Somatic embryogenesis from leaf explants of hermaphrodite Carica papaya: A new approach for clonal propagation

Carica papaya L. is an economically relevant fruit crop in some tropical and subtropical countries. Though this species shows three polygamous sexual types, commercial production of the fresh fruit is mainly established from hermaphrodite lines. As a result of the cross-pollinating reproductive mechanism of hermaphrodite C. papaya, cultivation areas also show unwanted female plants. Comparatively, hermaphrodite plants exhibit considerable variation in regards to yield, fruit quality, and susceptibility to pathogens. In this context, the present work aimed at establishing a somatic embryogenesis protocol to provide regenerants from leaves of hermaphrodite C. papaya plants. Leaf explants, collected in the rainy season, provided high frequency of friable embryogenic calli (FEC). In culture medium supplemented with 2,4-dichlorophenoxyacetic, FEC overgrew into a yellowish friable mass that fully covered the leaf explants. The somatic embryogenesis process occurred asynchronously, with new globular embryos continuously forming from the FEC. Torpedo and early cotyledonary somatic embryos matured in medium containing polyethylene glycol, activated charcoal and abscisic acid. These embryos were  germinated, and normal seedlings were recovered. Based on these outcomes, the tissue culture protocol presented here may be considered a successful alternative for large-scale and clonal propagation of adult hermaphrodite plants of C. papaya.Keywords: Carica papaya, clonal propagation, hermaphrodite plants, leaf explant, somatic embryogenesisAfrican Journal of Biotechnology Vol. 12(18), pp. 2386-239

Reproduction in Brachiaria spp.: SERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) in anther and ovary development and in embryogenesis

Gramíneas do gênero Brachiaria apresenta importância econômica como forrageiras no Brasil. O melhoramento genético, entretanto, é restrito devido a diferenças de ploidia entre os genótipos e a reprodução por apomixia. A indução de haplóides e duplo-haploides pelo cultivo in vitro de anteras ou de micrósporos isolados é relatada em diversas espécies. Além da criação de novos genótipos linhagens duplo-haplóides podem ser utilizadas como ferramentas para o estudo de marcadores moleculares. Dentre os fatores essenciais para o sucesso da técnica está o estádio do desenvolvimento do micrósporo, sendo o estádio uninucleado, geralmente, o mais responsivo. Um dos objetivos deste trabalho foi contribuir para a definição de parâmetros para a cultura de haplóides em Brachiaria. Flores e anteras isoladas de B. brizantha (B105) em diferentes estádios de desenvolvimento foram processadas para microscopia de luz. Os eventos observados foram associados a marcadores morfologicos. Experimentos de cultura de anteras foram estabelecidos e estruturas semelhantes a calos ocorreram em pouco explantes. Secções histológicas revelaram a ocorrência de micrósporos com divisão simétrica, indicando um possível desvio da rota do micrósporo para a rota esporofítica. Genes definidos como marcadores de embriogênese como SERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE), podem ser utilizados para identificar o potencial embriogênico de células ou grupo de células, possibilitando monitorar a resposta in vitro. Duas seqüências parciais de cDNA isoladas de B. brizantha apresentaram alta identidade com homólogos de SERK1 e SERK2 de monocotiledôneas. Análise filogenética confirmou a alta similaridade sugerindo serem os possíveis ortólogos de SERK em Brachiaria, sendo nomeados como BbrizSERK1 e BbrizSERK2. Clones parciais da seqüência genômica foram obtidos. Análise de Southern blot identificou duas cópias deste gene tanto na planta apomítica como na sexual. A análise da expressão de BbrizSERK2 por RT-qPCR na planta apomítica e sexual e durante a embriogênese somática revelou que este gene não é específico de tecidos reprodutivos. Expressão diferencial de BbrizSERK entre ovários apomíticos e sexuais, especialmente durante a megasporogênese, foi observada por hibridização in situ. Detectou-se forte sinal na célula-mãe do megásporo em ovários sexuais e ausência de sinal em ovários apomíticos. Após a antese, a expressão na planta apomítica foi observada apenas na região micropilar e proembriões. Na