Ilse Fernanda Ferrari
“Caracterização anatômica e molecular da
embriogênese somática direta e indireta de Coffea
arabica.”
“Anatomical and molecular characterization of somatic
embryos in Coffea arabica by direct and indirect
pathways”
Campinas 2016
UNIVERSIDADE ESTADUAL DE CAMPINAS
INSTITUTO DE BIOLOGIA
Ilse Fernanda Ferrari
“Caracterização anatômica e molecular da embriogênese somática
direta e indireta de
Coffea arabica.”
“Anatomical and molecular characterization of somatic embryos in
Coffea arabica by direct and indirect path
ways”
Orientador: Jorge Mauricio Costa Mondego Coorietadora: Juliana Lischka Sampaio Mayer
Campinas 2016
Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do título de Doutora em GENÉTICA E BIOLOGIA MOLECULAR na área de Genética Vegetal e Melhoramento.
Thesis presented to the Biology Institute of the University of Campinas as a partial fulfillment of requirements for the degree of Doctor, in the area of GENETICS AND MOLECULAR BIOLOGY in the area of Plant genetics and Breeding.
ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA ILSE FERNANDA FERRARI E ORIENTADA PELO ORIENTADOR JORGE MAURICIO COSTA MONDEGO.
Campinas, 30 de Novembro de 2016.
COMISSÃO EXAMINADORA
Dr. Jorge Mauricio Costa Mondego (Orientador)
Prof. Dr. Rafael Silva Oliveira
Profa. Dra. Alexandra Christine Helena Frankland Sawaya
Dra. Alexandra Bottcher Marchesini
Dr. Walter José Siqueira
Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno.
“A ciência nunca resolve um problema sem criar pelo menos outros dez”.
AGRADECIMENTOS
Primeiramente, gostaria de agradecer ao meu marido Felipe e meus pais Osmar e Meire, por todo apoio, carinho, compreensão e dedicação nesse período da minha vida, me motivando a seguir em frente e superar todas as dificuldades.
Aos meus orientadores Dr. Jorge Mondego e Profa. Dra. Juliana Mayer, por aceitar um projeto de doutorado em tão pouco tempo e, principalmente pela orientação e oportunidade de realização das atividades em seus laboratórios, incentivando e enriquecendo minha formação acadêmica.
Ao Prof. Dr. Marcelo Menossi, pela excelente coordenação e por se mostrar prestativo durante sua gestão na Pós Graduação em Genética e Biologia Molecular.
A CAPES pela concessão da bolsa.
Aos amigos que cultivei durante esse período, em especial, aos amigos da Fisiologia Vegetal, Laboratório de Anatomia vegetal da Unicamp e do departamento de Genética do IAC, pela troca de experiência na bancada e pela paciência e disponibilidade em me ensinar. E também por me ajudarem a encaram com leveza todos os períodos conturbados do Doutorado.
Aos estagiários, Giovanna A. Marques e Ton Sachetti pelo grande auxílio prestado no desenvolvimento do trabalho e pela amizade.
Aos técnicos de Laboratório: Eduardo Kiota, Luciano Pereira, Pedro Araujo, Elzira e Sebastião Militão Jr, pelo imenso auxílio prestado durante minhas atividades em laboratório;
A todos aqueles que contribuíram direta ou indiretamente para a realização desse trabalho.
Resumo
A embriogênese somática (ES) tem um papel fundamental na micropropagação in vitro de Coffea, proporcionando a produção de mudas em larga escala e em um menor tempo. A ES pode ser realizada pela via direta (ESD) e indireta (ESI). Na primeira os embriões formam-se diretamente de células do explante que se tornam determinadas e competentes através da formação da massa pró-embriogênica, para o desenvolvimento do embrião. Na segunda, o desenvolvimento dos embriões somáticos acontece a partir de calos. O conhecimento sobre a transição de células somáticas para células embriogênicas é fundamentado em análises histológicas, as quais permitem acompanhar as características diferenciadoras entre esses tipos celulares. Sabe-se que durante a formação e diferenciação dos embriões somáticos, os genes LEC1, BBM e
WUS são diferencialmente expressos e atuam como reguladores positivos da indução e
maturação. Considerando a aplicação biotecnológica da ES, o estudo da ontogênese dos embriões somáticos, bem como dos genes ligados a esse processo, pode ser uma importante ferramenta de apoio aos programas de melhoramento, conservação de germoplasma e na identificação e avaliação de novos genótipos. Assim, o presente trabalho teve por objetivo caracterizar a ontogênese dos embriões somáticos regenerados via ESD e ESI em explantes foliares de Coffea arabica cultivar Mundo Novo, analisar a expressão dos genes envolvidos na formação e diferenciação dos embriões e avaliar a influência dos compostos fenólicos na formação de embriões durante o processo in vitro. A partir da caracterização da ES e ontogênese dos embriões, verificamos que a ESI, utilizando explantes de plantas mantidas in vitro, é mais rápida e eficiente quando comparada às demais condições e que na ESD o desenvolvimento dos embriões se dá a partir da massa pro-embriogênica que é essencial para a formação destes. Os genes analisados durante a embriogênse apresentaram uma maior expressão durante a diferenciação das células que dão origem ao calo e a massa pro-embriogênica e no desenvolvimento dos embriões.
Palavras chaves: Embriogênse somática, Coffea arabica, Mundo Novo, LEC1, BBM,
Abstract
Somatic embryogenesis (SE) process enhance seedlings production at reduced time on
in vitro micropropagation of Coffea. SE is divided into two distinct ways: indirect (ISE)
and direct (DSE). DSE promotes embryos formation directly from pro-embryogenic mass from determined and competent explant cells. ISE develops somatic embryos from callus. The transition from somatic cells to embryogenic cells is based in histological analysis, allowing to identify the differentiation features among cell types. Some genes play a key role during the formation and differentiation of somatic embryos: LEC1, BBM and WUS. They are differentially expressed and act as regulators of embryogenesis induction and maturation. Somatic embryogenesis has biotechnological potential to breeding programs, germplasm conservation, identification and evaluation of new genotypes. Therefore, the ontogenesis of somatic embryos and associated genes can revaels new insights and increase our background. Our study intends to characterize the ontogenesis of somatic embryos from Coffea arabica Mundo Novo regenerated by DSE and ISE from leaf explants. We analyzed the gene expression in the formation and differentiation of embryos and, at the same time, evaluated the influence of phenolic compounds. ISE was clearly faster than DSE for somatic embryogenesis from explants
in vitro.
