A method for seedling recovery in Jatropha curcas after cryogenic exposure of the seeds.

Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collections. Under this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought and weather damage. Besides, field genebanks are costly to maintain and with important requirements of trained personnel. Thus, the development of efficient techniques to ensure its safe conservation and regeneration is therefore of paramount importance. In this work we describe a method for Jatropha curcas seeds cryoexposure and seedling recovery after thawed. In a first experiment, an efficient protocol for in vitro plant recovery was carried out using zygotic embryo or seeds with or without coat. In a second experiment, desiccated seeds with or without coat were exposed to liquid nitrogen and evaluated after cryoexposure. Germination percentages were variable among treatments, and seeds demonstrated tolerance to liquid nitrogen exposure under certain conditions. Seeds of J. curcas presented up to 99.6% germination after seed coat removal. Seeds with coat cultured in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the ½ MS medium (93.1%) than in WPM medium (76.2%), but from zygotic embryo, abnormal seedlings reached up to 99%. Seeds with coat exposed to liquid nitrogen showed 60% germination in culture after coat removal with good plant growth, and seeds cryopreserved without coat presented 82% germination, but seedlings showed a reduced vigor and a significant increase in abnormal plants. Seeds cultured in vitro with coat did not germinate, independently of cryoexposure or not. This study reports the first successful in vitro seedling recovery methodology for Jatropha curcas seeds, after a cryopreservation treatment, and is recommended as an efficient procedure for in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures.Actualmente, el germoplasma de las especies de Jatropha ssp. se conserva como plantas enteras en las colecciones de campo. Bajo este método de almacenamiento, los recursos genéticos están expuestos a enfermedades, plagas y desastres naturales tales como el error humano, la sequía y las inclemencias del tiempo. Además, los bancos de germoplasma de campo son costosos de mantener y requieren bastante personal capacitado. Por lo tanto, el desarrollo de técnicas eficientes para asegurar su conservación segura así como su regeneración, es de suma importancia. En este trabajo se describe un método de recuperación para semillas y plántulas crioexpuestas de Jatropha curcas después de descongeladas. En un primer experimento, se llevó a cabo un protocolo eficiente para la recuperación de plantas in vitro mediante el uso de embriones cigóticos o semillas con o sin testa. En un segundo experimento, las semillas disecadas, con o sin testa fueron expuestas a nitrógeno líquido y se evaluaron después de la crioexposición. Los porcentajes de germinación fueron variables entre los tratamientos, y las semillas demostraron tolerancia a la exposición del nitrógeno líquido bajo ciertas condiciones. Las semillas de J. curcas presentaron hasta un 99.6% de germinación después de la eliminación de la testa. Las semillas con la testa cultivadas in vitro no germinaron, y el 60% se contaminaron. La germinación de los embriones cigóticos fue significativamente alta en el medio ½ MS (93.1%) en comparación con el medio WPM (76.2%), pero desde los embriones zigóticos, las plántulas anormales alcanzaron más del 99%. Semillas con la testa inmersa en nitrógeno líquido mostraron un 60% de germinacion en cultivos despúes de la remoción de la testa con un buen crecimiento de la planta, y las semillas criopreservadas sin testa presentaron un 82% de germinación, pero las plántulas mostraron un reducido vigor y un incremento significativo de plantas anormales. Semillas con testa cultivadas in vitro no germinaron, independientemente de la criopreservación o no. Este estudio reporta el primer éxito in vitro de una metodología de recuperación de plántulas para semillas de Jatropha curcas, después de un tratamiento de criopreservación, que se recomienda como un procedimiento eficaz para la recuperación de plantas in vitro, cuando las semillas se conservan en bancos de germoplasma a bajas o crio-temperatura

A method for seedling recovery in Jatropha curcas after cryogenic exposure of the seeds

Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collections. Under this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought and weather damage. Besides, field genebanks are costly to maintain and with important requirements of trained personnel. Thus, the development of efficient techniques to ensure its safe conservation and regeneration is therefore of paramount importance. In this work we describe a method for Jatropha curcas seeds cryoexposure and seedling recovery after thawed. In a first experiment, an efficient protocol for in vitro plant recovery was carried out using zygotic embryo or seeds with or without coat. In a second experiment, desiccated seeds with or without coat were exposed to liquid nitrogen and evaluated after cryoexposure. Germination percentages were variable among treatments, and seeds demonstrated tolerance to liquid nitrogen exposure under certain conditions. Seeds of J. curcas presented up to 99.6% germination after seed coat removal. Seeds with coat cultured in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the ½ MS medium (93.1%) than in WPM medium (76.2%), but from zygotic embryo, abnormal seedlings reached up to 99%. Seeds with coat exposed to liquid nitrogen showed 60% germination in culture after coat removal with good plant growth, and seeds cryopreserved without coat presented 82% germination, but seedlings showed a reduced vigor and a significant increase in abnormal plants. Seeds cultured in vitro with coat did not germinate, independently of cryoexposure or not. This study reports the first successful in vitro seedling recovery methodology for Jatropha curcas seeds, after a cryopreservation treatment, and is recommended as an efficient procedure for in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures

