Miriam E. Arena

Accumulation patterns of phenolic compounds during fruit growth and ripening of Berberis buxifolia, a native Patagonian species

The accumulation patterns of phenolic compounds during fruit growth and ripening of Berberis buxifolia and their correlations were studied to determine the optimal time and conditions needed to obtain maximum phenolic content. Anthocyanin content increased from 1.7 to a maximum of 752.7 mg·100 g−1 FW at the end of ripening, while the flavonoid content was maximal in unripe fruits [604.0 mg (+)-catechin equivalents·100 g−1 FW]. Total phenolic compounds decreased from 968.1 to a minimum of 746.3 mg gallic acid equivalents·100 g−1 FW just as soluble solids started to accumulate; a maximum of 1522.9 mg gallic acid equivalents·100 g−1 FW was attained at the end of ripening. Both variables (1,1-diphenyl-2-picrylhydrazyl scavenging effect and reducing power) were maximum in unripe fruits, and decreased during the ripening period, although the 1,1-diphenyl-2-picrylhydrazyl scavenging effect increased again towards the end of this period. The accumulation patterns of phenols varied depending on the specific group of compounds considered, and could be correlated with fruit quality.

Polyamines and inhibitors used in successive culture media for in vitro rooting in Berberis buxifolia

Abstract Although Berberis buxifolia has been reported to propagate in vitro, rooting is difficult to achieve during subcultures, with reduced quantity and quality of roots, as is the case with many other woody species. Several cofactors of rhizogenesis have been proposed, and the role of polyamines in rooting has recently acquired relevance. Polyamines can stimulate or inhibit microshoot rooting depending on the type and concentration of polyamine and rooting phase, while polyamine inhibitors can improve rooting in some cases. A study of the in vitro rooting of B. buxifolia using polyamines and a mix of polyamine inhibitors in a two‐step culture medium is described here, in which a new successive rooting medium was successfully implemented. While polyamine inhibitors sometimes improved rooting, nutrient medium containing a low polyamine concentration (i.e., 1 μM) enhanced rooting compared with the control medium. The best micro‐shoot rooting response resulted from the addition of 1 μM spermidine during the expression phase in the absence of inhibitors in both rooting phases. Overall, incorporating polyamine in the successive media enhances the quality and quantity of roots in B. buxifolia, thus confirming their role during the induction and expression of the radicle primordia differentiation.

In vitro propagation of Ribes magellanicum poiret

Abstract A simple micropropagation system was developed for Ribes magellanicum . Significant differences between strength of Murashige and Skoog medium macronutrient salts and 6-benzylaminepurine (BA) levels were observed on total number of new axillary shoots, shoot length and total number of leaves. After 3 weeks in culture, a maximum of four new axillary shoots per original explant were obtained in medium which contained half-strength macronutrient salts plus 0.5 mg l −1 BA. After subculture on the same macronutrients without indole-3-butyric acid (IBA) rooting percentages were > 95%. Rooted shoots were then successfully acclimatized to field conditions.

Peroxidase and polyamine activity variation during the in vitro rooting of Berberis buxifolia

Abstract Berberis buxifolia is a Patagonian shrub with great economic potential for tinctorial, pharmacological, and food industries. Clonal propagation is possible through in vitro culture and is also useful for metabolite production. However, this species is difficult to root, and to improve this, more knowledge of rhizogenesis processes is needed. Polyamines and peroxidases are useful biochemical markers during analysis of rooting phases for correlation with tissue morphological changes. Therefore, endogenous polyamine (putrescine, spermidine, spermine) changes, peroxidase activity evolution, and morphological development were studied to characterise the in vitro rhizogenesis of microshoots of B. buxifolia and, thus, to define the rooting phases. Polyamine and peroxidase changed significantly during the rooting period, and had opposite behaviours which were directly related to the IBA media content. The lower polyamine concentration and the higher peroxidase activity were found in a treatment with a dark period during the first four days and with IBA in the culture medium. Putrescine was the most abundant polyamine found in B. buxifolia tissues, 14‐ to 18‐fold more than spermidine and spermine, respectively. Therefore, these compounds were used to define the rooting phases: an induction phase (0 to 4–7 days) followed by an expression phase (4–7 to 28 days). The observed changes in the biochemical markers could be correlated with microscopic and macroscopic tissue observations in the microshoots, and the time course of rooting percentage. Successive culture media can be developed including polyamines, or other compounds and environmental conditions, which positively modify the studied biochemical markers behaviour.

Successive media to improve the in vitro rhizogenesis of Nothofagus nervosa (Phil.) Dim. et Mil.