Lista de Figuras
Capítulo I ... 20 Figure 1: Scanning electron micrograph of somatic embryogenesis in Coffea arabica cv Mundo Novo ... 35 Figure 2: Somatic embryogenesis in Coffea arabica cv Mundo Novo. (A, C) Direct
somatic embryogenesis (DSE). ... 36 Figure 3: Process of direct somatic embryogenesis in Coffea arabica cv Mundo Novo ... 38
Figure 4: Process of indirect somatic embryogenesis in Coffea arabica cv Mundo
Novo ... 40 Figure 5: Process of indirect somatic embryogenesis in Coffea arabica cv Mundo
Novo ... 42 Figure 6: Process of indirect somatic embryogenesis in Coffea arabica cv Mundo
Novo, embryo development detail during ontogenesis of explants from in vitro plants ... 43 Figure 7: Representation of the ontogenesis of Coffea arabica cv Mundo Novo ... 44
Figure 8: Somatic embryogenesis in Coffea arabica under different histochemical tests . 45
Capítulo II ... 46 Figura 1. Tabela adaptada de Li et al. (2004), seleção dos iniciadores otimizados ... 52
Figura 2. Expressão relativa dos genes BBM2, LEC1, WOX4 durante a embriogênise somática ... 56
Lista de Tabelas
Capítulo II ... 46 Tabela 1. Sequência dos iniciadores desenhados ... 54 Tabela 2. Níveis transcricionais dos genes homeólogos (CaFt, CaCc, CaCe) de
BBM2, expresso pela média entre os Ct ao longo da embriogênese somática direta ... 54
Tabela 3. Níveis transcricionais dos genes homeólogos (CaFt, CaCc, CaCe) de
Lista de Figuras Suplementar
Capítulo II ... 46 Figura suplementar 1. Alinhamento das sequências transcritas traduzidas do gene
BBM2 ... 65
Figura suplementar 2. Alinhamento das sequências transcritas traduzidas do gene
LEC1 ... 66
Figura suplementar 3. Alinhamento das sequências transcritas traduzidas do gene
LISTA DE ABREVIATURAS E SIGLAS
2 iP Isopentenyl adenine
2,4-D 2,4-dichlorophenoxyacetic acid ANA Naphthalene acetic acid
BBM2 Baby Boom2
DSE Direct somatic embryogenesis ESD Embriogênese somática direta ESI Embriogênese somática indireta HCl Ácido clorídrico
IAC Instituto Agronômico de Campinas ISE Indirect somatic embryogenesis
LEC1 Leafy Cotyledon 1
NaOH Hidróxido de sódio
PCR Reação em cadeia da polimerase
qRT-PCR Reverse transcription polymerase chain reaction SE Somatic embryogenesis
SNPs Polimorfismo de nucleotídeo único
Sumário AGRADECIMENTOS ... 6 Resumo ... 7 Abstract ... 8 Introdução ... 15 Objetivos ... 19 Capítulo I ... 20
ORIGIN AND DEVELOPMENT OF SOMATIC EMBRYOS IN COFFEA ARABICA BY DIRECT AND INDIRECT PATHWAYS. ... 20
Artigo submetido a revista - Plant Cell, Tissue and Organ Culture (PCTOC) ... 20
Abstract... 20
1. Introduction ... 21
2. Methodology: ... 22
2.1. Biological Material: ... 22
2.2. Somatic Embryogenesis in Coffea: ... 22
2.3. Morphological and anatomical analyzes:... 23
3. Results: ... 24
4. Discussion: ... 27
5. References: ... 30
Capítulo II ... 46
CARACTERIZAÇÃO MOLECULAR DOS GENES BBM, LEC E WOX DURANTE A EMBRIOGÊNESE SOMÁTICA DIRETA E INDIRETA DE COFFEA ARABICA CV. MUNDO NOVO. ... 46
Resumo: ... 46
1. Introdução: ... 47
2. Metodologia: ... 49
2.2. Embriogênese somática em Coffea: ... 49
2.3. Análises in silico: ... 50
2.4. Desenho dos iniciadores homeólogos específicos: ... 51
2.5. Extração de RNA total e síntese de cDNA: ... 52
2.6. PCR quantitativo em tempo real (qRT-PCR): ... 52
3. Resultados: ... 53
4. Discussão: ... 57
5. Referências: ... 60
6. Material Suplementar: ... 65
Introdução Geral
O cafeeiro
O agronegócio cafeeiro tem um papel importante no PIB nacional e no comércio internacional. A estimativa de produção para 2016 é de 49,64 milhões de sacas de café beneficiado, sendo 83% da produção nacional de café de Coffea arabica, o que representa um acréscimo de 14,8% em relação a sacas produzidas em safra 2015 (Conab 2016).
O cafeeiro é uma planta perene, pertencente à família Rubiaceae, tribo
Coffeeae, gênero Coffea (Davis et al. 2006). Teve seu centro de origem na África,
contêm as três espécies utilizadas na produção e bebida do café: C. arabica, C.
canephora e C. liberica (Davis et al. 2006). Coffea L. é caracterizado como
dicotiledônea, de folhas persistentes e flores hermafroditas, porte arbustivo ou arbóreo e caule lenhoso (Carvalho 2008).
Os plantios comerciais no Brasil em sua grande maioria são de C. arabica, principalmente pela qualidade superior da bebida, característica aromática e baixo conteúdo de cafeína. A cultivar Mundo Novo, genótipo 38817-6, é altamente plantada no Brasil, apresentando porte alto, com maior diâmetro de copa, frutos vermelhos de maturação média e apresenta ótima qualidade de bebida (Carvalho 2008).
Embriogênese somática
A embriogênese somática (ES) em Coffea é um importante método de multiplicação in vitro de plantas elite, em larga escala, capaz de maximizar a propagação do cafeeiro, tanto de cultivares já recomendadas para plantio como de híbridos vindos de programas de melhoramento genético. Além disso, é de grande interesse em trabalhos de transformação genética de plantas (Ahmed et al. 2013; Almeida et al. 2014; Ibrahim et al. 2013). A ES é o processo pelo qual células ou tecidos somáticos se desenvolvem até a formação completa de uma planta, através de uma série de desenvolvimentos embriogênicos que apresentam características semelhantes ao desenvolvimento de embriões zigóticos (Wann 1988; Williams and Maheswaran 1986). Este processo inicia-se com a transição de células somáticas para um estado embriogênico onde a escolha da fonte apropriada de células competentes e estímulo por auxina, em condições in vitro, são pré-requisitos para a competência embriogênica nas células somáticas (Gaj 2004; Gaj et al. 2005). A ES apresenta eventos semelhantes
àqueles da embriogênese zigótica, portanto, fornece um atraente modelo experimental para o estudo molecular e de mecanismos celulares envolvidos nos processo de desenvolvimento in vivo, que vai do zigoto ao embrião (Gaj 2004; Mordhorst et al. 2002). A primeira fase da ES é a fase globular, em que o embrião é esférico, ligado através do suspensor ao tecido materno.
Após a formação dos primórdios cotiledonares, o embrião desenvolve a forma de coração e, a partir da expansão longitudinal dos cotilédones, o embrião adquire a forma de torpedo que, na fase final, desenvolve-se no embrião cotiledonar (Gaj et al. 2005). A propagação através da técnica de ES pode ocorrer pela via direta e indireta, sendo promissora na produção de mudas em Coffea arabica (Ibrahim et al. 2013). Na embriogênese somática direta (ESD) os explantes passam por uma mínima proliferação celular, com o surgimento da massa pro-embriogênica, antes da formação dos embriões (Vasconcellos et al. 2009). Já na embriogênese somática indireta (ESI), acontece a redeterminação de células diferenciadas para indiferenciadas, com a proliferação de calos, antes da formação do embrião (Berthouly and Michaux-Ferriere 1996; Sondahl et al. 1985).
Genes ligados à embriogênese
As pesquisas sobre engenharia genética do café têm contribuído de maneira decisiva para o desenvolvimento da cultura no país, ajudando a elucidar a função, regulação e interação de genes com importância para a produção agrícola (Mishra and Slater 2012). A investigação sobre os mecanismos de transição de células somáticas para embriogênicas está centrada em análises histológicas e na regulação hormonal (Rose and Nolan 2006). No entanto, sabe-se que durante a formação e diferenciação dos embriões somáticos, uma série de genes são diferencialmente expressos. O gene
Baby Boom (BBM), isolado a partir de culturas de embriões de micrósporos de Brassica napus, codifica um fator de transcrição pertencente à família gênica AP2/ERF,
preferencialmente expresso no desenvolvimento de embriões e sementes (Boutilier et al. 2002). Todos os genes da família AP2/ERF codificam proteínas que apresentam um domínio conservado AP2 compreendendo de 60-70 resíduos de aminoácidos (Sakuma et al. 2002). O domínio AP2 é etileno-responsivo e se liga ao fator de transcrição com domínio ERF. Este domínio, por sua vez, se liga ao GCC-box, que é uma sequência de DNA envolvida na resposta ao etileno (Ohme-Takagi and Shinshi 1995).
