Antonio Troncoso

Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them

The influence of sucrose or mannitol on in vitro zygotic embryo germination, seedling development and explant propagation of olive tree (Olea europaea L.) was compared. Embryos germinated without sucrose in the medium but for adequate development of the seedlings to yield viable plants, a carbohydrate supply was necessary; both sucrose and mannitol were equally suitable for this purpose. However, when explants obtained from in vitro germinated embryos were cultured with mannitol or sucrose, then the polyalcohol promoted significantly more growth than sucrose by increasing shoot length, pairs of leaves formed, and breaking apical dominance. This improved the in vitro culture of olive plant material, thus allowing new olive clonal lines to be obtained in shorter times. This will assist in future breeding experiments with the species.

Tropane Alkaloid Distribution in Atropa baetica Plants

Fifteen different tropane alkaloids and derivatives were identified by GC-MS in various plant organs of Atropa baetica. The main root and leaves displayed the largest number of tropane alkaloids, most of which were also present in lateral roots. In contrast, only five of these alkaloids appeared in stem tissue. Quantitative analysis by HPLC showed the presence of the two major tropane alkaloids, atropine and scopolamine, in all the samples studied. Atropine was more abundant, with the highest concentration in the main root (ca. 10.0 mg/g dry wt) followed by leaves (ca. 3.0 mg/g dry wt); scopolamine was present in highest concentration in the main root (0.6 mg/g dry wt) followed by leaves (0.4 mg/g dry wt). The lowest concentrations of these compounds were detected in stem tissue, followed by the lateral roots. The main root constitutes the major tropane alkaloid storage site; moreover, the distribution of these compounds does not appear to be organ dependent. These latter two characteristics are in contrast to closely related Atropa genera.

Growth responses of micropropagated cassava clones as affected by Glomus Intraradices colonization

ABSTRACT This study reports the effectiveness of an arbuscular mycorrhizal (AM) fungus Glomus intraradices on three clones (SOM-1, 05 and 50) of cassava (Manihot esculenta). Arbuscular mycorrhizal inoculation increased plant resistance to transplant stress from “in vitro” to “ex vitro” conditions and plant biomass (shoot and root) production was greatly enhanced by AM-colonization. The magnitude of AM growth stimulation over control clones was: 861% (SOM-1), 1042% (05) and 854% (50). Arbuscular mycorrhizal colonized cassava plants increased cassava water uptake in terms of percentage, 62% in clone SOM-1, 24% in clone 05, and 157% in clone 50. The highest effect of AM-colonization on water content in root of clone 50 was correlated with the greatest increment in leaf tissue production (1218% over control) and with the maximum shoot/root ratio determined. The biomass distribution between shoot and root was changed by AM symbiosis and such effect varied for each clone that may be caused by mycorrhizal changes in macro/micro-nutrients translocation/compartimentation. Cassava dependence on AM symbiosis was greatest in clone SOM-1 since AM-colonization provided the highest stem (weight, length, and diameters), leaf (weight and number), bud number, and root weight. These results lead to practical applications because AM inoculation is crucial for improving cassava yield (shoot and root) and nutrition irrespective of the clone involved. Thus, importance of AM symbiosis in micropropagated cassava clones is of great practical interest in agriculture and allows the selection of the most suitable clone for dry environments due to the particular effect on root water content that improves drought adaptation.

Arbuscular-Mycorrhizal Contributes to Alleviation of Salt Damage in Cassava Clones

ABSTRACT This study determined how arbuscular-mycorrhizal (AM) colonization by Glomus intraradices affected plant biomass and salt tolerance (in terms of growth) of three cassava clones (SOM-1, 05, and 50). Survival, root, stem and leaf production, and nutrient accumulation were determined in AM-inoculated and non-inoculated cassava clones under a range of sodium chloride (NaCl) levels (0, 68.4, or 136.8 mM) in the medium. The AM colonization stimulated plant growth and increased survival at 136.8 mM of salt. Clone SOM-1 showed to be the most salt tolerant of the three clones tested. G. intraradices-inoculation was important not only for growth promotion, but also played a crucial role in protecting cassava clones against salt (particularly the most salt sensitive clones). Mycorrhizal clones growing under 136.8 mM of NaCl showed greater dry weight than non-mycorrhizal clones growing without salt. Results show that AM-colonization provides a biological mechanism by which cassava clones increased plant biomass and salt tolerance being required for the best cassava clone development under non-stress and stress conditions.

