Half-seed method

Mutants of G. fujikuroi provide a useful means to investigate the biosynthesis of GAs and related diterpenes, the broad specificity of the enzymes mediating secondary metabolism, and the factors controlling the enzymes of individual biosynthetic steps. Their usefulness lies mainly in the ease of analysis of metabolic products from added substrates which normally are formed after the metabolic block. The main disadvantage lies in the acquisition of mutants and, although the halo-half seed bio-assay provides a rapid and sinq>le method of screening for presumptive mutants, the low frequency with which mutants are found with metabolic blocks along the pathway is a major draw-back. 

Then, in the Pascal method, a melt of Graham s salt is allowed to cool to 600°C, the seed crystals are strewn on its surface, and the melt is kept for another half hour at 550°C, during which it solidifies to a completely pure material. 

The principle of the method consists of rapidly extracting the anthocyanins from the skins, gently at first and then under more extreme conditions, where the diffusion barriers are broken down. Acidity is used as a vector to facilitate extraction. In addition, it is recommended that the grapes should be roughly crushed and the resulting flesh diluted by half. Crushing the seeds also results in partial extraction of their tannins, which is necessary to assess the characteristics of the grapes. The solutions are aqueous, pH 1 (HCl n/10) and pH 3.2 (solution with 5 g/1 tartaric acid neutralized by 1/3). 

Seeds from a wide range of species contain GAs detectable by a variety of methods ranging from bioassay to MS and about half of the known GAs and many GA conjugates have been found in developing seeds [29]. 

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