Browsing by Author "Verdeil, J.L."
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Item Androgenic potential in coconut (Cocos nucifera L.)(2008) Verdeil, J.L.; Hocher, V.; Perera, P.I.P.; Yakandawala, D.M.D.; Bandupriya, H.D.D.; Weerakoon, L.K.Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38 C) or cold (4 C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38 C for 6 days. Modified Eeuwens Y3 liquid medium supplemented with 100 lM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y3 medium containing 66 lM 2,4-D, followed by transfer to Y3 medium without plant growth regulators and finally to Y3 medium containing 5 lM 6-benzyladenine (BA) and 0.35 lM gibberellic acid (GA3), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthersItem Androgenic potential in coconut (Cocos nucifera L.)(2008) Perera, P.I.P.; Hocher, V.; Verdeil, J.L.; Yakandawala, D.M.D.; Bandupriya, H.D.D.; Weerakoon, L.K.Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38 C) or cold (4 C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38 C for 6 days. Modified Eeuwens Y3 liquid medium supplemented with 100 lM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y3 medium containing 66 lM 2,4-D, followed by transfer to Y3 medium without plant growth regulators and finally to Y3 medium containing 5 lM 6-benzyladenine (BA) and 0.35 lM gibberellic acid (GA3), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthers.Item Coconut clones through somatic embryogenesis(2010) Verdeil, J.L.; Buffard-Morel, J.; Rival, A.; Grosdemange, R.; Huet, C.; Pannetier, C.Item Coconut(Cocos nucifera L.) somatic embryogenesis:obtention of several clone ramets(1992) Verdeil, J.L.; Huet, C.; Grosdemanges, F.; Rival, A.; Buffard Morel, E.T.J.Item Cryopreservation as a Tool for the Management of Coconut Germplasm(2011) Malaurie, B.; N'Nan, O.; Bandupriya, H.D.D.; Borges, M.; Verdeil, J.L.; Tregear, J.We present hereafter a review of different collaborative research studies carried out on the cycopreserntion of coconut plumules excised from the zygotic embryo. Plumule (shoot apical meristem and leaf primordia) tissues have shown different degrees of success in cropresen ation depending on th combination of alginate encapsulation and osmotic or evaporative dehydration used. The percentage of regrowth was progressi\\ Cly impro\\ ed from vitrification (0%), to osmoprotection (10%), and subsequently the encapsulation-dehydration technique allowed 20% regrowth level into leafy shoots. Addition of abscisic acid (20 to 40 JI:\\I) boosted recovery growth after freezing (up ta 40%). Histological studies have clearly shown that the addition of lower amounts of ABA (10 JIM) allows cells to maintair. the structural characteristics of control cells for immersion into liquid nitrogen without dehydration. The effect of plant material conditioning, for transport from collecting site to laboratories, was studied to identify possible effects of unc( ntrolled factors on tissue tolerance to cryopreservation. Three conditioning methods lemb o set in endosperm core (ALB); emb,·yo transferred onto of solidified agar (5W); emb, o immersed into KCI solution (KCI)I were u~ed. 5W performance is clearl~ more efficient when combined with dehydration and freezing. giving 40% recovc . These results are interesting as they show that the medium surrounding the ~mb 1 (endosperm or medium supply) can be replaced by agar alone. without nutritive factors. This approach should also facilitate germrlasm exchanf e. As a result of the absence of phloem vascular bundles in the plumular tissues, the approach described here should increase the scope for obtai.ling material free of pathogenic agents, an essential prerequisite for the conserntion and exchange of germ plasm. This approach should be a strong point in the fight lIgainst Lethal Yellowing Disease. the most serious disease affecting coconut plantations.Item Effect of growth regulators on microspore embryogenesis in coconut anthers(2009) Perera, P.I.P.; Yakandawala, D.M.D.; Hocher, V.; Verdeil, J.L.; Weerakoon, L.K.The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid— NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 lM NAA in combination with 100 lM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 lM 2,4- D alone. Kinetin and 2-iP enhanced the production of calli/ embryos when 100 lM 2,4-D was present in the culture medium. Both cytokinins at 10 lM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 lM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were subcultured in somatic embryo induction medium (Y3 medium containing 66 lM 2,4-D), followed by maturation medium (Y3 medium without growth regulators) and germination medium (Y3 medium containing 5 lM-6-benzyladenine— BA and 0.35 lM gibberellic acid—GA3), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).Item Effect of medium sucrose on the photosynthetic capacity of coconut vitroplants formed from zygotic embryos(2007-02-08) Santamaria, J.M.; Talavera, C.; Lavergne, D.; Trabelsi, S.; Verdeil, J.L.; Huet, C.; Rival, A.; Hamon, S.; Nato, A.Item Effect of plant growth regulators on ovary culture of coconut (Cocos nucifera L.)