Claudio Stasolla

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.

Purine and pyrimidine nucleotide metabolism in higher plants.

Purine and pyrimidine nucleotides participate in many biochemical processes in plants. They are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products, such as sucrose, polysaccharides, phospholipids, as well as secondary products. Therefore, biosynthesis and metabolism of nucleotides are of fundamental importance in the growth and development of plants. Nucleotides are synthesized both from amino acids and other small molecules via de novo pathways, and from preformed nucleobases and nucleosides by salvage pathways. In this article the biosynthesis, interconversion and degradation of purine and pyrimidine nucleotides in higher plants are reviewed. This description is followed by an examination of physiological aspects of nucleotide metabolism in various areas of growth and organized development in plants, including embryo maturation and germination, in vitro organogenesis, storage organ development and sprouting, leaf senescence, and cultured plant cells. The effects of environmental factors on nucleotide metabolism are also described. This review ends with a brief discussion of molecular studies on nucleotide synthesis and metabolism.

The Effects of Polyethylene Glycol on Gene Expression of Developing White Spruce Somatic Embryos

Somatic embryogenic cultures of white spruce (Picea glauca) represent a valuable system to study molecular mechanisms regulating embryo development because many embryos of defined developmental stages can be generated. The inclusion of polyethylene glycol (PEG) in the maturation medium can improve the number and quality of embryos produced. To learn more about the mechanism of action of PEG, we analyzed transcript profiles of stage-specific embryos matured without (control) or with (PEG treated) PEG. RNA extracted from maturing spruce embryos was analyzed on DNA microarrays containing 2,178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has been documented previously (L. van Zyl, S. von Arnold, P. Bozhkov, Y. Chen, U. Egertsdotter, J. MacKay, R. Sederoff, J. Shen, L. Zelena, D. Clapham [2002] Comp Funct Genomics 3: 306–318). Several pine genes, including the apparent homologs to the Arabidopsis genes ZWILLE, FIDDLEHEAD, FUSCA, and SCARECROW, increased in expression after PEG treatments. These genes are known to be involved in the formation of the embryo body plan and in the control of the shoot and root apical meristems. The increased transcript levels of these genes in immature PEG-treated embryos suggest that PEG may improve the quality of spruce somatic embryos by promoting normal differentiation of the embryonic shoot and root. Changes in the transcript levels of many genes involved in sucrose catabolism and nitrogen assimilation and utilization were also observed between control and PEG-treated embryos.

Maturation of somatic embryos in conifers: Morphogenesis, physiology, biochemistry, and molecular biology

SummaryIn the past 15 years tremendons progress has been made towards the development of systems for the induction and development of somatic embryos of coniferous species. Since the first report in 1985, several species have been induced to produce somatic embryos. This has been rendered possible by the development of rational media and improvement of culture conditions, which have resulted in increased embryo quality and higher conversion frequency. Understanding the physiological and biochemical events occurring during in vivo embryogenesis has been fundamental in the design of new protocols for improving the somatic embryogenic process. Specifically, the inclusions of abscisic acid (ABA) and osmotic agents, such as polyethylene glycol (PEG), have been shown to be necessary for the functional development of somatic embryos. In the past few years, physiological and biochemical investigations have been useful in increasing our knowledge on the mode of action of ABA and PEG during embryo development. In comparison with the flowering plants, our understanding on the molecular mechanisms regulating the embryogenic process in coniferous species is still very limited. The application of new molecular techniques is therefore fundamental towards this end. The emphasis of this review is on recent information dealing with the maturation of conifer somatic embryos.

The role of polyamines during in vivo and in vitro development

Polyamines are ubiquitous polycationic compounds that mediate fundamental aspects of cell growth, differentiation, and cell death in eukaryotic and prokaryotic organisms. In plants, polyamines are implicated in a variety of growth and developmental processes, in addition to abiotic and biotic stress responses. In the last decade, mutant studies conducted predominantly in Arabidopsis thaliana revealed an obligatory requirement for polyamines in zygotic and somatic embryogenesis. Moreover, our appreciation for the intricate spatial and temporal regulation of intracellular polyamine levels has advanced considerably. The exact molecular mechanism(s) through which polyamines exert their physiological response remains somewhat enigmatic and likely serves as a major area for future research efforts. In the following review, we discuss recent advances in the plant polyamine field, which range from metabolism and mutant characterization to molecular genetics and potential mode(s) of polyamine action during growth and development in vitro and in vivo. This review will also focus on the specific role of polyamines during embryogenesis and organogenesis.

Modulation of embryo-forming capacity in culture through the expression of Brassica genes involved in the regulation of the shoot apical meristem.

Somatic embryogenesis in Arabidopsis is achieved by culturing bending-cotyledon embryos on a 2,4-D-containing induction medium for 14 d followed by a transfer on to a hormone-free development medium. Several genes orthologous to Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL) were isolated from Brassica oleracea (Bo), B. rapa (Br), and B. napus (Bn), and ectopically expressed in Arabidopsis to assess their effects on somatic embryogenesis. Ectopic expression of BoSTM, BrSTM, and BnSTM increased the number of somatic embryos, whereas a different effect was observed in lines overexpressing BnCLV1 in which somatic embryo formation was severely repressed. The introduction of BnZLL did not have any effects on Arabidopsis somatic embryogenesis. The increased embryo-forming capacity observed in lines overexpressing Brassica STM was associated with a lower requirement for the inductive signal 2,4-D, and a higher expression of WUSCHEL (WUS) which demarcates the formation of embryogenic cells. This was in contrast to the 35S::BnCLV1 lines which showed the highest requirement for exogenous 2,4-D and a reduced WUS expression. Microarray studies were conducted to monitor global changes in transcript levels during Arabidopsis somatic embryogenesis between the wild-type (WT) line and a BoSTM-overexpressing line, which showed the most pronounced enhancement of somatic embryo yield. The introduction of BoSTM affected the expression of many genes involved in hormone perception and signalling, as well as genes encoding DNA methyltransferases and enzymes of glutathione metabolism. Pharmacological experiments performed to confirm some of the microarray results showed that Arabidopsis somatic embryogenesis is encouraged by a global hypomethylation of the DNA during the induction phase and by a switch of the glutathione pool towards an oxidized state during the subsequent development phase. Both events occurred in the 35S::BoSTM line, but not in the WT line. Altered expression of Brassica STM also had profound effects on B. napus microspore-derived embryogenesis. The yield of microspore-derived embryos increased in lines overexpressing BnSTM and significantly decreased in antisense lines down-regulating BnSTM.