Genes que apresentam o domínio conservado AP2/ERF podem desempenhar papéis distintos no diferentes estágios de desenvolvimento, assim como, apresentam interações com genes redundantes e induzem diferentes vias de desenvolvimento dependendo do ambiente genético e celular (Srinivasan et al. 2007). A expressão ectópica de BBM em Arabidopsis e Brassica levou à formação espontânea de embriões somáticos e mudanças fenotípicas adicionais, incluindo o crescimento neoplásico e regeneração de explantes sem adição de hormônios (Boutilier et al. 2002; Passarinho et al. 2008).
A suprexpressão do BBM pode ser aplicada em espécies com baixa eficiência de regeneração in vitro, estudos com Populus e Theobroma cacao, demostraram que a superexpressão do BBM induz a formação de embriões somáticos sem passar pelo estádio de calo, melhorando a eficiência da regeneração de plantas transgênicas (Deng et al. 2009; Florez et al. 2015). A expressão ectópica do BBM resultou na indução de ESI em tabaco transgênicos (Srinivasan et al. 2007). Mutantes de tabaco com perda de função para BBM apresentaram perda completa da capacidade de ES (El Ouakfaoui et al. 2010).
O gene LEC1 (Leafy cotyledon 1) desempenham um papel central no controle da embriogênese zigótica, atuando como regulador da morfogênese (Chugh and Khurana 2002). A expressão ectópica de LEC1 confere características embrionárias e resulta na formação de estruturas parecidas com embriões, na superfície da folha, indicando assim, que o gene desempenha um papel em conferir competência embriogênica nas células (Lotan et al. 1998).
Este gene codifica uma proteína com a subunidade HAP3 do fator de transcrição CCAAT, que age como regulador transcricional (Lotan et al. 1998). Estudos demonstram que a expressão ectópica dos genes LEC de Arabidopsis (AtLEC1 e
AtLEC2) em plantas transgênicas de tabaco promovem embriogênese somática (Guo et
al. 2013). Em C. canephora, o gene LEC1 atua como regulador da ES, sendo expresso em diferentes estádios de desenvolvimento do embrião (Nic-Can et al. 2013). Segundo Gaspari-Pezzopane et al. (2011), o gene LEC1 é requerido para a especificação da identidade cotiledonar e completa maturação de embriões zigóticos em Coffea arabica.
Estudos com plantas de Zea mays L. (milho) demonstram que a expressão do gene LEC1 foi fortemente detectada na fase precoce do desenvolvimento do embrião e diminuindo nas fases posteriores, indicando que este gene pode ser usado como
marcador molecular do inicio da embriogênese somática (Zhang et al. 2002). Assim, o gene LEC1, pode ser utilizado como ferramenta para definir mecanismos moleculares subjacentes que controlam a fase de maturação e o início de ES (Braybrook and Harada 2008).
O gene que atua mais precocemente durante o desenvolvimento embrionário é o Wuschel-Related Homeobox 4 (WOX4), pertencente a superfamília de fatores transcrição homeobox (HB) (Gehring et al. 1990). Esta família gênica de fatores transcrição é diferentemente expressa nas fases iniciais da embriogênese e no desenvolvimento de órgãos laterais em plantas (Haecker et al. 2004).
Membros da família WOX cumprem funções especializadas em processos de desenvolvimento chave nas plantas, como a padronização embrionária, manutenção de células-tronco e formação de órgãos (van der Graaff et al. 2009). Estas funções podem estar relacionadas com a promoção da divisão celular e/ou prevenção de diferenciação celular prematura (Wu et al. 2007). Estudos indicam que alguns genes da superfamília WOX podem ser reguladores importantes da embriogênese somática, reduzindo a característica recalcitrante de algumas cultivares (Gambino et al. 2010). Em Arabidopsis, a combinação da atividade dos genes da família WOX, pode regular diferentes aspectos da proliferação de tecido no desenvolvimento embrionário (Wu et al. 2007).
Considerando que a indução de embriões somáticos de cafeeiro, através de técnicas de cultura de tecidos vegetais, permite não apenas a propagação vegetativa em larga escala, como também pode ser usado na formação e manutenção de bancos de germoplasma e em programas de melhoramento através da transformação genética. Sendo assim, o estudo da ontogênese dos embriões somáticos, bem como dos genes ligados a esse processo, pode ser uma importante ferramenta de apoio aos programas de melhoramento, na criação de novas cultivares e na identificação e avaliação de novos genótipos.
Objetivos
- Caracterizar a ontogênese dos embriões somáticos regenerados via embriogênese somática direta e indireta de explantes foliares de Coffea arabica cultivar Mundo Novo, com o propósito de verificar se a origem e as fases de desenvolvimento nos dois processos são semelhantes.
- Comparar o desenvolvimento dos embriões somáticos via embriogênese direta e indireta a partir de explantes foliares de plantas adultas mantidas no campo e de explantes foliares de plantas mantidas in vitro.
- Avaliar a expressão dos genes envolvidos nos diferentes estádios da embriogênse somática, com o intuito de, melhorar a compreensão sobre os genes envolvidos durante a formação e diferenciação dos embriões oriundos de explantes foliares em Coffea
Capítulo I
ORIGIN AND DEVELOPMENT OF SOMATIC EMBRYOS IN COFFEA ARABICA BY DIRECT AND INDIRECT PATHWAYS.
Artigo submetido a revista - Plant Cell, Tissue and Organ Culture (PCTOC)
Ilse Fernanda Ferrari1,2, Giovanna Arcolini Marques2, Welington Luis Sachetti Junior2, Bárbara Borti Biazotti2, Julieta Andrea Silva de Almeida3, Jorge Maurício Costa Mondego1, Juliana Lischka Sampaio Mayer2,†,‡
1
Centro de Pesquisa e Desenvolvimento de Recursos Genéticos Vegetais, Instituto Agronômico, Campinas, São Paulo, Brazil
2
Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil
3
Centro de Café Alcides Carvalho, Instituto Agronômico, Campinas, São Paulo, Brazil
† These authors share senior authorship. ‡ Correspondence: mayerju@unicamp.br
Abstract
In vitro micropropagation is an important way for coffee multiplication, performed by
somatic embryogenesis (SE). SE can be indirect (ISE) or direct (DSE). The first one consists of two phases: re-determination of explant cells, followed by formation of callus and development of somatic embryos from callus. In DSE, embryos form directly from explant cells that are determined and competent to embryogenic development. This work characterizes the ontogenesis of somatic embryos regenerated by indirect and direct way from leaf explants of Coffea arabica cultivar Mundo Novo, comparing the development of these embryos from leaf explants of adult plants grown in the field and leaf explants of plants maintained in vitro. It was found that both ways of somatic embryogenesis there were anatomical features not yet described. ESD the formation of pro-embryogenic mass is essential for the formation of the embryos and their anatomical characteristics differ greatly from the callus. ESI depending on the source of explant, callus development presents differences in anatomy. Explants from in vitro plants have the formation of layers of cells with intense cell divisions. In addition to producing embryos in less time than other conditions evaluated.
Key Words: Anatomy, callus, ontogenesis, pro-embryogenic mass, somatic embryos, Tissue culture.
1. Introduction
Micropropagation is an important method of multiplication able to maximize the in vitro propagation of coffee for elite and hybrid cultivars from breeding programs (Ahmed et al. 2013; Almeida et al. 2014; Rezende et al. 2012). It can be accomplished through somatic embryogenesis (SE), a process that allows high plant production genetically identical to the mother plant in a reduced space and time. SE is based in the concept of cell totipotency - somatic cells of the plant tissue contain all the genetic information needed to originate a complete and functional plant (Williams and Maheswaran 1986). This process presents similar events to those of zygotic embryogenesis, and can be used for most species in laboratory condition (Gaj 2004), thus it is an important approach to study the development of plant embryos (Quiroz-Figueroa et al. 2002a). Initially, there is the explant cell induction acquiring embryogenic characteristic, followed by expression of somatic embryo (Jiménez 2005), which is a bipolar structure, without connection to the original vascular tissue, and has unicellular or multicellular origin (Carman 1990; Dodeman et al. 1997; Feher et al. 2003; Quiroz-Figueroa et al. 2006; von Arnold et al. 2002).
