Browsing by Author "Hocher, V."
Now showing 1 - 11 of 11
Results Per Page
Sort Options
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 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 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 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 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 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 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.