M. Salomé Pais

Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.

Applicability of extracts from Centaurea calcitrapa in ripening of bovine cheese

Abstract Aqueous extracts obtained from cell suspension cultures of Centaurea calcitrapa were used as proteolytic additive in the manufacture of a commercial bovine cheese, coagulated with animal rennet and typically ripened for 28xa0d. The cheese was assessed in comparison to standard cheese for two levels of addition of said extract, viz. 0.61 and 1.22xa0mg of total protein mL −1 . The qualitative and quantitative evolutions of the nitrogen fractions were monitored in the experimental cheeses throughout the whole ripening period. In general, the chemical compositions of the cheeses were different depending on the amount of extract used, but no significant differences could be detected in the ripening index. With regard to electrophoretic profiles, the two types of cheese could be distinguished until up to ca. 7xa0d of ripening, but differences did essentially vanish by 28xa0d.

A scanning electron microscopy and X-ray microanalysis study during induction of morphogenesis in Camellia japonica L.

Abstract A scanning electron microscopy (SEM) study during the induction of direct and indirect morphogenesis (root and embryo formation) from leaves of Camellia japonica L. showed that the induction of morphogenesis is linked to the appearance of a layer of fibrillar material covering the surface cells of induced leaves and calli. Before embryo formation a 2nd deposition of material of smooth texture occured being the embryogenic regions of calli and globular embryos totally covered by a layer of this material. A positive reaction to aniline blue staining suggests that a deposition of ‘callose’ precedes embryo formation. Results of electron probe microanalysis (EPMA) during direct morphogenesis show higher levels of Ca, K, Na, Fe and S in leaf-induced parenchyma cells which decrease with the onset of morphogenesis. Results suggest a correlation between X-ray spectra and the in vitro response obtained (calli, embryogenesis, rhizogenesis). Identical X-ray spectra were obtained for both globular somatic and zygotic embryos. Roots also presented identical X-ray spectra independently of their origin. Can X-ray microanalysis be used to diagnose cell competence and determination?

Explant region-specific embryogenic competence and plant recovery inCamellia japonica

The culture conditions for direct, indirect, and repetitive embryogenesis were established forCamellia japonica cv. Elegans and cv. Ville de Nantes. Direct embryo production from leaves averaged 15.3 embryos per responsive leaf on Murashige and Skoog medium (MS) with 1.0 mg·liter−1 N6-benzyladenine and 0.5 mg·liter−1 2,4-dichlorophenoxyacetic acid. Plantlet production was 7.1 (±1.5) plantlets per leaf. Direct embryo production from stems averaged 5.7 embryos per shoot, and 2.7 embryos per stem portion, on MS medium supplemented with 1.0 mg·liter−1 N6-benzyladenine and 0.1 mg·liter−1 indolbutyric acid (MS28). Conversion was only obtained after repetitive embryogenesis. Embryogenesis from leaf-derived callus occurred in all callus after transfer to MS/2–25 medium (half strength MS medium with 25 g·liter−1 D-glucose) (production stage). Plantlet production was 16.3 (±3.6) plantlets per callus. Repetitive embryogenesis increased embryo population by 2.3- to 3.6-fold every 4 wk. Conversion of secondary embryos was obtained on MS medium supplemented with 2.0 mg·liter−1 N6-benzyladenine, 0.2 mg·liter−1 indolbutyric acid, 5 mg·liter−1 gibberellic acid (MS56). Direct embryo formation from leaves, stems, and cotyledons, and embryogenic callus formation from leaves were restricted to specific regions of the explant.

Enzymes of Opuntia ficus-indica (L.) Miller with potential industrial applications-I

We report on the screening of different enzymes such as arylamidases, lipases, proteinases, and glucosidases in plant extracts of the Cactaceae family, genus Opuntia, as well as on a newly purified plant proteases from O. ficus-indica fruit extracts. These proteinases showed the maximum activity at pH 5.2 and 55°C and FTC-casein was the best of the escreened substrates. Proteolytic activities were activated by anti-oxidant compounds and by some divalent cations. These proteinases were efficiently inhibited by cystein proteinase inhibitors and by 1,10-phenanth roline. The estimated Mt for the main proteolytic activity was about 23.2 kDa. The results on milk clotting characteristics suggest a potential use of the fruit cystein enzymes of this plant in dairy industries.

Induction of microspore embryogenesis in Camellia japonica cv. elegans

Three methods of microspore culture were tested for the induction of microspore embryogenesis in Camellia japonica L. cv. Elegans. Culture was performed on 17 different media consisting of Murashige and Skoog (MS) and N6 basal media with different combinations of carbon, growth regulators, serine and glutamine. Microspore suspensions plated over solid MS medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 0.5 μM kinetin, with sucrose (MS6) or glucose (MS9) were seen as the best culture conditions for induction of embryogenesis. The development of microspore derived proembryos was obtained in MS medium supplemented with 2.2 μM N6-benzyladenine (MS10) and reached the highest level when the microspores were cultured in MS6 inducing medium. The development of microspore-derived embryos ceased at the maturation stage.

Adventitious shoot formation and plant regeneration from Pinus pinaster Sol. ex Aiton

SummaryPinus pinaster plants were regenerated from cotyledons excised from in vitro germinated seeds and axenically cultured on induction medium (GMD). 6-Benzyladenine (2.2 µM) induced the highest frequency of direct bud formation from cotyledons. An average of 13.1 ± 2.1 elongated shoots per cotyledon was obtained. Germination time influenced shoot induction, and the organogenic potential decreased with explant age. Cotyledons remained for 21 d on induction medium, and in order to promote adventitious shoot elongation, they were transferred to Gupta and Durzan’s DCR medium without growth regulators, containing 0.5% (wt/vol) activated charcoal and 3% (wt/vol) sucrose. Rooting was achieved by application of an indole-3-butyric acid, (396.6 µM) pulse (24 h at 4° C), followed by transfer to a sterile mixture of peat plus perlite (1:1 vol/vol). Ninety-eight to 100% of the regenerated plants were successfully acclimatized. All plants have survived after transfer to the field.

Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine sodium caseinates

The rates and extents of hydrolysis of αS‐ and β‐caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficus‐indica, (L.) Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo‐first‐order enzymatic reaction, in the presence of first‐order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet exhibited higher enzymatic efficiency than the fruit extract, irrespective of the source (i.e., bovine, caprine, or ovine) and the type (i.e., αS‐ or β‐casein) of substrate. The enzymatic efficiency (kcat/Km) for αS‐casein ranged from 72 to 220 and from 43 to 65 L g−1 h−1, and for β‐casein from 242 to 742 and from 55 to 164 L g−1 h−1, for the animal rennet and the enzymatic extract of O. ficus‐indica, respectively. Finally, it was observed that β‐casein from caprine and ovine caseinates was degraded by O. ficus‐indica faster than its αS counterpart, but the reverse was observed for bovine caseinate.

Proteases from cell suspension cultures of Cynara cardunculus

Abstract Cell suspension cultures established from hypocotyl segments of Cynara cardunculus have shown different growth patterns in B 5 medium and TNO 3 − medium. The former medium is adequate for biomass production, while the latter promotes the synthesis of proteases. These proteases have shown different properties from those of flower cynarases. They are inhibited by leupeptin and cystatin at low inhibitor concentrations, and do not react with cynarase 3 antibodies. A high heterogeneity was found in the cell suspension culture proteases. Western blot analysis together with Northern hybridization experiments have shown that the flower-specific cynarases are not expressed in these suspension cultured cells.

Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.