SE may occur indirectly (ISE) or directly (DSE) (Williams and Maheswaran 1986). ISE consists of two phases: first occurs re-determination of differentiated cells, followed by division and proliferation activation leading to development of a cell mass called callus. The second stage is related to initiation and development of somatic embryos from certain callus cells (Almeida et al. 2005; Berthouly and Michaux-Ferriere 1996; Santana-Buzzy et al. 2007). In DSE, embryos are directly formed from explant cells that are determined and competent to embryogenic development, not needing callus formation (Dublin 1981; Emons 1994; Motoike et al. 2007; Vasconcellos et al. 2009; Yasuda et al. 1985).
SE has been successfully applied to Coffea arabica, but some genotypes respond only to indirect or direct way, while others respond to both. Most studies of C.
arabica comprise which application is better; usually ISE stands out generating a greater
number of somatic embryos than DSE (Vieira and Kobayashi 2000). The development pattern of somatic embryo has many similar morphological characteristics to the zygotic embryo. The various embryonic stages of development, from embryo in globular stage, going through heart and torpedo, are morphologically and anatomically similar to the formations related in Coffea canephora zygotic embryogenesis (Moens 1965).
Leaf tissue is the target tissue when generating embryos (Clarindo et al. 2012; Landey et al. 2013; Ribas et al. 2011; Silva et al. 2014). Genetic engineering studies have reported that low viability of embryonic tissues and low transformation efficiency of this type of tissue are major limitation in the genetic transformation of coffee (Mishra et al. 2010; Ribas et al. 2011). Thus, it is necessary studies to provide a clear distinction between embryogenic and non embryogenic tissues, demonstrating the best method for obtaining embryos with high cell viability.
This study aimed to characterize the ontogenesis of somatic embryos regenerated via direct and indirect somatic embryogenesis from leaf explants of Coffea
arabica cultivar Mundo Novo, in order to verify if the origin and stages of development in
both cases are similar, besides comparing the development of these somatic embryos from leaf explants of adult plants grown in the field and leaf explants of plants maintained
in vitro.
2. Methodology:
2.1. Biological Material:
Young leaves from field plants and leaves of plants grown in vitro were used, obtained from Coffea arabica cultivar Mundo Novo produced in the experimental area of Santa Eliza Farm, IAC, Campinas-SP.
2.2. Somatic Embryogenesis in Coffea:
Asepsis of young leaves of plants under field conditions was performed by washing the leaves in detergent solution, rinsing in running water, then subjecting them to sodium hypochlorite solution (2%) for 25 minutes and rinsing three times in autoclaved distilled water. The disinfected leaves were kept in a humid chamber (80% humidity) for twenty-four hours, and thereafter, subjected again to sodium hypochlorite solution (2%) (Ramos et al. 1993). Leaves were cut in laminar flow cabinet, removing the midrib and edges, obtaining rectangular explants of 1cm2, which were inoculated with the adaxial side in contact with the culture medium, then kept in dark in a temperature of 25° C ± 2°C.
In vitro plants obtained by DSE were maintained on half strength MS medium
(Murashige and Skoog 1962), with photoperiod of 12 hours light at 25° C ± 2° C. Leaves were used to obtain the explants in a laminar flow cabinet without the need of asepsis. Explants, rectangular with 1cm2 were directly inoculated in vitro.
Both conditions were subjected to ISE and DSE. For ISE induction it was used two culture media. First the Murashige and Skoog (1962) culture medium for induction of embryogenic callus with addition of 30 g L-1 sucrose, 2.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D), and 5 µM kinetin. Then for embryo induction it was used the modified MS medium (Murashige and Skoog 1962) with half the concentration of macronutrients and micronutrients with the addition of 20 g L-1 sucrose, 0.5 uM naphthalene acetic acid (NAA) and 2.5 µM kinetin. For DSE induction modified MS culture medium was used (Murashige and Skoog, 1962), with half the concentration of macronutrients and micronutrients, added with 20 g L-1 sucrose and 10 µM of isopentenyl adenine (2 iP). All culture media were solidified with the addition of 5 g L-1 agar and pH adjusted to 5.8 using NaOH or 0.1N HCl prior to autoclaving at 121 ° C and 1.5 atm for 20 minutes.
2.3. Morphological and anatomical analyzes:
Direct and indirect analyses were performed from samples of leaf explants collected at the time of in vitro inoculation (control) and in different development stages, at the time intervals of 2, 8, 12, 16, 18, 28, 62 and 72 days after inoculation in culture medium, somatic embryos were collected from their complete development, presenting torpedo shape embryos.
In the anatomical analysis, samples were fixed in FAA 50 solution (formaldehyde, acetic acid and 50% ethanol, 5: 5: 90) (Johansen 1940), then subjected to vacuum pump to remove the air contained in the tissues, being dried in series of ethanol and infiltrated in plastic resin (Leica Historesin®) according to manufacturer's instructions. Samples were sectioned in manual rotary microtome (Leica®) with C-type knife, 5 micrometers thick. Sections were stained with toluidine blue 0.05% (Sakai 1973) in phosphate and citrate buffer, pH 4.5 (McIlvaine 1921) and mounted in "Entellan®" (Merck®) synthetic resin. Different histochemical tests were used: Lugol reagent to highlight the presence of starch (Berlyn et al. 1976); Xylidine Ponceau reagent for proteins (Vidal 1969); ferric chloride 3% reagent for phenolic compounds (Johansen 1940); Red ruthenium for mucilage (Gregory and Baas 1989); Nadi reaction for terpenoid (David and Carde 1964) and Sudan IV for lipid substances (Pearse 1985). Image capture for result documentation was performed with Olympus DP71 video camera attached to the Olympus BX 51 microscope.
To analyze the surface of the samples in scanning electron microscopy (SEM), the botanical material was fixed in FAA 50 solution (formaldehyde, acetic acid and 50% ethanol, 5: 5: 90) (Johansen 1940), dehydrated in series of ethanol and critical point dried with CO2 in Balzers model CPD 030 equipment. Then the material was mounted on metal supports and coated with colloidal gold for 220 seconds in Bal-Tec SCD model 050 equipment. Analysis and micrograph recording was performed with LEO scanning electron microscope model VP 435 operated at 10 kV, at the Institute of Biology / Unicamp.
3. Results:
The emergence of pro-embryogenic mass in the DSE (Fig. 1A) and callus in ISE (Fig. 1D) occurs first at the end of the opposite side to the vascular bundle in contact with the culture medium. The pro-embryogenic mass in DSE has compact appearance and smooth surface (Fig. 1B-C; 2A). It appears that the emergence of embryos occurs in a restricted manner in those areas of pro-embryogenic mass (Fig 1B; 2C) and at the same time embryos at different stages of development (globular, heart and cotyledon stage) are observed (Fig. 1C; 2C).
The callus development in ISE has many elongated and disorganized cells with large intercellular spaces and covered in part by a secretion (Fig. 1E, 2B). Embryos originate exclusively from callus cells (Figure 1F; 2D) and it is possible to observe embryos at various stages of development (globular, heart, torpedo and cotyledon) in the same callus region (Fig 1H-I; 2D). Both in DSE as in ISE is possible to clearly check the presence of a suspensor, connecting the embryo to the pro-embryogenic mass or callus, respectively (Fig. 1C and 1G).
