Overcoming biological barriers to control pollinated seed production in Eucalyptus.
The overall aim of this PhD study was to develop protocols to improve the efficiency of eucalypt controlled pollinations (CPs) in order to make it more cost-effective for forestry companies to perform them on a commercial scale on small-flowered species. To achieve this, three research areas were explored, namely pollen handling, breeding systems and controlled pollination technique. Study species were Eucalyptus grandis, E. dunnii, E. smithii, E. nitens, E. urophylla and E. macarthurii. The first specific aim of the study was to identify a suitable liquid in vitro germination medium for reliably testing pollen viability of all six study species. Six levels of sucrose [0, 10, 20, 30, 40 and 50% (w/v)] were tested, both with (0.15 mg l-1) and without boric acid. The optimal sucrose concentration was found to be 30% (w/v), with boric acid stimulating pollen tube growth. A second aim was to determine temperatures suitable for the short-, medium- and long-term storage of E. smithii, E. nitens and E. grandis pollen. Pollen samples were stored at room (25oC), refrigerator (4oC), freezer (-10oC) and liquid nitrogen (-196oC) temperatures, and pollen viability tested every two months over a 12-month period. There was a rapid decline in the germination of pollen stored at 25oC, while temperatures cooler than 4oC appeared to maintain pollen viability for the duration of the 12-month study. Recommendations were thus to use a refrigerator for short-term (< 2 months), a freezer for medium-term (up to 10 months) and cryopreservation for longer-term storage. In the second part of the study, breeding systems of E. urophylla and E. grandis were examined by studying pollen-tube growth in the style after single-donor self- and cross-pollinations. Results showed that, in addition to both species exhibiting reduced seed yields following self-pollination, pollen tubes from self-pollen took significantly longer than those from cross-pollen to grow to the base of the style. This suggested the presence of both late-acting self-incompatibility and cryptic self-incompatibility (CSI) as possible mechanisms responsible for outcrossing in these two species. In a follow-up study, the siring ability of self- and cross-pollen was examined after single- and mixed-donor pollinations were performed on E. grandis. Once again, single-donor cross-pollinations resulted in a significantly higher number of seeds compared to self-pollinations. In addition, microsatellite molecular markers revealed that 100% of the progeny from mixed (self + outcross) pollinations were outcrossed, confirming the competitive advantage of cross-pollen. To date, CSI has never been associated with Eucalyptus, making this the first study to suggest its presence in the genus. For the final study area, three CP-techniques were compared, namely the Conventional method, One Stop Pollination (OSP) and Artificially Induced Protogyny (AIP), in E. grandis, E. smithii and E. macarthurii maternal parents. Although the AIP technique produced the highest seed yields in all three species, it also resulted in high self- and foreign-pollen contamination (determined using microsatellite markers). This necessitated exploration of different methods of isolating the pollinated flower, and this led to the identification of a novel method which uses sodium alginate gel. Flowers hand-pollinated and isolated with sodium alginate produced progeny that were 100% outcrossed with the applied pollen, confirming the superiority of this innovative isolation technique compared to the currently used exclusion bag. Sodium alginate isolation also increased the efficiency of CPs as the gel was naturally shed, removing the need for operators to return to the tree to remove the isolation material, and thereby reducing the cost per seed. Application of these results should make commercial CP-seed production of small-flowered eucalypts a practical reality.