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An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 mM) and kinetin (9 mM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 mM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 mM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18X0^0X70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets 8X00 X89% when cultured on basal MS medium with 5 mM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N 6 -benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 mM.

High production of viable somatic embryos was obtained from cultured anthers in the second phase of meiosis, using microscopic level observations of tetrads. The medium with the greatest embryogenic efficiency was H6, composed of Murashige and Skoog (MS) medium with 2 mg l À1 of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l À1 of kinetin. All (100%) of the somatic embryos obtained germinated and produced 63% green and 37% albino seedlings. In general, embryogenic calli had a higher ion concentration than non-embryogenic calli, with the exception of calcium whose concentration was higher in non-embryogenic calli. The calli induced in the different media differed in their sucrose and starch compositions. The most embryogenic medium H6-induced calli with the highest sucrose concentration and the lowest starch concentration, before visible embryos were observed. In the leaves of the albino seedlings, sucrose concentrations were very high while those of starch were very low. Ion concentrations were also lower in albino plants than in the leaves of green seedlings, with the exception of calcium, whose concentration was higher. Most of the albino individuals were homozygous, even when their progenitors were heterozygous, thereby confirming their haploid nature.

The embryonic axis plays an essential role in the mobilization of the main reserves of the cotyledons of seeds of Cicer arietinum L. cv Castellana. This control by the axis of the metabolism of the storage products of the cotyledons largely takes place through the cytokinins, which are transported from the embryonic axis to the cotyledons where the mobilization of reserves begins. The principal regulatory role of the endogenous cytokinins concerns the metabolism of carbohydrates and proteins; there is less influence on lipid metabolism. However, each cytokinin seems to have a different role in the mobilization processes. The glucosides, glucosyl zeatin riboside, and glucosyl zeatin act only as storage forms of the hormones. Zeatin riboside affects mainly the mobilization of carbohydrates and has less effect on protein mobilization. Zeatin regulates both the mobilization of carbohydrates and that of proteins and is more marked in the latter case.possibility that an endogenous hormone might be involved (3).Numerous workers have confirmed the role of cytokinins in reserve mobilization in cotyledons from dicotyledoneous seeds (9, 17); however, in most studies the endogenous level of these hormones have not been taken into account, and the application of endogenous hormone could alter the response.The aim of the present work was to provide a clearer idea of the role of cytokinins in reserve mobilization, carrying out all studies after reproducing the endogenous level of cytokinins. To do so we used chick-pea seeds from which the embryonic axis had been removed, so that we knew that there would be no detectable cytokinins (16). Then, by reproducing the endogenous levels ofthe different cytokinins present under normal conditions one can gain a clear idea of the role played by each of these substances in reserve mobilization.Most research on the control of reserve mobilization has been conducted in cereals (3,31). This is possibly due to the difficulty of isolating component tissues and organs of other species for investigation, a process which is straightforward with cereal grains as a result of the spatial separation of the different tissues.Reserve mobilization in cereal seeds appears to be controlled directly by the embryo, via the production of gibberellines which initiate the synthesis and secretion of hydrolytic enzymes from the aleurone layer into the endosperm (31).In dicotyledoneous plants, the regulation of food reserve mobilization is less well understood (1 1) and many conflicting results exist in the literature concerning the role ofthe embryo or embryonic axis in the breakdown of seed reserves (3,22,26).Stimulated, no doubt, by the elucidation of the mechanism in cereals, several attemps have been made to demonstrate that a factor from the embryo or axis is also implicated in other seeds. This has been approached in two ways. First, the promotive capacity of extracts or diffusates of embryos and axes has been investigated; second, it has been argued that if a known hormone such as gibberellin or c...

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The present study analyzes some effects of nano-CeO2 particles on the growth of in vitro plantlets of Medicago arborea when the nanoceria was added to the culture medium. Various concentrations of nano-CeO2 and bulk ceric oxide particles in suspension form were introduced to the agar culture medium to compare the effects of nanoceria versus ceric oxide bulk material. Germination rate and shoot dry weight were not affected by the addition of ceric oxide to the culture media. Furthermore, no effects were observed on chlorophyll content (single-photon avalanche diode (SPAD) measurements) due to the presence of either nano- or micro-CeO2 in the culture medium. When low concentrations of nanoceria were added to the medium, the number of trifoliate leaves and the root length increased but the root dry weight decreased. Also the values of maximum photochemical efficiency of PSII (F(v)/F m) showed a significant decrease. Dark-adapted minimum fluorescence (F 0) significantly increased in the presence of 200 mg L(-1) nanoceria and 400 mg L(-1) bulk material. Root tissues were more sensitive to nanoceria than were the shoots at lower concentrations of nanoceria. A stress effect was observed on M. arborea plantlets due to cerium uptake.

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12 h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.

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