Vespasiano Borges de Paiva Neto

Carbon sources and their osmotic potential in plant tissue culture: does it matter?

Plant cell, tissue and organ cultures require a carbohydrate supply in order to satisfy energy demands. Experiments to define type and concentration of carbon source that allow culture establishment and development are usually conducted. Several studies published in the literature have inappropriately used percentages as reference concentrations to determine the quantities of the different types of sugars contained in the treatments leading to conflicting results and conclusions. The use of percentage references adds an undesirable osmotic variable among different treatments and some authors fail to consider this aspect. Therefore, molar concentrations should be used to carbohydrate concentrations reference because it isolates the osmotic variable influence that acts concomitantly with the nutritional variable.

Direct organogenesis from hypocotyl-derived explants of annatto (Bixa orellana)

The relative importance of explant, cytokinin type, carbon source and gelling agent for annatto organogenesis was studied. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments and rooted hypocotyls were cultured onto MS medium supplemented with 4.56 μM zeatin, 87.6 mM sucrose, and 2.8 g l−1 Phytagel®. Adventitious shoots derived from hypocotyl segments were less frequent and more difficult to elongate than those derived from rooted hypocotyls. Thidiazuron (TDZ) promoted a higher organogenic response in rooted hypocotyls, resembling a rosette-like structure, but impaired shoot elongation. Histological investigation showed that zeatin-induced meristemoids originated mainly from wounding tissues, and that TDZ induced a high level of mitotic division resulting in several proliferation zones nearby the epidermis and outer cortical tissues. Rhizogenesis efficiency (rooting frequency and root number) was greater at the highest indole-3-butyric acid (IBA) concentration (5.0 μM) employed, although calli occurred at the basal end of shoots. Eighty percent of rooted plantlets survived after acclimatization. This optimized regeneration protocol may enable further development of an efficient genetic transformation protocol for this species.The relative importance of explant, cytokinin type, carbon source and gelling agent for annatto organogenesis was studied. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments and rooted hypocotyls were cultured onto MS medium supplemented with 4.56 μM zeatin, 87.6 mM sucrose, and 2.8 g l−1 Phytagel®. Adventitious shoots derived from hypocotyl segments were less frequent and more difficult to elongate than those derived from rooted hypocotyls. Thidiazuron (TDZ) promoted a higher organogenic response in rooted hypocotyls, resembling a rosette-like structure, but impaired shoot elongation. Histological investigation showed that zeatin-induced meristemoids originated mainly from wounding tissues, and that TDZ induced a high level of mitotic division resulting in several proliferation zones nearby the epidermis and outer cortical tissues. Rhizogenesis efficiency (rooting frequency and root number) was greater at the highest indole-3-butyric acid (IBA) concentration (5.0 μM) employed, although calli occurred at the basal end of shoots. Eighty percent of rooted plantlets survived after acclimatization. This optimized regeneration protocol may enable further development of an efficient genetic transformation protocol for this species.

Somatic embryogenesis from immature zygotic embryos of annatto (Bixa orellana L.)

SummaryIn order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl−1 activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.

Crescimento de mudas de pau-ferro sob diferentes níveis de sombreamento

Obtaining native seedlings requires information about seeds harvest time, storage and luminosity conditions, germination and types of substrate, among others. This study aimed at evaluating the luminosity demands for the initial growth of Caesalpinia ferrea Mart. ex. Tul. var. leiostachya Benth (Fabaceae-Caesalpinioideae) seedlings submitted to different shading levels, at 60 days after transplant (DAT). The experimental design was completely randomized, in 3x5 factorial scheme (three shading levels: 0%, 50% and 70%; and five evaluation times: 60, 90, 120, 150 and 180 DAT), with four replications of 10 seedlings per experimental unit. Plants grown under the shading level of 70% presented the highest contents for total chlorophyll (38.79 ?g cm -2 ), leaf area ratio (51.54 g cm -2 ), relative growth rate (0.0322 g g -1 day -1 ) and leaf weight ratio (0.2708 g g -1 ). The highest values for stem height (110.34 cm) and base diameter (9.3 mm), and root (11.13 g), leaf (5.59 g) and shoot (16.05 g) dry matter, as well as leaf area (705.25 cm 2 ), were observed in the plants cultivated under the shading level of 50%. No differences were observed for root growth, specific leaf weight, net assimilation rate and specific leaf area, concerning the different shading levels tested (averages of 9.3 cm, 0.0082 g cm -2 , 0.0006 g cm -2 day -1 and 186.24 g cm -2 , respectively). The Caesalpinia ferrea seedlings presented a better growth rate when cultivated under the shading level of 50%.

Somatic Embryogenesis in Annatto (Bixa orellana L.)

