Helena Lipavská

Somatic embryogenesis in conifers: The role of carbohydrate metabolism

SummarySomatic embryogenesis represents a promising tool for mass propagation of elite genotypes of conifers. The efficiency of the technique strongly depends on cultivation conditions, with the exogenous saccharide supply being one of the most important factors. Different types and concentrations of saccharides have been empirically evaluated with respect to production of acceptable numbers and quality of somatic embryos for particular conifer species. Only a few recently published papers have focused on deeper studies of carbohydrate metabolism, enabling insight into the physiological background of the crucial effects of carbohydrates. Generally, saccharides are known to serve as carbon and energy sources, osmotic agents, stress protectants, and signal molecules in plants. This review collects and critically discusses the experimental data on exogenous saccharide supplies, resulting endogenous levels, and key enzyme activities obtained from the most thoroughly described genus Picea. In conclusion, it stresses the necessily to broaden the studies and consider the unltiple roles of saccharides during conifer somatic embryogenesis.

Uptake of mannitol from the media by in vitro grown plants

Higher plants grown in vitro are very seldom fully autotrophic. Therefore, such cultures are usually supplied with exogenous sugars. However, at higher sugar concentration a decrease in dry matter accumulation is observed which can be explained by a decrease in osmotic potential of the medium.To test this explanation a series of experiments with mannitol, a sugar alcohol often used for simulation of osmotic stress, were performed with excised wheat embryos, rape seedlings and potato stem segments grown in vitro. As the presence of mannitol in the medium caused a significant decrease in dry matter accumulation, the content of mannitol in the shoot tissues was determined using HPLC analysis to estimate the uptake and transport of mannitol from roots to shoots. Mannitol contents up to 30% of dry weight in wheat and 20% in rape and potato shoots were found, indicating that mannitol is easily taken up by in vitro plants and transported to shoots. There were no large changes in the content of glucose, fructose and sucrose caused by the presence of mannitol in the tissues. These data show that mannitol can not be used as an inert osmoticum in in vitro studies.

Carbohydrate status during somatic embryo maturation in Norway spruce.

SummaryThe development of Norway spruce (Picea abies (L.) Karst.) somatic embryos on a maturation medium was accompanied by changes in nonstructural carbohydrate status. During embryo maturation, the content of total soluble sugars in the embryonal suspensor mass decreased and the partitioning between sucrose and hexoses changed considerably in favor of sucrose. Developing somatic embryos were mainly responsible for these changes. Osmotic stress caused by the presence of 3.75% polyethylene glycol (PEG) in the maturation medium (decrease in osmotic potential by 52.5 kPa) resulted in dramatic changes in the content of endogenous saccharides. There was a lower total carbohydrate content in the embryonal suspensor mass grown on the medium containing PEG in comparison with the untreated control. Isolated embryos from later stages of embryo development contained mainly sucrose with a small amount (20%) of fructose and nearly no glucose. A further increase in PEG concentration in the medium (7.5%; decrease in osmotic potential by 112.5 kPa compared to the maturation medium) led to a large increase in the total endogenous sugar content. This increase in sugars was a result of the enhanced content of sucrose, fructose, and glucose. The increased glucose content was in contrast to embryos grown on the medium with lower or no PEG content.

Annual dynamics of the content of non-structural saccharides in the context of structural development of vegetative buds of Norway spruce.

Summary Apical buds are very important organs as they determine further growth and development of tree species. Bud physiological state, including saccharide metabolism, determines their growth activity, and thus the development of the whole crown architecture. The present study focused on annual dynamics of the contents of non-structural saccharides (NSS) related to structural development in terminal vegetative buds of Norway spruce ( Picea abies L. Karst.) during 1995. Two types of material were analysed: 1) four-year-old nursery-planted trees; and, 2) adult individuals from two mountain sites in the Czech Republic. Sugar and starch contents were determined, and starch was localised histochemically. Generally, the dynamics of the NSS content reflects the major morphogenetic and developmental changes occurring during the annual cycle. The highest content of sugars corresponds with a bud dormant state. Bud break is accompanied by the lowering of sugar content, reflecting the transition of buds to metabolically active sinks. During bud cold hardening in autumn, a massive gradual sugar accumulation takes place. The most pronounced change during the annual cycle is found in the content of raffinose, with the highest values observed during autumn and winter. The possible role of raffinose in bud frost tolerance is discussed. The results obtained from the buds sampled at the mountain sites are both qualitatively and quantitatively similar to the more detailed NSS-dynamics of young trees, thus, giving the justification to generalisation of the above described pattern for vegetative buds of Norway spruce.

Sucrose metabolism during somatic and zygotic embryogeneses in Norway spruce: content of soluble saccharides and localisation of key enzyme activities

Changes in soluble carbohydrate compounds and their amounts were analysed during the somatic embryo maturation and seed development of Norway spruce (Picea abies (L.) Karst.). The data were correlated to the localisation of key enzymes of carbohydrate metabolism (invertase, EC 3.2.1.26; sucrose synthase, EC 2.4.1.13; phosphoglucomutase, EC 5.4.2.2). The decrease in total carbohydrate content as well as the accumulation of sucrose in later stages was a common feature in both systems. At the beginning of somatic embryo maturation the activity of sucrose synthase was low, and it gradually increased during development. At the final cotyledonary stage the SuSy localisation in somatic embryos resembled the pattern observed in cotyledonary stage zygotic embryos. Activity of invertase (pH 6.0) was detected in the early stages of somatic embryo development. Afterwards the activity decreased to almost undetectable levels in mature cotyledonary embryos. Very low invertase activity was detected during the period of seed development studied. The activity of phosphoglucomutase was high throughout the whole process of maturation in both zygotic and somatic embryos. The results are discussed in respect to the transition of developing embryos from metabolic to storage sink and the possible signalling potential of the changes.

