L'embryogenèse somatique en milieu liquide du palmier à huile : Elaeis guineensis Jacq. et E. guineensis x E. melanococca : premiers résultats

Des structures de type embryoïdes ont été obtenues en milieu liquide à partir de cultures à croissance rapide (CCR) et de cals primaires appartenant à plusieurs clones de palmier à huile (#E. guineensis et d'hybrides interspécifiques). Seuls des embryoïdes obtenus à partir de cals primaires ont poursuivi leur développement jusqu'au stade plantule. Des structures polarisées de type embryoïdes sont apparues après le transfert des cultures en milieu liquide (96 jours pour l'hybride interspécifique, 51 pour #E. guineensis). L'une d'entre elles, issue de cal primaire de l'hybride interspécifique s'est développée ultérieurement en pousse feuillée. Ce caractère d'embryoïde des formations obtenues a été confirmé par des examens histologiques. Ceux-ci ont permis ailleurs de mettre en évidence un phénomène d'embryogenèse secondaire ou adventive à partir des tissus du limbe cotylédonaire de l'embryoïde. (Résumé d'auteur

Influence of meristem-tip size and location on morphological development in Dioscorea cayenensis-D. rotundata complex 'Grosse Caille' and one genotype of D. praehensilis

Les effets de la taille et de la position apicale ou axillaire du méristème sur la viabilité et le développement morphologique, après 3, 8 et 11 mois de culture in vitro, ont été étudiés. La plus grande taille des méristèmes a donné le meilleur enracinement et la meilleure formation de plantule tandis que la plus petite taille a donné le plus fort taux de production de bourgeons. Les bourgeons axillaires ont présenté une callogenèse et un enracinement plus intense que pour les méristèmes d'origine apicale quand les méristèmes de petite taille du clone "Grosse Caille" ont été utilisés; l'utilisation de méristèmes caulinaires de plus grande taille n'a pas permis de mettre en évidence plus de différences entre les bourgeons d'origine apicale et ceux d'origine axillaire. La production de plantules enracinées a été réalisée avec succès, après 11 mois de culture, avec un taux de 82 à 39 % de survivants, respectivement pour le clone "Grosse Caille" et pour le génotype #D. praehensilis$. (Résumé d'auteur

Ultrastructural changes during cryopreservation of plumules and embryos of coconut (Cocos nucifera L.)

This article aims to show the changes occurring during cryopreservation of embryos and plumules of coconut which are responsible of their death or survival. Embryos have been cryopreserved by preculture-dehydration for 24h, 28h, 30h, 34h, 38h and 48h on agar medium containing 600g/L of glucose combined with silica gel. The plumules were cryopreserved by encapsulation dehydration on solid medium containing 0.5 M, 0.75 M and 1M followed by dehydration with 40g of silica gel for different durations before rapid freezing. This study indicates that the damages undergone by seed samples can be divided into three types. The first stage of changes concerned the plasmolysis of cells with small vacuoles, condensation of chromatin, changing in the conformation of the DNA and the nucleus and stopping of mitosis. These types of changes are described in general in the context of a desiccation tolerance. The second degree of the changes was the retraction of the cytoplasm inside the cell, the increase in the periplasmic volume. The third degree of modification concerned the deformation of the walls, the invagination or the lysis of the plasma membrane resulting in the observation of distorted cells and and the bursting of the nucleus. These two types of modifications are irreversible and correspond to an absence of regrowth of the samples. Understanding the damage or changes that occur in cryopreserved cells is an important part of understanding how dehydration and frozen affect the viability of recalcitrant plants cells. These changes are made by dehydration and accentuated by freezing

Isolation of phytoplasma DNA from the coconut palms (Cocos nucifera L.) collected from Ghana

This study aimed to verify the presence of the causative agent of Lethal Yellowing which is phytoplasma in samples provided from infected coconut trees. Study was carried out by using various samples like zygotic embryo, young leaves and immature & mature inflorescences. These materials were collected from trees at the stage 1 and 2 of the disease development.. Stage 1 of disease development is characterized by leaf yellowing and the start of the falling nuts while at the stage 2 of disease development, the trees has not bear nuts longer. From infected material, DNA was extracted by three different processes and isolated DNA was amplified by PCR. 16S rRNA gene was amplified by two specific primers of phytoplama viz P1/P2 and Ghana 813/AKSR. Among the various tested materials presence of phytoplasma was reported from the mature inflorescences while the presence of the phytoplasma was not reported from the leaves and embryos of the coconut

Medium-term and long-term in vitro conservation and safe international exchange of yam ( Dioscorea spp.) germplasm

