MICROPROPAGATION AND ACCLIMATIZATION OF Aegiphila verticillata Vell.: AN ENDANGERED WOODY SPECIES

The objective of this work was to establish an efficient protocol for in vitro multiplication and rooting, as well as ex vitroacclimatization of Aegiphila verticillata, a woody species found in Brazilian rocky fields. Aseptic cultures were established by seeds and two multiplication analyses were performed. In the first, we employed 6-benzylaminopurine (BAP – 0, 2.5, 5 and 7.5 μM) + α-naphthalene acetic acid (NAA – 0, 0.2, 0.4 and 0.6 μM) and, in the second, were studied adenine sulfate, kinetin and thidiazuron (0, 5, 7.5, 10 and 12.5 μM). After 90 days, we assessed the quantitative and qualitative shoot propagation. There were more than 90% seed germination and low contamination (2%). In multiplication phase, the culture medium that promoted the best quantitative and qualitative culture development was supplemented with 7.5 μM BAP + 0.4 μM NAA. In the rooting assay, were used NAA, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (0, 0.1, 0.2, 0.3 or 0.4 μM). After 90 days, the root number and rooting quality were evaluated. In this analysis, differences were not found between the control and the other treatments. Rooted plantlets were acclimatized in styrofoam trays for 30 days, after which they were transferred to pots in the greenhouse. Only 3% of the plants subjected to initial acclimatization died and 70% of the plants transferred to the field conditions survived and showed normal development. The results founded in this work are the first involving in vitro propagation and ex vitroacclimatization of Aegiphila verticillata and provide a continuous supply of this medicinal native species, endangered due anthropogenic activities.

Rooting and acclimatization of the Japanese plum tree, cv. América

Rooting and acclimatization are limiting steps in plant micropropagation, especially in woody plant species. This study aimed to evaluate the IAA and IBA effect on the in vitro rooting and acclimatization of micropropagated shoots of Japanese plum (Prunus salicina Lindl.) cv. América. Shoots from 3 to 4 cm long were inoculated in MS medium with half salt and vitamin concentrations (MS/2) added with IAA and IBA (0, 0.25, 0.5, 0.75 and 1 mg L-1). After a 20-day period in in vitro cultivation, the shoots were evaluated, and then transferred to a greenhouse, and evaluated after 30 days. At the end of the in vitro cultivation period, no significant interactions were observed for number of roots per shoot and rooting percentage, but a significant effect was recorded for auxin type only, for which shoots grown in media added with IBA showed high values - 0.87 and 41.95%, respectively. A linear increase response from 1.45 to 5.75 cm was verified for root length of shoots cultivated in IBA medium; however, no significant effect was observed, and a 0.86 cm average root length per shoot grown in medium added with IAA was found. After 30 days of acclimatization period, the largest survival percentage was obtained from shoots cultivated in medium with 1 mg L-1 of IBA and IAA (88% and 92%, respectively). Although, IBA provided the highest in vitro rooting, most of the surviving shoots were those originated in IAA-added medium, probably because IBA promoted longer fibrous roots, less appropriate for transplant and soil fixation, as they are easily damaged. It was concluded that in vitro rooting with the addition of the highest IAA concentration (1 mg L-1) provided the greatest plant survival during the acclimatization period of the Japanese plum cv. América.

In vitro development and acclimatization of dendezeiro (Elaeis guineensis)

Fruits and almond from the dendezeiro, oil palmbelonging to the Elaeis genus,are widely used for the production of cookingoils or for the pharmaceutical and cosmetic industries.In the last decade, this oil palm also emerged as a promising source for commercialbiofuel production. This study evaluated the effect of different culture media, MS (MURASHIGUE AND SKOOG) and Y3 (EEUWENS)and carbohydrates duringin vitro germination of zygotic embryos, the effect of growth regulators GA3, NAA and BA Ponin vitro seedling development, and the survival rate of acclimatized seedlingsof Manicoré hybrid (Elaeis oleifera x E. guineensis). Zygotic embryos were inoculated on MS and modified Y3 media, supplemented with different sucrose concentrations (30, 45, and 60 gL-1) or sorbitol (36 gL-1), and the germination rate was evaluated after 30 days. Subsequently, seedlings were transferred to modified Y3 culture medium supplemented with differentGA3 concentrations (3.5 and 7 mgL-1) or without it, combined or not with 1 mgL-1 of NAA, 5 mgL-1 of BAP.The highest germinationpercentage of germinated embryos (92%) was observed in MS medium supplemented with 36 gL-1 sorbitol. Culture media supplemented with growth regulatorsGA3, NAA and BAP promoted greater shoot lengththan control media. Rooted seedlings showed high survival percentage (85%) during acclimatization.

