Michael M. Yeoman

Phenylpropanoid metabolism during growth and development of Capsicum frutescens fruits

Abstract The production of capsaicinoids in fruits of Capsicum frutescens takes place after the increase in fruit length has ceased. However, before the ons

The synthetic potential of immobilised cells of Capsicum frutescens Mill cv. annuum.

Cells of Capsicum frutescens Mill. cv. annuum, immobilised in reticulate polyurethane foam, produced higher yields of capsaicin, the pungent principle of Chilli pepper fruits, than did freely-suspended cells, when batch-cultured in a medium conducive to culture growth. In the absence of specific precursors to capsaicin, immobilised cells produced between two and three orders of magnitude higher yields than did suspended cells over 5-d or 10-d culture periods (typically up to 4 or 5 mg capsaicin g-1 dry weight l-1 medium compared with up to 30 μg g-1l-1, respectively). These results were reflected by an increased rate and extent of incorporation of L-[U-14C]phenylalanine into capsaicin in immobilised as compared with freely-suspended cells, and evidence is presented for an inverse relationship between incorporation of [14C]phenylalanine into protein and capsaicin. The accumulation of capsaicin can be experimentally manipulated and increased by supplementing the medium with precursors of capsaicin such as phenylalanine and isocapric acid and by reducing the growth rate of immobilised cells by omitting growth regulators from the medium. The importance of these observations is discussed.

Early Development in Callus Cultures

Publisher Summary This chapter discusses the early development in callus cultures. Whatever their origin, the initiation and development of calluses involve vigorous cell division. Calluses are commonly observed in nature as a result of mechanical wounding or the interference of microorganisms or insects. The usual technique in promoting the formation of a callus from a fragment of plant tissue is to bring the explant into contact with a medium containing stimulatory chemicals. Excision and culture together promote the growth of particular tissues in the explant. If the explant is uniform and consists of one cell type, then the callus that develops initially is also uniform because it arises from only one cell type. A characteristic of developing callus cultures derived from excised tissue is that cell division is not induced throughout the tissue but tends to be restricted to the peripheral layers. The initiation of cell division in the outer layers of the tissue may be related to a complex of factors, including the wound response at the cut surface, greater availability of oxygen, more rapid release of carbon dioxide, greater availability of nutrients, and more rapid release of a volatile inhibitor, and light.

Plant regeneration from encapsulated embryoids and an embryogenic mass of pistachio, Pistacia vera L.

Pieces of an embryogenic mass (EMS) induced in culture from immature fruits of pistachio, Pistacia vera L., were encapsulated into calcium alginate beads. Somatic embryos were also encapsulated individually into calcium alginate beads to produce synthetic seeds. The viability of the encapsulated EMS and somatic embryos was investigated immediately following encapsulation, and after storage for 60 days at 4°C. The encapsulated-stored EMS fragments recovered their original proliferative capacity after two months storage following two sub-cultures, but non-encapsulated-stored EMS failed to recover. The conversion frequency of synthetic seeds to seedling plants was 14% after storage for 60 days at 4°C, from which it may be concluded that encapsulation is a practical procedure for short-term storage of embryogenic pistachio tissue, and may be applicable to the preservation of desirable elite genotypes.

Somatic embryogenesis in cultured immature kernels of Pistachio, Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl−1 casein hydrolysate, 114 μM 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 μM benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 μM benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.

Mechanism of nicotine N-demethylation in tobacco cell suspension cultures

Abstract Radioactive feeding experiments, in which dl -[pyrrolidine-2′- 14 C]-nicotine was added to 10-day-old cultures, confirmed that the kinetic pattern of this N -demethylation was similar to that of non-radioactive nicotine, and that nornicotine, the major product, was produced intracellularly with a maximum percentage conversion of about 70%. The appearance of nornicotine paralleled the disappearance of the added nicotine, although small amounts of radioactive metabolites other than nornicotine were also observed. One of these metabolites was tentatively identified by GC-MS as N -formyl-3′-nornicotine which is probably a side-product rather than an intermediate in nicotine N -demethylation. Nornicotine produced from added (−)-nicotine by the cultures was determined using both polarimetry and chiral GC, and shown to be exclusively one enantiomer. This provided strong evidence that the mechanism of this reaction in cultured cells was unlikely to involve opening of the pyrrolidine ring and, therefore, differed from that previously hypothesized for the tobacco plant. The possible mechanism for this reaction is also discussed.

