The composition of fatty acids in the endosperm and embryo lipids of <Emphasis Type="Italic">Pinus sibirica</Emphasis> and <Emphasis Type="Italic">P. sylvestris</Emphasis> seeds

The fatty acid (FA) composition of storage lipids in the seed endosperms and embryos of two pine species, Pinus sibirica and P. sylvestris, and possible biosynthetic pathways of these acids were studied by the GLC method. Linoleic acid predominated in the embryo and endosperm lipids of both P. sibirica (43.5 and 42.6%) and P. sylvestris (44.8 and 46.8%); this was evidently determined by a high expression of the gene encoding stearoyl-Δ9 acyl-lipid desaturase and the fad2 gene encoding microsomal ω6 acyl-lipid desaturase. P. sibirica lipids of the embryo and endosperm contained more oleic acid (22.0 and 24.0%, respectively) than corresponding P. sylvestris lipids (18.7 and 14%). Storage lipids of conifer seeds contain Δ5-unsaturated FAs: taxoleic (18:2Δ5,9), ephedrenic (18:2Δ5,11), pinoleenic (18:3Δ5,9,12), skiadonic (18:3Δ5,11, 14), and coniferonic (18:4Δ5,9,12,15). In the endosperm and embryos of P. sylvestris, the content of pinolenic acid was higher (22.1 and 19.6%) than in P. sibirica seeds (19.1 and 18.6%).     

Seasonal changes in the fatty acid composition of Pinus sylvestris needle lipids

The fatty acid (FA) composition of common pine (Pinus sylvestris L.) needle lipids was studied. It was shown that FA composition of needle lipids changed during the entire growth period (from March to October) under the influence of environmental factors (temperature, solar radiation) affecting the biosynthesis of chloroplast membrane lipids in pine needles. Among needle lipid FAs, unsaturated polymethylene-interrupted FAs (Δ5-UPIFA) were identified: pinolenic, skiadonic, coniferonic, and other); in March and April, their content attained 16.1 and 16.9% of total FAs; it decreased in June to 6.0% and increased again in September to 20.4%.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

The Composition of fatty acids and aldehydes of the marine bryozoans <Emphasis Type="Italic">Berenicea meandrina</Emphasis> and <Emphasis Type="Italic">Dendrobeania flustroides</Emphasis> (Bryozoa: Gymnolaemata)

This study examines the composition of lipids, fatty acids, and fatty aldehydes in two marine bryozoan species, Berenicea meandrina and Dendrobeania flustroides, from the Sea of Okhotsk. The share of neutral lipids was up to 57.3% in D. flustroides and 54.9% in B. meandrina; the share of polar lipids was 33.2 and 40.4%, respectively. In all, 57 fatty acids (FA) and 9 aldehydes were identified in total lipids. The main FAs were 16:0, 18:0, 22:6n-3, and 20:5n-3. The content of branched saturated FA in bryozoans was on the average 6.4%. Three isomers of 16:1 (n-9, n-7, and n-5), five isomers of 18:1 (n-13, n-11, n-9, n-7, and n-5), four isomers of 20:1 (n-13, n-11, n-9, and n-7), as well as 22:1n-9 and 22:1n-13 were found; the presence of 7-methyl-6-hexadienoic acid (on the average, 3.0% of total FAs) was demonstrated. Non-methylene-inter-rupted FAs contributed 8.9 and 1.6% of the total FAs in D. flustroides and B. meandrina, respectively, and were identified as 20:2(5,11), 20:2(7,13), 20:3(5,11,14), 22:2(7,13), and 22:2(7,15). In B. meandrina, minor amounts of 24:0, 24:1, 25:0, 26:0, 24:4n-3, 26:3(5,9,19), and 28:3(5,9,19) were found, suggesting sponge biofouling on some bryozoan colonies. Aldehydes (branched saturated and unsaturated C_16–19 homologues) did not exceed 10.3 and 1.9% of the total FAs in D. flustroides and B. meandrina, respectively. The presence of the FA markers that are characteristic of microalgae, protozoans, and detritus in bryozoan lipids agrees well with data on polytrophic feeding of these bryozoans.     

The role of genetic and chemical variation of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> seedlings in influencing slug herbivory

This study investigated the genetic and chemical basis of resistance of Pinus sylvestris seedlings to herbivory by a generalist mollusc, Arion ater. Using feeding trials with captive animals, we examined selective herbivory by A. ater of young P. sylvestris seedlings of different genotypes and correlated preferences with seedling monoterpene levels. We also investigated the feeding responses of A. ater to artificial diets laced with two monoterpenes, Δ³-carene and α-pinene. Logistic regression indicated that two factors were the best predictors of whether seedlings in the trial would be consumed. Individual slug variation (replicates) was the most significant factor in the model; however, α-pinene concentration (also representing β-pinene, Δ³-carene and total monoterpenes due to multicollinearity) of needles was also a significant factor. While A. ater did not select seedlings on the basis of family, seedlings not eaten were significantly higher in levels of α-pinene compared to seedlings that were consumed. We also demonstrated significant genetic variation in α-pinene concentration of seedlings between different families of P. sylvestris. Nitrogen and three morphological seedling characteristics (stem length, needle length and stem diameter) also showed significant genetic variation between P. sylvestris families. Artificial diets laced with high (5 mg g⁻¹ dry matter) quantities of either Δ³-carene or α-pinene, were eaten significantly less than control diets with no added monoterpenes, supporting the results of the seedling feeding trial. This study demonstrates that A. ater selectively feed on P. sylvestris seedlings and that this selection is based, in part, on the monoterpene concentration of seedlings. These results, coupled with significant genetic variation in α-pinene concentration of seedlings and evidence that slug herbivory is detrimental to P. sylvestris fitness, are discussed as possible evidence for A. ater as a selective force on the evolution of defensive chemistry in P. sylvestris.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of mature <Emphasis Type="Italic">Pinus sylvestris</Emphasis> buds stored at freezing temperatures

