Shooting control by brassinosteroids: metabolomic analysis and effect of brassinazole on Malus prunifolia, the Marubakaido apple rootstock.

To help unravel the role of brassinosteroids (BRs) in the control of shooting, we treated the shoots of Marubakaido apple rootstock (Malus prunifolia (Willd.) Borkh cv. Marubakaido) with brassinolide and Brz 220, an inhibitor of BR biosynthesis. Brassinolide differentially affected elongation and formation of main and primary lateral shoots, which resulted in reduced apical dominance. Treatment of shoots with increasing doses of Brz 220 led to a progressive inhibition of main shoot elongation. Eight different BRs were also identified in the shoots of M. prunifolia. Progressive decline in 6-deoxocathasterone, 6-deoxotyphasterol and castasterone was related to increased doses of Brz 220. Analysis of the metabolic profiles between a fluoro-containing derivative of 28-homocastasterone (5F-HCS) using treated and untreated shoots demonstrated that no 5F-HCS-specific metabolite was identified. However, 4 weeks after the treatment, fructose, glucose and the putatively identified gulonic acid were higher in 5F-HCS-treated shoots, compared to untreated shoots. These results indicate that the previously reported 5F-HCS-induced stimulation of shoot elongation and formation of new shoots in the Marubakaido shoots is under the control of changes in the endogenous BR pool. In addition, the results presented in this report also indicate that the 5F-HCS-induced shooting likely involves a variety of different mechanisms and consequently does not result from changes in the endogenous levels of any single metabolite.

Preparation of betulinic acid derivatives by chemical and biotransformation methods and determination of cytotoxicity against selected cancer cell lines.

Several novel 2,4-dinitrophenylhydrazone betulinic acid derivatives have been prepared by chemical and biotransformation methods using fungi and carrot cells. Some compounds showed significant cytotoxicity and selectivity against some tumor cell lines. The most active, 3-[(2,4-dinitrophenyl)hydrazono]lup-(20R)-29-oxolupan-28-oic acid, showed IC50 values between 1.76 and 2.51 µM against five human cancer cell lines. The most selective, 3-hydroxy-20-[(2,4-dinitrophenyl)hydrazono]-29-norlupan-28-oic acid, was five to seven times more selective for cancer cells when compared to fibroblasts. Cell cycle analysis and apoptosis induction were studied for the most active derivatives.

A galactomannan-driven enhancement of the in vitro multiplication rate for the Marubakaido apple rootstock (Malus prunifolia (Willd.) Borkh) is not related to the degradation of the exogenous galactomannan.

Agar is a complex mixture of gel-forming polysaccharides. Gelling agents are very often used to provide proper support for plants grown in semisolid culture media. And agar is the most frequently used gelling agent in plant tissue culture media. Galactomannans, another group of gel-forming polysaccharides, consists of a (1 → 4)-linked ß-D: -mannopyranosyl backbone partially substituted at O-6 with D: -galactopyranosyl side groups. In this work, we demonstrate that a statistically significant 2.7-fold increase on the multiplication rate (MR) for in vitro-grown Marubakaido (Malus prunifolia) shoots was associated with a 12.5% replacement of agar in the semi-solid culture media for a galactomannan obtained from seeds of Schizolobium paraybae. This increase on MR was due mainly to a 1.9-fold increase in the number of main branches and an 8.6-fold increase in the number of primary lateral branches. Gas liquid chromatography and thin layer chromatography analyzes demonstrated that the galactomannan-driven enhancement of the in vitro multiplication rate of the Marubakaido apple rootstock was not related to the galactomannan degradation. To the best of our knowledge, this is the first report on the successful use of partial replacement of high quality agar by a galactomannan from S. paraybae in a micropropagation system for a tree species.

Thermally induced changes in shoot morphology of Hancornia speciosa microcultures: evidence of mediation by ethylene.

A shoot microculture protocol for the tropical fruit tree Hancornia speciosa Gom. was developed based on high temperature stimulation of axillary branching. An increase in the culture temperature from 25 to 31 degrees C (continuous over a 4-week period) stimulated the elongation of newly subcultured micro-shoots. Increases in culture temperature from 31 to 35 degrees C suppressed elongation but induced branching of all shoots. Increasing temperature to 37 degrees C reduced both shoot elongation and branching severely. Thermoperiodic regimes involving daily exposure for 8 or 16 h at 35 degrees C followed by 31 degrees C for the remainder of the day were only moderately effective in stimulating branching. However, maintaining cultures at 35 degrees C for 2 weeks followed by growth at a constant temperature of 31 degrees C led to vigorous branching. We hypothesize that thermally induced branching is mediated by a reduction in ethylene biosynthesis. In cultures of nodal segments grown at 31 degrees C, ethylene evolution peaked 12 to 16 days after subculture, and then gradually decreased until the end of the culture cycle. Compared with cultures held at 31 degrees C, those grown at 35 degrees C showed an earlier peak and a reduced rate of ethylene evolution throughout most of the culture cycle, and less 1-aminocyclopropane-1-carboxylate (ACC) synthase mRNA transcript and ACC oxidase activity. An inhibitor of ethylene biosynthesis (L-(2-aminoethoxyvinyl)-glycine) at 4.5 &mgr;M mimicked the effect of elevated temperature (35 degrees C) in stimulating lateral branching. The branching pattern of shoots grown at 35 degrees C in the presence of 1 &mgr;l l(-1) ethylene gas resembled that of shoots grown at 31 degrees C. We conclude that a reduction in ethylene evolution has a role in thermal induction of branching in H. speciosa. Heat induced release of axillary buds may be useful in the microculture of trees with strong apical dominance that cannot be overcome by cytokinin treatment.