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Microspores were cultured on the modified B₅ liquid medium containing 2.4D (0.1 mg L⁻¹), NAA (0.1 mg L ⁻¹), L-glutamine (500 mg L⁻¹), L-serine (100 mg L⁻¹), and sucrose (100 g L⁻¹). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B₅ regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.
Isozyme, RAPD and AFLP markers were evaluated and compared for their ability to determine genetic similarity in a set of 18 parental lines of winter oilseed rape F1 hybrids developed using CMS ogura. Five isozyme systems, 64 RAPD starters and 23 EcoRI+3/MseI+3 AFLP primer combinations generated 597 polymorphic markers. These polymorphic fragments were chosen for estimation of genetic similarity. Of the total number of polymorphic products, polymorphic zymograms constituted only 3.0% of the markers, 57 RAPD primers 37.7%, and 23 AFLP primer combinations 59.3%. The size of RAPD polymorphic products ranged from 564 to 2100 bp. On average there were four amplified bands per primer, with 61.0% polymorphism. The AFLP polymorphic fragments ranged from 72 to 1352 bp in size. AFLP assays generated 15 bands per primer pair on average and detected roughly four times more bands than with RAPD analysis. The genetic similarity coefficients based on all marker data range from 0.52 to 0.84. The correlation of genetic similarities based on RAPD and AFLP markers was 0.58. Estimated genetic similarity based on isozyme data was not correlated with genetic similarity derived from the two DNA-based markers. The dendrogram constructed with the three types of markers taken together grouped all the winter oilseed rape parental lines into several similar clusters. The genomic distribution and frequency of the RAPD and AFLP markers can serve well as estimators of genetic similarity between parental lines of F1 CMS ogura hybrids.
The use of 2,4-D as a post pollination treatment to fertilize florets instead of GA₃ provided a two-fold improvement in seed set, culturable embryos and hence green plant production from wheat and maize pollination. The efficiency of the wheat by maize pollination method for haploid production was equal to the anther culture method in F₁ hybrid combinations that were responsive to anther culture. However, in recalcitrant combinations obtained from non-responsive genotypes the maize pollination method was far superior.
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