Regeneration and transformation of rice (Oryza sativa L.) haploid cells
Abstract
The objective of the present study was to develop a transformation and regeneration system for rice (Oryza sativa L.) using haploid cells as the materials in an attempt to obtain homozygous transgenic plants quickly. The study was divided into two parts, (1) attempt to establish an effective procedure for regeneration of fertile (double-haploid) rice plants from haploid cells, i.e. protoplasts, and to evaluate the phenotypic features of the regenerated Ro plants and their R1 progenies; (2) to genetically transform rice haploid cells and obtain fertile (double-haploid) transgenic plants, and to confirm homozygosity of the transgenes in the progeny. A regeneration system has been established for isolating haploid protoplasts of both japonica and indica rice varieties. Conditions have been modified and optimized for selection of donor explants (immature panicles), induction of anther-derived calli, initiation of haploid embryogenic suspension cultures, culture of isolated protoplasts, and regeneration of fertile rice plants. The regenerated Ro plants generally had a short stature and low seed fertility; however, their selfed R1 progenies had comparable phenotypic traits with the seed-grown plants. High phenotypic uniformity in the R1 progenies indicated the Ro plants were doubled-haploid plants. Fertile indica and japonica transgenic plants have been produced via either PEG-mediated transformation of protoplasts or microprojectile bombardment of haploid suspension cell clusters. Conditions and physical parameters involved in both transformation systems were optimized. Haploid protoplasts were found to be very sensitive to the PEG treatment and resulted in a relatively low efficiency of callus recovery. The plant regeneration frequency of PEG-treated protoplasts was about 0.3 to 4.0 per million protoplasts plated. Haploid suspension cells were also effective for obtaining fertile transgenic rice plants following gene delivery by particle bombardment. Multiple gene integrations and rearrangements in T$\sb{\rm o}$ plants were obtained by this method, and the frequency of co-transformation of two separate genes was about 60%. The T$\sb{\rm o}$ plants had phenotypic characteristics comparable to seed-grown plants, and the Southern blot analysis of T$\sb{\rm o}$ plants showed that the two separate transgenes were co-integrated in the plant genome. Inheritance of hpt gene activity in T1 progenies showed two near-homozygous transgenic lines. Based on results obtained in this study, the feasibility of using rice haploid protoplasts for producing homozygous transgenic plants is unlikely. However, particle bombardment of haploid suspension cells or microspore-derived callus to obtain fertile homozygous transgenic plants showed promising results.
Degree
Ph.D.
Advisors
Hodges, Purdue University.
Subject Area
Plant propagation|Botany|Genetics
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