Abstract
Haploids provide valuable material, for fundamental research and plant breeding. However, their utilization depends on the effectiveness of the chromosome doubling method. The use of two unconventional tissue culture methods from leaf explant contributed to the induction of doubled haploid.
The first method known as the long-term callus culture was based on the embryonic callus induction. The hypothesis is that it brings about endomitosis in callus cells. In this experiment 200 large leaf explants, from four genetically different haploid clones were used. They were cultured on modified MS medium [1] with enhanced NH4NO3 without KNO3, and 0.8 mg/l 2,4 D and 0.4 mg/l 2iP of growth regulators. The regeneration ability was estimated on the bases of the number of plantlets obtained per explant. The total of 36 explants exhibited regeneration and 10 produced viable plants. The rooted plants in the amount of 306 were transferred to greenhouse and 108 of them were tested for their ploidy level. As many as 74% of plants were diploid, 1% was triploid and 25% tetraploid
The second method employed the direct regeneration from the haploid leaf micro-explants (2–3 mm2) from the first and second juvenile leaves. The explants were cultured on modified MS medium [2]. This method was applied to five genetically different haploid genotypes. In total, 21.1% of explants from the first leaf and 12.1% of the second leaf produced plantlets. As a result 388 plants from the first leaf and 210 from the second leaf were obtained. Out of the first population 93 plants and 78 from the second were tested with respect to their ploidy level. All the plants obtained from the first leaf proved to be haploid, while in the population obtained from the second leaf there were 70.5% of haploids 28.2% of diploids and 1.3% of mixoploids.