Changes of morphogenic competence in mature P. sylvestris L. buds due to frozen storage were investigated. The highest callus formation was registered on explants stored at –18°C for three months, but on explants stored for five months, it was also higher than in the control. Budding and development of needles in vitro was observed only for buds frozen three to five months. Peroxidase activity was lowest in these buds. In contrast, polyphenol oxidase activity in bud tissues continually increased during frozen storage. Within 10 months of frozen storage the content of starch and sugars in resting buds changed. It may be concluded that changes in composition of non-structural sugars in pine buds after five months of frozen storage are part of metabolic changes leading to loss of morphogenic capacity.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Characteristics of Lipid Fatty Acids in Calli of Two Geographic Populations of <Emphasis Type="Italic">Larix gmelinii</Emphasis>

The fatty acid (FA) composition of lipids of two geographic populations of larch (Larix gmelinii (Rupr.) Rupr.) was determined. One of these populations was grown on soil with a naturally high content of fluorides. This population exceeded the control one in the content of unsaturated fatty acids (FAs) in the lipid fraction of its calli. These FAs were characterized by the presence of 5-unsaturated FAs with irregular positions of double bonds, such as taxoleic, pinolenic, and sciadonic acids. It was established that the representatives of the larch fluoride population exceeded those of the control population in the content of these FAs in the total lipid fraction (8.1 and 6.7%, respectively).__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 343–348.Original Russian Text Copyright © 2005 by Makarenko, Konstantinov, Shmakov, Konenkina, Khotimchenko.     

Plant regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of two elite aspen hybrid clones from <Emphasis Type="Italic">in vitro</Emphasis> leaf tissues

Summary An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens × P. grandidentada and P. tremuloides × P. davidiana). Callus was induced from in vitro leaf explants on modified Murashige and Skoog medium (MSA) and woody plant medium (WPM) containing four different combinations of cytokinins and auxins. Callus tissues regenerated into shoots on WPM medium supplemented with 2.0 mgl⁻¹ (9.12 μM) zeatin or 0.01 mgl⁻¹ (0.045 μM) thidiazuron. P. canescens × P. grandidentata exhibited the higher callus and shoot production. In vitro leaf explants from the two hybrid clones were cocultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uidA gene encoding for β-glucuronidase (GUS) and the npt II gene encoding for neomycin phosphotransferase II. Transformation was confirmed by GUS assays, polymerase chain reaction, and Southern blot analyses. Agrobacterium concentration, acetosyringone, and pH of the cocultivation medium were evaluated for enhancing transformation efficiency with the clone P. canescens × P. grandidentata.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Seasonal dynamics of chlorophyll and microelement content in developing conifer needles of <Emphasis Type="Italic">Abies sibirica</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis>

Composition of microelements and photosynthetic pigment content (chlorophylls (Chl) a and b) were monitored in growing needles of spruce (Picea abies (L.) Karst.) and Siberian fir (Abies sibirica Ledeb.) during spring-autumn vegetation period. The dynamics of fresh weight and needle length for the first-year needles of spruce and fir revealed a number of shared and species-specific features in growth patterns of photosynthetic organs. In the beginning of growth period (in May), the needles elongated rapidly, while June–July were marked by the increase in needle weight. In P. abies the needle weight accumulated rapidly (specific growth rates μ_max up to 0.20 day⁻¹) over a short period (14 days), while in A. sibirica the needle weight increased slower (μ_max ≤ 0.11 day⁻¹) but over a longer period (≥30 days). The dynamics of Chl a and Chl b content and their ratio were identical in needles of both species over the growth period, although changes in Chl a were pronounced stronger than those in Chl b. In spring (May), a relatively high total Chl content per needle dry weight was noted. In summer (June–August), the total Chl content declined appreciably. In autumn (September–November), the total chlorophyll content in first-year needles increased slightly. Microelements were classified into two groups according to seasonal dynamics of their relative content in first-year needles. The first group includes Ba, Mn, Zn, B, Cu, Co, Cr, Pb, and Mo, whose relative content had a distinctive maximum in July, coincident with the peak in Chl content. The second group comprises Ni, V, Ag, Be, Cd, and As, whose relative content was minimal at this period. Seasonal changes in microelement composition were similar for the two conifer species examined, which is likely due to different physiological values of various microelements for photosynthetic organs.     

