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The chemical content and composition of the lipophilic extracts from seeds of some fir species: Abies alba, A. cephalonica, A. concolor, and A. koreana, as well as of a few spruce species: Picea abies, P. orientalis, and P. pungens, were examined. The amount of lipophilic extractives is diverse among the tree species and it varies from 9.8% to 41% of seeds. The chemical characterization showed significant differences, not only in the content, but also in the composition of extractives. However, most of the identified compounds like resin alcohols, -aldehydes, and -acids, as well as fatty acids, were detected in the seed extracts of all the examined tree species. The dominating identified compound group was esterified fatty acids (2.5 - 55.4% w/w of dry extract), occurring mainly as tri- and diglycerides, as well as free acids. The main representatives of this group were linoleic and oleic acids. The resin acids, among which the main were abietic, neoabietic, dehydroabietic, and palustric acids, were also detected at high levels, from 1.8% to 16.9% of the dry seed extracts. Phytosterols, tocopherols, resin hydrocarbons, and resin esters, as well as fatty alcohols were also identified. The coniferous tree seeds, as a renewable natural material, could represent a prospective raw material for producing valuable chemicals., (© 2016 Wiley-VHCA AG, Zürich.)

In seed extracts of five oilseed species, in bran extracts of three cereal species, and in seed and/or whole berry extracts of 10 berry species, the concentrations of a large number of lignans and the enantiomeric composition of selected lignans were determined. In the case of sesame and hemp seeds, the lignan content and composition of the whole seeds was compared to that of the hulled seeds. The results showed that cloudberry seeds are the third most lignan-rich food source after linseeds and whole sesame seeds, and that most of the berry species analysed were more lignan-rich than the cereal brans. The lignans are concentrated in the hull of the oilseeds and in the seeds of the berries. In most samples, secoisolarici-, pino-, medio-, and syringaresinol were present as a mixture of two enantiomers., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cross-linked enzyme aggregates (CLEAs) of β-glucosidase were prepared and characterized. Under the optimum conditions, the activity recovery of CLEAs reached 84 %. The reduction by NaBH(4) resulted in slightly lower activities of CLEAs, while their thermostability was enhanced. CLEAs were more thermally stable than free enzyme (half lives, 973 vs. 518 min at 50 °C), while less stable than seed meal (half life, 1,090 min). In 90 % (v/v) t-butanol, the half lives of CLEAs and free enzyme were 53 and 6.7 h, respectively. Besides, the catalytic efficiency (V (max)/K (m)) of CLEAs was comparable to free enzyme (0.42 vs. 0.47 min(-1) mg(-1)). This carrier-free immobilized enzyme had a network structure with multiple layers. The productivity of salidroside using CLEAs reached 150 g/l g catalyst, while being 6.3 g/l g with seed meal.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (LP, Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue and to continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment may decrease this recalcitrance. Sugar and sugar alcohol analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiologic concentrations (Pullman, G.S. and M. Buchanan. 2008. Identification and quantitative analysis of stage-specific carbohydrates in LP (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol. 28:985-996). Major differences in stage-specific sugars were observed. A simple bioassay was used to evaluate the potential growth promotion of individual carbohydrates added to initiation or multiplication media at physiologic concentrations. Seventeen sugars were screened. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase the initiation of LP. d-xylose and d-chiro-inositol produced statistically significant increases in early-stage embryo growth. When tested for improved initiation in P. taeda, Pseudotsuga menziesii (mirb) Franco and Picea abies L., Karst., d-xylose increased the averages of initiation by 6.5%, 7.3% and 16.7%, respectively. d-chiro-inositol increased the initiation in P. taeda by 7.3% in one test but not in the other, whereas in P. menziesii the initiation increases averaged 8.4% in two tests. Analyses of sugars and sugar alcohols in the seed environment coupled with a bioassay to screen potential media supplements for protocol improvement resulted in statistically significant increases in embryogenic tissue initiation for several coniferous species.

Stage-specific analyses of starch and 18 sugars, including pentoses, hexoses, disaccharides, trisaccharides, oligosaccharides and sugar alcohols, were made throughout seed development for zygotic embryo and female gametophyte (FG) tissues of loblolly pine (Pinus taeda L.). Tissue was most often analyzed in triplicate from two open-pollinated families grown in different locations and sampled in different years. Carbohydrates were analyzed by enzymatic assay, high performance liquid chromatography or gas chromatography/mass spectrometry. For all carbohydrates quantified, peak concentrations were higher in embryo tissue than in FG tissue. Significant changes in starch and sugar concentrations occurred over time, with both seed collections showing similar trends in temporal changes. Although concentrations were not always similar, embryo and FG tissues generally showed similar patterns of change in starch and sugar concentrations over time. Total starch concentration was highest during early seed development and decreased as development progressed. The major sugars contributing to osmotic potential during early seed development were D-pinitol, sucrose, fructose and glucose. During mid-seed development, D-pinitol, sucrose, fructose, glucose, melibiose and raffinose provided major contributions to the osmotic environment. During late seed development, sucrose, raffinose, melibiose, stachyose and fructose were the major contributors to osmotic potential. These data suggest stage-specific media composition for each step in the somatic embryogenesis protocol.

