In vitro seed maturation in Brassica rapa L.: Relationship of silique atmosphere to storage reserve deposition

The Brassica rapa L. silique is a self-contained environment that maintains hypoxia around the developing seeds, and in which carbon dioxide accumulates to very high concentrations (>30,000 ppm). How the silique microenvironment modulates the composition and amount of storage reserves in the seeds is of interest because of the important agricultural role played by canola (B. rapa and Brassica napus) as an oilseed. Because of the small volume and dynamic nature of this microenvironment in Brassica, a standardized system was needed to study the environmental role played in storage reserve deposition. For this purpose we have developed a silique culture system that permits maturation of seed in vitro. Siliques excised from plants just 11 days after pollination complete the ripening of their seeds after 20 days of culture in light (200 mu mol/m(2)/s) on MS medium containing 30 g/l sucrose, 0.25 mg/l BAP, and 0.025 mg/l NAA. Cytochemical localization and biochemical analyses revealed that storage reserves were affected by the in vitro maturation system. Although following a comparable ripening timeline to that occurring on the plant, and producing fully germinable seeds, in vitro maturation resulted in a 40% reduction in seed weight and the mature seeds contained decreased lipid, but increased protein, starch and soluble carbohydrates. To study the internal atmosphere surrounding the seeds, we developed a method to capture silique gases in helium with subsequent quantification of O-2 and CO2 in the sample by gas chromatography. Analysis of the internal silique atmosphere showed that in vitro siliques provided seeds with a less oxygenated environment than they experience attached to the plant. Carbon dioxide concentrations remained high later into the maturation sequence in vitro than on the plant. When sampling gases from siliques attached to plants, we found multiple samples from the same plant resulted in higher variance than when only a single silique was sampled, suggesting that connection to the plant directly influences internal silique gases. Lower O-2 in the in vitro siliques was correlated with depressed lipid content in their mature seeds, supporting the conclusion that oxygen availability limits lipid accumulation. Previous studies showed how environmental factors influence Brassica embryos grown in tissue culture. These systems fail to preserve the component of metabolic regulation that is enforced by the silique wall tissues. Our in vitro maturation system provides a useful tool for specialized investigations since both the gaseous and hormonal environments can be readily manipulated. (C) 2007 Elsevier B.V. All rights reserved.