Study of intergeneric hybridization in Hippeastrum

Hippeastrum is a bulbous genus within the family Amaryllidaceae which is of horticultural importance as a cut flower and potted plant crop. The colour range available in commercial hybrids is dominated by white and red. The commercial potential of Hippeastrum as a floriculture crop is significant, but production of the cut flowers is limited to warm climatic regions or glasshouse production in temperate regions. Improved cold tolerance, along with extended colour range and flower fragrance, are key breeding objectives for Hippeastrum hybrids. One breeding strategy which may facilitate the introduction of these characteristics is intergeneric crossing. This strategy has recently been used successfully in other important bulbous genera including Lilium, and has some potential for Hippeastrum breeding as a number of intergeneric hybrids within family Amaryllidaceae have been reported previously. This study therefore aimed to develop methods for intergeneric hybridization of Hippeastrum based on detailed investigation of flowering physiology, reproductive biology and in vitro plant growth techniques. Rapid development of the flower bud and at least two new growth units was observed in bulbs which flowered following planting whereas none or only one growth unit was initiated in bulbs which produced only vegetative growth. A decrease in dry weight suggested that the stored reserves from the bulb were utilized during the flowering process. Starch and fructans were found to be the major storage carbohydrates in H. hybridum bulbs and fructans appeared to be involved in scape elongation and floret growth. The transport carbohydrate sucrose was found in lower concentrations in non-flowering bulbs than in flowering bulbs, implying a lower rate of carbon mobilization in non-flowering bulbs. ' 4C-sucrose translocation studies showed that under glasshouse conditions sucrose originating from the outermost scale tended to be partitioned towards the roots and the emerging flower bud, in contrast to carbon originating from the youngest expanded leaf which was directed predominantly towards the inflorescence. Carbohydrate partitioning from the bulb scales to the emerging flower bud was increased by repotting, a treatment shown to stimulate flower emergence. Pollen viability of H. hybridum, Brunsvigia orientalis and Amaryllis belladonna was high from 0-6, 0-2 and 0-2 days after anthesis respectively. Long term storage of pollen was necessary to overcome lack of synchronisation in flowering for controlled crosses and the results for this study showed that pollen of these three plant genera can be stored for at least 1 year at 2°C, -18°C or -80°C. Pollen viability of H. hybridum decreased rapidly after 64 weeks storage at 2°C but remained high after 104 weeks storage at -18°C and -80°C. The period of maximum receptivity of H. hybridum, B. orientalis and A. belladonna stigmas was two days after anthesis. Slow growth rate of pollen tubes were recorded in all intergeneric crosses with H. hybridum, suggesting a pre-fertilization incompatibility barrier based on rate of pollen tube growth in the style. Despite this barrier, pollen tubes were detected in the base of H. hybridum styles following crosses with A. belladonna and swelling of ovaries was observed in these crosses. The cross-pollinated pods, however, often died 10-18 days after pollination, suggesting that a post-fertilization bather was present. Ovule and ovary culture were used to overcome the post-fertilization incompatibility in the crosses H. hybridum x A. belladonna and H. hybridum x B. orientalis. Excised ovules or ovaries were cultured on MS medium supplemented with 60 g/L sucrose. Small bulblets were obtained from the cross H. hybridum x A. belladonna using ovule and ovary culture. Cut-style pollination, heat treatment of the style prior to pollination and in vitro pollination were used in an attempt to overcome the pre-fertilization barrier in the style. Application of cut-style pollination and heat treatment had no effect on pollen tube growth rate in H. hybridum styles. Small bulblets, however, were obtained from the crosses H. hybridum x B. orientalis, and H. hybridum x A. belladonna using in vitro pollination combined with ovule culture. The hybridity of these bulblets was confirmed using isozyme electrophoresis. The techniques developed in this study thus provide the basis for the incorporation of desirable traits from other genera of family Amaryllidaceae into H. hybridum.