Plantlet Regeneration from Unfertilized Ovule of Mandarin (Citrus reticulata)

The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide, many genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches of cultivar improvement. in this objective We aime at studying the effects of various culture media on the induction and the development of citrus somatic embryos.Callus cultures were initiated from the infertlized ovules of six varieties of mandarin (Anana, Lee, Murcott, Ortanique, Temple, and Wilking) within 3 media: MT (Murashig and Tuker, 1969) without hormones, MT + 1 mg/l BAP, MT + 1 mg/l Kinetin, the experiments show a highly significant effect (P < 0.001) of the culture media and genotype. No reactivity was observed on the MT environment in the absence of growth regulator, while the culture media MT in addition to 1 mg/l BAP gave the best results of induction of embryogenic callus induction. The induction of somatic embryogenesis was obtained on MT media without hormones. For the plantlets regeneration the favorable media was MT without hormones or added to ANA and active coal.

Date palm micropropagation: Advances and applications / Micropropagação de tamareira: Avanços e aplicações

ABSTRACT Date palm (Phoenix dactylifera L.) is a fruit tree resilient to adverse climatic conditions predominating in hot arid regions of the Middle East and North Africa. The date fruit contains numerous chemical components that possess high nutritional and medicinal values. Traditional propagation by offshoots is inefficient to satisfy current demands for date palm trees. Alternatively, micropropagation provides an efficient means for large-scale propagation of date palm cultivars. Both somatic embryogenesis and organogenesis, either directly or indirectly though the callus phase, have been demonstrated in date palm in vitro regeneration. Culture initiation commonly utilizes shoot-tip explants isolated from young offshoots. Recently, the immature inflorescences of adult trees were utilized as an alternative nondestructive source of explants. In addition to the nature of the explant used, successful plant regeneration depends on the cultivar, composition of the culture medium and physical status. Challenges of date palm micropropagation include long in vitro cycle, latent contamination, browning, somaclonal variation as well as ex vitro acclimatization and transplanting. A remarkable amount of research investigating these factors has led to optimized protocols for the micropropagation of numerous commercially important cultivars. This has encouraged the development of several international commercial tissue culture laboratories. Molecular characterization provides an assurance of genetic conformity of regenerated plantlets, a key feature for commercial production. This article describes date palm micropropagation protocols and also discusses recent achievements with respect to somaclonal variation, molecular markers, cryopreservation and future prospects.

RESUMO A tamareira (Phoenix dactylifera L.) é uma arvore frutífera adaptada à condições climáticas adversas predominantemente em regiões áridas do Oriente Médio e Norte Africano. As tâmaras possuem vários componentes químicos com alto valor medicinal e nutricional. A propagação tradicional por estacas não é suficiente para satisfazer a demanda por mudas e assim, a micropropagação apresenta-se como uma alternativa eficiente para a produção de mudas em larga escala. Embriogênese somática e organogênese, tanto direta quanto indireta via calos, tem sido usada para obter a regeneração in vitro de tamareira. O inicio do cultivo in vitro normalmente utiliza meristemas excisados de brotações jovens. Recentemente, inflorescências imaturas de árvores adultas são usadas como fonte alternativa de explantes não destrutiva. Além da origem do explante, o sucesso da regenerção depende do cultivar, da composição do meio de cultura e de condições físicas. Desafios na micropropagação de tamareira incluem um longo ciclo in vitro, contaminação, escurecimento do tecido, variação somaclonal além do enraizamento e aclimatização ex vitro. Diversos estudos investigando esses fatores tem conduzido à otimização de protocolos de micropropagação de inúmeros cultivares comerciais proporcionando o estabelecimento de vários laboratórios de cultura de tecidos de plantas. A caracterização molecular permite uma segura conformidade genética do material regenerado, considerado uma característica chave na produção comercial. Essa revisão descreve protocolos de micropropagação de tamareira e aborda as mais recentes conquistas relacionadas à variação somaclonal, marcadores moleculatres, criopreservação e perspectivas futuras.

Somaclones em batata identificados pelos descritores mínimos de broto / Potato somaclones identified by minimum descriptors sprout

Este estudo objetivou verificar a estabilidade fenotípica das cultivares de batata 'Asterix' e 'Macaca', avaliar o efeito do tipo de explante (organogênese direta e indireta) e do tempo de subcultivo (12 e 70 meses) em meio nutritivo MS sobre a ocorrência de somaclones nas duas cultivares na produção de batata semente, mediante o emprego de sete descritores mínimos de broto. Em 'Asterix' e 'Macaca' ocorreram somaclones em quatro dos sete descritores, contudo, apenas no formato e pubescência da base do broto houve variação, simultaneamente, em ambas. Os dois genótipos são suscetíveis à ocorrência de variação somaclonal. Registrou-se somaclonesnos dois tempos de subcultivo nas duas cultivares. Diferente do amplamente registrado, identificaram-se somaclones em segmentos apicais caulinares e nodais originados de organogênese direta em 'Asterix' e 'Macaca'.