FEASIBILITY AND ANATOMICAL DEVELOPMENT OF AN IN VITRO OLIVE CLEFT-GRAFT

SummaryThe feasibility and anatomical development of an in vitro olive cleft-graft method were studied. Grafting survival after 60.d in vitro was 85% which then dropped slightly to 67% after hardening. Three days after grafting, callus formation was observed along the cut surfaces of the rootstock and scion, after 6.d the first healing cellular unions were observed, and by 12.d after grafting a strong union developed. The first cellular differentiation to form vascular tissues was observed 12.d after grafting and continued rapidly until a total connection was reached 10.d later. Thus a complete graft union of the in vitro olive cleft-grafts was achieved in three weeks, and vigorous plants were established after 60.d of in vitro culture followed by 10.d of hardening. The in vitro olive cleft graft is suggested as an effective and useful method for germplasm multiplication.

Heavy metals and trace element concentrations in intertidal soils of four estuaries of SW Iberian Peninsula

Soils from four estuaries of SW Iberian Peninsula, affected by anthropogenic influence (urban, industrial and agricultural activities), were analyzed for the occurrence of a variety of metals and trace elements including Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, S and Zn. The soils presented very high levels of salinity (high concentrations of Na, K and Mg), organic matter and, consequently, of C and N concentrations. In contrast, very low values of CaCO3, Ca and P were found. In addition, it should be highlighted that in certain localities (Piedras 1 and 2 and Guadiana in Huelva, Spain, and Ria Formosa, Faro, Portugal) the concentrations of Pb, S and Zn were extremely high, reaching levels of pollution.

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced.

Some propagation methods for cloning holm oak (Quercus ilex L.) plants

Holm oak (Quercus ilex L.), a typical evergreen tree of the Mediterranean area, is very important due to its ecological and economical values. Propagation of this species is extremely difficult and traditionally carried out only by seed germination. In this work, mature acorns were germinated in vitro and in peat substrate in aseptic and non-aseptic conditions. Explants from the seedlings obtained were propagated in vitro in WPM plus 4 µM BA. Plant regeneration was achieved from hypocotyls and root segments cultured in vitro on modified Gamborg medium plus 20 µM BA and 20 µM NAA. 13.8% of the hypocotyls and approximately 30% of the root segments developed both shoots and roots after 30 days of culture. Rooting of stem segments was obtained both in vitro and ex vitro by basal dipping in IBA solutions. Within ex vitro rooting, mother plant age had major influence on the percentage of rooting of the cuttings as the younger plants showed higher ability to root. In this way, Q. ilex plants could be propagated and cloned. The procedure described here would be a very useful tool for breeding programs since vegetative propagation of selected individuals can be achieved.

The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L−1 of BA and 0.036 mg L−1 of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L−1 IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.

In vitro development and germination of immature olive embryos

Summary In vitro culture methods were used to germinate olive embryos prior to maturation. Fruit, seed and embryo development were established with consecutive sampling from 20 to 100 days after bloom. For that same period, embryo development and germination success were determined by in vitro culture trials using one-third strength MS medium with or without the addition of zeatin. For early developmental stages, when isolation of the embryo was difficult, a cut portion of the seed containing the embryo was used for culture. The embryos cultured within the cut seed portions germinated and formed normal plantlets. Histological observations indicated a close similarity between the natural and in vitro immature embryo differentiation pattern, progressing through preglobular, globular, heart-shaped and torpedo-shaped stages. In some cases, however, the in vitro immature embryos developed or germinated abnormally. The presence of zeatin (0.25 mg l–1) in the culture medium and the use of a cut seed-portion containing the immature embryo allowed in vitro germination sooner after bloom than previously obtained. On the contrary, zeatin was a handicap for mature olive embryo in vitro germination, which reached 100% seedling formation when no plant growth regulators were used.