Optimisation des conditions d’etablissement de suspensions cellulaires embryogenes a partir de cals d’apex caulinaire de mil (Pennisetum glaucum (L.) R.)

L’isolement de protoplastes en vue de la régénération de plantes transgéniques chez les monocotylédones dépend de la qualité des suspensions cellulaires embryogènes. Ce travail a pour objectif d’établir les conditions optimales d’établissement de suspensions cellulaires embryogènes régénérables en plantes pour permettre une transformation génétique ultérieure. Ce travail réalisé, pour la première fois, sur des variétés de Pennisetum glaucum de Côte d’Ivoire, a révélé les facteurs qui influent sur l’établissement et la croissance des suspensions cellulaires embryogènes à partir de cals d’apex caulinaires. En effet, l’établissement de suspensions cellulaires embryogènes a été obtenu au bout de 8 semaines, lorsque la première filtration a été faite après 2 semaines et que les amas de cals ont été broyés à chaque filtration. En début de culture, lorsque le volume du milieu de culture a été faible (30 ml), la croissance de la suspension cellulaire embryogène a été d’autant meilleure que l’intervalle de temps entre les subcultures a été plus court (3 jours). Inversement, un intervalle de temps plus long (6 jours) entre les subcultures s’est montré meilleur pour de grands volumes de milieu de culture (60 ml). La croissance de la suspension cellulaire embryogène a été comparable sur les deux milieux de culture utilisés (MS et N6).Mots clés : In vitro, suspension cellulaire, cal embryogène, Pennisetum glaucum, Côte d’Ivoire.EFFICIENT CONDITIONS FOR ESTABLISHING EMBRYOGENIC CELL SUSPENSIONS DERIVED FROM STEMAPEX CALLUS IN PEARL MILLET (Pennisetum glaucum (L.) R.)Isolation of protoplasts for the regeneration of transgenic monocotyledonous plants depends on the quality of embryogenic cell suspensions. This work aims to establish the optimal conditions for the establishment of regenerable embryogenic cell suspensions of plants to enable further genetic transformation. This work released for the first time, on varieties of Pennisetum glaucum from Côte d’Ivoire revealed the factors that influence the establishment (the date of the first filtration and the grinding of the callus) and the growth (the mass of the initial heap of callus, the volume of the culture medium and the time interval between subcultures) of embryogenic cell suspensions derived from stem apex callus. It appears that the establishment of embryogenic cell suspension is obtained after 8 weeks when the first filtration takes place after two weeks and heaps of callus are ground at each filtration. During the first days of culture, when the volume of the culture medium is low (30 ml), the growth of embryogenic cell suspension is better if the time interval between the subcultures is shorter (3 days). Conversely, a longer period of time (6 days) between subcultures is needed for larger volumes of culture medium (60 ml). The growth of embryogenic cell suspension is similar on both culture media used (MS and N6).Keywords : Tissue culture, embryogenic cell suspension, callus, Pennisetum glaucum, Côte d’Ivoire

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

Optimisation des conditions de régénération de plantes fertiles à partir de suspensions cellulaires issues de cals embryogenèse d’apex caulinaire de mil (Pennisetum glaucum (L.) R.)

Ce travail a pour objectif d’établir les conditions optimales de régénération de plantes fertiles à partir de suspensions cellulaires embryogènes en vue de leur utilisation pour une transformation génétique par bombardements de protoplastes. Ce travail réalisé sur des variétés de Pennisetum glaucum de Côte d’Ivoire a permis de mettre en évidence les facteurs qui influent (taille et âge des cals, milieu de conditonnement, et âge de la suspension cellulaire) sur la régénération de plantes fertiles. Il ressort de cette étude que la taille des cals et les milieux de conditionnement utilisés n’ont aucun effet sur le taux de régénération de plantes fertiles. Cependant, l’âge de la suspension cellulaire obtenue à partir de cals embryogènes et l’âge du cal initiateur de la suspension cellulaire influent sur le taux de régénération. Le taux de plantes normales régénérées est élevé durant les deux premiers mois de culture de la suspension cellulaire initiée à partir des cals de 2-3 mois (60 à 70%). Les plantes régénérées présentent les mêmes caractéristiques de croissance végétative (taille, longueur de la chandelle, nombre de barres, longueur de l’épi) que les plantes témoins. Pour le rendement, les plantes régénérées portent moins de grains (412) que les plantes témoins (715).Mots clés : Plantes fertiles, suspension cellulaire, cal embryogène, Pennisetum glaucum, Côte d’Ivoire