Anatomical analysis showed that leaf explants from plant controls grown in field (Fig. 3A) and in vitro (Fig. 3B) have structural differences. The main differences between them are that in vitro explants have: i) greater intercellular space in spongy parenchyma; ii) palisade cells are shorter and rounded; iii) chlorophyll parenchyma cells, in general, have little dense cytoplasm.
In the early stages of DSE, eight days after explant inoculation, these in vitro conditions have an intense cell division in the mesophyll from the spongy parenchyma cells (Fig. 3D), this is the first indication of pro-embryogenic mass formation. As for the explant derived from leaves of plants field grown, the onset of cell division occurs at eight days (Fig. 3C), but only becomes more intense 18 days after in vitro inoculation (Fig.
3E). In both growing conditions there was proliferation of mesophyll cells presenting cells with evident nucleus, dense cytoplasm and little intercellular space (Fig. 3D-E).
Formation of pro-embryogenic mass can be seen on the edges of in vitro explants, near the vascular bundle, 18 days after inoculation (Fig. 3F), and its surface region has intense cell division, the same happens in the vascular bundle surrounding region. Superficial mass cells have meristematic characteristics with dense cellular content and evident nucleus. After 28 days, in field explants (Fig. 3G) occurs the emergence of pro-embryogenic mass, more delayed and with less intense mitotic activity when compared to in vitro explants (Fig. 3H).
When the mass appears externally in the explants, 62 days after inoculation, field explant (Fig. 3I) seem to have lower pro-embryogenic mass than in vitro explants (Fig. 3J), and this mass has few regions containing cells with dense cytoplasmic contents on the surface. In vitro explant present masses with larger projection and the innermost cells are rounded, vacuolated and larger when compared to the smaller peripheral cells with dense cytoplasmic contents, forming meristem regions. The same is observed in subsequent samplings of both materials 72 days after in vitro inoculation.
In the initial ISE process, two days after in vitro inoculation, it is possible to observe presence of greenish phenolic compounds in the chlorenchyma cells, especially in the palisade, both in field and in vitro (Fig. 4A-B) . With eight days of culture, explants have regions with a high mitotic activity in the spongy parenchyma, mainly near the edge of the explant and next to the vascular bundles, both in field (Fig. 4C) and in vitro (Fig. 4D).
The beginning of callus formation can be seen on in vitro explants after 12 days, cells are have high mitotic activity in the entire explant length, these cells do not have intercellular spaces and form meristems containing cells with dense cytoplasm and evident nucleus (Fig. 4F). The field explant, 12 days after inoculation (Fig. 4E), remained similar to that observed after eight days.
Calluses are already visible at the edge of the explant 16 days after inoculation, noting that in both types of explants there were no changes in the skin. Cells separate leaving room for callus growth in the palisade, and the callus arises exclusively from the spongy parenchyma cells (Fig 4G-H; 5A-B). At this stage of callus development, size and structure of callus are the biggest difference between field and in vitro explants. Field explants have larger, vacuolated and rounded callus cells (Fig. 4G). In vitro explant
have more bulky innermost callus cells when compared to the surface cells which are smaller and with dense cytoplasmic contents, besides exhibiting strong mitosis (Fig. 4H).
At 28 days of cultivation, explant callus from in vitro and field leaves have very different structures (Fig. 5A-B). Field explants have more elongated, vacuolated and with larger intercellular spaces callus cells (Fig. 5A). In the edge of the explant is possible to observe projection of the vascular bundle toward the callus central portion (Fig. 5A-C). In vitro explant present callus with greater and vacuolated cells in its inner region and these cells are lined with smaller cells arranged in continuous layers in intensive cell division. More externally to these layers, cells become elongated, vacuolated, and are coated with a continuous cell layer without intercellular spaces (Fig. 5B). The arrangement of these cells with meristematic characteristics in layers becomes more evident after 30 days of culture (Fig. 6A-B), and it is possible to identify alignment of these continuous cells with expanding outer layers and with less dense cytoplasm cells (Fig. 6B). During the callus development, explants coming from field material showed no obvious anatomical changes, only increasing in size, with more spaced, elongated, vacuolated and irregularly shaped cells, giving a friable aspect to the callus (Fig 5C; 5E). In explants of plants grown in vitro, samples of 62 days (Fig. 5D) and 72 days (Fig. 5F) presented larger calluses when compared to the earlier stages of development, but they still have regions presenting cells with meristematic characteristics and intense mitotic activity. These meristem cells contribute to the continued callus growth. Only surface cells of the callus are bulky, have large vacuoles and intercellular spaces (Figure 5D; 5F).
Embryo formation begins with 62 days of in vitro culture (Fig. 6C). This pro-embryo comprises few dividing cells in the upper portion that result in the pro-embryo itself and its basal portion has a set of triangular-shaped cells, which give rise to the suspensor. The explant with 72 days presents embryos in globular stage, with well-defined protoderm and presence of the suspensor in the embryo base (Fig. 6D). When the explant reaches 176 days of culture (Fig. 6E-G), it is possible to observe embryos at different stages of development. During the transition from embryonic stages (globular to heart stage) there is a longitudinal elongation (Fig. 6E). The embryos in the torpedo and heart stages have well defined protoderm and procambium (Fig. 6F) remaining connected to the callus by the suspensor present in the embryo base (Fig. 6G).
During DSE and ISE, the embryo development process is similar for all studied conditions, regardless of the explant origin (field or in vitro), with a significant variation in the time which happens each ontogeny stage (Fig. 7).
Histochemical tests carried out during callus (Fig. 8B) and pro-embryogenic mass development (Fig. 8A; 8C-I) detected the presence of phenolic compounds in DSE (Fig. 8A) and ISE (Fig. 8B), and lipids in the vacuoles of the palisade parenchyma cells and in scattered cells bordering the callus and mass (Fig. 8D). Lipids are characterized as acidic in nature because of their light blue color with Nile Blue sulfate reagent (Fig. 8E).
After 28 days of culture and callus formation, it was possible to verify the accumulation of starch grains in chlorophyll parenchyma cells and in some callus cells (Fig. 8C). In DSE starch was seen in vitro. In callus after 72 days of culture, presence of terpenes was observed in ISE; DSE had terpenes only in the field condition at 16 days (Fig. 8F). Tests that indicate presence of proteins (Fig. 8G-H) and mucilage (Fig.8I) demonstrated that proteins accumulate in callus and in pro-embryogenic mass cells (Fig. 8I).
4. Discussion:
Somatic embryogenesis is the process that involves the concept of cell totipotency, wherein somatic cells from plant tissue undergo a restructuring to generate embryonic cells, going through a series of developmental stages similar to those of zygotic embryogenesis. During somatic embryogenesis, cells undergo morphological and biochemical changes that result in the formation of a somatic embryo. This process involves hormonal actions, transcription factors and epigenetic regulation (Yang and Zhang 2010).
Analysis of pro-embryogenic mass development during DSE and callus development during ISE demonstrated that both originate from mitotically active cells present in the spongy parenchyma, while epidermal and palisade parenchyma remained without anatomic changes. During DSE in coffee, embryo emergence does not occur directly from the explant cells, as previously described for other species (Dublin 1981; Emons 1994; Motoike et al. 2007; Vasconcellos et al. 2009; Yasuda et al. 1985). Embryo development occurred indirectly from pro-embryogenic mass, independently of the explant source. Initially there is the formation of pro-embryogenic mass in the spongy
parenchyma, and from the mass there is the embryo development as reported by Menéndez-Yuffá and de García (1997) and Quiroz-Figueroa et al. (2002b).
Pro-embryogenic mass and callus protruded out of the explants and the mechanical pressure exerted by them pushed away the palisade parenchyma and the epidermis. Several studies describe similar events during ISE, however, using field material as a source of leaf explants (Berthouly and Michaux-Ferriere 1996; Menéndez-Yuffá and de García 1997; Pierson et al. 1983).