Micropropagation of Maclura tinctoria L.: an endangered woody species

Some native species produce seeds with low germination percentage and in most cases with dormancy, which makes the appearance of new individuals by sexual propagation difficult. The Maclura tinctoria has been considered an endangered species due to the indiscriminate use of its wood and low rate of seed germination. In this context, the objective of the present study was to establish an in vitropropagation methodology for this species. Combinations of NAA + BAP, different concentrations of GA3 and combinations IBA + activated charcoal were evaluated for shoot induction, shoot growth and root formation, respectively. The results indicated that the maximum shoot formation was obtained when 5.37 µM NAA + 4.45 µM BAP was used. The use of 5.48 µM GA3 promoted shoot growth. Root formation was observed on explants inoculated in WPM with a pH adjusted to 7.0 and supplemented with 23.62 µM IBA + 4.7 g L-1 activated charcoal. The use of a 70% light screen for 7 days followed by the use of 50 and 30% light screens also for 7 days each provided 97% plantlet survival.

Acclimatization and leaf anatomy of micropropagated fig plantlets

The survival of micropropagated plants during and after acclimatization is a limiting process to plant establishment. There is little information on how the anatomy of vegetative organs of Ficus carica can be affected by culture conditions and acclimatization. The present research aimed to study the effects of time on culture medium and substrates during the acclimatization of fig tree plantlets produced in vitro, characterizing some leaf anatomy aspects of plantlets cultured in vitro and of fig trees produced in field. Plantlets previously multiplied in vitro were separated and transferred into Wood Plant Medium (WPM) where they were kept for 0, 15, 30, 45 and 60 days. Different substrates were tested and studies on leaf anatomy were performed in order to compare among plantlets grown in vitro, plantlets under 20, 40 and 60 days of acclimatization, and field grown plants. Keeping plantlets for 30 days in WPM allowed better development in Plantmax during acclimatization. Field grown plants presented higher number of stomata, greater epicuticular wax thickness and greater leaf tissue production compared to in vitro ones. The leaf tissues of in vitro plantlets show little differentiation and have great stomata number compared with acclimatized plants, which reduce the number of stomata during the acclimatization process.

In vitro culture of Phyllanthus stipulatus (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus stipulatus (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication rates (8-9 shoots per explant) was achieved on MS media supplemented with either 2.5-5.0 muM IBA. The best basal media for axillary shoot proliferation when 0.62 muM BA was supplemented were MS, MS/2 and AR (4-5 shoots per explant). Rooting was achieved with 100% of the microshoots on MS medium without growth regulators. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed in 81% of the ex vitro grown plantlets after 12 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated either in the vertical position, in the light on MS medium supplemented with 5.0 muM NAA or horizontally oriented, in the dark on MS supplemented with 5.0 muM NAA or 1.25-5.0 muM BA or 2iP. Root cultures were successfully established on MS medium containing 1.1 muM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/organ culture techniques for vegetative propagation and secondary metabolism studies.

Molluscicide activity of the "Avelós" plant (Euphorbia tirucalli, L. ) on Biomphalaria glabrata, the mollusc vector of schistosomiasis

An aqueous solution of the latex of Euphorbia tirucalli collected at sites receiving large amounts of sunlight showed molluscicide action on Biomphalaria glabrata, with LD50 obtained at the concentration of 28,0 ppm and LD90 at the concentration of 85,0 ppm. The toxicity of the product for fish was similar to that of Bayluscide and of copper sulfate used for comparison. However, the wide distribution of the plant, its easy propagation and the simple procedure for extraction of the active substance, which is biodegradable, favor "avelós" as a promising agent in the control of schistosomiasis.

