Cold stress on Araucaria angustifolia embryogenic cells results in oxidative stress and induces adaptation: implications for conservation and propagation.

Araucaria angustifolia (Bert.) O. Kuntze is a species critically endangered of extinction and its development and propagation is strongly affected by abiotic stress. We have previously shown the activation of uncoupling protein in A. angustifolia embryogenic stem cells subjected to cold stress. Now, we have furthered those studies by exposing these cells to cold stress (4 ± 1 °C for either 24 or 48 h) and evaluating parameters associated with oxidative stress and alterations in the cellular and mitochondrial responses. Cold stress affect the H2O2 levels and lipid peroxidation increased after both stress condition, an effect associated with the decrease in the activities of peroxidases, catalase and ascorbate/dehydroascorbate ratio. On the other hand, the activities of ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases increased as an indication of adaptation. Another important impact of cold stress conditions was the decrease of external alternative NAD(P)H dehydrogenases activity and the increase of mitochondrial mass. These results show that cold stress induces oxidative stress in A. angustifolia embryogenic cells, which results in activation of the glutathione-ascorbate cycle as a compensation for the decrease in the activities of catalase, peroxidases, and external NAD(P)H dehydrogenases. Our results contribute to the understanding of the pathways that gymnosperms employ to overcome oxidative stress, which must be explored in order to improve the methods of conservation and propagation of A. angustifolia.

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized.

Early harvest increases post-harvest physiological quality of Araucaria angustifolia (Araucariaceae) seeds

ResumenAraucaria angustifolia es una conífera nativa de Brasil y una especie en peligro de extinción. Sus semillas tienen un corto período de viabilidad, factor que contribuye a su vulnerabilidad. Este estudio tuvo como objetivo evaluar la calidad fisiológica durante el período de desarrollo y post-almacenamiento de semillas de A. angustifolia. Conos de A. angustifolia fueron recolectados en poblaciones naturales en Curitibanos, Santa Catarina, Brasil, en marzo, abril, mayo y junio y clasificados en los estadios de desarrollo cotiledonar i, ii y iii de acuerdo con el mes de recolecta. Un total de 10 conos fueron recolectados para cada estadio. Las semillas fueron almacenadas en refrigerador durante 60 y 120 días y posteriormente sometidas a pruebas de germinación (25 °C - fotoperiodo de 12 h) siendo evaluados el contenido de humedad, tetrazolio y el vigor (conductividad eléctrica [75 mL de agua destilada a 25 °C], índice de velocidad de germinación, y la longitud de la parte aérea y de la raíz). Durante el desarrollo de las semillas, el contenido de humedad se redujo desde el estadio cotiledonar (66.54 %) al estadio iii (47.44%), y el vigor aumentaron en el último estadio. Durante el almacenamiento, el contenido de humedad en el estadio cotiledonar y estadio i fue estable. Entretanto, las semillas almacenadas mostraron una reducción en el contenido de humedad después de 120 días en los estadios ii y iii. La calidad fisiológica en el estadio cotiledonar mostró un aumento de 86 % y 93 % de germinación después de 60 y 120 días de almacenamiento, respectivamente, a diferencia de los estadios ii y iii, los cuales mostraron una disminución en la viabilidad de las semillas y en el vigor después del almacenamiento. La conductividad eléctrica fue mayor para las semillas en estadio cotiledonar recién recolectadas que para aquellas almacenadas durante 60 y 120 días. Sin embargo, en otras estadios, el contenido de lixiviados después de 120 días de almacenamiento aumentó con el avance del período de recolecta. El índice de velocidad de germinación y la longitud de la parte aérea y raíz después del almacenamiento eran más altos para las semillas en el estadio cotiledonar y el estadio i, a diferencia del estadio ii y iii, los cuales tenían raíz y parte aérea de menor longitud durante el almacenamiento. Por lo tanto, el mantenimiento del contenido de humedad de la semilla durante el almacenamiento fue variable y depende del período de recolecta. Además, la calidad fisiológica de las semillas difiere entre los estadios más tempranos o tardíos. Una recolección precoz favoreció la calidad fisiológica de las semillas, y puede ser una estrategia para aumentar la conservación de semillas de A. angustifolia.

