The biochemical and cytokinin changes in the developing and germinating seeds of Podocarpus henkelli stapf.
A review of the literature revealed that there is a lack of depth in our knowledge of gymnospermous seeds with regard to the development and germination processes. The phytohormones, particularly the cytokinins have been implicated in these processes. The seeds of Podocarpus henkelii were thus selected as experimental material for studying the biochemical and cytokinin-like changes associated with development and germination. The development of these seeds was also followed at the ultrastructural level. These studies revealed that cellular detail within the female gametophyte only began .to form in December (early summer), approximately six weeks after fertilization had taken place. At this time some reserve protein was evident and the embryo sporophyte consisted of only a few pro-suspensor and pro-embryo cells. Concurrently, the cytokinin levels were fairly high in the female gametophyte but low in the epimatium. In both seed components two cytokinin- like compounds predominated which co-chromatographed with the free base cytokinin zeatin and its ribonucleoside. The second sample was taken in late January (mid-summer) by which time the embryo sporophyte had developed rapidly into a readily distinguishable seed component. The cellular detail indicated that much cell division had recently taken place and that the cells were currently increasing in size and accumulating starch and lipid. In the female gametophyte the soluble sugars were at the maximum level recorded during these experiments and the level of starch was increasing. The extractable cytokinin content of the seed was high at this time, particularly in the embryo sporophyte. In all three seed components cytokinin-like compounds which co-chromatographed with zeatin and ribosylzeatin were present. These high levels of cytokinin coincided with the rapid increase in both fresh and dry mass of the embryo sporophyte and female gametophyte. Ultrastructural studies of the third sample collected in mid-March (early autumn) showed that cellular changes were associated mainly with increases in cell size and the accumulation of food reserves, particularly starch. The cytokinin levels had decreased in all three seed components at this time. There was an increase in the cytokinin which co-chromatographed with glucosylzeatin in the female gametophyte. The seeds matured in late April (autumn) and had the unusual features of not drying out during maturation. Fresh seeds collected from the ground had a moisture content of ca. 62 per cent. The main food reserve was starch with relatively small amounts of protein and lipid also present. The seeds of Podocarpus henkelii germinated readily after scarification in the absence of water provided that their moisture content remained ca. 60 per cent. Seeds in which the moisture content had fallen below ca. 54 per cent required additional water for germination. The moisture content of the seeds fell rapidly under natural conditions and viability was lost below a moisture content of ca. 34 per cent. Unscarified seeds of 52 per cent moisture content placed under moist conditions at a constant 25°C took 23 weeks to achieve 68 per cent germination. These experiments showed that although the epimatium limited water uptake by the seeds it did not prevent moisture loss to the atmosphere. This appears to be the main factor contributing to the seed's inefficiency as a propagule. A small degree of after-ripening was recorded with the embryo sporophyte increasing in size with storage. This appeared to contribute to the increased rate of germination of the scarified seeds. An interesting feature of the seeds of Podocarpus henkelii is that they have the ability to fix atmospheric carbon, which is subsequently translocated from the epimatium to the female gametophyte and embryo sporophyte. The mature seeds were stored at 4°C for six weeks during which time little change had occurred at the ultrastructural level. Protein vacuoles in the embryo sporophyte had disappeared and in all three seed components cytokinin levels were low. Three days after scarification and the start of incubation, little change in cellular detail was apparent as limited rehydration was necessary due to the high moisture content. The cytokinin levels in the embryo sporophyte and epimatium had increased, whilst the levels in the female gametophyte had decreased at this time. In the embryo sporophyte lipid mobilization had commenced with these reserves apparently being metabolized within vacuoles. The rate of respiration measured in terms of increases in CO evolution, increased 60 hours after the start of the incubation period, just 12 hours before ten per cent germination was recorded. Germination was accompanied by a large increase in the levels of cytokinins in the female gametophyte and embryo sporophyte. The cytokinins detected co-chromatographed with the free base cytokinin zeatin and its riboside, ribosylzeatin. Concurrently, marked ultrastructural changes were recorded with increases in the amounts of dictyosomes, endoplasmic reticulum and the formation of polyribosomes, all of which are indicative of increased metabolic activity. Similar increases in the female gametophyte were ofa lower order and occurred only after nine days of incubation. By this time the levels of cytokinins had decreased considerably. After 12 days of incubation 65 per cent of the seeds had germinated. As much of the food reserves in the female gametophyte and embryo sporophyte remained, it is suggested that these reserves are utilized for subsequent seedling establishment rather than for germination. The actual role that cytokinins play in the development and germination of these seeds is not clear. High levels of this phytohormone coincide with periods of food deposition and mobilization suggesting that they play an important part in these processes. The results of the biochemical, cytokinin and ultrastructural studies are discussed in relation to the developmental and germination processes and are compared to the data of other seeds.