Biomedical subjects

A M Vieitez

Publications and source records attributed to A M Vieitez.

10 recordsLinked to original sources

PubMed2006

Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis.

Somatic embryogenesis was induced in expanding leaf explants excised from epicormic shoots forced from branch segments taken at four different times of year from a mature oak (Quercus robur L.). Branch segments 2-4 cm in diameter produced most shoots when collected in March. Somatic embryos were induced on explants derived from branches of all collection dates, although collection in November seemed to afford the best results. Germination and conversion ability of embryos of embryogenic lines derived from six oak trees depended heavily on genotype, conversion rates ranging from 0 to 70%. RAPD analyses found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryo derived plantlets and the trees of origin. The embryogenic system used in this study appears to be suitable for true-to-type clonal propagation of mature oak genotypes.

Culture Media↗

PubMed2004

Agrobacterium-mediated transformation of European chestnut embryogenic cultures.

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical beta-glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.

Acetophenones↗

PubMed2003

Proliferation, maturation and germination of Castanea sativa Mill. Somatic embryos originated from leaf explants.

Experiments were performed to determine the influence of proliferation medium on the maintenance of embryogenic competence and on repetitive embryogenesis in Castanea sativa Mill. somatic embryos derived from leaf explants. Somatic embryo proliferation was carried out by both direct secondary embryogenesis and by the culture of nodular callus tissue originated from cotyledons of somatic embryos. Both systems led to the production of cotyledonary somatic embryos on Murashige and Skoog proliferation medium supplemented with 0.1 mg l-1 benzyladenine and 0.1 mg l-1 naphthaleneacetic acid. Carbon source and concentration had a marked influence on maturation and subsequent germination ability of chestnut somatic embryos. Plantlet conversion was achieved in embryos matured on media with 6 % sucrose, and on 3 or 6 % maltose, whereas mean shoot length, root length and leaf number of produced plants were not significantly affected by these maturation media. Overall, the best results were obtained with 3 % maltose-matured somatic embryos, giving rise to 6 % plant recovery in addition to 33 % of embryos exhibiting only shoot development. The application of a 2-month cold treatment at 4 degrees C to somatic embryos matured on medium with 3 % maltose was necessary for achieving plant conversion, while partial desiccation did not appear to influence this response. A total of 39 % of embryos eventually produced plants either through conversion to plantlets or indirectly through rooting of shoots. Shoots formed by somatic embryos could be excised, multiplied and rooted following the micropropagation procedures previously developed for chestnut.

Culture Media↗

PubMed2003

Cryopreservation of embryogenic cultures of Quercus robur using desiccation and vitrification procedures.

Oak embryogenic cultures are generally maintained by repetitive embryogenesis. To facilitate management of embryogenic lines and limit the risks of somaclonal variation and contamination a cryopreservation protocol should be developed. In this work we investigated the ability of several pre-treatments to enable 4-6mg clumps (1.0-1.5mm) of globular-heart stage somatic embryos of Quercus robur to withstand freezing in liquid nitrogen. In the best of the two embryogenic culture lines used, 56% of clumps resumed embryogenesis after cooling when they had been pre-treated by successive pre-culture on 0.3 and 0.7M sucrose supplemented media followed by desiccation in the air flow of a laminar flow cabinet to water contents of 24-34%. In both lines, embryogenesis resumption rates of about 70% were achieved by pre-culture on 0.3M sucrose medium followed by application of a vitrification solution (PVS2) for 60-90min prior to rapid plunging in liquid nitrogen.

Cell Line↗

PubMed2003

Developmental stages during the rooting of in-vitro-cultured Quercus robur shoots from material of juvenile and mature origin.

In-vitro-cultured shoots of clones initiated from shoots of the basal parts (BS) and the crown (C) of two mature Quercus robur L. trees were subjected to rooting experiments to relate rooting with shoot topophysical origin. The BS shoots exhibited morphologically juvenile characteristics and rooted more easily after indole-3-butyric acid (IBA) treatment than C shoots. When naphthylphthalamic acid (NPA) was applied to BS shoots, rooting capacity decreased and root emergence was delayed at least 2 days compared with shoots treated with IBA only. During the first days of the rooting process, endogenous indole-3-acetic acid (IAA) concentration was higher in C shoots than in BS shoots, regardless of whether the shoots were treated with NPA. Mitotic figures were observed in cells from the basal part of both BS and C shoots 24 h after IBA treatment. After 4 days of IBA treatment, the occurrence of histological events differed between BS shoots and C shoots. Cells of BS shoots became meristematic, giving rise to meristemoids and root primordia, whereas no differentiation of root meristemoids occurred in cells of C shoots. Thus, although adult oak material (C shoots) is capable of responding to the initial stimulus of auxin during the adventitious rooting process, the endogenous IAA concentration is not the factor limiting rooting in adult material.

Indoleacetic Acids↗

PubMed2002

Somatic embryogenesis in elm.

