We have developed an effective protocol for in vitro micropropagation in order to obtain large numbers of identical plants and another protocol for in vitro polyploidization of Ajuga reptans, based on the use of oryzalin. Two donor plants of A. reptans (AR 4, AR 7) were treated with 0, 1, 5, 10 μM oryzalin for 2 weeks. The analysis of the ploidy level of these plants was verified by flow cytometric analysis using the internal standardization method. The effects of polyploidization on growth as well as morphological and stomatal size were also measured. After in vitro polyploidization, some plants became tetraploids or octoploids. The most efficient conditions for inducing tetraploidy were the treatments with 10 μM oryzalin.
We used germination tests to assess the frequency of polyembryony in 9 asparagus cultivars with a high propensity to produce double embryos with different ploidy levels: Alpha, Andreas, Boonlim, Cipres, Eposs, Helios, Limbras, Ravel and Sartaguda. Twin embryos inside a single seed were found in 3 cultivars: Eposs 2n, Ravel 2n and Sartaguda 2n, at 0.60% frequency (15 seeds with twin embryos out of 2500 seeds). Of 30 obtained seedlings, 14 were separated diploid-diploid twins, 6 were conjoined diploid pairs, 8 were separated diploid-haploid and 2 were diploid-haploid pairs conjoined in the hypocotyl region. Some embryos showed unilateral dominance of one embryo (size and shape). The haploid status of the smallest embryo was confirmed by chromosome number (n=x=10) and flow cytometry (nuclear C DNA amount 1.95 pg). The haploid obtained in this manner possessed enough vegetative vigor to undergo chromosome doubling.