Genetic analysis of plant regeneration and transformation of indica rice (Oryza sativa L.) via direct DNA uptake into protoplasts
Abstract
A diallel study involving reciprocal crosses of four rice (Oryza sativa L.) genotypes (IR8, 36, 54, and 64) was carried out to understand the genetic control of plant regeneration from immature embryo-derived calli in rice. Regeneration frequency (% of calli that produced plants) varied from a high of 86% for IR54 to a low of 0% for IR36, while regenerated plants per embryo numbered from 0 to 7 when these same IR lines and the F$\sb1$ hybrids were compared. Combining ability analysis revealed that both nuclear (with both additive and dominant effects) and cytoplasmic genes were important in controlling plant regeneration in rice. High general combining ability (GCA) parental lines and high specific combining ability (SCA) F$\sb1$ hybrids were identified. Protoplasts were isolated from suspension cultures of an indica rice variety, IR54 for transformation studies. The regeneration potentials of newly established suspension lines IR54-5 and IR54-6 were tested through protoplast culture. A PEG-mediated transformation protocol was used to transiently transform the protoplasts with the gusA gene (encodes for $\beta$-glucuronidase), and $\beta$-glucuronidase (GUS) activity was used as an indirect measure of transformation frequency. Heat shock treatment and various media were evaluated for their effect on transient GUS activity. Stable transformation with both the neo (encodes for neomycinphosphotransferase II) and gusA genes were carried out using optimized transformation protocols and selection on kanamycin or G-418. It was demonstrated that PEG-mediated to co-transformation of protoplasts with two genes coupled with selection on kanamycin was an effective way of transferring foreign gene(s) into the indica rice genome. Transgenic indica rice plants were regenerated from calli following transformation with the neo gene and selection on G-418. The presence of the neo gene in the plant genome was confirmed by Southern blot analysis. Many transgenic plants were obtained from separate experiments and grown in soil to flowering. Two plants were co-transformed with both the neo and the gusA gene as evidenced by the Southern blot analysis. The gusA gene driven by the cauliflower mosaic virus 35S promotor was actively expressed in all the tissues assayed at various times during the growth period. (Abstract shortened with permission of author.)
Degree
Ph.D.
Advisors
Hodges, Purdue University.
Subject Area
Botany|Genetics|Botany
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.