Topworking of 'Bengal' lychee trees in the state of São Paulo, Brazil

This study aimed to evaluate the performance of 'Feizixiao', 'Nuomici', 'Heiye', 'Kway May Pink' and 'Bengal' lychee cultivars topworked onto bearing 'Bengal' trees. The experiments were carried out in commercial orchard at Rio das Pedras Farm in Jundiaí, state of São Paulo. The experimental design consisted of randomized blocks in a subdivided parcel arrangement, in which the parcel consisted of five cultivars and the sub-parcel consisted of six evaluation periods. Five replicates of three plants per parcel and six grafts per plant, (with two grafts per main branch) were used. The trees were approximately 10 years old propagated by air layering and planted with 6 x 4 m (416 plants/ha) spacing. The following characteristics were evaluated: success rate of the grafts, number of shoots, shoot length, number of nodes, number of leaves and chlorophyll content (SPAD). The control ('Bengal'/'Bengal') had the highest grafting success rate with 89.9%. On the other hand, 'Heiye', 'Feizixião', 'Nuomici' and 'Kway May Pink' had, respectively, 60.6, 55.0, 52.6 and 47.0% grafting success rates. All of the shoot grafts had normal growth with no signs of incompatibility. Therefore, the cultivars studied are compatible with 'Bengal' and may be used on commercial orchards.

Variability of the 3' terminal of the polymerase gene of Grapevine leafroll-associated virus 3 isolates from Vale do São Francisco, Brazil

Many viral diseases, including leafroll, which is of great economic importance, affect grapevines (Vitis spp.). A complex of eight viruses [Grapevine leafroll-associated virus (GLRaV) -1 to 8] is associated with this disease. The objective of this study was to compare the variability of the 3' terminal region of the polymerase gene of three isolates of GLRaV-3 (Grapevine leafroll-associated virus-3), from Submédio do Vale do Rio São Francisco (Petrolina-PE) with that of other isolates available at the GenBank, including an isolate from North America and another from Southern Brazil. The viral RNA was extracted from three infected ELISA reactive plants and a fragment of 340 bp was amplified, by RT-PCR, using primers that recognize that portion of the polymerase gene found between nucleotides 8267 and 8606. The three isolates from Vale do Rio São Francisco named Pet-1, Pet-2 and Pet-3, showed similarities ranging from 98% and 94%, respectively to the isolates from North America (AF037268) and Southern Brazilian (AF438411). Considering the whole genome, the main variation found was one amino acid change at position 2766 (F2766Y). These preliminary data indicate the existence of a natural variation among GLRaV-3 isolates from grapevines. This could be due to the vegetative propagation and long cycle of the plant, associated with the error-prone nature of RNA-dependent RNA polymerase.

Using aster multispectral imagery for mapping woody invasive species in pico da vara natural reserve (Azores Islands, Portugal)

This paper aims to assess the effectiveness of ASTER imagery to support the mapping of Pittosporum undulatum, an invasive woody species, in Pico da Vara Natural Reserve (S. Miguel Island, Archipelago of the Azores, Portugal). This assessment was done by applying K-Nearest Neighbor (KNN), Support Vector Machine (SVM) and Maximum Likelihood (MLC) pixel-based supervised classifications to 4 different geographic and remote sensing datasets constituted by the Visible, Near-Infrared (VNIR) and Short Wave Infrared (SWIR) of the ASTER sensor and by digital cartography associated to orography (altitude and "distance to water streams") of which the spatial distribution of Pittosporum undulatum directly depends. Overall, most performed classifications showed a strong agreement and high accuracy. At targeted species level, the two higher classification accuracies were obtained when applying MLC and KNN to the VNIR bands coupled with auxiliary geographic information use. Results improved significantly by including ecology and occurrence information of species (altitude and distance to water streams) in the classification scheme. These results show that the use of ASTER sensor VNIR spectral bands, when coupled to relevant ancillary GIS data, can constitute an effective and low cost approach for the evaluation and continuous assessment of Pittosporum undulatum woodland propagation and distribution within Protected Areas of the Azores Islands.

