Morphogenesis in vitro from hypocotyl and root explants of annatto (Bixa orellana L.)

A espécie Bixa orellana (Bixaceae) é conhecida como popularmente como urucum, sendo uma lenhosa nativa da América Tropical e amplamente distribuída em todas as regiões do Brasil. As sementes de urucum são ricas em carotenóides bixina e norbixina, muito utilizados nas indústrias alimentícia, cosmética e farmacêutica, despertando imenso interesse econômico. O desenvolvimento de protocolos de morfogênese in vitro de urucum é importante por gerar novas alternativas para obtenção de plantas transformadas geneticamente com genes de interesse agronômico e aqueles associados à biossíntese de pigmentos carotenóides, permitindo o melhor entendimento e a manipulação das rotas de sua produção. Os objetivos desse trabalho foram avaliar a influência de pulsos de zeatina, testar diferentes meios de cultivo na organogênese de urucum, além de testar diferentes concentrações de Pluronic® F-68 e de analisar as combinações de manose:sacarose na morfogênese de urucum. Foram testados diferentes tempos de exposição de segmentos de hipocótilo. Observou-se em média, 8,8 brotos por explantes e freqüência de organogênese de 80 % quando os explantes permaneceram no meio de indução (meio MS suplementado com zeatina 4,56 µM) por 6 dias e depois transferidos para meio MS sem o regulador de crescimento. Contudo, freqüências de brotações adventícias em torno de 60 % foram obtidas para os explantes que permanecer 2 ou 3 dias no meio de indução. Foram testadas formulações de meios de cultivo na indução de organogênese em urucum. Os meios MS e JADS propiciaram as maiores freqüências de organogênese para o genótipo M2 (98 e 90 %, respectivamente), enquanto o uso do meio WPM levou a índices de regeneração mais baixos (6 %). Quanto ao meio DKW observaram-se freqüências intermediárias de 70 a 80 %. Para Piave Vermelha observou-se que meio JADS proporciona as freqüências mais elevadas, em comparação aos demais meios (68 %). Notaram-se respostas diferenciadas entre os genótipos. O surfactante não-iônico Pluronic® F-68 foi testado em diferentes combinações com a zeatina 4,56 µM, tanto em explantes de hipocótilos quanto em segmentos de raízes. Não foram observadas grandes variações em relação às concentrações de Pluronic® F-68 utilizadas para M2 , porém as freqüências mais baixas foram observadas na concentração 0,001 % (82 %). Já para Piave Vermelha as baixas concentrações do surfactante (0,001 e 0,1 %) mostraram as maiores freqüências de brotações em segmentos de hipocótilo (28 e 12 %, respectivamente). As concentrações 0,001 e 0,5 % de Pluronic® F-68 para explantes radiculares no genótipo M2 promoveram os maiores números de brotações adventícias (43 e 40, respectivamente); o genótipo Piave Vermelha não respondeu aos tratamentos. A possibilidade da utilização da fonte de carbono manose como agente de seleção em experimentos de transformação genética de urucum como gene pmi (fosfomanose isomerase) foi testada com segmentos de hipocótilo e de raízes. O urucum, aparentemente, não foi capaz de metabolizar a manose presente em diferentes combinações com sacarose no meio de indução JADS suplementado com zeatina 4,56 µM. Dessa forma não foram observadas brotações adventícias quando a manose era a única fonte de carbono do meio, e a presença de sacarose, mesmo na concentração mais baixa (21,9 mM), foi capaz de promover a morfogênese. Os explantes radiculares de Piave Vermelha não responderam aos tratamentos, enquanto os de M2 emitiram o maior número médio de brotações (7,2) quando 75 % da manose foram substituídos por sacarose. Portanto, analisando-se o comportamento geral dos dois genótipos conclui-se que a variação interespecífica, a constituição mineral, a adição de substâncias orgânicas/ inorgânicas, reguladores de crescimento e agentes gelificantes, assim como também os tipos de explantes analisados são fatores que devem ser considerados quando se estuda a morfogênese in vitro de urucum, tendo em vista a busca por melhores resultados e conhecimento melhor fundamentado do comportamento demonstrado pela espécie em cultura de tecidos.Bixa orellana (Bixaceae) popularly known as annatto is a wood species native from Tropical America, and widely distributed in all Brazilian territory. The seeds are a wealthy source of the carotenoids bixin and norbixin, largely used in food, cosmetic, and pharmaceutical industries, and presenting high economic interest. The development of in vitro regeneration protocols is important as a mean to generate new alternatives for obtainment of genetically modified plants with genes of agronomic interest and those associated to the biosynthesis of carotenoids, allowing a better understanding and manipulation their biosynthetic pathways. The goals of this work had been to evaluate the influence of zeatin pulses, to test different medium of culture in organogenesis of annatto, beyond testing different concentrations of Pluronic® F-68 and to analyze the combinations of mannose: sucrose in morphogenesis of annatto. In the present work, different exposure periods to growth regulators using hypocotyl segments were assayed. In average, 8.8 shoots were obtained per explant and high organogenesis frequency (80 %), when explants were cultured on induction medium (MS supplemented with 4.56 µM zeatin) during 6 days, and transferred to MS medium devoid of growth regulators. However, adventitious regeneration frequencies around 60 % were achieved when explants remained for 2 or 3 days on induction medium. Regarding media formulation used, MS and JADS propitiated the highest organogenic frequencies for genotype M2 (98 and 90 %, respectively), whereas WPM led to the lowest regeneration frequencies (6 %). DKW medium enabled intermediate regeneration frequencies ranging from 70 to 80 %. For Piave Vermelha it was observed that JADS medium provided the highest frequencies, as compared to other media (68 %). It was noticed genotype-dependent regeneration responses. The non-ionic surfactant Pluronic® F-68 was tested in different combinations with zeatin 4.56 µM, for both hypocotyl and root segment explants. No significant variations were observed for Pluronic® F-68 used for M2 , but the lowest frequencies were obtained at 0.001 % (82 %). However, for Piave Vermelha the lower surfactant concentrations (0.001 and 0.1 %) led to greater shooting frequencies in hypocotyl segments (28 and 12 %, respectively). For M2 root explants, Pluronic® F-68 at 0.001 e 0.5 %, promoted the highest adventitious shoot numbers (43 e 40 %, respectively); root explants of Piave Vermelha did not display any response to the same surfactant treatments. The use of an alternative carbon source (mannose) as a selective agent in annatto genetic transformation by means of pmi (phosphomannose isomerase) gene was evaluated for both hypocotyl and root explants. Annatto explants were not able to metabolize the mannose present alone or in combination with sucrose, on zeatin- supplemented (4.56 µM) JADS medium. This way, no regenerative response was achieved when mannose was the sole carbon source; sucrose, even at the lowest concentration (21.9 mM) promoted shoot morphogenesis. Root explants from Piave Vermelha did not show any response to the different carbon source treatments, however M2 root explants showed the higher number of shoots (7.2) when 75 % of the mannose were replaced by sucrose. Therefore, analyzing the overall behavior of both genotypes it can be concluded that interespecific variation, the mineral composition of the medium, the supplementation with organic/inorganic substances, growth regulators, gelling agents, and the type of explants must be taken into account when targeting the in vitro morphogenesis of annatto, aiming at the optimization of in vitro responses of this species.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Morphoanatomical and histochemistral characterization, and expression of the SERK genes during somatic embryogenesis in Bixa orellana L. (Bixaceae)

