Induction of polyploidy in Eucalyptus species and interspecific hybrids.
A large sector of the forestry industry of South Africa comprises Eucalyptus species, covering approximately 49% of the forestry plantation area. Polyploidy induction has become an attractive tool to increase yield and reduce invasiveness in forestry species. Polyploidy induction in Eucalyptus using colchicine treatments on seed and axillary buds was undertaken to produce tetraploids that could be used in breeding programmes; specifically to increase yield and decrease species invasiveness through the production of triploids after crossing with diploid parents. Eight seedlots of E. urophylla and seven of E. grandis were treated with four colchicine concentrations (0.00, 0.01, 0.03, 0.05%) at two exposure times (18 h and 24 h), treating two seeds per treatment, repeated eight times. For axillary bud induction, 20 buds of two E. grandis clones and three E. grandis × E. urophylla hybrids and one E. grandis × E. nitens hybrid were treated with four colchicine concentrations (0.0, 0.5, 1.0, 1.5%) for three consecutive days. A known tetraploid hybrid E. grandis E. camaldulensis and its corresponding diploid were included as reference material. Seedlings and bud sports were pre-screened by determining stomatal guard cell lengths. Seedlings and bud sports displaying cell lengths significantly (p<0.0001) larger than the diploid were selected as putative polyploids. Polyploidy was then confirmed by quantifying the DNA content using flow cytometry. Stomatal frequencies and guard cell chloroplast frequencies were also determined in the induced tetraploid seedlings to evaluate their suitability to discern between ploids. All putative polyploidy seedlings, identified in the pre-screening process, were confirmed, using flow cytometry, as either tetraploids or mixoploids. Of the 17 E. urophylla putative polyploids, from various seedlots, six were tetraploid and 11 mixoploid. In E. grandis one of the five putative polyploids, from various seedlots, was tetraploid and four mixoploid. Pre-screening of bud sports was less accurate; only four of the 12 E. grandis hybrid putative polyploids were mixoploid and only three of the six E. grandis putative polyploids were mixoploid. E. urophylla seedlings were more sensitive to colchicine than E. grandis seedlings displaying a lower survival rate (52%) than E. grandis (63%). Extreme treatments that caused the lowest survival rates were also responsible for most of the polyploidy successful inductions; 0.05%/18 h and 0.05%/24 h for E. urophylla and 0.03%/24 h and 0.05%/24 h for E. grandis. Phenotypic effects of colchicine included shorter, thicker roots and hypocotyls; darker leaves; longer and narrower leaves in some tetraploids; and asymmetrical leaf margins in many mixoploids and tetraploids compared with the controls. In the tetraploids, stomata were significantly larger (p<0.0001) and less frequent (p<0.001). A significant (p<0.001) increase in the number stomatal chloroplasts was also ascertained. Confirmed mixoploid seedlings all displayed tetraploid leaves based on stomatal size and thus classified as periclinal chimeras. In bud sports, only leaves with islands of diploid and tetraploid stomata in the confirmed mixoploids were encountered. Mixoploid bud sports were thus either sectional or mericlinal chimeras. Stomatal size proved to be a suitable pre-screening method, especially in polyploidy induction in seedlings. Additionally confirmed tetraploids exhibited significantly different stomatal frequencies and stomatal chloroplast frequencies compared with the diploids, thus proving to be suitable detection methods for polyploidy screenings. Polyploidy induction in seed was effective, however, less effective in axillary buds which requires further research to refine methods.