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<i>Oryza officinalis</i> Wall is a potential genetic resource for rice breeding; however, its distant genome limits its crossing ability with cultivated rice. The interspecific hybridization of <i>O. officinalis</i> and cultivated rice, establishment of its tissue culture, and induction of polyploidy are ways to improve <i>O. officinalis</i>’s poor crossability. We developed an interspecific hybrid and studied its reproductive pollen development process in this work, and the results showed that abortive pollens (81.94%) and embryo sac abnormalities (91.04%) were the key causes of its high sterility. In order to induce callus formation in interspecific hybrid explants, two different culture media, namely Chu’s N-6 medium (N6) and 1/2 Murashig and Skoog medium (1/2 MS), were employed. Additionally, two plant growth regulators (PGRs), namely 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), along with L-proline (Pro) and acid hydrolyzed casein, were utilized in the experiment. The optimal N6 medium, supplemented with 2.0 mg·L⁻¹ 2,4-D, produced the highest induction rate (80.56 ± 5.44)%. For callus differentiation and proliferation, the MS medium supplemented with 2.0 mg·L⁻¹ BA + 0.2 mg·L⁻¹ NAA produced the highest differentiation rate (75.00 ± 4.97)% and seedling emergence ratio (28.97 ± 4.67)%. The optimal combination for seedling rooting was the 1/2 MS medium supplemented with 2.0 mg L⁻¹ NAA and 0.2 mg L⁻¹ BA, which produced an average of 13.95 roots per plant. For polyploidy induction in the interspecific hybrid, the concentration of colchicine treatment at 400 mg·L⁻¹ for three days is an ideal protocol. We devised tissue culture and interspecific hybrid polyploidy induction to overcome <i>O. officinalis</i>’ poor crossability and introduce its favorable features into cultivated rice.