It was examined the phenotypic stability of potato cultivars 'Asterix' and 'Macaca', evaluated the effect of explant type (direct and indirect organogenesis) and subculture time (12 and 70 months) in MS nutrient medium on the occurrence of somaclonal variation in both cultivars in seed potato production through the use of seven minimum descriptors sprout. In 'Asterix' and 'Macaca' somaclones have occurred in four of the seven descriptors, however, only in the shape and in the base of the bud pubescence that somaclonal variation occurred simultaneously in both cultivars. Both genotypes are susceptible to the occurrence of somaclonal variation. It was identify the occurrence of somaclones both at 12 months and 70 months of subculture in both genotypes. Unlike the widely recorded, somaclones were identified in shoot apical segments and nodal segments derived from direct organogenesis in 'Asterix' and 'Macaca'.

In vitro plant regeneration of Spartina argentinensis Parodi / Regeneración in vitro de Spartina argentinensis Parodi

Spartina argentinensis Parodi es la especie dominante en comunidades halófitas que ocupan alrededor de 20.000 km2 en la Provincia de Santa Fe, Argentina. El objetivo de este trabajo fue desarrollar un método simple para la regeneración de plantas in vitro de S. argentinensis que podría ser utilizado para la investigación básica y aplicada. Se utilizaron como explantes, segmentos basales de hojas de plantas jóvenes y maduras, puntas de raíces e inflorescencias inmaduras. Los medios de cultivo utilizados para callos, brotes e inducción de raíces consistió en la base salina de Murashige y Skoog suplementado con diferentes reguladores del crecimiento vegetal (2,4-D; BAP o ANA). La mayoría de los explantes (con la excepción de puntas de raíces) mostró una proliferación celular y formación de callos después de 30 días de cultivo. Solo las inflorescencias inmaduras regeneraron brotes y raíces cuando los callos se incubaron en sales MS con 2,4 -D y BAP (0,1 y 0,01 mg.L-1, respectivamente), posteriormente los callos se transfirieron a medio de inducción de brotes (sales MS, 0,5 mg. L-1 BAP) y luego a medios de inducción de raíz ( MS y 0,5 mg.L-1NAA). Las plantas regeneradas se evaluaron para detectar anomalías morfológicas y el contenido de lignina de sus hojas. El análisis histológico de los callos mostró que los brotes y las raíces se originaron vía organogénica. Un bajo porcentaje de las plantas regeneradas mostraron deficiencia de clorofila (plantas albinas) y otras anomalías morfológicas. Entre las plantas regeneradas se detectó variaciones significativas en el contenido de lignina. El protocolo que se describe en este trabajo podría ser utilizado para la regeneración in vitro de plantas de S. argentinensis y la selección de variantes somaclonales para futuros planes de mejoramiento.

Spartina argentinensis Parodi is the dominant species of the temporally-flooded halophyte communities of the Santa Fe Province, Argentina. It occupies around 20,000 km2 and it is mainly used as low-cost impute forage for cattle production. The objective of this work was to develop a simple method for in vitro plant regeneration of S. argentinensis that could be used for fundamental and applied research. Leaf-basal segments from both young and mature plants, roots tips and immature inflorescences were used as explants. Culture media for calli, shoot, and root induction consisted of Murashige & Skoog salts supplemented with different plant growth regulators (2,4-D; BAP or ANA). Most of the explants (with the exception of root tips) showed cell proliferation and calli formation after 30 days of culture. However, only immature inflorescences responded to shoot and root induction when calli were incubated on MS salts plus 2,4_D and BAP (0.1 and 0.01 mg.L-1, respectively), transferred to shoot inductionmedia (MS salts plus BAP, 0.5 mg.L-1) and then to root induction media (MS slts plus NAA 0.5 mg.L-1). Regenerated plants were evaluated for morphological abnormalities and lignin content. Historical analysis of regenerating calli showed that shoots and roots originated via organogenesis. A low proportion of regenerated plants resulted with deficiency in chlorophyll (albino plants) and other morphological abnormalities. Interestingly, significant variations in the lignin content were detected between regenerated plants. The protocol described in this work could be used ordinarily for S. argentinensis in vitro plant regeneration and selecting somaclonal variants for breeding purposes.