AbstractAraucaria angustifolia is a conifer native to Brazil and is an endangered species. Since this species seeds have a short period of viability, its vulnerability is higher. Thus the aim of this study was to evaluate the physiological quality of A. angustifolia seeds during the development and post-storage periods. For this, cones of A. angustifolia were collected from a natural population in Curitibanos, Santa Catarina, Brazil, in March, April, May and June 2012. The collected seeds were classified into developmental stages of cotyledonary, i, ii and iii according to the month of collection; a total of 10 cones were collected for each stage. Seeds were stored in a refrigerator for 60 and 120 days, and were submitted to a chamber germination test (25 °C-photoperiod 12 h). Additionally, seeds were tested for moisture content (105 °C for 24 hours), tetrazolium (0.1 % for 1 hour) and vigor (electric conductivity [75 mL distilled water at 25 °C], germination speed index, and shoot and root length). Our results showed that during seed development, moisture content decreased from the cotyledonary stage (66.54 %) to stage iii (49.69 %), and vigor increased in the last stage. During storage, moisture content at cotyledonary stage and stage i was stable. On the other hand, stored seeds exhibited a decrease in moisture content after 120 days at stages ii and iii. Physiological quality at the cotyledonary stage resulted in an increased germination rate of 86 % and 93 % after 60 and 120 days of storage, respectively; unlike stages ii and iii exhibited a decrease in seed viability and vigor after storage. Electrical conductivity was higher for fresh seeds at the cotyledonary stage, than for those stored for 60 and 120 days. However, in other stages, released leachate content after 120 days of storage, increased with the advance of the collection period. Germination speed index and shoot and root lengths after storage were highest for seeds at the cotyledonary stage and stage i; unlike stages ii and iii which had short root and shoot lengths during storage. Thus, the maintenance of seed moisture content during storage was variable and dependent on the period of collection. Furthermore, the physiological quality differed among earlier and later stages. Early collection favored seed physiological quality, and may be a strategy for better conservation of A. angustifolia seeds. Rev. Biol. Trop. 64 (2): 885-896. Epub 2016 June 01.

Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae).

Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 µM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants.

Early harvest increases post-harvest physiological quality of Araucaria angustifolia (Araucariaceae) seeds.

Araucaria angustifolia is a conifer native to Brazil and is an endangered species. Since this species seeds have a short period of viability, its vulnerability is higher. Thus the aim of this study was to evaluate the physiological quality of A. angustifolia seeds during the development and post-storage periods. For this, cones of A. angustifolia were collected from a natural population in Curitibanos, Santa Catarina, Brazil, in March, April, May and June 2012. The collected seeds were classified into developmental stages of cotyledonary, I, II and III according to the month of collection; a total of 10 cones were collected for each stage. Seeds were stored in a refrigerator for 60 and 120 days, and were submitted to a chamber germination test (25 °C-photoperiod 12 h). Additionally, seeds were tested for moisture content (105 °C for 24 hours), tetrazolium (0.1 % for 1 hour) and vigor (electric conductivity [75 mL distilled water at 25 °C], germination speed index, and shoot and root length). Our results showed that during seed development, moisture content decreased from the cotyledonary stage (66.54 %) to stage III (49.69 %), and vigor increased in the last stage. During storage, moisture content at cotyledonary stage and stage I was stable. On the other hand, stored seeds exhibited a decrease in moisture content after 120 days at stages II and III. Physiological quality at the cotyledonary stage resulted in an increased germination rate of 86 % and 93 % after 60 and 120 days of storage, respectively; unlike stages II and III exhibited a decrease in seed viability and vigor after storage. Electrical conductivity was higher for fresh seeds at the cotyledonary stage, than for those stored for 60 and 120 days. However, in other stages, released leachate content after 120 days of storage, increased with the advance of the collection period. Germination speed index and shoot and root lengths after storage were highest for seeds at the cotyledonary stage and stage I; unlike stages II and III which had short root and shoot lengths during storage. Thus, the maintenance of seed moisture content during storage was variable and dependent on the period of collection. Furthermore, the physiological quality differed among earlier and later stages. Early collection favored seed physiological quality, and may be a strategy for better conservation of A. angustifolia seeds.

Ultra-low temperature conservation of Brazilian pine embryogenic cultures.

This study aimed to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, enabling the ex situ conservation of the species. Embryogenic cultures were established from immature seeds and treated with variations of the cryoprotectant solutions SuDG, SoD and PVS2 prior to immersion in liquid nitrogen. Cell viability was evaluated after 30, 60 and 90 days of re-growth. The highest re-growth without morphological alterations and with normal biochemical composition was obtained with the PVS2 solution with 40 min immersion in ethanol (-20 °C). This procedure opens new horizons for the ex situ conservation of the species genetic.

Polyamines affect the cellular growth and structure of pro-embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines.

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro-embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal-bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H(+) -ATPase P- and V-types and H(+) -PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.

The involvement of PUMP from mitochondria of Araucaria angustifolia embryogenic cells in response to cold stress.

In this study, the responses of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX) in mitochondria from embryogenic cells of A. angustifolia subjected to cold stress (4°C for 24 h or 48 h) is reported. In the mitochondria of stressed cells, PUMP activity increased by approximately 45% (at 24h and 48 h), which was determined by measuring the oxygen consumption after the addition of linoleic acid and the inhibition by BSA and ATP. PUMP activation was confirmed using transmembrane electrical potential (Δψ) assays. Immunoblot assays showed an increase of PUMP expression by 40% and 150% after 24h and 48 h of cold stress, respectively. AOX activity, measured under conditions similar to those of the PUMP assays, was only slightly increased in the mitochondria from stressed cells (at 24h and 48 h), as demonstrated by oxygen consumption experiments. Cell viability was unaffected by cold stress, indicating that the effects on PUMP and AOX were not caused by cell death. These results show that the main response of this gymnosperm to cold stress is the activation of PUMP, which suggests that this protein may be involved in the control of reactive oxygen species generation, which has been previously associated with this condition.

Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission.

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia.

Female parthenogenetic apomixis and androsporogenetic parthenogenesis in embryonal cells of Araucaria angustifolia: interpolation of progenesis and asexual heterospory in an artificial sporangium.

Cell fate, development timing and occurrence of reproductive versus apomictic development in gymnosperms are shown to be influenced by culture conditions in vitro. In this study, female parthenogenetic apomixis (fPA), androsporogenetic parthenogenesis (mAP) and progenesis were demonstrated using embryonal initials of Araucaria angustifolia in scaled-up cell suspensions passing through a single-cell bottleneck in darkness and in an artificial sporangium (AS). Expression was based on defined nutrition, hormones and feedforward-adaptive feedback process controls at 23-25 °C and in darkness. In fPA, the nucleus of an embryonal initial undergoes endomitosis and amitosis, forming a diploid egg-equivalent and an apoptotic ventral canal nucleus in a transdifferentiated archegonial tube. Discharge of egg-equivalent cells as parthenospores and their dispersal into the aqueous culture medium were followed by free-nuclear conifer-type proembryogenesis. This replaced the plesiomorphic and central features of proembryogenesis in Araucariaceae. Protoplasmic fusions of embryonal initials were used to reconstruct heterokaryotic expressions of fPA in multiwell plates. In mAP, restitutional meiosis (automixis) was responsible for androsporogenesis and the discharge of monads, dyads, tetrads and polyads. In a display of progenesis, reproductive development was brought to an earlier ontogenetic stage and expressed by embryonal initials. Colchicine increased polyploidy, but androspore formation became aberrant and fragmented. Aberrant automixis led to the formation of chromosomal bouquets, which contributed to genomic silencing in embryonal initials, cytomixis and the formation of pycnotic micronucleated cells. Dispersal of female and male parthenospores displayed heteromorphic asexual heterospory in an aqueous environment.

Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine)

Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz.,ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semiquantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia.

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes, oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed.

Monozygotic cleavage polyembryogenesis and conifer tree improvement.

The mass cloning of elite genotypes of commercially important conifers has led to the establishment of an industrial forest of two of the most important softwood species in the USA. Embryonal-suspensor masses, produced by monozygotic cleavage polyembryony (MCP), are rescued from controlled-pollinated seeds in tree breeding orchards. MCP is scaled up as cell suspensions and grown into mature somatic embryos. The embryos serve as a source for the production of various artificial and manufactured seeds used in replicated field trials to test genotype x environmental interactions. For the capture of genetic gains, early selections are based on correlations with known traits. This reduces the costs of years of tree improvement. Mass cloning and genotype cryopreservation enables field testing under a wider range of sites. Process-controlled bioreactors are proposed as artificial ovules to impose nutritional variables from the mother tree, and to simulate environmental factors that are known to affect the performance of the new generation. Comparisons among extant and modern conifer genotypes would provide new insights regarding their latent potentials for apomixis, the alternation of generations, and adaptive plasticity.

