The goal of the present study was to evaluate the germination, initial growth, and in vitro co-cultivation of Comanthera curralensis Moldenke, a "sempre viva" native of the Chapada Diamantina state of Bahia. Full strength (MS) and half-strength MS (MS1/2) growth media supplemented with two different sucrose concentrations (15 and 30g L-1) were tested for germination and initial plant growth. Three different plant densities were tested by in vitro culture (8, 10 and 12 plants per container). MS1/2 medium with 15g L-1 sucrose resulted in a higher percentage of germination and plant growth for the in vitro establishment of C. curralensis. The use of 12 plants per container is indicated for cost reduction in C. curralensis in vitro production.

Bromeliads are the target of predatory extractivism and consequently many species are included in the red list of threatened species, such as those belonging to the genus Neoregelia. Although Neoregelia mucugensis has not been evaluated for the degree of threat, its exploitation is exclusively extractive and its occurrence in Chapada Diamantina-BA is subject to the action of fires that affect the region annually. In this context, applying techniques aimed at protecting this genetic resource is fundamental for both the maintenance of its natural populations and the ex situ conservation of this genetic material. Plant tissue culture techniques have been successfully applied for the conservation of several bromeliad species. One of the methods used is slow growth, which consists in reducing plant metabolism and consequently decelerating its growth, which allows the maintenance of in vitro plant collections without the need for subculture. In this context, the objective of this study was to test the reduction of salts in the culture medium and the addition of osmoregulators on the induction of slow growth of N. mucugensis. Plants were subjected to treatments composed of different concentrations of MS medium and mannitol for a period of 12 months, when then analyses were conducted to evaluate growth, chlorophyll content and regeneration capacity of shoots in vitro. It was found that the treatment containing MS ½ and 7.8 g.L-1 of mannitol is indicated for in vitro conservation of N. mucugensis with maintenance of the regenerative capacity of its tissues.

Sincoraea mucugensis (Wand. & A.A. Conc.) LOUZADA & WAND, an endangered bromeliad, is confined to the central region of the Chapada Diamantina, in the municipality of Mucugê, Brazil. From various researches, it is evident that for the propagation of this species, the in vitro technique is a feasible option. However, due to the low multiplication rates reported in various papers, this study aimed to establish a micropropagation protocol of direct organogenesis for S. mucugensis. First, the inoculation of the stem explants was done in MS ½ culture medium which contained different levels of BAP (0.00; 6.66; 8.88; 11.10; 13.20 µM) and NAA (0.00; 2.60; 5.20 µM). These shoots were then subjected to a couple of distinct rooting periods (of 30-and 60-day duration) using activated charcoal; finally, these microplants were transferred to a greenhouse for acclimatization, and covered with transparent plastic cups, as a water loss prevention test method. All the data were submitted to the analysis of variance (ANOVA), and the means were subjected to regression analysis or compared using the Tukey test. The findings revealed that the S. mucugensis stem explants raised in the NAA-rich medium (6.42 to 7.43 shoots/explants) showed high multiplication rates; the shoot rooting was done for 30 days using activated charcoal with the medium. Acclimatization, which was performed by directly exposing the microplants to the ex vitro environment, showed 95% survival rate. RESUMO: Sincoraea mucugensis (Wand. & A.A. Conc.) LOUZADA & WAND é uma bromélia vulnerável de ocorrência restrita ao município de Mucugê, Chapada Diamantina. Estudos indicam que a cultura de tecidos é uma alternativa viável para a propagação in vitro destaespécie. Contudo, em função das baixas taxas de multiplicação obtidas em estudos anteriores, objetivou-se estabelecer um protocolo de micropropagação via organogênese direta para S. mucugensis. Explantes caulinares foram inoculados em meio de cultura MS ½ contendo diferentes concentrações de BAP (0,00; 6,66; 8,88; 11,10; 13,20 µM) e ANA (0,00; 2,60; 5,20 µM). Os brotos obtidos foram submetidos a diferentes períodos de enraizamento (30 e 60 dias) com carvão ativado; posteriormente as microplantas foram aclimatizadas em casa de vegetação, testando-se o efeito da cobertura com copos plásticos transparentes como estratégias contra perda de água. Os dados foram submetidos à análise de variância (ANOVA) e as médias analisadas por regressão ou comparadas pelo Teste de Tukey. Os resultados demonstram que altas taxas de multiplicação de S. mucugensis são geradas a partir de explantes caulinares cultivados em meio contendo ANA (6,42 a 7,43 brotos/explantes); o enraizamento dos brotos é realizado por 30 dias em meio com carvão ativado e a aclimatização é feita com exposição direta das microplantas ao ambiente ex vitro com 95% de sobrevivência. Palavras-chave: propagação in vitro, aclimatização, Sincoraea mucugensis, ANA.