Our research group has pioneered the work on somatic embryogenesis of Bixa orellana (annatto), and since then we have directed efforts in understanding several aspects of this morphogenic pathway in annatto. Here, we present a synthetic description of such works, emphasizing anatomical analyzes and the characterization of the cellular alterations that occur in the process, and the association of the SERK gene expression and somatic embryogenesis. These results are unprecedented and contribute to a better understanding of the processes involving somatic embryogenesis in the species. Advances in this area will facilitate the improvement of the mass propagation, genetic manipulation of the carotenoid biosynthetic pathway, and the overall breeding perspectives of the genus.

Biochemical and morpho-anatomical analyses of strawberry vitroplants hyperhydric tissues affected by BA and gelling agents

ABSTRACTRESUMOAnalises bioquimicas e morfo-anatomicas em vitr oplantas de morangueir o hiper -hidricasafetadas pelo BA e agentes geleificantes A propagacao in vitro tem se destacado como uma tecnica efetiva na producao em larga escala de plantas sadiasde morangueiro. Neste trabalho estudamos a hiperhidricidade associado a capacidade de multiplicacao in vitro napropagacao de duas variedades de morangueiro ( Fragaria x ananassa Duch. “Dover” e “Burkley”). Plantas mantidasem meio de cultura MS, suplementadas com 1.0 mg L -1 de BA fora individualizadas e transferidas para o mesmo meiocom Agar (6.5 g L -1 ) ou Phytagel ® (2,5 g L -1 ) e BA em diferentes concentracoes (0; 0,5; 1,0; 2,0 e 3,0 mg L -1 ). Foramrealizadas analises bioquimica e anatomicas, alem da caracterizacao morfologica do material hiper -hidrico. A analise Received: 05/03/2012; Accepted: 04/03/2013. 1 Biologist, Master of Science. Departamento de Biologia Vegetal, Universidade Federal de icosa, Campus V Avenida Peter Henry Rolfs, s/n, 36570-000, icosa, MinasGerais, Brasil. leticiampb@yahoo.com.br

In vitro behaviour of Aspasia variegata, an epiphytic orchid from the Brazilian Cerrado

Aspasia variegata occurs naturally in the savanna of the Mato Grosso do Sul State, Brazil and it has been widely collected for its beautiful flowers. Additionally, its habitat has been greatly reduced and little or no investigation of its spread has been performed. Aiming to establish a protocol to obtain seedlings of the orchid A. variegata, different compositions of culture medium were tested to identify which one provided better in vitro growth and development and to assess the influence of these media in seedling acclimatisation. Thus, seeds obtained from mature capsules were inoculated in Knudson culture medium for 120 days until the protocorm stage. They were transferred to different culture media formulations, including MS and Knudson with half or full formulation, and 3.0 and 6.0g L-1 activated charcoal were added to them or not. After 180 days of protocorm inoculation, seedlings were evaluated for length of roots and shoots, number of roots and leaves, and chlorophyll contents. After that, seedlings were transferred to trays containing a mixture of Plantmax® and coconut fibre (1:1) for acclimatisation. Best results for the in vitro growth of A. variegata were obtained with the use of MS medium supplemented with 6.0g L-1 activated charcoal. Higher levels of chlorophyll were obtained, however, in treatments containing MS salts without activated charcoal presence, and lower levels in media containing Knudson salts with the presence of activated charcoal. The seedlings originated by higher chlorophyll levels during in vitro cultivation presented the highest survival rates and better development in the acclimatisation phase.

A orquídea ornamental Bletia catenulata é autocompatível, mas dependente de polinizador para reprodução

Bletia Ruiz & Pav. is a neotropical orchid genus consisting of about 40 species (Brown 2005). However, in Brazil, only two species have been registered (Barros et al. 2015). Bletia catenulata Ruiz & Pav. is a common species in the Brazilian Savannah, Amazonia and Atlantic Forest, being also found in the seashore of the Rio de Janeiro State, at the Massambaba sandbank (Cepemar 2004). Some authors reported that B. catenulata was found in different Brazilian States, such as Tocantins and Maranhão (Silva et al. 1995), Minas Gerais (Araújo et al. 2002), Distrito Federal (Batista et al. 2005) and São Paulo (Ferreira et al. 2010), and also in other countries, such as Bolivia (Vásquez et ABSTRACT

Ação inibitória do endosperma na germinação in vitro de embrião zigótico de pinhão manso

Researches on physic nut micropropagation have resulted in reports about difficulties in the process of in vitro germination of fresh seeds, and we hypothesized that this dormancy is related to endosperm presence. Aiming to investigate the influence of the endosperm presence on in vitro development of zygotic embryos of physic nut, pure and filtered extracts of the endosperm of newly harvested and stored seeds for up to 100 days were added to MS medium, in which zygotic embryos were added alone or attached to the endosperm. It was observed that the endosperm presence attached to the embryo seems to establish dormancy to the restart of the embryo development. Moreover, addition of endosperm extracts into the MS medium greatly reduced the embryos development, supporting the idea that in addition to probable inhibition physics, the endosperm may contribute to chemical inhibitors in the germination process. All these idea were supported by perfect embryo development when it was added to MS medium without endosperm structure or extract.