Ricardo Sánchez Tamés

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

Abstract A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field.

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

Abstract A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions.

In vitro filbert (Corylus avellana L.) micropropagation from shoot and cotyledonary node segments.

Plantlets were successfully regenerated from shoot and cotyledonary node segments excised from 20 day old filbert seedlings. In both cases the optimum initiation and elongation of shoot buds was obtained after 15 days culture in 1/2K(h) medium plus BAP (25 μM) followed by 20 days culture in the same medium in presence of a reduced BAP concentration (0.5 or 2.5 μM).Maximum of functional roots were readily formed after 5 days of submersion of the basal end of shoots in 1/2K(h) liquid medium plus IBA (50 μM), then transferred to a fresh 1/2K(h) solid medium for a further 15 days. Following these two consecutive steps, root initiation and development was achieved in 80% of the explants.The histological origin of neoformed organs was studied.

Regulation of asexual embryogenesis in Filbert cotyledonary nodes. Morphological variability

Abstract Filbert cotyledonary nodes were used as explants in order to study the effect of auxins, cytokinins and culture period on embryoid induction and development. Initiation processes took place during the first 20-day culture. Embryoid development rarely surpassed the globular stage independently of the added indole-3-butyric acid/6-benzylaminopurine (IBA/BAP) combination. Conversely, in the first subculture, different developmental stages coexisted in the same area of the formed embryogenic clusters, and structures with very heterogeneous morphology were observed. Indole-3-butyric acid seems to promote embryoid initiation in both cultures, while BAP promoted development during the first subculture. The state of embryoid development could be controlled with 2,4-dichlorophenoxyacetic acid (2,4-D) in such a way that most of the embryoids were in the globular stage. Benzylaminopurine acts as the main agent in restoring polarized development. A detailed anatomical and histological study of embryogenesis in each culture period was carried out.

Ethylene biosynthesis and endogenous polyamines in relation to development of in vitro cultured kiwifruit explants

The relationship between polyamines and ethylene is controversial because the degree of interference of one pathway with the other may differ according to species, stage of development and experimental procedure. In this paper, we modify ethylene biosynthesis by the addition of aminoethoxyvinylglicine (AVG) or 1-aminocyclopropane-1-carboxylic acid (ACC) and study how it affects polyamine content and development of kiwifruit explants (Actinidia deliciosa CS Liang. & AR Fergusson). Cultured under ventilation where ethylene did not accumulate in the culture vessels, kiwi explants had higher ACC synthase activity and lower polyamine content than those grown without ventilation. In explants cultured in the reference medium, putrescine was the more abundant polyamine and spermine was only detected in the free fraction irrespective of ventilation. Under ventilation, addition of ACC to the culture medium inhibited organogenesis, there was less spermidine and spermine was not detected. Addition of AVG to the culture medium increased both the number of shoots and the amount of polyamines, and inhibited ACC synthase, so S-adenosylmethionine (SAM) led to increasing synthesis of spermidine and spermine. The increase in putrescine is more difficult to explain on the basis of inhibition of ethylene biosynthesis. The increase in the number of shoots in kiwi explants due to AVG addition may be attributed to the lack of ethylene in the atmosphere of the vessels or the increase in free polyamines.