No abstract

The term “sempre-viva” denotes plants whose structures retain their natural form and color after being cut and dried. For these reasons, they are commercially valuable for ornamental purposes. However, due to extractive overexploitation of their inflorescences, some of these species are considered endangered. The genus Comanthera includes the sempre-vivas species with greatest economic importance in Brazil. Previous studies have shown that tissue culture is a workable strategy for in vitro propagation and conservation of species of this genus. However, these studies are still incipient. Therefore, the objective of this review is to summarize the findings on the in vitro cultivation of species of the Comanthera genus, to serve as the basis for future research. The text is structured in two main topics: micropropagation and in vitro conservation.

Physalis ixocarpa Brot. ex Horm. is considered the most economically important species of the genus. Tissue culture is pointed out as a strategy for its propagation, but researches indicate that in vitro responses are genotype-dependent. This study aimed to evaluate the in vitro morphogenesis of the P. ixocarpa green and purple varieties, in view of the massive propagation of the species. The morphogenic capacity of the explants cotyledonary node, hypocotyl and cotyledon was evaluated in Murashige & Skoog medium supplemented with benzylaminopurine - BAP (0.00, 2.5, 5.0, 7.5 or 10.0 μM) and naphthaleneacetic acid - NAA (0.00 or 2.5 μM), using a completely randomized experimental design, in a 3 x 5 x 2 factorial scheme, with 30 treatments for each variety. The number of shoots per direct and indirect organogenesis and the percentage of explants with callus were analyzed. The in vitro morphogenetic expression of P. ixocarpa is influenced by the type of explant and by the plant regulators BAP and NAA. The cotyledonary node explant is efficient for the production of shoots via direct organogenesis in the two varieties studied.

The “sempre-viva” species Comanthera mucugensis is endemic to the municipality of Mucugê, Bahia, Brazil, where it was widely exploited through extractivism to be commercialized as an ornamental, causing a drastic reduction of its population, so that it is now classified as endangered. Although its main area of occurrence is now protected for being within Chapada Diamantina National Park, the continued risks of inclement conditions and anthropic actions make it necessary to develop alternative methods for ex situ conservation of the species, such as in vitro conservation. Therefore, the objective of this study was to test the effect of different concentrations of salts and sucrose on the in vitro conservation of Comanthera mucugensis. Two salts concentrations of the medium MS (½ and ¼) and two sucrose levels (7.5 and 15.0 g L-1) were tested, and the experimental design was completely randomized with four treatments. After 365 days, the survival, growth and regeneration of the conserved plants were analyzed, achieving up to 100% survival, reduced shoot growth and maintenance of regenerative capacity. Reduction of the concentration of salts and sucrose in the culture medium is indicated to conserve the plants in vitro for a period of one year.

Amburana cearensis is an arboreal legume of the Fabaceae family, with high phytotherapic and medicinal potential due the presence of secondary metabolites. The objective of this study was to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-2,5,6- trichloro-2-pyridinecarboxylic acid (picloram) on the in vitro induction of callogenesis of A. cearensis and analyze the biochemical and phytochemical potential of these calluses. For callus induction, leaf and cotyledon segments were used as explants, which were inoculated in woody plant medium (WPM) supplemented with different concentrations of 2,4-D (0, 5, 10, 20, 40 μM) or picloram (0, 5, 10, 20, 40, 80 μM). The callus growth curve was estimated based on fresh weight, measured at 7-day intervals until 28 days after inoculation. The calluses were analyzed by biochemical tests to quantify the reducing sugars and total proteins. Phytochemical screening and high-performance liquid chromatography were performed to establish the phytochemical profile of extracts from calluses. The concentrations of 21.94 μM and 26.46 μM of 2,4-D induced the greatest formation of compact and friable calluses from the leaf and cotyledon segments, respectively. The growth curve had two distinct phases (lag and exponential) for both types of calluses evaluated. The maximum levels of reducing sugars and total proteins in the calluses from leaf and cotyledon segments were obtained on the day of inoculation and after 28 days of cultivation, respectively. The results of the phytochemical analysis identified the presence of coumarin in all the extracts evaluated, this secondary metabolite has high pharmacological potential.