Yam edible tubers feed million of peoples in the intertropical area, where they represent 12% of human feeding. However, as a vegetatively propagated crop, yam is seriously affected by an accumulation of pathogens. Establishing in vitro germplasm collection is a process that cleans the plants from all diseases but viruses. It gives a good control on the preservation of the yam genetic resources and facilitates international exchanges of healthy plant material. Two kinds of in vitro germplasm preservation were considered : slow growth condition culture for mid-term preservation, and cryopreservation using the encapsulation/dehydration technique for long-term preservation. Virus eradication was approached by meristem culture and chemo and thermotherapy. Production of virus-free plants was controlled by ELISA. We succeeded in the introduction and maintenance of 20 yam species, under slow growth conditions. Cryopreservation was applied successfully on two edible yam species, Dioscorea. alata L and D. bulbifera L. Virus-free plants were obtained by meristem culture in D. cayenensis-D. rotundata complex and D. praehensilis. Indexation allowed the detection of different virus (poty-, potex-, badna- and cucumovirus), where the most important potyvirus was YMV. Mid-term conservation of yam germplasm is used routinely, and from these conditions a direct acclimatization is possible. On the cryopreservation aspect, experiments are under way to apply the optimized protocol to genotypes which are more representative of the diversity, to insure a routinely use. More work can be conducted now on virus eradication, based on knowledge accumulated on potyvirus diversity, on several tests available for yam indexing (ELISA, rt/PCR, monoclonal antibodies) and on new sanitation techniques

In vitro conservation of Dioscorea alata L. clon caraqueño (Dioscoreaceae)

El trabajo tuvo como objetivo establecer, para Dioscorea alata L. clon caraqueño, un método eficiente para la conservación in vitro de durante 9 y 12 meses basado en la modificación del medio de cultivo (MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1) con distintos niveles de manitol y BAP. Los tratamientos consistieron en la adición en el medio de cultivo de manitol (0; 0,5 y 1,5%) y BAP (0; 0,1 mg.L-1). A los 9 y 12 meses de conservación in vitro se realizaron las siguientes evaluaciones: supervivencia (%), senescencia foliar (%), numero de nudos, longitud del vástago, número de microtubérculos. A las 8 semanas de la regeneración de los segmentos nodales (procedentes de vitroplantas conservadas a 9 y 12 meses) en el medio de cultivo (MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1) se determinó la regeneración de plantas completas (%), número de nudos, número de hojas y longitud del vástago. A las 5 semanas durante la micropropagación convencional se determinó el número de nudos, número de hojas y la longitud del vástago. Las variantes de cultivo formadas por el medio MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1 y el MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1 + BAP 0,1 mg.L-1 permitió de manera efectiva la conservación de vitroplantas a partir de segmentos uninodales de D. alata clon caraqueño durante 9 y 12 meses con altos porcentajes de supervivencia, un número significativo de microtubérculos, los menores porcentajes de senescencia foliar y 100% de regeneración en plantas completas con un crecimiento normal en condiciones de micropropagación.In this study, we report an efficient method for conservation in vitro of Dioscorea alata L. clone caraqueño during 9 and 12 months based on the culture medium modification (MS to 75% + vitamins MS + sucrose 30 g.L-1 + activated charcoal 2 g.L-1) with different manitol (0; 0,5 and 1,5%) and BAP (0; 0,1 mg.L-1) levels. At 9 and 12 months of in vitro conservation, the following evaluations were determined: survival (%), leaf senility (%), shoot length, buds and microtuber explant count. Plant regeneration (%), shoot length, leaf number and bud explant count were determined at 8 weeks of the nodal cutting regeneration (from vitroplants conserved for 9 and 12 months) in the culture medium (MS to 75% + vitamins MS + sucrose 30 g.L-1 + activated charcoal 2 g.L-1). .Shoot length, leaf number and bud explant count were evaluated at 5 weeks during the conventional micropropagation. The cultivation variants formed by media MS to 75% + vitamins MS + sucrose 30 g.L-1 + activated charcoal 2 g.L-1 and MS to 75% + vitamins MS + sucrose 30 g. l-1 + activated charcoal 2 g.L-1 + BAP 0, 1 mg.L-1 allowed an effective way the conservation of in vitro plants from nodal cutting of clone caraqueño yam during 9 and 12 months with high survival percentages, a significant number of microtubers, smallest leaf senility percentages and 100% regeneration in whole plants with a normal growth in micropropagation conditions

Anatomía comparada de plantas de Dioscorea alata L. clon Caraqueño cultivadas en tres ambientes de crecimiento in vitro