O objetivo do presente estudo foi analisar o processo de embriogênese somática a partir de embriões zigóticos imaturos de Bixa orellana L. (Bixaceae) sob aspectos morfoanatômicos e histoquímicos, além de análises moleculares da expressão de genes SERK. Embriões zigóticos imaturos foram colocados em meio MS suplementado com 2,26 μM de 2,4-D, 4,52 μM de cinetina e 0,1 % de carvão ativado. O início da embriogênese somática foi observado após 10 dias no meio de indução, com divisões celulares alterando o eixo embrionário e os cotilédones. Após 20 dias, foram notados embriões somáticos em estádio globulares iniciais, originados da protoderme dos embriões zigóticos, limitados por cordões de compostos fenólicos na camada abaixo da protoderme. Aos 52 dias do início da indução embriogênica, embriões somáticos em estádios iniciais, tardios e secundários foram observados. Análises histoquímicas demonstraram a mobilização de reservas durante o processo. Foram observados carboidratos totais no embrião zigótico durante o início do processo embriogênico, porém, compostos lipídicos não foram notados nessa fase. Corpos proteicos estavam presentes no embrião zigótico após 15 dias de indução. Análises moleculares durante a embriogênese somática levaram à clonagem e identificação de três possíveis membros da família SERK (BoSERK1, BoSERK2 e BoSERK3). Análises filogenéticas das proteínas indicaram que o agrupamento dos diferentes membros SERK ocorre de acordo com a função desempenhada na planta. Experimentos de hibridização in situ comprovaram a expressão de SERK em células do tecido vascular e da epiderme nos embriões zigóticos imaturos. Sinal de intensidade variável foi observado nos embriões somáticos em diferentes fases de desenvolvimento, desde globulares a cotiledonares. Os resultados do presente trabalho esclarecem os processos morfoanatômicos da embriogênese somática em B. orellana e contribuem com o primeiro registro da expressão de genes SERK para a espécie, subsidiando futuros estudos de expressão com mutantes.This study aimed to analyze the process of somatic embryogenesis from immature zygotic embryos of Bixa orellana L. (Bixaceae) by means of morphoanatomical and histochemical aspects of regeneration, as well as molecular analysis of SERK gene expression. The embryos were placed onto MS-based induction medium supplemented with 2.26 μM 2,4-D, 4.52 μM kinetin and 0.1 % activated charcoal. The initiation of somatic embryogenesis was observed as early as 10 days in the induction medium, with cell divisions altering the embryonic axis and the cotyledons. After 20 days, somatic embryos at early globular stage were visualized, originating from the protodermis of zygotic embryos, bound by a sheath-like of phenolic compounds in the subepidermal layer. After 52 days of onset of embryo induction, somatic embryos at early and late stages, as well as secondary somatic embryos were observed. Histochemical analyses demonstrated the mobilization of reserves during the process. Total carbohydrates were observed in zygotic embryo during early embryogenic process, but lipid compounds were not perceived at that stage. Protein bodies were present in the zygotic embryo after 15 days of induction. Molecular analysis during somatic embryogenesis led to the cloning and identification of three possible SERK family members (BoSERK1, BoSERK2, and BoSERK3). Phylogenetic analysis of the proteins indicated that the different members of the SERK group occur according to the function performed in the plant. In situ hybridization experiments confirmed the expression of SERK in cells of vascular tissues and in the epidermis of immature annatto zygotic embryos. Signals of variable intensity were observed in somatic embryos at different stages of development, from globular to cotyledonary. The results of this work clarify morphoanatomic aspects of somatic embryogenesis in B. orellana and contribute as the first record on the cloning and expression of SERK genes for the species, supporting future expression studies with mutants.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