Plant regeneration protocol of medicinally important <Emphasis Type="Italic">Andrographis paniculata</Emphasis> (Burm. F.) Wallich <Emphasis Type="Italic">ex</Emphasis> Nees via somatic embryogenesis

Summary In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice cv. ADT 43

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T₀ plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l⁻¹ (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l⁻¹ timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Shoot regeneration from organogenic callus of sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

A complete method to regenerate adventitious shoots and to produce field-ready trees from three commercial cultivars of sweet cherry (Prunus avium L.) is described. The effects of explant types, pre-treatments, basal media, and phloroglucinol on cultivars Bing, Sweetheart, and Lapins were investigated. Callus developed on four explant types: apical shoot tips isolated from orchard trees; and punctured shoot tips, stem sections, and shoot bases of in vitro shoot cultures. Callus formed on Bing (5%), Sweetheart (8%), and Lapins (20%) shoot tips from orchard trees after 4 months on Murashige and Skoog medium (MS) at half-strength with 3 μM benzylaminopurine (BA). In vitro-derived explants formed callus after 3 months on Woody Plant Medium with 3 μM BA (W3B): punctured shoot tips (Sweetheart and Lapins 67%), stem sections (Sweetheart 31%, Lapins 27%), and shoot bases (Sweetheart 10%, Lapins 17%). Pre-treatment of shoot cultures on MS with 3 μM BA and 1 mM phloroglucinol increased callus formation three-fold on shoot base explants. Callus was separated from parental explants and maintained on MS with 3 μM BA. Shooting was induced by transferring callus to W3B. At 2 weeks, shoot development approached 100%. By 4 weeks, 7–17 shoots had formed on each explant. Callus was maintained for 1.5 years with no decrease in shoot production. Shoots were grafted onto Mazzard (P. avium) rootstocks with 54% (Sweetheart), 57% (Lapins), and 21% (Bing) success after 5 weeks.     

Field-based artificial crossings indicate partial compatibility of reciprocal crosses between <Emphasis Type="Italic">Pinus sylvestris</Emphasis> and <Emphasis Type="Italic">Pinus mugo</Emphasis> and unexpected chloroplast DNA inheritance

Crossability relationships between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was studied, using artificial pollination approach. Partial compatibility of the reciprocal crossings of these species was proved experimentally, validating the idea of a spontaneous formation of their hybrid swarms under natural conditions. The hybrids were validated using organellar DNA markers and nuclear DNA microsatellites. Based on the percentage of filled seeds, the interspecific crossings were less efficient than the intraspecific cross-pollinations of P. sylvestris and P. mugo individuals. Both species were found to intercross readily with individuals of their putative hybrid swarm, P. mugo exhibiting a higher hybridological affinity towards putatively hybrid individuals than P. sylvestris. Validation of the hybrids confirmed the paternal inheritance of chloroplast DNA (cpDNA) in the combination P. sylvestris × P. mugo only. Surprisingly, in the reciprocal crossing P. mugo × P. sylvestris, maternal inheritance of cpDNA was revealed. Obtained results offer a new insight into the direction and intensity of gene flow within the hybrid swarms of Scots pine and mountain dwarf pine.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

On the Physiological Role of Anaerobically Synthesized Lipids in <Emphasis Type="Italic">Oryza sativa</Emphasis> Seedlings

The objective of this work was to elucidate a possible adaptive role of lipid biosynthesis and unsaturated fatty acids (FAs), esterified to lipids, as terminal acceptors of electrons, alternative to molecular oxygen, in the shoots of rice seedlings (Oryza sativa L.) under conditions of strict anoxia. Biosynthesis of lipids and their accumulation, as well as the reduction of double bonds in unsaturated FAs, were studied by electron microscopic observation of the accumulation of lipid bodies in the cytoplasm and by the biochemical analysis of FAs in shoot lipids before and after anaerobic incubation of the shoots. The experiments were carried out with intact coleoptiles after 5 and 8 days of anaerobic germination of seeds (primary anoxia) and with detached shoots, preliminarily grown in air and then subjected to anoxia in the presence of 2% glucose for 48 h (secondary anoxia). In these experiments, lipid bodies did not accumulate in the cytoplasm under anoxic conditions. Lipid bodies appeared only during 48-h anaerobic incubation of detached coleoptiles in the absence of exogenous glucose, when mitochondria degraded. There was no change either in the double bond index of FAs, or in the qualitative and quantitative composition of FAs during shoot anaerobic incubation. We conclude that neither lipids synthesized under anaerobic conditions nor esterified unsaturated FAs are involved in plant adaptation to anaerobiosis as terminal acceptors of electrons, alternative to molecular oxygen. Lipid biosynthesis under anoxic conditions, which was demonstrated for anoxia-tolerant seedlings of Oryza sativa and Echinochloa phyllopogon in experiments with radioactive precursors, ¹⁴C-acetate and ³H-glycerol, is only the manifestation of a turnover of saturated FAs and various classes of lipids, which stabilizes cell membranes under adverse conditions of strict anoxia.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 540–548.Original Russian Text Copyright © 2005 by Generosova, Vartapetian.