sexual, foi observada nas antípodas e aparato da oosfera. Em anteras o mesmo padrão de expressão foi observado entre a planta apomítica e sexual, com forte expressão no tapete e nas células-mãe do grão de pólen, o que sugere um importante papel deste gene na esporogênese em Brachiaria. Em calos embriogênicos forte sinal foi observado em massas proembriogênicas, em embriões globulares e também em embriões somáticos mais desenvolvidos, confirmando a função de SERK como marcador de embriogênese em Brachiaria. Em anteras cultivadas in vitro, forte sinal foi observado em micrósporos binucleados com divisão simétrica e em alguns micrósporos vacuolados. Experimentos futuros com genes desta família poderão contribuir para monitorar processos in vitro em Brachiaria, indicando células ou grupos de células com potencial embriogênico, ajudando na definição das melhores condições de cultivo para a embriogênese e também para o estudo da reprodução neste gêneroBrachiaria, gramineae present economic importance as forage in Brazil. The genetic breeding, however, is restricted by differences in ploidy among the genotypes and the apomixis. The development of a haploid and double haploid production has been reported for several species. Besides the production of new genotypes the development of double haploid lineages may be used for studies with molecular markers. Among important factors for the success of haploid plant development relies on the microspore developmental stage, with the uninucleate stage being usually the most responsive. One of the objectives of this work was to contribute for the definition of parameters for the development of haploid cultures in Brachiaria. B. brizantha (B105) flowers and isolated anthers were collected in different stages of development and processed for light microscopy. The events observed were associated to morphological markers. Anther culture experiments were established and calli formation ocurred to a few explants. Histological sections of anthers cultivated in vitro, however, revealled the occurrence of microspores with symmetrical divisions, indicating a possible alternative to the sporophytic route. Genes defined as markers of embryogenesis, such as SERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE), may be utilized to identify the embryogenic potential of cells, or cell clusters, in an attempt to monitor the in vitro response. Two partial sequences of cDna isolated from B. brizantha (B30) showed high identity of these two sequences to SERK1 and SERK2 homologs from monocots. The phylogenetic analysis confirmed high similarity with these genes, indicating that these sequences are possible orthologs of SERK in Brachiaria, hence these were named BbrizSERK1 and BbrizSERK2. Partial clones of genomic sequences were obtained. Southern blot analysis suggested two copies of this gene in both apomictic and sexual B. brizantha. The expression analysis of BbrizSERK2 by RT-qPCR revealed that this gene is not specific to reproductive tissues. Differential expression of BbrizSERK between apomictic and sexual ovaries, especially during megasporogenesis, was observed by in situ hybridization. A strong signal was detected in the megaspore mother cell in sexual ovaries, with absence in apomictic ovaries. After anthesis, the expression in the apomictic plant was observed only in the micropylar region and proembryos, however, in the sexual plant, the expression was observed in the antipodals and egg apparatus. In anthers, the same in situ pattern was observed in the apomictic and the sexual plant, with a strong expression in the tapetum and the pollen grain mother cell, suggesting an important role of this gene in the sporogenesis in Brachiaria. In embryogenic calli, strong signal was observed in proembryogenic masses, globular embryos and somatic embryos in later stages of development, confirming the role of SERK as an embryogenic marker in Brachiaria. In cultivated anthers, a strong signal was observed in binuclear microspores with symmetric division and vacuolated microspores. Further studies with this gene family may contribute to monitoring the in vitro processes in vitro in Brachiaria, defining cells or cell clusters with an embryogenic potential, helping for the definition of culture conditions for embryogenesis and also for reproductive studies in this genu