Morphology and structure of the pollen cone and pollen grain of the Araucaria species from Argentina.

The pollen cone and the pollen grain of the two Argentinean species of Araucaria are described with LM, SEM and TEM. Primordia of pollen cones are formed in April and May and reach maturity by mid-October in A. angustifolia (Bert.) O. Kuntze and by mid-November in A. araucana. (Mol.) K. Koch. Characters of the mature pollen cones and microsporophylls between both taxa are clearly differentiated. Pollen grains are spheroidal-subspheroidal, inaperturate, and asaccate with granulate exine and a subequatorial annular area that corresponds to the sexine thickness. Sculpturing consists of irregularly dispersed granules that are sometimes fused to each other (A. angustifolia) or forming microrugulae (A. araucana). Microgranules and microspinules are also present. The pollen wall ultrastructure is formed by a granular ectexine and lamellated endexine. Granular elements in A. angustifolia are more loosely disposed, form more interstices, and are gradually smaller towards the endexine than in A. araucana. To asses the probable relationships within the family, we compared the pollen grains of the two Araucaria species with those of other extant genera (Agathis, Wollemia) and also with fossil pollen (Araucariacites, Balmeiopsis, Cyclusphaera, Dilwynites) attributed to Araucariaceae.

Morphology and structure of the pollen cone and pollen grain of the Araucaria species from Argentina

The pollen cone and the pollen grain of the two Argentinean species of Araucaria are described with LM, SEM and TEM. Primordia of pollen cones are formed in April and May and reach maturity by mid-October in A. angustifolia (Bert.) O. Kuntze and by mid-November in A. araucana. (Mol.) K. Koch. Characters of the mature pollen cones and microsporophylls between both taxa are clearly differentiated.Pollen grains are spheroidal-subspheroidal, inaperturate, and asaccate with granulate exine and a subequatorial annular area that corresponds to the sexine thickness. Sculpturing consists of irregularly dispersed granules that are sometimes fused to each other (A. angustifolia) or forming microrugulae (A. araucana). Microgranules and microspinules are also present. The pollen wall ultrastructure is formed by a granular ect exine and lamellated endexine. Granular elements in A. angustifolia are more loosely disposed, form more interstices, and are gradually smaller towards the endexine than in A. araucana. To asses the probable relationships within the family, we compared the pollen grains of the two Araucaria species with those of other extant genera (Agathis, Wollemia) and also with fossil pollen (Araucariacites, Balmeiopsis, Cyclusphaera, Dilwynites) attributed to Araucariaceae.

Morphology and structure of the pollen cone and pollen grain of the Araucaria species from Argentina

The pollen cone and the pollen grain of the two Argentinean species of Araucaria are described with LM, SEM and TEM. Primordia of pollen cones are formed in April and May and reach maturity by mid-October in A. angustifolia (Bert.) O. Kuntze and by mid-November in A. araucana. (Mol.) K. Koch. Characters of the mature pollen cones and microsporophylls between both taxa are clearly differentiated.Pollen grains are spheroidal-subspheroidal, inaperturate, and asaccate with granulate exine and a subequatorial annular area that corresponds to the sexine thickness. Sculpturing consists of irregularly dispersed granules that are sometimes fused to each other (A. angustifolia) or forming microrugulae (A. araucana). Microgranules and microspinules are also present. The pollen wall ultrastructure is formed by a granular ect exine and lamellated endexine. Granular elements in A. angustifolia are more loosely disposed, form more interstices, and are gradually smaller towards the endexine than in A. araucana. To asses the probable relationships within the family, we compared the pollen grains of the two Araucaria species with those of other extant genera (Agathis, Wollemia) and also with fossil pollen (Araucariacites, Balmeiopsis, Cyclusphaera, Dilwynites) attributed to Araucariaceae.(AU)

The cellular and molecular biology of conifer embryogenesis.

