Nieves Villalobos

Differences in the contents of total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from Medicago arborea L.

The total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from explants (cotyledons, petioles, hypocotyls and leaves) obtained from Medicago arborea L. seedlings were evaluated. Total sugars were the major components in the calli and no significant differences between embryogenic and non-embryogenic calli were observed. In contrast, important differences between the embryogenic and non-embryogenic calli were observed for reducing sugars, the highest levels being observed in embryogenic calli. The highest starch levels were found in non-embryogenic calli developed in MS medium. During the development of somatic embryogenesis very low starch levels in the callus were found. During the first months of culture, no significant differences in the sucrose content were found between calli that produced embryos and those that did not. The most important differences in sucrose were seen between calli transferred to medium F0, which had the greatest embryogenic capacity, and those transferred to medium F6, which inhibited embryogenesis. In the latter case, an increase in sucrose was observed.

Improvement of somatic embryogenesis in Medicago arborea

A number of medium constituents were evaluated in an attempt to improve somatic embryo production in Medicago arborea ssp. arborea, using cotyledons, petioles and leaves as explants. Two culture steps were applied: in the first stage (2 months), Murashige–Skoog (MS) medium was used, containing 2,4 dichlorophenoxyacetic acid (9μM 2,4-D) and kinetin (9 μM KIN) together with different nitrogen sources (alanine, glutamine, proline or tryptophan (2.5 and 5 mM); casein hydrolysate (100, 500 and 1000 mg l−1; nitrate (4.69 and 9.39 mM) or casein hydrolysate (100 mg l−1) and nitrate (4.69 mM)), polyalcohols (mannitol at 164 and 328 mM or sorbitol at 219 and 438 mM), sucrose (43.8 and 175.4 mM) or calcium (1.5 and 6 mM). In the second stage (3 months of cultivation), calli were transferred to a kinetin-free MS medium with 2,4-D (2.25 μM) only. The inclusion of proline (2.5 mM) was the most effective treatment for the induction of somatic embryos, with the petiole being the best explant. Treatment with casein hydrolysate (100 mg l−1) also improved the embryonic efficiency. The rest of the treatments neither affect nor inhibit the embryonic response.A special treatment with sorbitol (219 mM) in the second stage of cultivation produced a slight increase in embryogenesis, but less than that obtained with proline.

Cytokinins in chick-pea seeds identification and transformation during germination and seedling growth

Summary Eight cytokinins were detected in germinated chick-pea ( Cicer arietinum L. var. Castellana) seeds. Treatments of extracts with β-glucosidase revealed the presence of two cytokinin glycosides: zeatin glucoside and glycosil zeatin riboside. Treatment with potassium permanganate allowed the differenciation of dihydrozeatin and dihydrozeatin riboside. These dihydro forms are present at advanced stages of germination.

Variation of the Levels and Transport of Cytokinins during Germination of Chick-pea Seeds

Summary Cytokinins detected in chick-pea seeds were present in the embryonic axes and first appeared in the cotyledons after 12 hours of germination. The appearance and initial increase of cytokinins in cotyledons is common to both the free and bound forms. However, from 24 hours of germination onwards the disappearance of bound cytokinins is much faster than that of the free forms, suggesting transformation of the bound forms into free ones during this stage of germination. The cytokinins detected in the cotyledons seem to originate from the embryonic axes, though there is no passage of these substances from the cotyledons to the axes. The β-glucosidase activity measured both in cotyledons and in embryonic axes, was seen to be positively correlated with the decrease in bound cytokinins and the increase in free forms, suggesting the transformation of cytokinin glycosides into their corresponding free forms.

Somatic embryogenesis and plant regeneration in Medicago arborea L.

SummaryAn 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 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) 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 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N6-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 μM.

Nitrate reduction in cotyledons of Cicer arietinum L.: regulatory role of cytokinins

Abstract Application of different concentrations of kinetin to chick-pea seeds inhibits the nitrate reductase ativity detected in cotyledons when the seeds are sown without a hormone. However, kinetin concentrations between 10 −6 and 10 −5 M, applied to excised cotyledons are able to induce nitrate reductase activity, which also becomes manifest in an active metabolism of nitrate and nitrite in the cotyledons. The development of nitrate reductase activity in excised cotyledons is due to synthesis of the enzyme, except when induction is carried out by applying low concentrations of cytokinins. In this case, apart from synthesis, an inactive nitrate reductase becomes activated.

Involvement of cytokinins in the germination of chick-pea seeds

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 12h 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.

Cytokinin levels during the germination and seedling growth of Cicer arietinum L.: effect of exogenous application of calcium and cytokinins

Abstract In chick-pea seeds, the exogenous application of calcium decreases the content of this ion in the embryonic axes, the decrease being inversely proportional to the calcium concentration applied. Application of cytokonins, in particular that of zeatin, however, increases the levels of endogenous calcium in the axes. The highest levels of calcium are found in the apical segment of the radicle and in the subapical zone of the epicotyl. Unlike calcium, the cytokinins applied increase the levels of the calcium ion in these segments. Such an increase also affects, in the case of zeatin, the apical segment of the epicotyl and the subapical zone of the radicle.

Effects of high temperature on the variation and transport of endogenous cytokinins during the germination of Chick-pea seeds

Summary Chick-pea seeds have an optimal germination temperature of 25°C. A temperature of 30°C induces a delay of approximately 24 hours in the germination of these seeds and also a 24–36 hours delay in the appearance of cytokinins in the cotyledon, the transformation of glycosides into free bases and of free bases into dihydroderivatives. The transport of cytokinins from the axis to the cotyledon also undergoes a similar delay and as at 25°C no passage of cytokinins from the cotyledon to the embryonic axis can be detected. On the 5th day of the period studied, the delay occurring in germination and levels of cytokinins as compared with normal conditions is less pronounced, there already being a diminished effect of high temperature. The cytokinins evaluated and identified when the seeds were germinated at 30°C were the same as those observed when germination took place at 25 °C.

Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of Medicago arborea L.

Analysis of cell wall polysaccharide composition of embryogenic and non-embryogenic calli obtained from hypocotyl and petiole explants from Medicago arborea L. revealed significant differences. For calli induced from both hypocotyls and petioles, levels of total sugars, pectins, and hemicelluloses were higher in embryogenic than in non-embryogenic calli. Whereas in the residual cellulose fraction, the highest levels of sugar were detected in non-embryogenic calli. When comparing the two donor sources of callus explants, the highest total sugar levels were detected in embryogenic calli induced from petioles, mainly in the pectin fraction and to a lesser extent in the hemicellulose fraction. Moreover, analysis of uronic acids revealed higher levels in embryogenic calli, primarily in the pectin fraction. Analysis of those sugars associated with cell walls of calli suggested that these polysaccharides consisted of pectic polysaccharides and glucans, and that their levels were higher in embryogenic than non-embryogenic calli.