Structure and function of homodomain-leucine zipper (HD-Zip) proteins

Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors unique to plants and are encoded by more than 25 genes in Arabidopsis thaliana. Based on sequence analyses these proteins have been classified into four distinct groups: HD-Zip Ι-ІV. HD-Zip proteins are characterized by the presence of two functional domains; a homeodomain (HD) responsible for DNA binding and a leucine zipper domain (Zip) located immediately C-terminal to the homeodomain and involved in protein-protein interaction. Despite sequence similarities HD-ZIP proteins participate in a variety of processes during plant growth and development. HD-Zip Ι proteins are generally involved in responses related to abiotic stress, abscisic acid (ABA), blue light, de-etiolation, and embryogenesis. HD-Zip ΙΙ proteins participate in light response, shade avoidance, and auxin signalling. Members of the third group (HD-ZipIII) control embryogenesis, leaf polarity, lateral organ initiation, and meristem function. HD-Zip ΙV proteins play significant roles during anthocyanin accumulation, differentiation of epidermal cells, trichome formation, and root development.

Molecular regulation of plant somatic embryogenesis

In vitro embryogenesis is an asexual reproduction process by which embryos are produced from either gametophytic (androecium/gynoecium) or sporophytic (somatic) tissues. Regardless of the type of explant used, the hallmark of this process is that the explant cells undergo dedifferentiation and acquire meristematic identity. The developmental program of such meristematic cells can then be redirected to form somatic embryos, depending on the imposed culture environment. Analysis of proteomes and transcriptomes has led to the molecular identification and functional characterization of many genes involved in the initiation and development of somatic embryos. These genes can be classified into three categories: embryonic induction, embryonic, and maturation. So far, few genes involved in early somatic embryogenesis have been characterized because isolation of early pure embryonic tissue is very difficult. This review focuses on genes regulating the induction process. Furthermore, we employed bioinformatic tools and pathway databases to identify genes that may play roles in regulating early somatic embryogenesis. A total of 51 proteins were identified that may function in early somatic embryogenesis. These proteins are predicted to be involved in hormone signal transduction, chromatin remodeling, cell cycle regulation, cellulose biosynthetic and metabolic activity, GTPase signal transduction, transcription regulation, meristem formation and maintenance, and/or apoptosis and microtubule organization. This review will help advance knowledge and promote research on molecular regulation of early somatic embryogenesis.

Transcriptional response of abscisic acid (ABA) metabolism and transport to cold and heat stress applied at the reproductive stage of development in Arabidopsis thaliana.

The phytohormone abscisic acid (ABA) plays an important role in developmental processes in addition to mediating plant adaptation to stress. In the current study, transcriptional response of 17 genes involved in ABA metabolism and transport has been examined in vegetative and reproductive organs exposed to cold and heat stress. Temperature stress activated numerous genes involved in ABA biosynthesis, catabolism and transport; however, several ABA biosynthesis genes (ABA1, ABA2, ABA4, AAO3, NCED3) were differentially expressed (up- or down-regulated) in an organ-specific manner. Key genes (CYP707As) involved in ABA catabolism responded differentially to temperature stress. Cold stress strongly activated ABA catabolism in all organs examined, whereas heat stress triggered more subtle activation and repression of select CYP707A genes. Genes involved in conjugation (UGT71B6), hydrolysis (AtBG1), and transport (ABCG25, ABCG40) of ABA or ABA glucose ester responded to temperature stress and displayed unique organ-specific expression patterns. Comparing the transcriptional response of vegetative and reproductive organs revealed ABA homeostasis is differentially regulated at the whole plant level. Taken together our findings indicate organs in close physical proximity undergo vastly different transcriptional programs in response to abiotic stress and developmental cues.

Ascorbic acid improves conversion of white spruce somatic embryos

SummaryThe effects of exogenous applications of ascorbic acid on white spruce somatic embryogenesis were examined. Increasing concentrations of ascorbate (1 µM to 100 µM) in the germination medium enhanced somatic embryo conversion in a linear fashion. At the optimal ascorbate level (100 µM) the number of embryos able to undergo normal conversion, i.e., emergence of both root and shoot, increased from 34% (control) to 58%. The effect of ascorbate had a more pronounced effect on shoot growth than on root emergence; and at 100 µM ascorbate, the percentage of embryos able to produce new leaf primordia increased from 47% (control) to 79%. Root emergence increased slightly from 64% in the control embryos to 74% in the presence of ascorbic acid. The ascorbate-treated embryos were characterized by an enlarged apical region, presumably due to a larger number of leaf primordia produced, and by dark green leaves. When allowed to grow further, these embryos were able to develop into normal plantlets.