1. Shoot Regeneration in Élite Genotypes of Prunus avium L
Shoot Regeneration in Élite Genotypes of Prunus avium L. for Wood Production: Recent Developments at Bosques Naturales SA
Ricardo J. Licea Moreno1 and Luis Gómez2
1 Bosques Naturales S. A., Ave. de la Vega, 1, Madrid, Spain
2 Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo. Km 38 de la Autovía M-40, Pozuelo de Alarcón, Madrid, Spain
The protocol defined by Petri et al. (2008) for shoot regeneration of P. armeniaca L. was used as reference. The influence of several factors were assessed: genotype; explant source; callus induction media (CIM); micropropagation culture media (MCM); shoot pre-induction and hormone type and concentration. Leaves from 4 weeks-old micropropagated explants were used as described in Pérez-Tornero et al. (2000) . The protocol had 2 main steps: induction in the dark and expression under light conditions. Shoots were considered regenerated if they had, at least, two leaves raising from a stem.
Bosques Naturales SA is the leading company for noble wood production in Spain, mostly from P. avium and hybrid Juglans. Over cherry trees are currently planted for this purpose in two different locations, providing an excellent material to identify individuals excelling in desirable characteristics (plus trees). Some of the most promising genotypes are now being tested by us for mass propagation through in vitro culture, as part of the company’s biotechnological agenda to increase current yields and improve the overall quality of its material
Pre-Induction in the Dark
Expression Under Light Conditions
During ontogenesis embryogenic capacity of a cell is normally lost. To induce embryogenesis and/or organogenesis the explant source has to be, firstly, dedifferentiated to, then, facilitate the rediferentiation processes: an adequate explant, suitable environmental conditions and a stimulus are needed (Neuman, 2000). The main aim of the first experiment was to determine if the defined protocol for P. armeniaca L. (Petri, 2005) would work with some of our élite genotypes (K11, K12, K17, K21, K58 and KC). At the same time we tested the hypothesis that an auxin shock in liquid media would be enough to induce shoot regeneration. The results showed that there was high genetic influence, ranging the regeneration percentage between for K17 and 0 for K11, K21 and K58. K12 and KC also had the ability to regenerate (16.5 and 8.2 % respectively). Having demonstrated that induction in liquid medium was better than in gelled one - immersion during 3 h in a culture medium supplemented with TDZ, NAA and 2, 4 D was the best treatment for shoot regeneration (SR) – was key to this trial. Another important conclusion is that when 2,4 D alone was used the explants failed to regenerate as well as when it was removed from the induction medium (CIM)
LIQ.: Regeneration Pre-Induction in Liquid Medium During 3 hours
GEL.: Regeneration Pre-Induction in Gelled Medium During 4 days
Organogenic Cluster
Although internodal segments form calli, they are not suitable for regeneration. Leaves´ calli began to form on the border of the wounded explants, spreading later to the central veins and to the rest of the leaf. The first regenerants appeared after 2 weeks in the light, mainly on the veins. It is difficult to determine if the shoots are regenerated directly from the explant or if they have arisen from a callus. Normally, only one shoot is regenerated per explant. The shoots were detached from the explants weekly as recommended by Petri (2005). However, better results were obtained when an organogenic cluster and not a single shoot was selected. The multiplication culture medium (MCM) has an important role on shoot regeneration. Despite QL (1977) medium and Phloroglucinol being needed for shoot recovery they are not suitable for the regeneration when used in MCM.
Spermidine
Internodal segment
Shoot Regenerated
Central Vein
Spermidine (2mM) did not increase the regeneration of P. armeniaca L. However, it produced healthy and dark-green shoots (Petri, 2005). Similar results were obtained in our investigation with the addition of Spermidine to the expression culture medium (ECM). Shoot regeneration was improved in clones K12 and K17 for we continued using it in the experiments.
Regenerated shoots were able to multiply in a QL (1977) medium, supplemented with Phloroglucinol, Adenine, BAP and IBA. The high rates of multiplication the shoots showed was probably a consequence of the use of TDZ in the regeneration medium
Paromomycin
The use of aminoglucosides as markers for genetic transformation is frequent. Nevertheless, they have a high influence on morphogenic response and finding the right dose is needed. Petri (2005) established that low doses of Kanamicine stimulate shoot regeneration and relatively high concentrations (40 mM) to inhibit it were necessary. On the other hand, Paromomycin 20 mM completely inhibited the shoot regeneration. Based on these results we used Paromomycin at the recommended dose which affected calli formation and shoot regeneration in K12 and K17 genotypes.
Bibliography:
Feeney, M.; Bhagwat, B.; Mitchell, J. S. and Lane, W. D Shoot regeneration from organogenic callus of sweet cherry (Prunus avium L.). Plant Cell, Tissue and Organ Culture. 90:
Neuman, K. H Some Studies on Somatic Embryogenesis, a Tool in Plant Biotechnology. 87th Indian Science Congress.
Petri, C Transformación del albaricoquero (Prunus armeniaca L.), mediada por Agrobacterium, y regeneración de plantas transformadas. Tesis Doctoral. Dpto. de Biología Vegetal, Universidad de Murcia. pp 194.
Pérez-Tornero, O.; Egea, J.; Vanoostende, A.; and Burgos, L Assessment of Factors Affecting Adventitious Shoot Regeneration from In Vitro Cultured Leaves of Apricot. Plant Science. 158:
These results could be useful for clonal propagation of élite genotypes and open a way for the improvement of sweet cherry through genetic engineering.
Workshop “SUSTAINABLE FOREST MANAGEMENT: GENOMIC AND BIOTECHNOLOGICAL RESOURCES” 28th to 30th September 2009