Millán Cortizo

Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro

Germination negatively affects adventitious shoot formation induced by cytokinins in pine cotyledons. To investigate the causes of this decrease in the organogenic response, uptake and metabolism of benzyladenine (BA) were studied in stone pine cotyledons (Pinus pinea) isolated from in vitro germinating embryos and cultured in bud induction medium. As embryos grew, cotyledons showed a progressive decrease in the amount of BA taken up from the medium. BA was barely metabolized; however, a BA metabolite previously undescribed in conifers was found. It was identified as a glucoside of the BA riboside, a type of metabolite recently described in other gymnosperms. Data revealed that differences in the organogenic capacity of P. pinea cotyledons associated with embryo germination are related primarily to their ability to absorb BA from the bud induction medium.

Improving plantlet yield in Pinus pinaster somatic embryogenesis

Abstract Somatic embryogenesis is expected to play a significant role in the future forest tree improvement programmes. The main bottleneck of this technique is still the progression from immature embryogenic cultures to mature cotyledonary embryos able to develop properly into well-growing plants. In this work, we present an improved protocol focused on increasing the maturation and conversion rate of Pinus pinaster Ait. embryogenic cultures. Results showed that the optimisation of the nutrient composition in the maturation medium increased the number of mature embryos by 25% (187.8 embryos per gram of fresh mass in average compared to 144.5 embryos in regular medium). It was also shown that 12-month cryostorage did not reduce viability or embryogenic ability of maritime pine cultures. A further increase in the yield of the protocol could be obtained by using benzyladenine in the conversion medium, promoting the bud-break of axillary buds that yielded 5.7 shoots in average per somatic embryo. Rooting of axillary shoots reached 84.3%. This methodology offers an alternative to overcome some problems associated with low somatic embryo production since the plantlet yield could be increased fivefolds.

CLAVATA1-LIKE, a leucine-rich-repeat protein receptor kinase gene differentially expressed during adventitious caulogenesis in Pinus pinaster and Pinus pinea

Molecular cloning and characterization of a CLAVATA1-LIKE gene in Pinus pinaster and Pinus pinea is reported. CLAVATA1 is a well-characterized gene in Arabidopsis integral to the balance between primordial differentiation and meristem proliferation. Currently, it is not known if the Arabidopsis model of in vitro caulogenesis is applicable to conifers. In this work, we study the putative role of the CLAVATA1-LIKE gene during caulogenic induction in cotyledons of P. pinea and P. pinaster after exposure to benzyladenine. Expression analysis showed that the gene was differentially expressed during the first phases of adventitious caulogenesis in cotyledons from both species, suggesting that CLAVATA1-LIKE may play a role in caulogenesis in conifers. A binary vector carrying CLAVATA1-LIKE promoter driven GFP:GUS expression was constructed. Results of genetic transformation showed GUS activity during somatic embryogenic mass proliferation and embryo maturation.

Characterization of a type-A response regulator differentially expressed during adventitious caulogenesis in Pinus pinaster

The molecular cloning and characterization of PipsRR1, a type-A response regulator in Pinus pinaster, is reported here. Type-A response regulators mediate downstream responses to cytokinin and act as negative feedback regulators of the signal transduction pathway. Some type-A response regulators in Arabidopsis have been related to de novo meristem formation. However, little information exists in Pinus spp. The PipsRR1 gene contains 5 exons, as do all type-A response regulators in Arabidopsis, and the deduced protein contains a receiver domain with the conserved DDK residues and a short C terminal extension. Expression analysis showed that the PipsRR1 gene is differentially expressed during the first phases of adventitious caulogenesis induced by benzyladenine in P. pinaster cotyledons, suggesting that PipsRR1 plays a role in caulogenesis in conifers. Additionally, a binary vector carrying the PipsRR1 promoter driving GFP:GUS expression was constructed to analyze the promoter activity in P. pinaster somatic embryos. The results of genetic transformation showed GUS activity during somatic embryo mass proliferation and embryo maturation.

Cloning and characterization of a type-A response regulator differentially expressed during adventitious shoot formation in Pinus pinea L.

Type-A response regulators play an important role in cytokinin-induced adventitious shoot formation, acting as negative regulators of cytokinin signal transduction. In this work, we obtained the full-length cDNA clone of a type-A response regulator from the conifer Pinus pinea, designated PipiRR1. The derived peptide sequence showed all the characteristic motifs found in angiosperms. Gene expression analysis showed that the gene was differentially expressed during adventitious shoot formation in P. pinea cotyledons, suggesting that PipiRR1 may play a role in caulogenesis in conifers. This is the first type-A response regulator identified in gymnosperms.

Cork Oak Trees (Quercus suber L.).

A transformation system for selected mature Quercus suber L. trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses are inoculated with AGL1 strain harbouring the plasmid pBINUbiGUSint, which carries the nptII and uidA genes. Evidence of stable transgene integration is obtained by polymerase chain reaction for nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos are germinated and successfully transferred to soil.