Identificação de variantes somaclonais em bananeiras 'Prata Anã', utilizando técnicas moleculares e citogenéticas / Identification of somaclonal variants in 'Prata Anã' banana using molecular and cytogenetic techniques

Variação somaclonal é uma variação fenotípica de origem genética, ou seja, uma variação cromossômica que se torna herdável nas gerações seguintes, ou epigenética, que é uma variação transitória devido ao estresse fisiológico que o material sofre, quando submetido ao cultivo in vitro. Um problema específico envolvendo a variação somaclonal em bananeiras 'Prata Anã' foi observado em Andradas, Minas Gerais, em plantas oriundas de micropropagação. A maior dificuldade na separação dos indivíduos normais e variantes é que os caracteres morfológicos, que são inerentes a este tipo de variação, só se tornam evidentes quando a planta está adulta, o que impossibilita a eliminação dos indivíduos variantes ainda em viveiro. Com o objetivo de identificar, ainda em viveiro aqueles indivíduos variantes somaclonais, técnicas moleculares (RAPD e SSR) e citogenéticas (contagem cromossômica e citometria de fluxo) foram utilizadas. Cento e três primers RAPD, 11 combinações de dois primers RAPD, e 33 pares de primers SSR foram utilizados na tentativa de se encontrar marcadores polimórficos capazes de distinguir os indivíduos normais dos variantes, além de distinguir bananeiras 'Prata Anã' de 'Prata'. O primer OPW-08 gerou um fragmento polimórfico que distinguiu uma planta variante de todas as demais, provando que a variação não ocorre de maneira uniforme no genoma dos indivíduos variantes e que não há um retorno à cultivar Prata. As análises com marcadores SSR e a contagem cromossômica não possibilitaram a distinção dos indivíduos variantes, nem a separação das cultivares Prata e Prata Anã. As análises de citometria de fluxo evidenciaram a grande instabilidade cromossômica das bananeiras, porém elas não foram eficientes na identificação de variantes somaclonais.

Somaclonal variation is a phenotypical variation of genetic origin, that is, a chromosomal variation that becomes inheritable in the generations to follow, or of epigenetic origin, in this case being a transitory variation due to the physiological stress suffered when the material is submitted to in vitro cultivation. A specific problem involving somaclonal variation in 'Prata Anã' banana was observed in Andradas, Minas Gerais, in plants originated from tissue culture. The main difficulty in the distinction between the normal and variant plants is the fact that the morphological characters that allow the separation of these two types are only visible and distinguishable when the plants are in their adult phase, which makes it impossible to eliminate the variant seedlings at the nursery stage. For the early distinction of the variants, molecular (RAPD - Random Amplified Polymorphic DNA and SSR - Simple Sequence Repeat) and cytogenetic (chromosome counting and flow cytometry) techniques were used. In the attempt to find polymorphic markers that distinguished the normal plants from the variants as well as the Prata cultivar from the Prata Anã cultivar, 103 RAPD primers, 11 combinations of two RAPD primers, and 33 pairs of SSR primers were used. Primer OPW-08 generated a polymorphic fragment that distinguished a variant from all of the other plants, proving that the variation does not occur uniformly in the genome of all variants, and that there is no return to Prata cultivar. Analyses with SSR markers and chromosome counting were not efficient in separating normal plants from variants or 'Prata' from 'Prata Anã'. Flow cytometry analyses showed an evident instability in the banana genome in terms of number of chromosomes, however, they were not efficient in identifying the somaclonal variants.

Genetic changes in grapevine genomes after stress induced by in vitro cultivation, thermotherapy and virus infection, as revealed by AFLP

The Amplification Fragment Length Polymorphism (AFLP) technique was employed to study genetic variations which can be induced in vines by the stress occurring during different aspects of viticulture (in vitro cultivation, in vitro thermotherapy and virus infection). Analysis of AFLP banding patterns, generated by using 15 primer combinations, pointed to negligible genetic variation among plants exposed to individual stress. The average of similarity coefficients between differently stressed plants of the cultivars Müller Thurgau and Riesling were 0.984 and 0.991, respectively, as revealed by AFLP analysis. The low incidence of observed polymorphism demonstrates the high level of genome uniformity in plants reproduced by in vitro micropropagation via nodes, those subjected to in vitro thermotherapy and virus-infected plants.

Micropropagation of selected somaclones of Begonia and Saintpaulia.

Begonia x elatior plantlets which regenerated from leaf disk callus showed variations in plant morphology, number of flowers per plant, and flower size. Variations in flowering period, number of flowers per plant, and flower morphology were observed in Saintpaulia ionantha L. plants directly regenerated from leaf disk explants. The cytokinins, benzylaminopurine and zeatin, tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. Micropropagation of selected somaclones having the desirable trait of high number of flowers per plant was stable in the MV2 and MV3 generations.