La conservación in vitro de Dioscorea alata L. clon Caraqueño es fundamental para garantizar la propagación y distribución de material de plantación sano a los productores, y disponer de un banco in vitro de un clon de gran valor agronómico y comercial en la región oriental de Cuba. Con el fin de evaluar las modificaciones anatómicas que se producen en plantas de ñame en tres condiciones de cultivo in vitro: plantas conservadas por métodos de mínimo crecimiento, plantas regeneradas y plantas en fase de multiplicación en el medio MS 75 %, se realizó un análisis de la anatomía foliar y caulinar a partir de cortes transversales de la lámina foliar y del tallo, y cortes longitudinales y transversales de microtubérculos formados durante el proceso de conservación. Las hojas de las plantas conservadas mostraron menor espesor del mesófilo y la epidermis y el área de los haces conductores del tallo también fue menor, debido al proceso de stress durante la conservación in vitro. Sin embargo, durante la recuperación del material conservado a través de la regeneración y la multiplicación in vitro se restablecieron de manera normal estos parámetros. También se evidenció que los microtubérculos formados en la conservación in vitro, poseen parénquima amilífero con abundantes gránulos de almidón, capa delgada de parénquima cortical, y haces conductores poco desarrollados, todo lo cual indica la presencia de actividad meristemática.Palabras clave: caracterización anatómica, conservación in vitro, microtubérculos, ñame, segmentos nodalesThe in vitro conservation of Dioscorea alata L. clone Caraqueño is fundamental to guarantee the propagation and distribution of healthy plantation material to the farmers and the establishment of one in vitro bank of this clone of great agronomic and commercial value in the Oriental Region of Cuba. With the purpose of evaluating the anatomical modifications that take place in yam plants under three in vitro culture conditions: conserved plants by slow growth, regenerated plants and in plants multiplication phase in MS 75% medium, was carried out an analysis of the foliar and caulinar anatomy from transversal cuts of the foliar sheet and of the stem, and longitudinal and transversal cuts of microtubers formed during the conservation process. Smaller thickness of the mesophyll and of the epidermis in the leaves of the conserved plants were showed and the conductive sheaves area of the stem were also smaller, due to the stress process during the in vitro conservation. However during the recovery of the conserved material through the regeneration and the in vitro multiplication were reestablished to their normal state these parameters. It was also evidenced that the microtubers formed in the in vitro conservation, have reserve parenchyma with abundant starch granules, thin cortical parenchyma and conductive sheaves little developed were determined. All this characteristics indicated the presence of meristematic activity.Key words: Anatomical characterization; In vitro conservation; In vitro tubers; Nodal cutting; Ya

Respuesta en campo de plantas in vitro de Dioscorea alata L. clon `Caraqueño' en distintos momentos de plantación

The use of biotechnological methods of in vitro plant tissue culture to produce categorized seed yams (Dioscorea spp.) has allowed increased agricultural yields of this crop. This study aimed to determine the response of in vitro plants of Dioscorea alata L. clone `Caraqueño' at different times of planting in the field. The treatments consisted in field planting of previously acclimatized in vitro plants, from June to November, once a month. In June were planted 125 g fresh weight fragments tubers as a control. After 30 days of culture in all the treatments the survival of plants was evaluated and at the time of harvest (January) 20 plants per treatment were taken and determined agronomic variables such us number of tubers per plant, fresh mass of tubers per plant (kg), tuber fresh weight (kg) and tuber dried mass (%). The results showed that the in vitro plants reached high survival rates (96.5 to 99.5%), with higher values for the number and fresh weight of tubers per plant in the months from June to August (3.3, 3.1 and 2.9), and June and July (3.5 and 3.1 kg), respectively in relation to the conventional plant material propagation. It was demonstrated that in vitro plants can be planted in field from June to November, with production of tubers that can reach the category of commercial (June to August) and the category of basic seed (September to November).Key words: in vitro culture, tubers production, yamLa utilización de métodos biotecnológicos del cultivo in vitro de tejidos vegetales para la producción de semilla categorizada de ñame (Dioscorea spp.) ha permitido el incremento de los rendimientos agrícolas de este cultivo. El presente trabajo tuvo como objetivo determinar la respuesta de plantas in vitro de Dioscorea alata L. clon `Caraqueño' en distintos momentos de plantación en campo. Los tratamientos consistieron en la plantación en campo de plantas in vitro previamente aclimatizadas, de junio a noviembre, una vez por mes. Como control se plantaron en junio fragmentos de tubérculos de 125 g de masa fresca. Después de 30 días de cultivo se evaluó en todos los tratamientos la supervivencia de las plantas (%), y en el momento de la cosecha (mes de enero) se tomaron 20 plantas por tratamiento a las cuales se les determinaron las variables agronómicas número de tubérculos por planta, masa fresca de tubérculos por planta (kg), masa fresca del tubérculo (kg) y masa seca de tubérculo (%). Los resultados demostraron que las plantas in vitro alcanzaron altos índices de supervivencia (96.5 a 99.5%), con los mayores valores para el número y masa fresca de tubérculos por planta en los meses de junio a agosto (3.3, 3.1 y 2.9), y junio y julio (3.5 y 3.1 kg) respectivamente en relación con el material vegetal de propagación convencional. Se demostró que las plantas in vitro pueden ser plantadas en campo desde junio hasta noviembre, con la producción de tubérculos que pueden alcanzar la categoría de comercial (junio hasta agosto) y la categoría de semilla básica (septiembre a noviembre).  Palabras clave: Dioscorea alata L., cultivo in vitro, producción de tubérculo