In the initial phase of development, an event that draws attention is the presence of callus and pro-embryogenic mass near the vascular bundle, showing that there is a correlation between presence of vascular bundles and early formation of these tissues. The beginning of cell divisions at the bundles end can be explained by the presence of pericycle cells with totipotent potential, which is the outermost layer of the vascular bundle (Xu and Huang 2014). The exposure of tissue in the explant edge due to sheet incision in regions near vascular bundles of small size facilitates the action of plant hormones placed in the culture medium, increasing callus and mass formation.
In direct somatic embryogenesis (DSE) tissue development occurs more rapidly in explant from in vitro plants and later in explant from field plants (Fig. 7). This occurs both at the beginning of cell division in the spongy parenchyma, and in the emergence of pro-embryogenic mass. Chlorenchyma material of explants from in vitro are less differentiated when compared to the parenchymal explants coming from the field plants, and possibly has a greater totipotency. The most significant difference between field and in vitro material was the time of embryo development, occurring with 270 and 86 days of culture, respectively (Fig. 7). Studies with cassava cultivars suggest that more juvenile tissue have a high efficiency in production of somatic embryos (Ravindran et al. 2015).
In vitro grown plants may present a rejuvenation of tissues, which explains
the anatomical and temporal differences of explants from field and in vitro material. According to Claudot et al. (1993), rejuvenation of mature trees can be obtained by in
vitro culture and is associated with the reappearance of the features found in plant
seedlings. Some cultures, when in vitro, have tissue rejuvenation along with the acquisition of juvenile morphology (Brand and Lineberger 1992; Ruaud et al. 1992). Reversion to the juvenile state has the potential to induce embryogenesis in cultures of species that have so far been recalcitrant (von Aderkas and Bonga 2000).
During ISE, initial stages of callus development have little time gap (Fig. 7). However, from the complete formation of callus it was observed an anatomical difference in ISE of explants from field and in vitro plants. Callus from field material resemble those described by Pierson et al. (1983), and callus from in vitro material were similar to the pro-embryogenic mass, as the presence of large regions of meristematic cells with single large nucleus and dense cytoplasm, similar structures were observed by Quiroz-Figueroa et al. (2002a) and Silva et al. (2014).
In histochemical studies, it was observed that in explants from field leaves, the presence of phenolic compound was more marked than in explants from in vitro leaves. This can be explained in part by the mechanical disinfection process and chemical and/or response to injury and strain in which the leaves are subjected to during
in vitro inoculation (Alemanno et al. 2003; van Boxtel and Berthouly 1996). Furthermore,
leaves of adult plants may exhibit greater accumulation of phenols, since accumulation of phenolic compounds is indicative of mature tissue (Claudot et al. 1993). In explants from
in vitro leaves the small amount of phenol may be related to controlled condition in which
plants are, providing little differentiation of tissues and low presence of products derived from the secondary metabolism.
Presence of starch detected in ISE was also described by Pierson et al. (1983) and (Berthouly and Michaux-Ferriere 1996). Starch accumulation may be associated with embryogenic potential of explants, starch is required for the formation of somatic embryo, being consumed during the development of meristematic tissue (Appezzato-da-Glória and Machado 2004; Cunha 2001). Protein reserve appears to be crucial for formation of embryogenic tissue during ontogenesis. Berthouly and Michaux-Ferriere (1996) showed that when the protein is absent, embryonic tissue does not develop.
Embryos, both in DSE and ISE were observed in three developmental stages (globular, heart and torpedo), which were connected to the callus or pro-embryogenic mass by the suspensor. Suspensor in the basal region of the embryo connects the embryo itself to the mother tissue, as cited in other varieties of Coffea by Quiroz-Figueroa et al. (2006). Although it was not observed unicellular formation in the beginning of the embryo development, the fact that embryos are attached to the mother tissue by the suspensor is an indication that it has unicellular origin (Quiroz-Figueroa et al. 2006; Williams and Maheswaran 1986). Quiroz-Figueroa et al. (2002a) showed that the somatic embryo in leaf explants of C. arabica, both by the direct route (DSE) and the
indirect (ISE) has a single-cell origin. In this study, even with the analysis of numerous explants and performing sequences sections it was not possible to confirm the unicellular origin.
In this work, it was found that among the process of somatic embryogenesis and ontogenesis of embryos, indirect somatic embryogenesis (ISE) using as explants leaves of in vitro plants proved to be far more promising than the other evaluated conditions, generating callus with 12 days and embryos with 62 days of culture. This was not observed in other conditions where the onset of mass and callus as well as embryo development occurred much later, for example, the explants derived from field plants in the DSE and ISE took at least 270 days of culture until the appearance of embryos. According to Vieira and Kobayashi (2000), indirect somatic embryogenesis (ISE), has the advantage of producing a larger number of somatic embryos over the direct route (DSE). Time reduction in tissue culture is a difference which can ensure a higher quality of embryos, decreasing somaclonal variation, which is a frequent problem in cellular aggregates maintained for long periods in tissue culture (Smith et al. 2000). It is an important tool to support breeding programs associated with gene regulation studies of ontogenesis.
Acknowledgment:
The authors thank the Agronomic Institute of Campinas (IAC), for providing the plant material used in this work and CAPES (Coordination for the Improvement of Higher Level Personnel) for granting the PhD student - IFF.
5. References:
Ahmed W, Feyissa T & Disasa T (2013) Somatic embryogenesis of a coffee (Coffea
arabica L.) hybrid using leaf explants. Journal of Horticultural Science and
Biotechnology(88):469-475
Alemanno L, Ramos T, Gargadenec A, Andary C & Ferriere N (2003) Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis. Annals of Botany 92(4):613-623
Almeida JAS, Leal RR, Carmazini VCB, Salomon MV & Guerreiro Filho O (2014) Effect of temperature and cytokinin on the capacity of direct somatic embryogenesis in
Coffea arabica L. genotypes. Coffee Science 9(3):394-399
Almeida JAS, Ramos LCS & Fazuoli LC (2005) Efeito das estações do ano na calogênese e embriogênese somática em oito genótipos de Coffea arabica.
Appezzato-da-Glória B & Machado SR (2004) Ultrastructural analysis of in vitro direct and indirect organogenesis. Brazilian Journal of Botany 27(3):429-437
Berlyn GP, Miksche JP & Sass JE (1976) Botanical microtechnique and cytochemistry. Berthouly M & Michaux-Ferriere N (1996) High frequency somatic embryogenesis in
Coffea canephora. Plant cell, tissue and organ culture 44(2):169-176
Brand MH & Lineberger RD (1992) In vitro rejuvenation of Betula (Betulaceae): biochemical evaluation. American Journal of Botany:626-635
Carman JG (1990) Embryogenic cells in plant tissue cultures: occurrence and behavior.
In vitro Cellular &Developmental Biology 26(8):746-753
Clarindo WR, Carvalho CR & Mendonça MAC (2012) Ploidy instability in long-term in
vitro cultures of Coffea arabica L. monitored by flow cytometry. Plant Growth
Regulation 68(3):533-538
Claudot A, Jay-Allemand C, Magel E & Drouet A (1993) Phenylalanine ammonia-lyase, chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid walnut tree. Trees 7(2):92-97
Cunha A (2001) Ontogénese lipídica associada à embriogênese somática e ao crescimento de culturas in vitro de linho (Linum usitatissimum L.).
