ABSTRACT
BACKGROUND: Pinus radiata is an economically important conifer, and somatic embryogenesis is being currently used for its propagation. But the embryogenic competence of cultures decreases with culture age. To cope with this, cryopreservation protocols have been developed lately for different Pinus species. Although cryopreservation reduces the costs associated with embryogenic cultures maintenance, the initial investment and the maintenance of cryotanks are expensive when dealing with somatic embryogenesis basic research issues. OBJECTIVE: To study the feasibility of storing embryogenic cell lines at -80 degree C for over a year. MATERIALS AND METHODS: The feasibility of the conservation method was assessed in terms of recovery, maturation and germination rates. RESULTS: The recovery rates were up to 77 percnt;, and maturation and germination rates were 86 percnt; and 83 percnt;, respectively. CONCLUSION: The work described here is a simple and low-cost protocol that enables successful conservation of embryogenic cell lines for over a year.
Subject(s)
Cryopreservation/methods , Pinus/embryology , Seeds , Cell Line , Germination/physiology , Seeds/cytology , Seeds/embryology , Time FactorsABSTRACT
BACKGROUND: Although somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation. FINDINGS: Shoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from in vitro-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant's origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1). CONCLUSIONS: While limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess some level of embryogenic character. Additionally, expression of vegetative meristem markers is consistent with their vegetative origin, as are differences in expression patterns as compared with EM.
Subject(s)
Gene Expression Regulation, Plant , Pinus/genetics , Plant Proteins/genetics , Seeds/genetics , Transcription Factors/genetics , Culture Media/chemistry , Culture Media/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genetic Markers , Hydroponics , Microarray Analysis , Pinus/growth & development , Pinus/metabolism , Plant Growth Regulators/pharmacology , Plant Proteins/metabolism , Plant Shoots/genetics , Plant Shoots/growth & development , Plant Shoots/metabolism , Plant Somatic Embryogenesis Techniques , Plant Stems/genetics , Plant Stems/growth & development , Plant Stems/metabolism , Seeds/drug effects , Seeds/growth & development , Seeds/metabolism , Transcription Factors/metabolism , Zygote/growth & development , Zygote/metabolismABSTRACT
The recently cloned rice transglutaminase gene (tgo) is the second plant transglutaminase identified to date (Campos et al. Plant Sci. 205-206 (2013) 97-110). Similarly to its counterpart in maize (tgz), this rice TGase was localized in the chloroplast, although in this case not exclusively. To further characterise plastidial tgo functionality, proteomic and transcriptomic studies were carried out to identify possible TGO-related proteins. Some LHCII antenna proteins were identified as TGO related using an in vitro proteomic approach, as well as ATPase and some PSII core proteins by mass spectrometry. To study the relationship between TGO and other plastidial proteins, a transcriptomic in vivo Dynamic Array (Fluidigm™) was used to analyse the mRNA expression of 30 plastidial genes with respect to that of tgo, in rice plants subjected to different periods of continuous illumination. The results indicated a gene-dependent tendency in the expression pattern that was related to tgo expression and to the illumination cycle. For certain genes, including tgo, significant differences between treatments, principally at the initiation and/or at the end of the illumination period, connected with the day/night cycling of gene expression, were observed. The tgo expression was especially related to plastidial proteins involved in photoprotection and the thylakoid electrochemical gradient.
