Optimization of Apex-Mediated DNA Transformation in Rice
Genetic transformation using the rice shoot apical meristem, derived from germinating seedlings, was established in this study for commercial varieties of Oryza sativa. To optimize apex-mediated DNA transformation in rice several parameters were tested to improve the efficiency and reliability of isolation of the shoot apices and regeneration of vegetative shoot apices. Results from these experiments indicated that certain factors were important in increasing the frequency of survival rate of the apex, co-cultivation and co-transformation. These factors included: the starting size of the apex had to range between 1.0 mm and 1.2 mm for maximum survival of the apex, concentration of the infecting bacteria (pCL3 and pCL4) at an OD of 0.6-0.8 at 600nm and pTOK233 at an OD of 1.5-1.8 at 600nm, the minimum lethal concentration of hygromycin B was 5 mg/L, and use of Rice Shoot Apex (RSA) medium. Furthermore, the presence of acetosyringone before and during co-cultivation at levels between 400 ppm and 1000 ppm increased the number of transformed apices by 40%. Vacuum infiltration at 20-21 Hg for 5 minutes did not have an effect on apex survival, however apex transformation rate was increased by 30¨C35%. Adhering to the development of the new protocol resulted in a 90% shoot apex culturing success rate and a 95% transformation rate of the apices.
Vectors containing the GUS gene with either the maize ubiquitin promoter or rice ubiquitin promoter resulted in > 85% transformation efficiency of the apices based on GUS assays. The vectors LBA4404 (pCL3) and LBA4404 (pCL4) proved to be more apex-specific than root-specific, an advantage over the super-binary vector LBA4404 (pTOK233). Preliminary vectors such as pRQGus, pRQHg and pSB41 were constructed for future use in experiments involving LBA4404 (pTOK233) and the results of the tissue culture experiments. Conducting experiments with both the results from tissue culture and vector construction could provide alternative routes to further enhance the efficiency of apex-mediated transformation.
Advisor:Charles Johnson; James Oard; Gerald Myers; Bobby Harville
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Date of Publication:01/31/2002