David R & Carde J (1964) Histochimie-coloration differentielle des inclusions lipidiques et terpeniques des pseudophylles du pin maritime au moyen du reactif NADI. Comptes rendus herdomadaires des seances de lacademie des sciences 258(4):1338-&
Dodeman VL, Ducreux G & Kreis M (1997) REVIEW ARTICLE Zygotic embryogenesis versus somatic embryogenesis. Journal of Experimental Botany 48(8):1493-1509 Dublin P (1981) Embryogenèse somatique directe sur fragments de feuilles de caféier
Arabusta. Café Cacao Thé 25(4):237-242
Emons AMC (1994) Somatic embryogenesis: cell biological aspects. Acta Botanica Neerlandica 43(1):1-14
Feher A, Pasternak TP & Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant cell, tissue and organ culture 74(3):201-228
Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regulation 43(1):27-47
Gregory M & Baas P (1989) A survey of mucilage cells in vegetative organs of the dicotyledons. Israel Journal of Botany 38(2-3):125-174
Jiménez VM (2005) Involvement of plant hormones and plant growth regulators on in
vitro somatic embryogenesis. Plant Growth Regulation 47(2-3):91-110
Johansen DA (1940) Plant microtechnique McGraw and Hill Book Company.
Landey RB, Cenci A, Georget F, Bertrand B, Camayo G, Dechamp E, Herrera JC, Santoni S, Lashermes P & Simpson J (2013) High genetic and epigenetic stability in Coffea arabica plants derived from embryogenic suspensions and secondary embryogenesis as revealed by AFLP, MSAP and the phenotypic variation rate. PloS one 8(2):e56372
McIlvaine T (1921) A buffer solution for colorimetric comparison. Journal of Biological Chemistry 49(1):183-186
Menéndez-Yuffá A & de García EG (1997) Morphogenic events during indirect somatic embryogenesis in coffee “Catimor”. Protoplasma 199(3-4):208-214
Mishra MK, Devi S, McCormac A, Scott N, Chen D, Elliott M & Slater A (2010) Green fluorescent protein as a visual selection marker for coffee transformation. Biologia 65(4):639-646
Moens P (1965) Developpement de l'ovule et embryogenese chez/Coffea canephora/Pierre. Cellule (Bélgica) 65(2):127-147
Motoike SY, Saraiva ES, Ventrella MC, Silva CV & Salomão LCC (2007) Somatic embryogenesis of Myrciaria aureana (Brazilian grape tree). Plant cell, tissue and organ culture 89(1):75-81
Murashige T & Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum 15(3):473-497 doi:10.1111/j.1399-3054.1962.tb08052.x
Pearse A (1985) Histochemistry: theoretical and applied vol2: analytical technology. Pierson E, Van Lammeren A, Schel J & Staritsky G (1983) In vitro development of
embryoids from punched leaf discs ofCoffea canephora. Protoplasma 115(2-3):208-216
Quiroz-Figueroa F, Fuentes-Cerda C, Rojas-Herrera R & Loyola-Vargas V (2002a) Histological studies on the developmental stages and differentiation of two different somatic embryogenesis systems of Coffea arabica. Plant Cell Reports 20(12):1141-1149
Quiroz-Figueroa F, Méndez-Zeel M, Sánchez-Teyer F, Rojas-Herrera R & Loyola-Vargas VM (2002b) Differential gene expression in embryogenic and non-embryogenic clusters from cell suspension cultures ofCoffea arabica. Journal of plant physiology 159(11):1267-1270
Quiroz-Figueroa FR, Rojas-Herrera R, Galaz-Avalos RM & Loyola-Vargas VM (2006) Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant cell, tissue and organ culture 86(3):285-301
Ramos L, Yokoo E & Gonçalves W Direct somatic embryogenesis is genotype specific in coffee. In: COLLOQUE Scientifique International sur le Café, 15. Montpellier (Francia), Juin 6-11, 1993.. 1993.
Ravindran BM, Winter S & Thangaraj M (2015) Influence of Age of Explants and Genotype on Somatic Embryogenesis in African and Indian Cassava Cultivars. Journal of Root Crops 40(2):21-27
Rezende JCd, Carvalho CHS, Santos ACR, Pasqual M & Teixeira JB (2012) Multiplication of embryogenic calli in Coffea arabica L. Acta Scientiarum. Agronomy 34(1):93-98
Ribas AF, Cenci A, Combes MC, Etienne H & Lashermes P (2011) Organization and molecular evolution of a disease-resistance gene cluster in coffee trees. BMC genomics 12(1):240
Ruaud JN, Bercetche J & Pâques M (1992) First evidence of somatic embryogenesis from needles of 1 year old Picea abies plants. Plant Cell Reports 11(11):563-566 Sakai WS (1973) Simple method for differential staining of paraffin embedded plant
material using toluidine blue O. Biotechnic & Histochemistry 48(5):247-249
Santana-Buzzy N, Rojas-Herrera R, Galaz-Ávalos RM, Ku-Cauich JR, Mijangos-Cortés J, Gutiérrez-Pacheco LC, Canto A, Quiroz-Figueroa F & Loyola-Vargas VM (2007) Advances in coffee tissue culture and its practical applications. In vitro Cellular & Developmental Biology-Plant 43(6):507-520
Silva AT, Barduche D, do Livramento KG, Ligterink W & Paiva LV (2014) Characterization of a Putative Serk-Like Ortholog in Embryogenic Cell Suspension Cultures of Coffea arabica L. Plant Molecular Biology Reporter 32(1):176-184
Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG & Waterhouse PM (2000) Gene expression: Total silencing by intron-spliced hairpin RNAs. Nature 407(6802):319-320
van Boxtel J & Berthouly M (1996) High frequency somatic embryogenesis from coffee leaves. Plant cell, tissue and organ culture 44(1):7-17
Vasconcellos RCdC, Almeida JASd & Silvarolla MB (2009) Indução de embriões somáticos em explantes foliares de genótipos de Coffea arabica em presença da citocinina 2-iP.
Vidal BC Dichroism in collagen bundles stained with Xylidine-Ponceau 2R. In: Annales d'histochimie, 1969. vol 15. p 289-296
Vieira LGE & Kobayashi AK (2000) Micropropagação do cafeeiro. Simpósio de Pesquisas dos Cafés do Brasil, Consórcio Brasileiro de Pesquisa e Desenvolvimento do Café. Brasil:147-167
von Aderkas P & Bonga JM (2000) Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiology 20(14):921-928
von Arnold S, Sabala I, Bozhkov P, Dyachok J & Filonova L (2002) Developmental pathways of somatic embryogenesis. Plant cell, tissue and organ culture 69(3):233-249
Williams E & Maheswaran G (1986) Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Annals of Botany 57(4):443-462
Xu L & Huang H (2014) Genetic and epigenetic controls of plant regeneration. Curr. Top. Dev. Biol 108:1-33
Yang X & Zhang X (2010) Regulation of somatic embryogenesis in higher plants. Critical Reviews in Plant Science 29(1):36-57
Yasuda T, Fujii Y & Yamaguchi T (1985) Embryogenic callus induction from Coffea
Figure 1: Scanning electron micrograph of somatic embryogenesis in Coffea arabica cv Mundo Novo. (A-C) Direct somatic embryogenesis; (D-I) Indirect somatic embryogenesis. (A) Beginning of pro-embryogenic mass formation. (B) Well developed pro-embryogenic mass, presence of embryos in globular phase (gl) and heart (he). (C) Embryo in cotyledon phase, connected to the pro-embryogenic mass by the suspensor (su). (D) Beginning of callus formation. (E) Well developed callus. (F) Embryos in globular phase (gl). (G) Suspensor detail (su), a structure that connects the embryo itself to the callus. (H) Embryos in globular (gl) and torpedo (to) phases. (I) Somatic embryos in torpedo stages (to) and cotyledon (cot). Bars: A-D, F-H = 100μm; E and I = 1mm.