Somatic embryogenesis in papaya (Carica papaya L.): anatomy, histochemistry and influence of AVG, ACC, STS and pulses of 2,4-D

O presente trabalho objetivou estudar alguns aspectos relacionados à histologia e histoquímica da embriogênese somática em Carica papaya L. ‘Improved Sunrise Solo Line 72/12’. Embriões zigóticos imaturos resgatados a partir de frutos imaturos com 80 a 90 dias após a antese foram cultivados em meio MS meia-força, suplementado com 6% de sacarose, 100 mg L -1 de mio- inositol, 400 mg L -1 de L-glutamina, 2 mg L -1 de ácido diclorofenoxiacético e solidificado com 2,8 g L -1 de Phytagel ® . Para os estudos anatômicos, amostras de calos embriogênicos foram analisadas em microscópio óptico e submetidas à microscopia eletrônica de varredura. Para análise histoquímica foram utilizadas amostras de material fresco ou incluídas em historesina. A análise histológica de amostras, coletadas em vários períodos de cultivo, mostrou que a reação calogênica induzida teve início nas células do meristema fundamental, próximas aos cordões procambiais e embriogênese somática direta ocorrendo a partir das células protodérmicas e subepidérmicas, ao vigésimo dia de cultivo, especialmente na região meristemática do domo apical do embrião zigótico. As células embriogênicas foram caracterizadas por apresentarem núcleos volumosos, nucléolos proeminentes e citoplasma denso com poucos grãos de amido. Aos 45 dias de cultivo, visualizou-se a formação de inúmeros complexos proembriogênicos, com variável número de células, isolados das demais por paredes espessas, típicos de um processo de embriogênese somática indireta. Grande quantidade de grãos de amido foi visualizada nos estádios iniciais da calogênese, especialmente nas células periféricas. A reação positiva ao teste com Sudan detectou uma grande quantidade de lipídeos, evidenciando sua importância como material de reserva. Proteínas em forma de grânulos foram evidenciadas pelo teste XP (Xylidine Ponceau), especialmente nos cotilédones intactos dos embriões zigóticos cultivados. Os efeitos do precursor do etileno 1-aminociclopropano-1- carboxílico (ACC) e dois inibidores – aminoetoxivinilglicina (AVG) e tiossulfato de prata (STS) durante o processo de indução da embriogênese somática foram analisados. Neste experimento o meio indutor foi suplementado com essas substâncias, nas concentrações de 3, 10 e 30 ìM. A presença do ACC não inibiu a formação de calos e a aquisição de competência embriogênica. Contudo, nos maiores níveis testados, observou-se redução no número de embriões somáticos diferenciados por explante. Em presença de AVG na concentração de 3 ìM verificou-se resultados semelhantes ao tratamento controle em relação a formação de calos embriogênicos. Contudo nos níveis de 10 e 30 μM foram observados altos índices de explantes não responsivos, sugerindo possível toxidez desses níveis. Nos tratamentos com STS observou-se intensa proliferação de calos, porém com pouca diferenciação embriogênica. Em outro experimento, verificou-se os efeitos de pulsos de 2,4-D sobre a morfogênese in vitro em mamoeiro, tendo como objetivo inicial determinar o período de exposição necessário para aquisição de competência embriogênica. Foram testadas altas concentrações (10 e 100 mg L -1 ) em tempos reduzidos de 0, 1, 6, 12, 24, 36, 48, 72 horas e a exposição contínua. Em seguida, os explantes foram transferidos para o meio MS, destituído de reguladores de crescimento. Para cada concentração e períodos de exposição foram observadas respostas morfogênicas diferenciadas como rizogênese, calogênese e embriogênese somática. Melhores respostas embriogênicas foram observadas na concentração de 10 mg L -1 durante 72 horas e na concentração de 100 mg L -1 no pulso de 48 horas.The present study was conducted to evaluate some aspects related to histological and histochemical aspects of somatic embryogenesis of C. papaya ‘Improved Sunrise Solo line 72/12’. Immature zygotic embryos derived from harvested fruits after 80-90d after anthesis were cultured onto a semi-solid half- strength MS-based medium supplemented with 6% sucrose, 100 mg L -1 myo- inositol, 400 mg L -1 L-glutamine, 2,4-D (2.0 mg L -1 ), and solidified with 2.8 g L -1 Phytagel, at pH 5.7 ± 0.1. For anatomical studies embryogenic calli samples were characterized under photonic and scanning electron microscopy. Histochemical analyses were carried out using both fresh and hystoresin embedded samples. Histological analyses carried out throughout several cultural periods, revealed that callusing responses initiated from fundamental meristem-derived cells, close to procambial strands, whereas direct somatic embryogenesis occurred from peripheric, protodermic and sub-epidermic cells, at 20 th d of culture, mainly from apical dome of the zygotic embryo. Embryogenic cells presented typical meristematic characteristics large nuclei, prominent vacuoles, densely cytoplasmic, and few starch grains. At 45 d of culture, several proembryogenic complexes with variable number of cells were visualized, isolated by thick cell-walls, typical of indirect somatic embryogenesis process. Large amounts of starch grains were detected at initial stages of callusing processes, especially in the periphery. Positive reactions with Sudan detected large amount of lipids, evidencing its importance as storage material. Protein granules were also evidenced by Xylidine Ponceau mainly in intact cotyledon of zygotic embryos. The effect of the ethylene precursor 1-aminocyclopropane-1-carboxylic (ACC) and two inhibitors – aminoethoxyvinylglyine (AVG) and silver thiosulfate (STS) during the induction process of somatic embryogenesis were analyzed. In this experiment the inductor medium was supplemented with these substances, in the concentrations of 3, 10 and 30 ìM. The presence of ACC did not inhibit the callusing responses and the acquisition of embryogenic competence. However, at higher tested levels, there was a significant decrease in number of somatic embryos per explant. In the presence of AVG in the concentration of 3 μM, similar results were verified as compared to the control treatment regarding to the formation of embryogenic calli. Therefore, in the levels of 10 and 30 ìM it was observed high percentage of explants without responses, suggesting possible toxicity in these levels. In the treatment with STS it was observed a high proliferation of callus although with few embryogenic differentiation. Another experiment it verified the effects of pulses of 2,4-D upon in vitro morphogenesis in papaya, having as initial goal determine the period of the exposition demanded for acquisition of embryogenic competence. The concentrations de 2,4-D (10 and 100 mg L -1 ) combined with pulsing times 0, 1, 6, 12, 24, 36, 48, 72 hours and the continuous exposition was evaluated. Following, the explants were transferred to the medium MS without growing regulators. For each concentration and exposition period it was observed morphogenic response differing as rhizogenesis, calogenesis and somatic embryogenesis. Better embryogenic response were observed in the concentration of 10 mg L -1 during 72 hours and in the concentration of 100 mg L -1 in the pulse of 48 hours.Conselho Nacional de Desenvolvimento Científico e TecnológicoDissertação importada do Alexandri

