Fertility Restoration of Buddleia Species by In Vitro Chromosome Doubling

Mitotic chromosome doubling using colchicines has been successful for many crop species. It was involved in the production of triploid watermelons, tetraploid grapes, and some autopolyploid ornamental species. Induced autopolyploids could enhance the crossability of two species, particularly if both are diploids. Amphiploids could be used to restore some fertility of a totally sterile F₁ hybrids thus to facilitate further backcrossing and introgression. In this study, chromosome doubling of three Buddleia sterile lines, B. marrubifolia x (B. davidii x B. crispa), B. marrubifolia x B. crispa, and B. marrubifolia x B. alternifolia, was carried out to restore their fertility. Based on the field observation, these three lines are sterile. In vitro shoot tips and nodes were treated with colchicine solution at 0, 0.01, 0.1, and 1 mM for 1, 2, and 3 d. After the treatment, the explants were washed in sterile distilled water and transferred into the Petri dishes containing MS medium plus 20 g l^-1 sucrose with 0.5-2.5 μM BA for shoot recovery. Results showed that the shoot recovery rate increased as the concentration of colchicines and treatment time decreased. Plant recovery was also affected by genotype in which B. marrubifolia x B. alternifolia was the most tolerant to colchicine treatment. The effect of colchicine was also observed during the in vitro rooting period with a decrease of rooting rate as the concentration and treatment time increased. Shoots with 3-4 opened leaves were rooted in vitro and then transferred into the potting mix. Acclimated plants were grown in the greenhouse for several months and eventually grown outside of the greenhouse. More than 500 plants have been developed from these three lines by in vitro chromosome doubling. Seeds were collected from these plants. The seed germination rate will be determined for fertility verification. Chromosome number of the plants with viable seeds will be counted for further verification of ploidy. Other traits will also be evaluated.