High responsiveness in de novo shoot organogenesis induction of Passiflora cristalina (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^−1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^−1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^−1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species

In vitro regeneration of triploid plants from mature endosperm culture of commercial passionfruit (Passiflora edulis Sims)

Due to the triploid nature of endosperm, an embryonic reserve tissue, in vitro culture of endosperm tissues has been considered a direct method for production of polyploids. In the present study, we report the establishment of an in vitro regeneration system from endosperm culture for production of triploid Passiflora edulis plants, the main commercial species of passionfruit. Surface-sterilized endosperms were cultured in MS medium with different concentrations of benzyladenine (BA), thidiazuron (TDZ), and kinetin (KIN). The cultures were maintained in a growth chamber under controlled conditions, for 60 days. Thidiazuron was the only type of cytokinin that induced shoot production in the endosperm tissues; the highest number of shoots was produced in the presence of 4.5 and 9.0 μM TDZ. Flow cytometry and chromosomal analysis confirmed that endosperm-derived plants were triploid. The internal standard, Pisum sativum, and the diploid control, seed-derived Passiflora edulis plants (2n = 2× = 18), showed average DNA quantities of 9.09 and 3.35 pg respectively. Endosperm-derived P. edulis plants showed an average DNA content of 5.10 pg and a chromosome count of 27 (3n = 3× = 27), the same ploidy level as the endosperm (triploid). Our data open new prospects for breeding of passionfruit by means of a stable and reproducible regeneration system from endosperm culture leading to generation of triploid plants

Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon

The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species

High responsiveness in de novo shoot organogenesis induction of Passiflora cristalina (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^−1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^−1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^−1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species

In vitro regeneration of triploid plants from mature endosperm culture of commercial passionfruit ( Passiflora edulis Sims)

Due to the triploid nature of endosperm, an embryonic reserve tissue, in vitro culture of endosperm tissues has been considered a direct method for production of polyploids. In the present study, we report the establishment of an in vitro regeneration system from endosperm culture for production of triploid Passiflora edulis plants, the main commercial species of passionfruit. Surface-sterilized endosperms were cultured in MS medium with different concentrations of benzyladenine (BA), thidiazuron (TDZ), and kinetin (KIN). The cultures were maintained in a growth chamber under controlled conditions, for 60 days. Thidiazuron was the only type of cytokinin that induced shoot production in the endosperm tissues; the highest number of shoots was produced in the presence of 4.5 and 9.0 μM TDZ. Flow cytometry and chromosomal analysis confirmed that endosperm-derived plants were triploid. The internal standard, Pisum sativum, and the diploid control, seed-derived Passiflora edulis plants (2n = 2× = 18), showed average DNA quantities of 9.09 and 3.35 pg respectively. Endosperm-derived P. edulis plants showed an average DNA content of 5.10 pg and a chromosome count of 27 (3n = 3× = 27), the same ploidy level as the endosperm (triploid). Our data open new prospects for breeding of passionfruit by means of a stable and reproducible regeneration system from endosperm culture leading to generation of triploid plants