MicroRNAs (miRNAs) are known to regulate plant development, but have not been studied in gymnosperm seed tissues. The presence and characteristics of several miRNAs were examined in zygotic embryos (ZEs) and female gametophytes (FGs) of Pinus taeda (loblolly pine). Evidence for miRNAs was obtained using northern analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mediated with poly(A) polymerase. Partial sequences of two miRNAs were verified. Three regions of putative mRNA targets were analyzed by qRT-PCR to monitor the occurrence of stage-dependent miRNA-mediated cleavage. Five miRNAs were identified in ZEs and FGs along with partial sequences of Pta-miR166 and Pta-miR167. Both miRNAs showed differing degrees of tissue-specific and stage-specific modulation. Analysis of HB15L mRNA (a potential Pta-miR166 target) suggested miRNA-guided cleavage in ZEs and FGs. Analysis of ARF8L mRNA (a potential Pta-miR167 target) implied cleavage in ZEs but not in FGs. Argonaute9-like mRNA (ptAGO9L) showed stage-specific modulation of expression in ZEs that appeared to be inverted in the corresponding FGs. MicroRNAs and argonaute genes varied spatiotemporally during seed development. The peak levels of Pta-miR166 in FGs and ptAGO9L in embryos occurred at stage 9.1, a critical transition point during embryo development and a point where somatic embryo maturation often stops. MicroRNAs identified in FG tissue may play a role in 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 process of embryogenesis in gymnosperms differs in significant ways from the more widely studied process in angiosperms. To further our understanding of embryogenesis in gymnosperms, we have generated Expressed Sequence Tags (ESTs) from four cDNA libraries constructed from un-normalized, normalized, and subtracted RNA populations of zygotic and somatic embryos of loblolly pine (Pinus taeda L.). A total of 68,721 ESTs were generated from 68,131 cDNA clones. Following clustering and assembly, these sequences collapsed into 5,274 contigs and 6,880 singleton sequences for a total of 12,154 non-redundant sequences. Searches of a non-identical amino acid database revealed a putative homolog for 9,189 sequences, leaving 2,965 sequences with no known function. More extensive searches of additional plant sequence data sets revealed a putative homolog for all but 1,388 (11.4%) of the sequences. Using gene ontologies, a known function could be assigned for 5,495 of the 12,154 total non-redundant sequences with 13,633 associations in total assigned. When compared to approximately 72,000 sequences in a collated P. taeda transcript assembly derived from >245,000 ESTs derived from root, xylem, stem, needles, pollen cone, and shoot ESTs, 3,458 (28.5%) of the non-redundant embryo sequences were unique and thereby provide a valuable addition to development of a complete loblolly pine transcriptome. To assess similarities between angiosperm and gymnosperm embryo development, we examined our EST collection for putative homologs of angiosperm genes implicated in embryogenesis. Out of 108 angiosperm embryogenesis-related genes, homologs were present for 83 of these genes suggesting that pine contains similar genes for embryogenesis and that our RNA sampling methods were successful. We also identified sequences from the pine embryo transcriptome that have no known function and may contribute to the programming of gene expression and embryo development.

Glucagon-like peptide-1 (7-36) amide (GLP-1) is the most potent physiological insulinotropic hormone in humans. We produced large amounts of a GLP-1 analogue, [Ser8, Gln26, Asp34]-GLP-1, which is resistant to trypsin-digestion, as part of a chimeric rice seed storage protein, a 26 kDa globulin, in genetically modified rice seeds. Junction sites between GLP-1 analogue and globulin were replaced by tryptic cleavage sites. The highest level of GLP-1 analogue accumulation was approximately 20-50 microg per seed. We found that GLP-1 analogue derived from trypsin-digested genetically modified rice seeds stimulated insulin secretion from a mouse pancreatic beta-cell line, MIN6.

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.

Loblolly pine ( Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7 micro M), silver nitrate (20 micro M), and guanosine 3',5'-cyclic monophosphate, 8-bromo-, sodium salt (10 micro M) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4-6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.

Clonal production of loblolly pine ( Pinus taeda L.) through somatic embryogenesis has the potential to meet the increasing industrial demands for high-quality uniform raw materials. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Twenty-five newly initiated loblolly pine genotypes were followed through the process of liquid culture establishment, embryo maturation, germination, and retrieval from cryogenic storage. A maturation medium, capable of promoting the development of loblolly pine somatic embryos that can germinate, is presented that combines 1/2 P6 modified salts, 2% maltose, 13% polyethylene glycol 8000 (PEG), 5 mg/l abscisic acid (ABA), and 2.5 g/l Gelrite. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also described. A set of somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed. The quality of the resulting embryos was examined and compared to that of zygotic embryos using such parameters as morphology, dry weight, germination performance, and gene expression. All of the observations that were made support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth.

In contrast to angiosperms, pines and other gymnosperms form well-developed suspensors in somatic embryogenic cultures. This creates a useful system to study suspensor biology. In a study of gene expression during the early stages of conifer embryogenesis, we identified a transcript, PtNIP1;1, that is abundant in immature loblolly pine (Pinus taeda) zygotic and somatic embryos, but is undetectable in later-stage embryos, megagametophytes, and roots, stems, and needles from 1 year-old seedlings. Analysis of PtNIP1;1 transcript in embryo proper and suspensor tissues by reverse transcription-polymerase chain reaction suggests preferential expression in the suspensor. Based on comparisons of derived amino acid sequences, PtNIP1;1 belongs to the nodulin-like members of the major intrinsic protein superfamily branch of the aquaporin (major intrinsic protein) superfamily. Through heterologous expression in Xenopus laevis oocytes and the yeast (Saccharomyces cerevisiae) fps1(-) mutant, PtNIP1;1 has been shown to be an active aquaglyceroporin.