Evidence in favour of an oxidative N-demethylation of nicotine to nornicotine in tobacco cell cultures

Summary Nicotine N -demethylation to nornicotine has been studied in cell-free preparations from cell suspension cultures of Nicotiana tabacum , in attempt to explore the mechanism of this enzymatic reaction. Subcellular fractionation of the homogenate using isopycnic differential centrifugation, together with TEM, showed that most of the enzyme activity (2.8 × 10 −5 U) was present in the intermediate pellet whilst the maximum specific activity of 8.8 × 10 −5 U per mg protein appeared to be associated with the microsomal fraction. The addition of selected putative methyl group acceptors, i.e. putrescine, glycine and ethanolamine, to either dialysed or undialysed enzyme preparations, did not promote enzyme activity. In addition, studies involving Sephadex G-25 gel filtration showed that molecules smaller than 5,000 MW are unlikely to be involved in this reaction. The current findings appear to support an oxidative nicotine N -demethylation and, together with the results from previous studies, provide some tentative evidence for the involvement of cytochrome P-450 in nicotine N -demethylation. The possible mechanism is also discussed.

Biotransformation of (−)-codeinone to (−)-codeine byPapaver somniferum cells immobilized in reticulate polyurethane foam

Papaver somniferum cells immobilized in reticulate-polyurethane foam biotransformed (−)-codeinone to (−)-codeine. A biotransformation ratio of 79% was found in immobilized cells whereas a ratio of 57% was found in suspended cells. Of the total amount of codeine formed only 12.2% was detected inside the cells, most of the product (87.3%) being released into the medium. When immobilized cells were cultivated in the absence of nitrate, only 40% of the cells remained alive and the biotransformation of codeinone was strongly reduced. When orthophosphate was omitted from the growth medium a bioconversion ratio of 86% was achieved.

The accumulation of phenylpropanoid and capsaicinoid compounds in cell cultures and whole fruit of the chilli pepper, Capsicum frutescens Mill

The accumulation of the phenylpropanoid precursors of capsaicin in suspended and immobilised cell cultures of C. frutescens has been studied and compared with accumulation in whole pepper fruit. The use of HPLC techniques has revealed that the phenolic precursors of capsaicin are present in chilli pepper cells at extremely low levels, irrespective of the source of tissue or the developmental state. Radioactive tracer studies have indicated that the majority of the phenolic derivatives of phenylalanine are ultimately bound to the insoluble fraction of the cells. Results from experiments where immobilised cell cultures were grown under conditions which enhance capsaicin yield would suggest that the ‘diversion’ of compounds into this bound fraction has a considerable influence upon capsaicin biosynthesis in this system.

Nicotine N-demethylase in cell-free preparations from tobacco cell cultures

Abstract The activity of the enzyme(s) catalysing the bioconversion of nicotine to nornicotine has been demonstrated, for the first time, in cell-free preparations from tobacco cell cultures. Using 14 C-assay, it has been shown that a maximal specific activity of ca 0.2 pkat mg protein −1 is present exclusively in the supernatant fraction after centrifugation at 8 800 g . The enzyme has a pH optimum between 9.0 and 9.5, and a temperature optimum between 25 and 30°, while the V max and the apparent K m are 7.6 × 10 −2 pkat and 7.4 μM of 14 C-nicotine, respectively. The decline in enzyme activity, following the removal of some endogenous co-factors and co-enzymes by dialysis of the crude enzyme preparation, can be restored and indeed enhanced by the addition of NADPH, suggesting that this enzyme may be NADPH dependent.