Following the track of “Híbrido de Timor” origin by cytogenetic and flow cytometry approaches

The supposedly first plant of the coffee cultivar “Híbrido de Timor” (HT) was found in 1927, being denoted as HT CIFC 4106. According to different researchers, this plant originated from a natural interspecific hybridation between Coffea arabica (4x = 44) and Coffea canephora (2x = 22). From HT CIFC 4106, other HT accessions were obtained and employed to establish germplasm banks in some countries. As HT has been widely used in Coffea breeding programs, this study aimed to characterize different HT accessions with regard to ploidy, nuclear DNA content and base composition. Based on these data, the ploidy of HT CIFC 4106 was determined, suggesting that this accession is an allotriploid formed from reduced reproductive cell of C. canephora and of C. arabica. All HT CIFC 4106 plants exhibited the same 2C-value, AT% and chromosome number, showing that vegetative propagation has enabled the multiplication and germplasm conservation of this cytotype since 1927. Further five analyzed HT accessions showed distinct nuclear 2C-value and AT%. Since HT CIFC 4106 has been considered the first HT, it is suggested that aneuploid reproductive cells of this HT originated the other plants. Considering that HT accessions are used in the development of C. arabica cultivars, the findings of this study are important for the design of strategies to obtain new cultivars for breeding programs. Moreover, these data represent the first step to understand the origin and genome evolution of the HT

Somatic embryogenesis and de novo shoot organogenesis can be alternatively induced by reactivating pericycle cells in Lisianthus (Eustoma grandiflorum (Raf.) Shinners) root explants

This study demonstrated that somatic embryogenesis and de novo shoot organogenesis-based systems of root-derived Lisianthus (Eustoma grandiflorum) explants can be alternatively induced by exogenous supply of auxin or cytokinin. Somatic embryogenesis was observed when root explants were cultured in the dark on Murashige and Skoog-based medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos were differentiated by transferring embryonic calluses to an embryo conversion phase medium containing 2 μM 6-benzyladenine (BA) to promote full plantlet development. Regarding de novo shoot organogenesis, the addition of 4 μM of either BA or zeatin was the most effective treatment for inducing adventitious shoot buds. A detailed histological characterization of somatic embryogenesis and de novo shoot organogenesis showed that both morphogenetic processes shared the same cellular origin. The formation of somatic embryos and adventitious shoot buds occurred through the reactivation of pericycle and vascular parenchyma cells into proembryos and meristemoids, respectively, which consisted of meristematic cells with similar characteristics. These results provide further evidence of optimization of in vitro propagation as a useful approach to improve this important ornamental species

De novo assembly and transcriptome of Pfaffia glomerata uncovers the role of photoautotrophy and the P450 family genes in 20-hydroxyecdysone production

Pfaffia glomerata is a medically important species because it produces the phytoecdysteroid 20-hydroxyecdysone (20-E). However, there has been no ready-to-use transcriptome data available in the literature for this plant. Here, we present de novo transcriptome sequencing of RNA from P. glomerata in order to investigate the 20-E production as well as to understand the biochemical pathway of secondary metabolites in this non-model species. We then analyze the effect of photoautotrophy on the production of 20-E genes phylogenetically identified followed by expression analysis. For this, total messenger RNA (mRNA) from leaves, stems, roots, and flowers was used to construct indexed mRNA libraries. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 164,439 transcripts were annotated. In addition, the effect of photoautotrophy in two genes putatively involved in the 20-E synthesis pathway was analyzed. The Phantom gene (CYP76C), a precursor of the route, showed increased expression in P. glomerata plants cultured under photoautotrophic conditions. This was accompanied by increased production of this metabolite indicating a putative involvement in 20-E synthesis. This work reveals that several genes in the P. glomerata transcriptome are related to secondary metabolism and stresses, that genes of the P450 family participate in the 20-E biosynthesis route, and that plants cultured under photoautotrophic conditions promote an upregulated Phantom gene and enhance the productivity of 20-E. The data will be used for future investigations of the 20-E synthesis pathway in P. glomerata while offering a better understanding of the metabolism of the species

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