(2009) Perera, P.I.P.; Vidhanaarachchi, V.R.M.; Gunathilake, T.R.; Yakandawala, D.M.D.; Hocher, V.; Verdeil, J.L.; Weerakoon, L.K.Coconut is a cross pollinating palm, propagated only by seeds. Tissue culture is the only vegetative propagation method available for coconut. Consistent callogenesis was obtained by culturing unfertilised ovaries at -4 stage in CRI 72 medium containing 100 lM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1% activated charcoal. Callusing was improved by application of 9 lM thidiazuron (TDZ). Embryogenic calli were subcultured onto somatic embryogenesis induction medium containing 66 lM 2,4-D. Stunted growth was observed in the somatic embryos after subculture onto CRI 72 medium containing abscisic acid (ABA). Maturation of somatic embryos could be achieved in Y3 medium without growth regulators. Conversion of somatic embryos was induced by adding gibberellic acid (GA3) to conversion medium containing 5 lM 6-benzyladenine (BA) while 2-isopentyl adenine (2iP) increased the frequency of plant regeneration. A total of 83 plantlets was produced from 32 cultured ovaries.Item Efficient Method of Transporting Coconut (Cocos nucifera L.) Zygotic Embryos for Cryopreservation of Plumules by Encapsulation/Dehydration(2014) Bandupriya, H.D.D.; Fernando, S.C.; Verdeil, J.L.; Malaurie, B.Coconut is both socially and economically important crop in tropical and subtropical countries, thus the conservation of existing diversity of its germplasm is vital to maintain biodiversity, sustain crop production and utilisation of germplasm for crop improvement strategies. The recalcitrant storage behavior and large size of the coconut seed make it impossible to use as a germplasm storage material. Cryopreservation is an ideal means of long-term storage of germplasm which offers long-term storage capability with minimal storage space and maintenance requirements. The coconut embryo has been now adapted by various researchers for the purpose of germplasm exchange and it is now being routinely applied in germplasm collection and exchange activities with sufficient germination rates. The aim of the present study was to determine the effect of different coconut embryo transport! storage methods [as solid endosperm plugs under cold temperature, embryos cultured in Solidified Agar Medium (SAM) or KCI solution under room temperature] on cryopreservation of plumules using encapsulation/dehydration method. The results revealed that plumules excised from embryos transported/ stored in SAM and pretreated with 1.0M sucrose could be cryopreserved with 71.8% survival and 56% recovery rates. The survival and recovery could be further increased up to 77.5% and 65% respectively by supplementation of I.OM sucrose with 20 uM ABA.Item Feasibility of Using the Expression of the Retinoblastoma Gene as a Marker for Assessing the Embryogenic Potential of Coconut Ovary Culture(2009) Perera, P.I.P.; Hocher, V.; Weerakoon, L.K.; Yakandawala, D.M.D.; Verdeil, J.L.Coconut is a monocotyledonous tree crop that is highly recalcitrant to in vitro culture conditions. Ovary culture is a promising technique for clonal propagation of coconut. A greater understanding of the fundamental aspects of somatic embryogenesis and plant regeneration is very important in achieving a break-through. Identification of tissues having a high embryogenic potential at an early stage is also very important to achieve a high regeneration efficiency and to avoid maintenance of non-responsive cultures for a prolonged period. In situ hybridization was employed to study the expression of CnRb gene in selected tissues, to identify a potential molecular marker to assess the embryogenic potential of coconut ovary cultures. The results revealed that in situ hybridization can be used to detect the expression of CnRb gene in the cells. It was possible to establish a relationship between the meristematic activity and expression of CnRb gene in the tissues tested. CnRb mRNAs were mainly localized in the meristematic cells and tissues such as calli and growing point of the developing shoots.Item Flow cytometric analysis of the cell cycle in different coconut palm (Cocos nucifera L.) tissues cultured in vitro(2003) Sandoval, A.; Hocher, V.; Verdeil, J.L.We conducted a study of the cell cycle of coconut palm tissues cultured in vitro in order to regulate regeneration. Coconut palm is a plant for which it is difficult to monitor the ability of the meristematic cells to actively divide. Cell nuclei were isolated from various types of coconut palm tissues with and without in vitro culture. After the nuclei were stained with propidium iodide, relative fluorescence intensity was estimated by flow cytometry. Characterization of the cell cycle reinforced the hypothesis of a block in the G0/G1 and G1/S phases of the coconut cells. A time-course study carried out on immature leaves revealed that this block takes place gradually, following the introduction of the material in vitro. Synchronization of in vitro-cultured leaves cells using 60 M aphidicholin revealed an increase in the number of nuclei in the S phase after 108 h of treatment. The significance of these results is discussed in relation with the ability of coconut tissue cultured in vitro to divide.Item Free Amino Acid Composition of Coconut (Cocos nucifera L) Calli under Somatic Embryogenesis Induction Conditions(1995) Mangaval, C.; Noirot, M.; Verdeil, J.L.; Blattes, A.; Huet, C.; Grosdemange, F.; Buffard-Morel, J.Item Free Amino Acid Composition of Coconut (Cocos nucifera L) Calli under Somatic Embryogenesis Induction Conditions(1995) Mangaval, C.; Noirot, M.; Verdeil, J.L.; Blattes, A.; Huet, C.; Grosdemange, F.; Buffard-Morel, J.Item Histological analysis of plant regeneration from plumule explants of Cocos nucifera(2003) Fernando, S.C.; Verdeil, J.L.; Hocher, V.; Weerakoon, L.K.; Hirimburegama, K.Plant regeneration was achieved from plumules excised from mature zygotic embryos of a local coconut cultivar (Sri Lanka Tall). A detailed histological study was undertaken to gain a better understanding of the cellular changes that occur during plant regeneration from plumule tissues. This study led to the identification of the cellular origin, specific cell characterization and development pattern of embryogenic calluses. It also revealed that abscisic acid induces plant regeneration through somatic embryogenesis. The presence of incomplete somatic embryos that lacked shoot poles was also observed.Item Improved somatic embryogenesis from cocos nucifera (l.) Plumule explants(Society for In Vitro Biology, 2006-02) Perez-Nunez, M.T.; Chan, J.L.; Saenz, L.; Gonzalez, T.; Verdeil, J.L.; Oropeza, C.Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embryogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenic callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.Item Morphological and Histological Changes During Somatic Embryo Formation from Coconut Plumule Explants(Society for In Vitro Biology, 2006-02) Saenz, L.; Azpeitia, A.; Chuc-Armendariz, B.; Chan, J.L.; Verdeil, J.L.; Hocher, V.; Oropeza, C.Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing. At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.Item Specific Nutritional Requirements of Coconut Calli (Cocos nucifera L) during Somatic Embryogenesis Induction(2007) Magnaval, C.; Buffard-Morel, J.; Beule, T.; Grosdemange, F.; Huet, C.; Blattes, A.; Verdeil, J.L.; Noirot, M.Coconut calli were cultivated on two somatic embryogenesis induction media (SEIMs), differing in their 2,4-D content. Gain in dry matter weight, composition of soluble sugars within calli, but also pH and contents of glucose and macroelements in media were analysed at 0, 15, 28, and 60 days of culture. Relationships between contents of endogenous sugars, on the one hand, and between contents of media macroelements, on the other hand, were analysed. Comparison was made with calli maintained on a control multiplication medium. Traits could be classified into 3 types of response with regard to condition of somatic embryogenesis induction (SEI condition). The first correspond to traits that were modified by the SHI condition and varying over time. Two phases were determined. During the first phase (T0-T15), soluble sugar contents within calli decreased over rime. The higher the 2,4-D content in SHIMs, the higher the sugar contents, Consumption of glucose and macroelements in media was negligible. However, strong relationships in the contents of chloride, nitrate, phosphate, and sulfatc were modified in the SF.I condition, During the second phase (TI5-1 60, growth became lower in the SEI condition. Requirements for glucose, nitrate and phosphate and acidification of media were higher. I he relationship, determined by changes in nitrate and phosphate (R>0.98), was modified by the SEI conditions, showing a preferential consumption for nitrate in this case. Endogenous sucrose content decreased to become lower in the SEI condition. The higher the 2,4-D content in SEIMs, the higher the requirements for media compounds, the higher the contents of sugars within calli, but the lower the growth. The second type of response corresponded to traits modified by the SEI condition, but constant over time. It concerned relationships between contents of some cations in the media. The third type of response corresponded to traits unchanges by the SEI condition and over time, It concerned the high relationship contents of endogenous glucose and fructose (R = 0,88), and between contents of chloride, ammonium, calcium, magnesium, and potassium.Item Use of SSR Markers to Determine the anther-derived Homozygous Lines in Coconut(Springer-Verlag, 2008-01) Perera, P.I.P.; Perera, L.; Hocher, V.; Verdeil, J.L.; Yakandawala, D.M.D.; Weerakoon, L.K.Anther culture was used to obtain dihaploid (DH) coconut plants and their ploidy level was determined by flow cytometric analysis. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. Ploidy analysis showed that 50% of the tested plantlets were haploid and 50% were diploid. Polymorphic fragments of the mother palm and their segregation patterns in anther-derived plantlets were used to determine the origin of the diploid plantlets. Using a diagnostic SSR marker (CNZ43), all the diploid plantlets tested were identified as being derived from microspores (i.e. were homozygous) and were thus candidates for use in coconut breeding programs.Item Use of SSR markers to determine the anther-derived homozygous lines in coconut(2008) Perera, P.I.P.; Verdeil, J.L.; Hocher, V.; Perera, L.; Yakandawala, D.M.D.; Weerakoon, L.K.Anther culture was used to obtain dihaploid (DH) coconut plants and their ploidy level was determined by flow cytometric analysis. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. Ploidy analysis showed that 50% of the tested plantlets were haploid and 50% were diploid. Polymorphic fragments of the mother palm and their segregation patterns in anther-derived plantlets were used to determine the origin of the diploid plantlets. Using a diagnostic SSR marker (CNZ43), all the diploid plantlets tested were identified as being derived from microspores (i.e. were homozygous) and were thus candidates for use in coconut breeding programsItem What are the possible applications for coconut (Cocos nucifera L.) micropropagation(1998) Verdeil, J.L.; Baudouin, L.; Hocher, V.; Bourdeix, R.; N' cho, Y.P.; Sangare, A.; Rillo, E.; Hamon, S.; Oropeza, C.