The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Artichoke (Cynara scolymus L.) flower extract was assayed with the aim of replacing animal rennet in the manufacture of Gouda-type cheeses from bovine milk. Floral extract coagulated milk within a suitable time for use on an industrial scale, while the yield of cheese obtained was equal to that achieved with bovine abomasum. Five proteolytic fractions with milk-clotting activity were isolated in a two-step purification protocol, three belonging to the cardosin group. Cheeses made with C. scolymus proteases must be brined for a longer period (40 h) to prevent overproteolysis and avoid the development of a background flavor. The type of coagulant (bovine or vegetable) had no significant effect on the cheeses' chemical parameters analyzed throughout ripening, and no significant organoleptic differences were detected between those manufactured with C. scolymus or animal rennet. The results indicate that C. scolymus flower extract is suitable for replacing animal rennet in the production of Gouda-type cheeses.

An alternative protocol was developed for in vitro propagation of photinia (Photinia x fraseri Dress), an ornamental shrub, using the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Azotobacter chroococcum during rhizogenesis. Shoot tips from four-year-old mature plants, cut in spring and summer, were used as initial explants. They were cultured on Murashige-Skoog (MS) medium with Gamborg's vitamins, N(6)-benzyladenine (BA: 11.1 microM) and gibberellic acid (GA(3): 1.3 microM), obtaining 63% of established explants. The highest shoot length (22.9 mm) and multiplication rate (4.3) was achieved by cultivating for four weeks in the same basal medium supplemented with 4.4 microM BA. Both auxin induction and bacterial inoculation were used for rooting. Elongated shoots were treated with two concentrations of indole-3-butyric acid (IBA: 4.9 or 49.2 microM) during 6 days for auxin induction. Then, the shoots were transferred to an auxin-free medium and inoculated with A. brasilense Cd, Sp7 or A. chroococcum (local strain). Bacterial inoculation induced earlier rooting of photinia shoots. A. brasilense Cd with 49.2 microM IBA pulse showed a significant increase (P

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Explants of jojoba (Simmondsia chinensis (Link) Schn.) from randomly selected plants were propagated in vitro. On modified Murashige-Skoog (MS) medium supplemented with 1 mg/litre BA (N 6 -benzyl-adenine) there was a 4.6-fold increase in shoot numbers at 30 days. After 15 days on modified MS medium supplemented with 3 mg/litre IBA (indolebutyric acid) c. 25% of the shoots had developed roots. The best exflasking survival rate of the rooted plantlets (90%) was observed when the plantlets were transferred to an organic mixture, Agrosoil™. Subsequent growth rates in Agrosoil™ were higher than in other potting media tested. On the other hand, the response of each clone was highly variable, some exhibiting 75% root formation at 60 days while others displayed little response.

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The anatomy of normal and hyperhydric shoots (leaves and stems) of in vitro Handroanthus impetiginosus was compared using light microscopy, scanning electron microscopy, and transmission electron microscopy. In contrast to normal shoots, hyperhydric shoots presented numerous anatomical abnormalities at the proliferation stage. Disorganized cortex, epidermal holes, epidermal discontinuity, collapsed cells, and other structural characteristics were observed in hyperhydric shoots. So, by using anatomical analysis of in vitro H. impetiginosus shoots at the proliferation stage, we can predict which plants will survive the rhizogenesis and acclimatization stages.

The anatomy of normal and hyperhydric in vitro shoots and leaves from micropropagated Simmondsia chinensis (Link.) Schn. (jojoba) was compared with that of seedlings (control plants). In vitro normal plantlets displayed good development and survived during the acclimatization stage. In vitro hyperhydric plantlets presented numerous anatomical defects, such as hypertrophy of the mesophyll and of the stem cortex, malformed non-functional stomata, epidermal discontinuity, and xylem hypolignification; they did not survive acclimatization. The study of the anatomical features of in vitro jojoba shoots and leaves allowed determination of the structural condition of the plantlets and prediction of which plantlet would survive the critical acclimatization stage.