We show that isolated zygotic embryos of Ulmus minor and U. glabra can produce embryogenic cultures provided they are isolated from immature seeds before storage proteins begin to accumulate. Rates of somatic embryogenesis were highest among zygotic embryos collected 6 weeks post-anthesis when they were at the midcotyledonary stage, were about 5 mm long and had a fresh weight of approx. 10 mg. At this time, induction was even possible in Murashige and Skoog basal medium with no plant growth regulators, but addition of 2,4-dichlorophenoxyacetic acid was necessary at earlier stages of zygotic development. In medium supplemented with benzyladenine (BA) only, no embryogenic induction was observed. The formation of callus was an essential step not only for the induction of embryogenic masses, but also for the maintenance of embryogenic competence through successive subculture of callus on induction media supplemented with 0.1 mg l(-1) BA. Nine embryogenic U. minor lines and 24 U. glabra lines have been maintained in this way for 3 years. However, conversion into plantlets has occurred only rarely.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1996

Requirements for in vitro rooting of Quercus robur and Q. rubra shoots derived from mature trees.

Stabilized shoot cultures initiated from crown material of six adult Quercus robur L. trees and from basal epicormic shoots of a Quercus rubra L. tree showed good in vitro rooting capacity. An initial five-day dark period generally improved the rooting response but was detrimental to plantlet quality. There were clonal differences in rooting capacity. The concentration and exposure time of the indolebutyric acid (IBA) treatment were critical for root induction. In both species, best rooting efficiency was achieved by culture in medium containing 25 mg l(-1) IBA for 24 h and subsequent transfer to an auxin-free medium containing 1% activated charcoal. For all clones tested, the charcoal benefited both shoot quality and root system development, the latter being enhanced by the formation of many lateral roots. Total root system area and length, measured with a digital image analyzer, were significantly greater in medium containing charcoal than in medium lacking charcoal. Because darkening the basal part of the shoots with aluminum foil during the rooting phase only caused a small increase in rooting, we conclude that the large effect of charcoal on rooting was the result of adsorption of inhibitory compounds from the medium or the explant or both, rather than of basal darkening. Other factors affecting the rooting response of Q. robur were: (a) the position on the tree of the material from which cultures were initiated (the topophysical effect); and (b) shoot quality. Recycling the same horizontally placed explant on multiplication medium allowed three successive crops of shoots to be obtained, and rootability was typically maintained from crop to crop.

Journal Article↗

PubMed1993

In vitro shoot proliferation determined by explant orientation of juvenile and mature Quercus rubra L.

Shoot cultures of Quercus rubra (L.) were established from both juvenile and adult plant material. Initial explants from epicormic shoots formed on the basal zone of the trunks had a greater capacity for in vitro establishment than explants from crown branches. The growth of vigorous axillary shoots was obtained by culturing decapitated shoots horizontally on Woody Plant Medium supplemented with 0.2 mg l(-1) of 6-benzylaminopurine. After 3 weeks of culture the shoots were transferred to fresh medium for two more weeks, giving a 5-week multiplication cycle. Efficient shoot production was achieved by combining three treatments favoring the growth of lateral buds: excision of the apex, horizontal culture and cytokinin treatment. The addition of indoleacetic acid or indolebutyric acid to the multiplication medium did not improve shoot proliferation rates, and naphthaleneacetic acid was detrimental. Recycling the same explant for several successive subcultures improved the efficiency of the propagation procedure. Using the optimal multiplication procedures, nine clones (six of juvenile origin and three from adult trees) were tested in vitro and it was found that genotype and age affected performance.

Journal Article↗

PubMed1991

In vitro morphogenetic competence of basal sprouts and crown branches of mature chestnut.

Basal shoots of five clones of mature chestnut tree (Castanea sativa Mill. and C. sativa x C. crenata Siebold & Zucc.) had a greater capacity for in vitro establishment, multiplication and rooting than crown branches of the same trees. Cultures from basal shoots were more responsive than crown-derived cultures in terms of in vitro reactivity (proportion of the explants with shoot development), the mean number of shoots formed per explant, the length of the tallest shoot in each culture, and the multiplication coefficient (defined as the product of the reactivity and the mean number of shoots per explant). Multiplication coefficients were greatest between subcultures 6 and 12, but subculturing failed to increase the rooting potential of shoots of crown origin. Multiplication and rooting rates were also determined for clones derived from seeds of mature trees. Genotype influenced the in vitro performance of clones of both adult and seedling origins.

Journal Article↗

PubMed1988

Factors affecting in vitro propagation of Quercus robur L.

Explants from five clones of Quercus robur (three of juvenile origin and two from adult trees) were cultured on Gresshoff and Doy medium supplemented with 0.2 mg l(-1) 6-benzylaminopurine. Shoot proliferation from apical and nodal segments was influenced by both clone and type of explant. To increase the efficiency of the propagation procedure, donor shoots (20-25 mm in length and with 2 mm removed from the tip) were recultured at 4-week intervals, and the newly formed shoots harvested before each transfer. Under this regime, the multiplication coefficient (proportion of explants forming axillary shoots multiplied by the mean number of new 8-mm stem segments per explant) was greatest for the second crop and declined sharply by the fourth or fifth crop, in three of the four clones tested. Successive additions of fresh liquid medium to old cultures was much less effective than transfer to fresh medium in promoting axillary shoot production. Elongation of shoots before rooting was increased significantly (P < 0.05) in one of two clones tested by transfer to a medium containing either 0.1 or 1.0 mg l(-1) of zeatin. Addition of fresh liquid medium containing zeatin to old cultures failed to improve shoot elongation or axillary shoot production. However, treatment for 15 days with liquid medium containing 0.1 or 1.0 mg l(-1) indol-3-yl-acetic acid increased subsequent rooting.

Journal Article↗