Figure 2: Somatic embryogenesis in Coffea arabica cv Mundo Novo. (A, C) Direct somatic embryogenesis (DSE). (B, D) Indirect somatic embryogenesis (ISE). (A, B, D) Explant of in vitro grown plants, (C) Explant of field grown plants. (A) Presence of pro-embryogenic mass in the end of the explant. (B) Well-developed callus on the edge of explant. (C) Somatic embryos at different stages of development, globular (gl) and Cotyledonary (cot) connected to the pro-embryogenic mass. (D) Embryos at heart (he), torpedo (to) and cotyledonary (cot) stages connected to callus. Bars: A-B = 1 mm; C-D = 2mm.
Figure 3: Process of direct somatic embryogenesis in Coffea arabica cv Mundo Novo. (A-B) explants controls, observed in both the presence of adaxial epidermis (AD), palisade parenchyma (Pp), spongy parenchyma (Sp), vascular bundle (Vb), abaxial epidermis (AB) and stomata (St). (A; C; E; G, I) Foliar explants of field plants (B, D, F, H, J) Leaf explants from in vitro plants. (C-J) Direct somatic embryogenesis: (C) 8 days, field, early cell division in the spongy parenchyma (*); (D) 8 days, in vitro, high mitotic activity in the spongy parenchyma (*); (E) 18 days, field, high mitotic activity in the spongy parenchyma (*); (F) 18 days, in vitro, presence of pro-embryogenic mass (Pm) with meristematic cells (Mc) in the mass peripheral region; (G) 28-day, field, beginning the formation of pro-embryogenic mass (Pm); (H) 28 days, in vitro, pro-embryogenic mass (Pm) is formed, the presence of surface meristematic cells (Mc). (I) 62 days, field, pro-embryogenic mass (Pm) formed, presence of sparse meristematic cells (Mc). (J) 62 days, in vitro, pro-embryogenic mass (Pm) with large size compared to the field, presence of meristematic cells (Mc) on the surface. Arrow: indicates the edge of the explant with the palisade parenchyma and epidermis without anatomical changes. Bars: A - F, H = 50μm; E, G, I, J = 100μm.
Figure 4: Process of indirect somatic embryogenesis in Coffea arabica cv Mundo Novo: (AB) 2 days, in the field explants (A) and in vitro (B), there is the presence of adaxial epidermis (AD), palisade parenchyma (Pp), spongy parenchyma (Sp), vascular bundle (Vb), abaxial epidermis (AB) and stomata (St) .; (C) 8 days, field, early cell division in the spongy parenchyma (*); (D) 8 days, in vitro, high mitotic activity in the spongy parenchyma (*); (E) 12 days, field, mitotic activity in scattered cells of the spongy parenchyma (*). (F) 12 days, in vitro, beginning of callus formation (CI) the palisade containing meristem cells inside. (G) Explant 16 days, field, presence of well-defined callus (cl). (H) 16 days, in vitro, callus (Cl) formed with peripheral regions containing meristematic cells (Mc). Arrow: indicates the edge of the explant with the palisade parenchyma and epidermis without anatomical changes. Bars: A- G = 50μm; H = 100μm.
Figure 5: Process of indirect somatic embryogenesis in Coffea arabica cv Mundo Novo. (A) 28 day, field, callus (C) formed originated near a vascular bundle (Vb); (B) 28 days, in
vitro , callus (C) with a band of meristematic cells (Mc); (C) 62 days, field, callus in
advanced stage of development (Cl), with fusiform cells, vacuolated and with few intercellular spaces (D) 62 days, in vitro , callus (Cl) in an advanced stage of development, containing cell mass with denser and smaller cells, has meristematic cells (Mc) in the outer callus; (E) 72 days, field, callus (Cl) in an advanced stage of development, containing spindle cells with large intercellular spaces; (F) 72 days, in vitro , callus (Cl) with spindle and spaced cells, in the central part of callus, presence of meristematic cells (Mc) in different regions. Arrow: indicates the edge of the explant with the palisade parenchyma and epidermis without anatomical changes. Bars: A-C = 50μm; D-F = 100μm.
Figure 6: Process of indirect somatic embryogenesis in Coffea arabica cv Mundo Novo, embryo development detail during ontogenesis of explants from in vitro plants. (A) 30 days, has a meristematic tissue (Mc), containing small cells with dense cytoplasm; (B) Detail of the meristem region (Mc), with intense cell divisions; (C) 62 days, beginning of embryo development, presence of suspensor (su); (D) 72 days, detail, presence of globular embryo in the callus end, has protoderm (Pd) and suspensor (su) well defined; (E, F, G) 172 days, at (E) in the presence of embryonic transition stage between globular and heart, has protoderm (Pd) well-defined; (F) Presence of embryos in stages heart and torpedo present protoderm (Pd) and procambium well developed; (G) suspender detail (su) at the base of the torpedo embryo. Arrow: indicates the edge of the explant with the palisade parenchyma and epidermis without anatomical changes. Bars: A-D = 50μm; E = 100μm; F-G = 100μm.
Figure 7: Representation of the ontogenesis of Coffea arabica cv Mundo Novo during the direct somatic embryogenesis (DSE) and indirect (ISE) of explants from plants cultivated in experimental field (condition - Field) and in vitro plant genebanks (condition - in vitro ).
Figure 8: Somatic embryogenesis in Coffea arabica under different histochemical tests. (A, D and F) Direct somatic embryogenesis of field grown plants; (C, E, G-I) Direct somatic embryogenesis of in vitro grown plants; (B) Indirect embryogenesis somatic of field grown plants. (A, B) Ferric chloride 3% positive of phenols, brown coloration; (C) Lugol positive of starch, dark blue color; (D) Sudan IV, positive of lipids, yellow coloring; (E) Nile Blue sulfate, positive for acid lipids, blue color; (F) Nadi reaction, positive of terpenoid, light blue color; (G, H) Red ruthenium, pectins and mucilage, red color; (I) Xylidine Ponceau reagent positive of protein, pink color. Bars: A,B = 100µm; C-I = 50µm.
Capítulo II
CARACTERIZAÇÃO MOLECULAR DOS GENES BBM, LEC E WOX DURANTE A EMBRIOGÊNESE SOMÁTICA DIRETA E INDIRETA DE COFFEA ARABICA CV.
MUNDO NOVO.
Resumo:
A embriogênese somática (ES) é um importante meio de multiplicação do cafeeiro, que permite a produção de plantas geneticamente idênticas à planta matriz, em menor tempo e espaço físico. A ES pode ocorrer pela via indireta (ESI) ou direta (ESD). Durante o processo de indução da embriogênese, ocorre a expressão diferencial de genes, conferindo às células somáticas a capacidade de manifestar um potencial embriogênico. O padrão de resposta dos genes durante a ES em Coffea ainda não está bem definido. Sabe-se que alguns genes podem influenciar positivamente a competência regenerativa das células durante a ES, dentre eles, os genes Baby Boom (BBM), Leafy Cotyledon 1 (LEC1) Wuschel-Related Homeobox 4 (WOX4). Neste estudo, foram apresentados os padrões de expressão dos genes BBM2, LEC1 e WOX4, que durante a ESD tiveram um aumento da expressão com 28 dias de cultura in vitro, coincidente ao inicio da formação da massa pró-embriogênica. Para os genes BBM2 e
LEC1 houve uma expressão aumentada no início do desenvolvimento do embrião. Já na
ESI, os genes BBM2 e LEC1 tiveram uma maior expressão no estádio de calo e nos períodos próximos ao surgimento do embrião. O gene WOX4 apresentou uma expressão aumentada no início da indiferenciação do explante. Sugerindo que os genes estudados desempenham um papel na promoção da proliferação celular e morfogênese durante a ES, sendo um provável regular do desenvolvimento do embrião.
Palavras chave: Embriogênses somática; Baby Boom; Leafy Cotyledon 1;