Growth promotion of pineapple 'vitória' by humic acids and burkholderia spp. during acclimatization

In vitro propagation of pineapple produces uniform and disease-free plantlets, but requires a long period of acclimatization before transplanting to the field. Quicker adaptation to the ex vitro environment and growth acceleration of pineapple plantlets are prerequisites for the production of a greater amount of vigorous, well-rooted planting material. The combination of humic acids and endophytic bacteria could be a useful technological approach to reduce the critical period of acclimatization. The aim of this study was to evaluate the initial performance of tissue-cultured pineapple variety Vitória in response to application of humic acids isolated from vermicompost and plant growth-promoting bacteria (Burkholderia spp.) during greenhouse acclimatization. The basal leaf axils were treated with humic acids while roots were immersed in bacterial medium. Humic acids and bacteria application improved shoot growth (14 and 102 %, respectively), compared with the control; the effect of the combined treatment was most pronounced (147 %). Likewise, humic acids increased root growth by 50 %, bacteria by 81 % and the combined treatment by 105 %. Inoculation was found to significantly increase the accumulation of N (115 %), P (112 %) and K (69 %) in pineapple leaves. Pineapple growth was influenced by inoculation with Burkholderia spp., and further improved in combination with humic acids, resulting in higher shoot and root biomass as well as nutrient contents (N 132 %, P 131 %, K 80 %) than in uninoculated plantlets. The stability and increased consistency of the host plant response to bacterization in the presence of humic substances indicate a promising biotechnological tool to improve growth and adaptation of pineapple plantlets to the ex vitro environment.

Chemical sterilization in in vitro propagation of Arundina bambusifolia Lindl. and Epidendrum ibaguense Kunth

There is a great demand for simpler and less costly laboratory techniques and for more accessible procedures for orchid breeders who do not have the necessary theoretical basis to use the traditional seed and clone production methods of orchids in vitro. The aim of this study was to assess the use of sodium hypochlorite (NaClO) as a decontaminant in the process of inoculating adult orchid explants of Arundina bambusifolia and Epidendrum ibaguenses. Solutions of NaClO (1.200, 2.400, 3.600, 4.800 and 6.000 mg L-1 - equivalent to 50, 100, 150, 200 and 250 mL L-1 of commercial bleach - CB) were sprayed on the explants (1.0 mL) and the culture medium (GB5), in the presence or absence of activated charcoal (2 g L-1). The explants used were nodal segments of field-grown adult plants. The procedures for inoculating the explants were conducted outside the laminar flow chamber (LFC), except for the control treatment (autoclaved medium and explant inoculation inside the LFC). The best results for fresh weight yield, height and number of shoots were obtained using NaClO in solution at 1.200 mg L-1 (equivalent to 50 mL L-1 commercial bleach) with activated charcoal in the culture medium. Fresh weight figures were 1.10 g/jar for Arundina bambusifolia and 0.16 g/jar for Epidendrum ibaguenses. Spraying the NaClO solutions controls the contamination of the culture medium already inoculated with the explants.

On the mechanism of protective action of cold acclimatization against carbon tetrachloride - and ethionine-induced fatty liver

In this study the hepatic lipoprotein lipase (LPL), activity was evaluated in adult female mice acclimatized at 5-C and submitted to carbon tetrachloride (CCI) or ethionine, in order to determine the possible role of this enzuyme in the fatty liver. The results were compared with those obtained in mice kept at room temperature (27-C) that the same hepatoesteatosis inducing agent. In contrast to animals kept at room temperature, in cold aclimatized mice neither the enhancement of the LPL-liver activity by the action of CCI or ethionine occurred nor the development of fatty infiltration in the liver was observed. We conclude that the low temperature induced a protective effect against CCI or ethionine-induced fatty liver that was correlated with the no-increase of the hepatic LPL activity.

Influence of substrates and in vitro preconditioning treatments on ex vitro acclimatization of Arachis retusa

The objective of this work was to evaluate the influence of substrate and preconditioning treatments on the acclimatization of in vitro plants of Arachis retusa. Plants were transferred to Plantmax or sand, and fertilized with Hoagland's nutrient solution. Plants maintained in sand, with or without fertilizer, showed the highest survival rates. In order to evaluate the influence of in vitro preconditioning treatments, stem segments were cultured on MS medium supplemented with different sucrose concentrations. The highest survival and developmental rates were observed in plants from two accessions cultured on MS supplemented with 1.5% and 3% sucrose. Flowering and fruit production were observed after five months.