| Anther culture has been demonstrated to be an effective and efficient way for rice breeding. Through rice anther culture several kinds of ploidy plants can be obtained, such as haploid, diploid, polyploid and usually based on the morphology to distinguish their ploidy during maturity. Nonetheless, the chromosome numbers of these haploids must be doubled before its application in rice breeding. The present study comparatively identified the chromosome doubling effectiveness of an antimicrotuble herbicide called Oryzalin with Colchicine. Flow Cytometry (FCM) was used for ploidy identification, and paraffin method was employed to comparatively study the stem anatomical structures in different ploidy plants. The main results are summarized as follows:1. In order to study the chromosome doubling effects of Colchicine and Oryzalin solutions on rice haploids, three groups of experiments have been designed. (1) Roots of haploid seedlings were soaked for 24h using Colchicine solutions with concentration of 1.25mmol/L, 2.50mmol/L, 5.00mmol/L, and Oryzalin with concentration of 10μmol/L, 25μmol/L, 50μmol/L, 100μmol/L, and 200μmol/L respectively(all solutions with 2% dimethyl surtoxide);(2) Roots of haploid seedlings were soaked for 12 h, 24 h, 48 h, using 2.50mmol/L Colchicines and 50μmol/L Oryzalin sulutions with 2% dimethyl surtoxide (DMSO) respectively;(3) Roots of haploid seedlings were soaked for 24 h using 2.50mmol/L Colchicine and 50μmol/L Oryzalin solutions with 0%, 1%, 2%, and 4% DMSO respectively. The results showed that for FC311, the effectiveness of chromosome doubling with 1.25mmol/L Colchicine and 25μmol/L Oryzalin with 2% DMSO for 24 h respectively was significant compared with other treatments, their doubling rates were reached to 30.6% and 13.9% respectively;whereas for FC322, the significant effectiveness were found to soak roots for 24hrs with 2.50mmol/L Colchicine and 50μmol/L Oryzalin with 2% DMSO (25.0% and 22.2% respectively), which indicated that Colchicine is still served as the most effective agent for rice haploids chromosome doubling.2. Six different buffers were used to prepare rice nuclei suspensions for flow cytometric analysis. And highly coefficients of variation (CV > 6%) of G0/1 peaks were found after theanalysis of nuclei isolated by Galbraith's buffer and Arumuganathan's buffers;and next was the Lysis buffer LB01, its CV of Go/i peak was 4.75%;whereas the CV of Go/i peaks of Otto buffers and Marie's nuclear isolation buffer were 3.57% and 3.41% respectively, but the composition and preparation condition of Otto buffers is the simplest of all. In addition, using Tris-MgCk buffer for preparation of rice nuclei suspension, much background debris can occur. Based on these results we introduce that the Otto buffer is better, convenient, and rapid for preparation of rice nuclei suspersions for flow cytometric analysis.3. Using anther culture-derived haploid, diploid, and triploid of FC311and haploid, diploid, and tetraploid of FC322 as materials, the anatomical structure of stems were observed and analyzed by the paraffin method. The results showed a general tendency of increase of ploidy with the increase of the number and size of vascular bundle, areas of stem wall and vascular bundle in internodes of tested materials. However, no obvious correlations were found between ploidy with the layer number and development of collenchyma cell under epiderm and in vascular bundle sheath of outer circle and inner circle, starch grains in ground tissue cells, the ratio of total area of vascular bundle and area of stem wall.4. A rice mutant termed as MTMS temporarily, having characteristics of multiple tilling numbers and male sterile was identified in the anther culture progenies from the Fi hybrid 2S258. Compared with diploid plants, this mutant shows specific characters, such as tall Culm, long leaves, multiple and big panicle, and sterile pollen. After examining its ploidy with FCM, it is found that this mutant belongs to diploid.
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