Resumo Condições ambientais de crescimento podem modificar a estrutura foliar e a produção de metabólitos. Plantas de nim produzem uma quantidade elevada de metabólitos medicinais e contém terpenóides biopesticidas com baixa toxicidade. Entretanto, a alta variação genética e a baixa qualidade do material, além das modificações do ambiente alertam para a necessidade de técnicas de biotecnologia para certificar a produção de metabólitos de elevada qualidade. O objetivo foi investigar a estrutura foliar e características histoquímicas de Azadirachta indica cultivadas in vitro, in vivo e em condições de aclimatização. Foram encontradas diferenças anatômicas entre os ambientes, com maior espessura foliar e estômatos mais uniformemente distribuídos associados a condições in vivo. Essas modificações não afetaram a produção de metabólitos e biopesticidas. Terpenos e taninos foram sintetizados em células especializadas chamadas de idioblastos, localizadas no mesofilo e na região da nervura central, respectivamente. Assim, em uma abordagem qualitativa, pode-se afirmar que os diferentes ambientes não modificaram a produção de metabólitos. O aumento da produção destes compostos poderia ser relizada por meio do isolamento e cultivo in vitro de idioblastos como nova fonte de pesquisa em biotecnologia vegetal .

Abstract Environmental conditions of grow can modify leaf structure and metabolite production. Neem plants produce a high amount of medicinal metabolites and contain biopesticide terpenoids with low toxicity. However, the high genetic variation and the low material quality, besides the environmental modifications warn to the need of biotechnological techniques to ensure the production of high quality metabolites. The aim was to investigate leaf structural and histochemical characteristics of Azadirachta indica grown in vitro, in vivo and acclimatized condition. It was found anatomical differences among the environments, with higher leaf thickness associated to in vivo conditions, as well as were more evenly distributed stomata. Those modifications did not qualitatively affect the production of medicinal metabolites and biopesticides. Terpenes and tannins were observed in specialized cells called idioblasts, located in the mesophyll and in the midrib region, respectively. Thus, in a qualitative approach, we can affirm that the different environments do not modify metabolites production. Increased production of these bioactive compounds could be achieved by isolation and in vitro culture of idioblasts as a new source of research in plant biotechnology.

The use of cell and plant tissues culture techniques to produce economically important active metabolites has been growing. Among these substances, azadirachtin (AZA), produced by the neem tree (Azadirachta indica), has received considerable attention due to its bioinsecticide action. The main goal of this work was to analyze the AZA levels in neem cotyledonary calli. The calli were grown in agitated Woody Plant Medium (WPM) liquid medium, supplemented with glucose (Gl), hydrolyzed casein (HC) and methyl jasmonate (MeJ) as elicitor agent. An interaction was observed between these substances, depending on in vitro cultivation time with orbital agitation. The highest concentrations (average of 0.2470 µg g-1) of AZA were produced in the first and second weeks of culture when the cell mass was grown in a medium with 2% Gl v/v, 500 mg L-1 HC and 100 µM of MeJ. This corresponded to approximately 57% of the AZA content stored in the donor plants seeds, used as a source of explants to induce in vitro callus formation. It was concluded that the nutrition, as well as the concentration of MeJ as signal transduction of secondary metabolism in neem cells, might influence the AZA content produced in vitro.

A propagação in vitro tem se destacado como uma técnica efetiva na produção em larga escala de plantas sadias de morangueiro. Neste trabalho estudamos a hiperhidricidade associado a capacidade de multiplicação in vitro na propagação de duas variedades de morangueiro (Fragaria x ananassa Duch. "Dover" e "Burkley"). Plantas mantidas em meio de cultura MS, suplementadas com 1.0 mg L-1 de BA fora individualizadas e transferidas para o mesmo meio com Ágar (6.5 g L-1) ou Phytagel® (2,5 g L-1) e BA em diferentes concentrações (0; 0,5; 1,0; 2,0 e 3,0 mg L-1). Foram realizadas análises bioquímica e anatômicas, além da caracterização morfológica do material hiper-hídrico. A análise dos dados mostrou: a) o incremento na concentração de citocinina aumentou a freqüência de hiper-hidricidade para ambas as variedades; b) concentrações maiores que 2,0 mg L-1 de BA, com a substituição do Ágar pelo Phytagel® induziu a maior formação de ramos hiper-hídricos; e c) a adição de BA induziu o estresse oxidativo, caracterizado pelo incremento da atividade antioxidante e peroxidação de lipídeos, bem como alterações a nível celular, como má formação dos estômatos e células epidérmicas. Concluindo, o meio de cultura contendo 0,5 mg L-1 de BA solidificado com Ágar promoveu menor porcentagem de hiper-hidricidade associado com maiores taxas de proliferação de gemas em morangueiro.

In vitro propagation has become an effective practice for large-scale production of strawberry plants. The objective of this study was to evaluate the hyperhydricity and the multiplication capacity of two strawberry varieties (Fragaria x ananassa Duch. 'Dover' and 'Burkley') propagated in vitro. Plants maintained in MS medium supplemented with 1.0 mg L-1 BA were individualized and transferred to the same medium solidified with Agar (6.5 g L-1) or Phytagel® (2.5 g L-1) and BA at different concentrations (0; 0.5; 1.0; 2.0 and 3.0 mg L-1). Biochemical and anatomical analyses were carried out, as well as the analysis of the morphological hyperhydricity characteristics. The analysis of data showed: a) the increase in cytokinin concentration increased hyperhydricity frequency in both varieties; b) at concentrations up to 2.0 mg L-1 BA, the replacement of Agar by Phytagel® induced a higher formation of hyperhydric shoots; and c) the addition of BA induced oxidative stress, which is characterized by increased antioxidant activity and lipid peroxidation, as well as alterations at the cellular level, such as malformation of stomata and epidermal cells. In conclusion, the culture medium containing 0.5 mg L-1 BA solidified with Agar provided lower hyperhydricity percentages in association with higher rates of shoot proliferation in strawberry.