Morphological and molecular characterization of a spontaneously tuberizing potato mutant: an insight into the regulatory mechanisms of tuber induction

BackgroundTuberization in potato (Solanum tuberosum L.) represents a morphogenetic transition of stolon growth to tuber formation, which is under complex environmental and endogenous regulation. In the present work, we studied the regulatory mechanisms and the role of different morphogenetic factors in a newly isolated potato mutant, which exhibited spontaneous tuberization (ST). The ST mutant was characterized in detail at morphological, physiological and biochemical levels.ResultsTuberization of the ST mutant grown in the soil was photoperiod-insensitive; predominantly sessile tubers formed directly from axillary buds even under continuous light. Single-node cuttings of the ST mutant cultured in vitro frequently formed tubers or basal tuber-like swellings instead of normal shoots under conditions routinely used for shoot propagation. The tuberization response of ST cuttings under light was dependent on sucrose, the concentration of which had to exceed certain threshold that inversely correlated with irradiance. Gibberellic acid prevented tuberization of ST cuttings, but failed to restore normal shoot phenotype and caused severe malformations. Carbohydrate analysis showed increased levels of both soluble sugars and starch in ST plants, with altered carbohydrate partitioning and metabolism. Comparative proteomic analysis revealed only a few differences between ST- and wild-type plants, primary amongst which seemed to be the absence of an isoform of manganese-stabilizing protein, a key subunit of photosystem II.ConclusionST mutant exhibits complex developmental and phenotypic modifications, with features that are typical for plants strongly induced to tuberize. These changes are likely to be related to altered regulation of photosynthesis and carbohydrate metabolism rather than impaired transduction of inhibitory gibberellin or photoperiod-based signals. The effect of gibberellins on tuberization of ST mutant suggests that gibberellins inhibit tuberization downstream of the inductive effects of sucrose and other positive factors.

Plant intelligence: Why, why not or where?

The concept of plant intelligence, as proposed by Anthony Trewavas, has raised considerable discussion. However, plant intelligence remains loosely defined; often it is either perceived as practically synonymous to Darwinian fitness, or reduced to a mere decorative metaphor. A more strict view can be taken, emphasizing necessary prerequisites such as memory and learning, which requires clarifying the definition of memory itself. To qualify as memories, traces of past events have to be not only stored, but also actively accessed. We propose a criterion for eliminating false candidates of possible plant intelligence phenomena in this stricter sense: an “intelligent” behavior must involve a component that can be approximated by a plausible algorithmic model involving recourse to stored information about past states of the individual or its environment. Re-evaluation of previously presented examples of plant intelligence shows that only some of them pass our test.

Comparing carbohydrate status during norway spruce seed development and somatic embryo formation

SummaryThe carbohydrate status of developing seeds of Picea abies was examined in order to provide a frame of reference for the evaluation of changes in carbohydrate content in maturing somatic embryos of the same species. Samples were taken at weekly intervals from 12 May 1998 (estimated time of pollination) until 20 October 1998. The total non-structural carbohydrate content was high (≈150–180 μg mg−1 dry weight) at the time of the first samples and the carbohydrate spectrum consisted of sucrose, glucose, fructose, and pinitol. A dramatic decrease in carbohydrate content took place from June 6 onwards, that was accompanied by changes in carbohydrate partitioning to favor sucrose over hexoses and the disappearance of pinitol. Raffinose and stachyose were first detected on July 28, and their content gradually increased thereafter. Isolated embryos and remaining megagametophytes were analyzed starting with September 1. Carbohydrate content was higher in isolated zygotic embryo than in the rest of the seed, with a slowly increasing fraction of raffinose and stachyose. Comparisons of presented data with the results of our previous study of somatic embryo carbohydrate status (Lipavská et al., 2000) revealed the following common features: (1) a decrease in total carbohydrate content and (2) an increase in sucrose:hexose ratios in developing seeds and embryonal suspensor mass. Marked differences were observed in carbohydrate spectra: (1) somatic embryo development was not accompanied by pinitol accumulation in any phase; (2) mature zygotic embryos, in contrast to mature somatic embryos, contained raffinose and stachyose. These observations will provide a solid basis for improvement of protocols for somatic embryogenesis in Picea.

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 μmol m−2 s−1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 μmol m−2 s−1 during further ex vitro growth (in the period of 7th – 17th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 μmol m−2 s−1 (3 % LL) or without sucrose either under PFD of 50 μmol m−2 s−1 or 200 μmol m−2 s−1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.

Mannitol utilisation by celery (Apium graveolens) plants grown under different conditions in vitro

Abstract Celery plants produce sucrose and mannitol as primary photosynthetic products and translocate both carbohydrates in the phloem and metabolise them in sinks. In vitro cultivation allows the supply of plants with different exogenous carbon and energy sources, thus influencing the endogenous equilibrium of carbohydrates and allowing study of resulting changes on growth and developmental processes. We investigated the growth and metabolic responses of celery ( Apium graveolens , var. dulce , cv. Afina) to the supply of carbohydrates under in vitro conditions. No differences in biomass accumulation were observed in sucrose or mannitol supported plants at lower carbohydrate concentrations in the media (1 and 3%). Higher concentrations of mannitol, however, did not support the growth as well as media supplemented with equivalent sucrose concentrations . Differences in the degree of responses of particular organs to changes in type and concentration of media carbohydrates occurred. HPLC analysis of the soluble carbohydrate contents revealed that celery grown on mannitol accumulated very high concentrations of mannitol (65–91% of total soluble carbohydrate content). The accumulated mannitol was utilised when exogenous carbohydrates were withdrawn by transferring the plants to media without carbohydrates and the osmotic potential of the media increased.