Efeito de agentes geleificantes alternativos no meio de cultura no cultivo in vitro de abacaxizeiro e bananeira Effect of alternative gelling agents in culture medium in the in vitro cultivation of pineapple and banana

Com este trabalho, objetivou-se avaliar a ação do ágar e sua substituição parcial e total pelo amido de mandioca na composição de meios de cultura de bananeira e abacaxizeiro. Gemas axilares de abacaxizeiro, cvs. Rio Branco e Quinari foram estabelecidas e avaliadas por quatro subcultivos quanto à multiplicação em meio de MS, suplementado com 2 mg.L-1 de BAP e 0,25 mg.L-1 de ANA, e os seguintes tratamentos: M1: ágar (5 g.L-1), M2: ágar (2,5 g.L-1) + fécula de mandioca (60 g.L-1), M3: fécula de mandioca (60 g.L-1), e M4: ágar (2,5 g.L-1) + fécula de mandioca (30 g.L-1). Num segundo experimento, por três subcultivos sucessivos brotações de bananeira da cv. Grand Naine foram avaliadas quanto à multiplicação em meio de cultura composto por: MM1: ágar (6 g.L-1) ; MM2: ágar (3 g.L-1) + fécula de mandioca (30 g.L-1); MM3: fécula de mandioca (60 g.L-1) e; MM4: meio de consistência líquida estacionário.Estes meios foram suplementados com 0, 2, 4 e 6 mg.L-1 de BAP. Para o abacaxizeiro, verificou-se quea substituição total do ágar pela fécula proporcionou resultados similares aos obtidos com o tratamento com ágar. Na combinação ágar + fécula os resultados foram inferiores aos obtidos com os solidificantes usados isoladamente. Para a bananeira, o uso isolado ou combinado da fécula com ágar não proporcionou melhora nas taxas de multiplicação. Os melhores resultados foram obtidos em meio com ágar e 2 mg.L-1 de BAP. O cultivo em meio líquido apresentou o menor índice de multiplicação.<br>The objective of this work was to test the agar action and its partial and total substitution for cassava starch as gelling agents in the culture media of banana and pineapple. Axillary buds of 'Rio Branco' and 'Quinari' cultivars were established and the multiplication rate evaluated by four subcultives in media with 2 mg.L-1 BAP and 0,25 mg.L-1 NAA, with the following combination of gelling agents: M1: agar (5 g.L-1), M2: agar (2,5 g.L-1) + cassava starch (60 g.L-1), M3: cassava starch (60 g.L-1), and M4: agar (2,5 g.L-1) + cassava starch (30 g.L-1). In a second experiment, for three successive subcultivation shoots of banana, 'Grand Naine' cultivar were evaluated in media composed with the following gelling treatments: MM1: agar (6 g.L-1); MM2: agar (3 g.L-1) + cassava starch (30 g.L-1); MM3: cassava starch (60 g.L-1) and; MM4: stationary liquid medium. To these media were added 0, 2, 4 and 6 mg.L-1 BA. To pineapple it was verified similar results when agar was completely substituted for cassava starch as gelling agent, but the combination agar + cassava starch provided the worse results when compared with gelling agents used separately. For banana shoots multiplication, the isolated or combined use of cassava starch did not improve the multiplication rates. The best results were obtained in medium with 2 mg.L-1 BA and agar as gelling agent. The shoot cultivation in liquid medium presented the lowest multiplication index

Preservação in vitro da batata com ácido acetilsalicílico e duas fontes de carboidrato In vitro storage of potato under acetyl salicylic acid and two carbohydrate sources

