty combination of AFLP primers produced 3000 fragments. Only sixteen combination primers reappeared the uniform results. Through separating these PCR products on a sequence gel, about 5 differentially expressed cDNA fragments were obtained.The fragments were cloned and sequenced and their functions were sought in the GenBank database. Four of them showed significant homology with the floral of Gossypium raimondii L. and the lint fiber of Gossypium hirsutum L. to GenBank database entries. The indentities were above ninety percent. The results indicated that the genes of whole plant had already expressed at the early time of embryogenesis.