In vitro rooting constitutes a difficult step during the micropropagation process of forest species. The successive media culture technique represents one way to overcome this barrier and includes modifying physical (e.g. photoperiod) and chemical (e.g. flavonoids) factors during the rooting phases. The aim of this study was to obtain a successive media protocol based on the incorporation of flavonoids during the in vitro rooting of Nothofagus nervosa. The factors evaluated were the type, concentration, and combination of flavonoids in relation to the rooting phases, the presence of IBA in the culture medium, the photoperiod, and the effect of flavonoids on total tissue peroxidase activity. The photoperiod used included a darkness period during the rooting induction stage and the presence of 0.61 µMIBA in the culture medium. The results showed that flavonoid incorporation at a concentration of 20 µM accelerated the appearance of roots and improved the quality of the already formed ones. Each type and concentration of flavonoid produced different responses, with (±)naringenin giving the best results. The latter caused a peak in the peroxidase activity that was absent in the control treatments. This work allowed identifying an optimized rooting protocol through a successive media culture technique that improved the speed of appearance, as well as the quantity and quality of roots for a single N. nervosa clone.

Histological events during in vitro rooting of Nothofagus nervosa (Fagaceae)

Abstract In vitro rooting protocols were previously performed on Nothofagus nervosa to optimise the culture medium and environmental conditions. A qualitative and quantitative tissue differentiation study during adventitious rooting in N. nervosa under different light/dark conditions was carried out to increase knowledge of the rhizogenesis process in shoots. The histological events through the rooting period were consistent with the proposed biochemical markers (peroxidases, tissue nutrient contents, polyamines, flavonoids). Development of parenchymatic tissues, shown by the staining pattern of the cell contents, and cambium activity were the best tissues to define the rooting phases. Knowledge of the histological changes could be used to optimise a protocol for micro‐propagation improving timing, quantity, and quality of roots, which would positively affect the survival rate during hardening.

Adventitious shoot induction from leaf explants of Ribes magellanicum cultured in vitro

Several factors were studied on adventitious shoot induction from leaf explants of Ribes magellanicum cultured in vitro: type of explant, position of the leaves along the stem and growth regulators. Explants with petiole and complete leaf lamina presented the highest percentage of organogenesis. The position of the leaf along the shoot strongly affected the induction capacity. The percentage of explants with adventitious shoots was maximum (53%) when using apical leaves, with an average of three shoots per explant. 6-benzylaminopurine (BA) and N-benzyl-9-(2-tetrahydropyranyl)-adenine (BPA) had a significantly higher promotive effect than 6-(τ-τ-dimethylallylamino) purine (2iP) or kinetin on induction of adventitious shoots. Cytokinin concentration significantly affected shoot production, best responses being obtained with 4.44 to 8.88 μM. No significant differences were observed between α-naphthaleneacetic acid (NAA) concentrations but 0.5 μM NAA usually promoted the highest number of non-vitrified shoots per explant. The results of this work show that R. magellanicum can be induced to form adventitious shoots from leaf explants cultured in vitro.

In Vitro Propagation of Juvenile Nothofagus leoni Espinosa (Fagaceae)

Nothofagus leoni has a restricted distribution in Chilean forests. This work determines suitable culture conditions forin vitro multiplication and rooting through shoots obtained from seedlings. Broadleaved Tree Medium was suitable for shoot multiplication. A medium with a pulse of 0.55 µM BA in the first subculture and two subcultures on BA-free medium resulted in a multiplication rate at day 63 of × 5.7, without callus growth or shoot neoformation. Rooted shoots of good quality (number and length of roots without callus growth) were obtained with 1.23 µM IBA (91.4% of rooting). The first roots appeared at day 11, reaching a higher speed of rooting at day 15.

In vitro propagation of Rubus geoides

Abstract Rubus geoides, a native herb of Patagonia, has fruit with a bitter‐sweet taste that can be eaten fresh or in marmalades and jams. The aim of this work was to develop a protocol for the in vitro propagation of R. geoides. The proposed protocol allowed a multiplication rate of 1:3.5 to be obtained at day 63 with Murashige & Skoog (MS) medium with 1.10 μM 6‐benzylaminopurine added. Most (80%) of the shoots rooted on MS with half‐strength macronutrient salts and 2.46 μM indol‐3‐butyric acid added and an initial period of darkness of 7 days. The root systems were of suitable quality to begin the acclimatisation stage. These results are the first for R. geoides from adult plants obtained in the field.

Genetic and morphological analysis of Berberis microphylla G. Forst. accessions in southern Tierra del Fuego

Abstract Calafate (Berberis microphylla G. Forst.) is a spontaneous shrub grown in the Patagonian region, from which berries are traditionally harvested for different purposes. This study aims to investigate on the spatial genetic structure of calafate populations grown in southern Tierra del Fuego and their morphological and genetic diversity. A first step of the research focused on 23 putative populations screened by a landscape genetic approach based on 82 geo-referenced and Random Amplified Polymorphic DNA marker characterized plants. The second phase regarded the analysis of the morphological characteristics of fruits, shoots and leaves observed on a subset of 39 plants. Taking into account multiple similarity between pairs and sub-sets of accessions, the observed differences have been associated to geographical and environmental conditions. The results allowed to postulate the existence of homogeneous populations within the studied plant sets and to formulate hypothesis on the evolution of B. microphylla in that area. No association between genetic and morphologic distances of the accessions has been observed.