Gymnosperms and angiosperms are thought to have evolved from a common ancestor c. 300 million yr ago. The manner in which gymnosperms and angiosperms form seeds has diverged and, although broad similarities are evident, the anatomy and cell and molecular biology of embryogenesis in gymnosperms, such as the coniferous trees pine, spruce and fir, differ significantly from those in the most widely studied model angiosperm Arabidopsis thaliana. Molecular analysis of signaling pathways and processes such as programmed cell death and embryo maturation indicates that many developmental pathways are conserved between angiosperms and gymnosperms. Recent genomics research reveals that almost 30% of mRNAs found in developing pine embryos are absent from other conifer expressed sequence tag (EST) collections. These data show that the conifer embryo differs markedly from other gymnosperm tissues studied to date in terms of the range of genes transcribed. Approximately 72% of conifer embryo-expressed genes are found in the Arabidopsis proteome and conifer embryos contain mRNAs of very similar sequence to key genes that regulate seed development in Arabidopsis. However, 1388 loblolly pine (Pinus taeda) embryo ESTs (11.4% of the collection) are novel and, to date, have been found in no other plant. The data imply that, in gymnosperm embryogenesis, differences in structure and development are achieved by subtle molecular interactions, control of spatial and temporal gene expression and the regulating agency of a few unique proteins.

The combined impact of pH and activated carbon on the elemental composition of a liquid conifer embryogenic tissue initiation medium.

This study considered the elemental composition of plant tissue culture media in response to pH and two different types of activated C (AC; tissue culture and non acid-washed grades) in liquid media. When tissue culture medium is supplemented with AC the method of AC addition and pH adjustment can greatly impact the final medium pH, in turn, altering mineral availability. Over the pH range of 4-7, Cu and Zn adsorbed (95% and 50%) onto the two physically different ACs to the same extent. As the pH exceeded 5.8, precipitation became pronounced, resulting in 50% reductions in Mn and Fe and smaller reductions in Ca (20%), and P (15%), independent of AC. Non acid-washed AC released significant levels of Mg (65% increase) and Ca (10% increase) at pH 5.8 compared to the no-AC control. No adsorption was indicated for inorganic anions. Low levels for Cu and Zn are a concern when using AC, and low levels of Fe and Mn are a concern when the pH of the medium exceeds 5.8. Due to its impurity content and difficulty associated with its neutralization, non-acid-washed AC may be a poor choice for use in tissue culture medium.

Overdominant lethals as part of the conifer embryo lethal system.

In pines, self-pollination rates can be as high as 34% yet only 5% of viable seed is a product of self-fertilization. This decline in selfed seed viability is the consequence of post-fertilization exclusion mechanisms operating via the embryo lethal system. Recent molecular marker dissection studies suggest that the embryo lethal system is composed of semilethal factors dispersed across the genome, but it is not clear whether overdominant lethal factors are rare or representative. The study objective was to determine if overdominance was rare for the embryo lethal system in conifers. Three cohorts of selfed offspring from a single Pinus taeda parent were genotyped for nuclear microsatellites. Maximum likelihood tests based on distorted segregation ratios for single markers and for interval mapping were used to infer the degree of dominance. Four hypotheses about overdominance lethal factors were tested: (1) overdominant lethal factors rarely occur within the embryo lethal system, (2) overdominant lethal factors are rarely detected because they are transient and display stage-specific expression, (3) overdominant lethal factors are rarely detected due to tight linkage with rare marker alleles and (4) dominance estimation is unbiased by gametic selection. Four out of the seven chromosomal segments were linked to an overdominant lethal factor. One of these four segments had symmetric overdominance, an effect which persisted from embryo maturity through germination. Four overdominant lethal factors were linked to common and rare marker alleles. Gametic selection was not a source of bias in dominance estimation. Overdominant or pseudo-overdominant lethal factors are a common component of the conifer embryo lethal system.

The impact of Gelrite and activated carbon on the elemental composition of two conifer embryogenic tissue initiation media.

The elemental composition of plant tissue culture media was studied in response to (1) different levels of Gelrite and activated carbon (AC) in semisolid media and (2) different levels and types of AC in liquid media. Doubling the level of Gelrite from 2 g/l to 4 g/l reduced available magnesium (20%), calcium (16%), zinc (17%) and manganese (24%) and increased potassium (6%). AC adsorbed copper (90-95%) and zinc (35-51%) from both liquid and semisolid media. Two significantly different ACs gave minor differences in adsorption. No adsorption was indicated for inorganic anions. Nonacid-washed AC released significant levels of magnesium (44% increase), calcium (16% increase) and silica (a 75% increase to 1.8 mg/l). The elemental composition of media may need to be adjusted when increasing the Gelrite level or adding AC.