Feasibility of High Molecular Weight DNA transfer into Tobacco Protoplasts

• Main Authors: Chia-Li Chen, 陳佳莉
• Other Authors: Yueh-Long Chang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/19092113942916991453

碩士 === 國立嘉義大學 === 農業生物技術研究所 === 94 === Abstract 　　Plant transformation is the introduction of a foreign piece of DNA into plant genome which then was stably inherited through generations by several methods. To date, DNA transfer to plants with less than 25 kb of DNA fragments has become a routine procedure. For genome research in large-scale gene function and map-based cloning after post-era genome, high-molecular-weight DNA transformation would be useful. Therefore, to develop a high-molecular-weight DNA transformation system is necessary. Recently binary bacterial artificial chromosome (BIBAC) has been developed for transferring about 100 kb of foreign DNA into plant genome by many methods such as Agrobacterium-mediated, biolistic, and protoplast system transformation. In this experiment, high-molecular-weight DNA transfer into tobacco protoplasts was conducted by electroporation, PEG, liposome, and liposome-PEG methods. For establishment of the four transformation methods, pCAMBIA 1301 (11 kb) and pCAMBIA 1302 (10 kb) vectors were used and then transgenic plants were regenerated. In the transfer of large DNA fragments, the BIBAC 3I5 clone with 83-kb Arabidopsis genomic DNA was used. At present, transformed protoplasts on selection medium have grown up to the stage of calli, which is necessary for further cultivation. As high molecular weight DNA transformation system is established, it could be beneficial for genetic engineering and genome studies. Keywords: Bacterial artificial chromosome ，Electroporation，High-molecular-weight DNA，Liposome，Protoplast，PEG