O objetivo deste trabalho foi avaliar o efeito de carboidratos e do ácido acetilsalicílico (AAS) na preservação in vitro da batata (Solanum tuberosum L.), cultivar Macaca. Brotações de 1,5 a 2,0 cm de comprimento foram transferidas para meio de MS, acrescido de mio-inositol (100 mg L-1) e ágar (6 g L-1). Testaram-se duas fontes de carboidrato, sacarose e manitol (87,6 mM), e cinco concentrações de AAS (0, 30, 60, 90 e 120 mg L-1). O delineamento foi em blocos casualizados com quatro repetições por tratamento e cada repetição formada por oito tubos de ensaio com uma brotação. O material foi mantido à temperatura de 25±2ºC, fotoperíodo de 16 horas e radiação de 19 miE m-2 s-1. O crescimento e o número de gemas nas hastes foram avaliados por três meses. Passados nove meses, a sobrevivência e o número de microtubérculos também foram avaliados. O uso de manitol, associado às concentrações a partir de 30 mg L-1 de AAS, proporcionou menor crescimento e formação de gemas nas hastes. No meio suplementado com sacarose, a sobrevivência e o número de microtubérculos foram maiores, independentemente das concentrações de AAS utilizadas, após nove meses de cultivo.<br>The aim of this work was to evaluate the effect of carbohydrates and acetyl salicylic acid (ASA) during the in vitro storage of potato (Solanum tuberosum L.), cultivar Macaca. Stems derived from in vitro cultures were cut into 1.5 to 2.0 cm segments and inoculated in a MS medium supplemented with myo-inositol (100 mg L-1) and agar (6 g L-1). Sucrose and mannitol 87.6 mM and five ASA concentrations (0, 30, 60, 90 and 120 mg L-1) were tested. The stems were cultured on 10 mL medium in test tubes (20 x 150 mm) and incubated in a growth chamber at 25±2ºC, 16 hour photoperiod and 19 muE m-2 s-1 radiation. The growth and the bud number formed in the stems for a period of three months were evaluated. Nine months later the survival percentage and the number of microtubers formed were also evaluated. It was observed that the mannitol, associated with 30 mg L-1 of ASA provided the smallest growth and bud formation after three months of storage. The survival and the microtubers amount were larger when the stems were maintained in the medium supplemented with sucrose, apart from the ASA concentration, after nine months of storage

Toxicidade de antibióticos no cultivo in vitro da batata em meios semi-sólido e líquido Antibiotics toxicity on the in vitro potato cultivation in semi-solid and liquid media

O objetivo deste trabalho foi avaliar os efeitos fitotóxicos de antibióticos no crescimento e na taxa de multiplicação in vitro da batata. Brotações da cultivar Baronesa foram cultivadas em meio de multiplicação de consistência semi-sólida e líquida. O meio de multiplicação foi formado pelos sais e vitaminas de MS ao qual adicionou-se um dos seguintes antibióticos: ampicilina, cloranfenicol, estreptomicina e tetraciclina, previamente selecionados em razão da ação bactericida sobre contaminantes da cultura, nas concentrações de 0, 32, 64, 128, 256, 512 e 1.024 mg L-1. Por 21 dias os materiais foram mantidos em sala de crescimento a 25±2°C, 16 horas de luz e fluxo de radiação de 35 µmol m-2 s-1. Nos tratamentos em que se utilizou meio de cultura líquido, os frascos foram mantidos sob constante agitação em mesa agitadora do tipo orbital. A ampicilina foi o único antibiótico que não afetou a sobrevivência e o desenvolvimento dos explantes de batata em meio de multiplicação, podendo ser indicada para trabalhos de descontaminação in vitro dessa espécie. O aumento das concentrações de cloranfenicol, estreptomicina e tetraciclina no meio de cultura apresentou efeitos fitotóxicos severos sobre o crescimento e taxa de multiplicação do material vegetal.<br>The objective of this work was to evaluate the effects of antibiotics on the in vitro growth and multiplication rate of potato. Potato explants, cv. Baronesa, were cultivated in semi-solid and liquid multiplication culture medium. The medium was formed by MS salts, vitamins and one of the following antibiotics: ampicillin, chloramphenicol, streptomycin or tetracycline, previously selected by the bactericidal action on some culture contaminants, at 0, 32, 64, 128, 256, 512 and 1,024 mg L-1. Each treatment had three replicates of ten explants. The materials were incubated for 21 days in a growth room at 25±2°C temperature, 16-hour-light and 35 µmol m-2 s-1 radiation. The flasks with liquid culture medium were kept under continuous agitation in an orbital shaker. Only ampicillin has not affected the survival and the normal development of potato explants in the culture medium, thereby being indicated for decontamination works in vitro of this specie. The increase of the concentrations of chloramphenicol, tetracycline and streptomycin presented severe phytotoxic effects on the growth and multiplication rate of potato explants