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

Use of plumules cryopreservation to save coconut germplasm in areas infected by lethal yellowing

Plumules excised from zygotic embryos through the largest representative diversity of four of the five different areas of coconut cash and food crops were used in a cryopreservation process using encapsulation-dehydration technique. Five accessions of coconut trees were used [Panama Tall (PNT/GPA), Brazilian Green Tall (BGD/NVB), Cameroon Red Dwarf (CRD/NRC), Vanuatu Tall (VTT/VNT/GVT), and Tagnanan Tall (TAGT/GTN)] in addition to the accession model [Malayan Yellow Dwarf (MYD)] from which an optimal protocol was obtained. A great variability of response was observed depending on accessions with survival and growth recovery rates varying from 6 to 66% and 0 to 24% after 2 and 7 months of culture, respectively

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

Morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured zygotic embryos

In this study, we compared the evolution of morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured embryos and from seeds over an 8-yr period. At the end of the second year after planting, the height and root collar diameter of plants originating from in vitro cultured embryos were significantly lower than those originating from seeds. However, palms from both categories had the same number of leaves. When palms originating from in vitro plantlets and from seeds were observed later in their development, they were similar for most morphological characteristics measured, except for minor differences in inflorescence morphology, which were still present 8 yr after planting. The flowering pattern, bunch, and fruit production were similar between the two categories of palms. These results indicate that in vitro culture of zygotic embryos does not adversely affect further development of palms in natural conditions

Méthode simple d’échange de germoplasme de cocotier (Cocos nucifera L.) par l’utilisation d’embryons zygotiques.

Objectif : Chez le cocotier, les transferts de matériels effectués sous forme de cylindres d’albumen contenant l’embryon, compte du poids et du volume de la graine, se heurtent à plusieurs difficultés notamment les problèmes phytosanitaires et les nombreuses contaminations. Ce présent travail vise à déterminer les meilleures conditions de transfert permettant une meilleur régénération des embryons dans les laboratoires d’accueil.Méthodologie et résultats : Pour cela les embryons issus de noix matures (10 à 12 mois) ont été conditionnés sur deux milieux. Un milieu de régénération contenant du sucre (culture direct) et un milieu de maintien en vie ralentie sans sucre (conservation préalable avant la mise en culture). Le taux de germination des embryons extraits de noix matures sur les deux milieux ont été évalués. Les résultats ont montré que les taux de germination des embryons sont similaires après leur transfert sur le milieu de germination pour les deux milieux utilisés. Il varie entre 74 % et 84 % comparativement aux noix entières utilisées comme témoin qui ont un taux de 58 %.Conclusion et application des résultats : Cette étude a montré qu’il est possible d’utiliser directement les embryons zygotiques pour les échanges de matériel chez le cocotier en les maintenant en vie ralentie sur un milieu de culture sans sucre. Le maintien des embryons en vie ralentie et leur transfert sur le milieu dépourvu de sucre n’affecte pas leur régénération ultérieure. Ce mode de transfert permettrait de réduire le taux de contamination élevé lors du transfert de matériel sous forme de cylindres d’albumen. La priorité des pays producteurs de cocotier étant la création de nouvelles collections et l’enrichissement des collections existantes, la disponibilité d’une méthode fiable de transfert de matériel constitue une avancé majeur. L’utilisation du milieu de régénération peut conduire à un épuisement des constituants minéraux et entrainer le mort d’embryons ou des malformations au cours de la croissance.Mots clés: Cocotier, cylindres d'endosperme, embryon, culture in vitro, échange de matérie

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