Elena Carneros

The GRAS gene family in pine: transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots

BackgroundAdventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown.ResultsHere, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata’s SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or non-existent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation.ConclusionsThe GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots.

Effect of different cryoprotectant procedures on the recovery and maturation ability of cryopreserved Pinus pinea embryogenic lines of different ages

Strategies for genetic improvement programs of Pinus pinea L, an important tree species of the Mediterranean ecosystem, are focused on increasing pine nut yield. Somatic embryogenesis and cryopreservation of elite genotypes are emerging as key components of advanced forest breeding programs. This study was carried out with embryogenic lines of different ages obtained from selected half-sib families of the species. The effect of three cryoprotectant procedures on the recovery and maturation ability was tested in embryogenic lines that showed different growth rate, two of them at different ages. In general, cryopreservation drastically reduced growth rates of frozen and rewarmed tissues; however, the use of 5% PEG–sucrose–DMSO dramatically increased growth rates of rewarmed embryogenic cultures. Overall, embryogenic lines of stone pine were suitable for cryopreservation. Seven out of eight lines were recovered, although the initial growth rates were variable. Five of six lines including the three oldest ones were recovered using 5% PEG–sucrose–DMSO. No relation was observed between age and growth rate of embryogenic lines and their response to cryopreservation. The line 2F47 showed the most stable response after long-term subculture and recovery after cryopreservation, at different ages. On the contrary, younger embryogenic lines either recovered after cryopreservation or did not, depending on the applied procedure. Maturation of some of the older lines was restored or enhanced after cryopreservation. Somatic embryos were obtained in three out of five tested embryogenic lines recovered from cryopreservation. However, only a few plantlets from cryopreserved lines were regenerated indicating the process must be optimized further before it is a practical adjunct to breeding.

Expression pattern of the GRAS gene family during somatic embryogenesis in pine

The GRAS protein family of putative transcription factors, which includes SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE (SCL) proteins, is involved in root development in Arabidopsisthaliana and other plant species [1]. In forest species, genes with homology to the A. thalianaSCR gene have been involved in the induction of somatic embryogenesis in Picea glauca (Moench) Voss [2] and Pinus taeda L. [3] as well as in the development of radial patterning of roots in Pinus sylvestris L. [4]. Schrader et al[5] also reported the expression of genes with homology to the A. thalianaSHR gene in cambial region of Populus tremula x tremuloides. Increased levels of mRNA of Pinus radiata SHR (PrSHR), Pinus radiata SCARECROW-LIKE1 (PrSCL1) and Castanea sativaSCARECROW-LIKE1 (CsSCL1) have been associated with the early stages of adventitious root induction in Pinus radiata D. Don and Castanea sativa Mill., respectively [6-9]. In addition to PrSHR and PrSCL1, we have identified 13 new GRAS genes belonging to the different GRAS clades in the pine genome. The objective of this work is the analysis of the spatiotemporal expression patterns of the pine GRAS gene family during somatic embryogenesis in Pinus radiata D. Don. Somatic embryogenesis has become the first biotechnology showing great potential for mass propagation of conifers for application in forestry, allowing the implementation of multivarietal forestry (MVF) [10,11]. Despite major advances in clonal regeneration by somatic embryogenesis or organogenesis, many forestry species are recalcitrant [12]. More knowledge of the regeneration process regulation is necessary to improve the capacity of vegetative regeneration. The expression pattern of the genes was analyzed by qRT-PCR following the methodology described by Sanchez et al[6] and Sole et al[7]. For expression analysis, total RNA was extracted from four stages of the somatic embryogenic process: proliferative tissue after 7 and 14 days from the last transference to proliferation medium, somatic embryos at the beginning of differentiation and cotyledonary somatic embryos. In general, the transcripts of the pine GRAS genes accumulated at the highest levels in cotyledonary somatic embryos. In addition, the transcript levels of PrSCR, PrSHR, PrSCL1, PrSCL6, PrSCL8, PrSCL11 andPrSCL12 showed an increase in somatic embryos at the beginning of differentiation. No differences in PrSCL10 transcript levels were found between the four stages analyzed. Transcript levels of PrSCL16 were undetectable at all stages. In situ hybridization for spatial expression analysis will confirm differential expression domains. This work has been funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR). Embryogenic lines were provided by C. Walter (Scion).

Stone Pine Pinus Pinea L.

Stone pine (Pinus pinea L.) is a Mediterranean native conifer species. It is used for protection against soil erosion, ecosystem restoration and farmland afforestation. The most important commercial application is the production of timber and pine nuts. The stone pine kernels are highly appreciated as part of the Mediterranean diet because of their high nutritional value. They are currently one of the most expensive dried fruits. Breeding programs of stone pine mainly focus on the improvement of cone production by planting selected genotypes in grafted orchards. Therefore the production of clonal rootstocks is desirable. This chapter describes protocols to regenerate stone pine plants by somatic embryogenesis from immature zygotic embryos, and for the cryopreservation of embryogenic lines. Protocols used in the attemps to induce somatic embryogenesis in tissues from adult trees are also described.

Gene expression patterns associated with developmental transitions during somatic embryogenesis in pine

The low regeneration capacity of forest species is one of the major limitations for vegetative propagation [1]. The molecular mechanisms that determine the efficiency of clonal propagation programs via either adventitious organogenesis or somatic embryogenesis have not been established. For clonal propagation via somatic embryogenesis, the success of the process depends on an initial reprogramming step and on further developmental transitions involved in the maturation of somatic embryos [2] . The identification of candidate genes involved in the regulation of key steps of the regeneration processes is essential to generate tools and strategies to improve the success of clonal propagation programs in forest species. The aim of this work is to identify new candidate genes potentially involved in the regulation of developmental transitions in somatic embryogenesis in pine. For that purpose, samples of embryogenic tissue from Pinus radiata D. Don at different stages of development were used: proliferative tissue (after 7 and 14 days from the last transference to fresh proliferation medium), somatic embryos at the beginning of differentiation and somatic embryos at cotyledonary stage [3,4]. Large-scale expression analysis using a microarray containing an EST collection enriched in auxin-induced genes, and several tissue-specific cDNA libraries from meristematic and embryonic tissues, were used for the identification of phase-specific candidate genes. Genes related to auxin signaling, regulation of gene expression, signal transduction, proliferation and embryo development were selected for further analysis. The expression of these candidate genes was confirmed by QRT-PCR. The information obtained from this work will open new ways of research on molecular mechanisms involved in developmental processes in conifers. This work was funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR). Embryogenic lines were provided by C. Walter (Scion).

Improvement of Eucalyptus sp for biomass and bioenergy production in the north of Spain

Eucalyptus globulus Labill. has been used in Spain for decades as a cellulose source for the paper and textile industry. Since 1997, Sniace group has tested new provenances and families from 29 different Eucalyptus species to explore their capacity for biomass and bioenergy production. Plus trees for growth, wood quality, rooting capacity and tolerance to Mycosphaerella sp have been identified by mass selection, and more than 300 controlled crosses among those trees have been carried out. However, the restrictions caused by the high susceptibility to Mycosphaerella leaf disease and the low rooting capacity of the species Eucalyptus globulus Labill. delay the application of the gains to a commercial scale. The objective of this project is to improve the production of Eucalyptus in the North of Spain focused on the improvement of two traits of economic importance: clonal/rooting capacity and tolerance to Mycosphaerella sp. To achieve this goal three partial objectives have been approached: