Dark Culture Research Articles

TESTES PRELIMINARES DE CRESCIMENTO COM UMA CEPA DA MICROALGA PRODUTORA DE ASTAXANTINA Haematococcus pluvialis (CHLOROPHYCEAE,VOLVOCALES)

Este trabalho descreve um experimento preliminar de crescimento com uma cepa da microalga produtora de astaxantina Haematococcus pluvialis (Chlorophyceae, Volvocales). Cultivos com 4 composições diferentes de células em termos de proporção e coloração de aplanósporos e macrozoóides, foram submetidos à 20°C ± 2°C, 70µE.m-2.s-1, fotoperíodo de 12 horas de luz : 12 horas de escuro e meio de cultura adequado por cerca de 900 horas. Alíquotas de 2 ml eram retiradas periodicamente ao longo do experimento para a determinação da densidade de células e construção das curvas de crescimento. Os resultados indicaram um padrão de crescimento similar para todos os frascos de cultivo, onde os macrozoóides predominavam nos primeiros dias sendo substituídos gradualmente por aplanósporos, que tornaram-se dominantes em torno de 300 horas. A densidade máxima de aplanósporos foi verificada em torno das 800 horas de experimento. O experimento como um todo, mostrou que a cepa testada é viável, podendo ser utilizada para experimentos específicos de determinação das condições ótimas de crescimento e produção de astaxantina.

Callus culture of Cyclocarya paliurus (Batal.) Iljinskaja leaves

The hormone combinations and dark culture of the callus induction for Cyclocarya paliurus (Batal.) Iljinskaja leaves were optimized by an orthogonal experiment. The effects of hormones on callus proliferation were also studied. The results show that the optimum medium for callus induction of C. paliurus leaves was MS + 1.0 mg·L−1 6-BA + 0.5 mg·L−1 NAA + 1.0 mg·L−1 2,4-D. The combination of 6-BA and NAA significantly affected the induction of callus, while the effect of 2,4-D was less. The maximum rate of callus induction was 95.4%. A 10-day darkness period was beneficial for callus induction. The best proliferation medium was 1/4 DKW + 0.5 mg·L−1 6-BA + 1.0 mg·L−1 2,4-D.

The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short‐term thermal stress of the alga

The dinoflagellate alga Symbiodinium sp., living in symbiosis with corals, clams and other invertebrates, is a primary producer in coral reefs and other marine ecosystems. The function of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in dinoflagellates is difficult to study because its activity is rapidly lost after extraction from the cell. We report procedures for the extraction of Rubisco from Symbiodinium cells and for stable storage. We describe a continuous assay for Rubisco activity in these crude cell extracts using the Mn(2+) chemiluminescence of Rubisco oxygenase. Chemiluminescence time courses exhibited initial transients resembling bacterial Form II Rubisco, followed by several minutes of linearly decreasing activity. The initial activity was determined from extrapolation of this linear section of the time course. The activity of fast-frozen cell extracts was stable at -80 degrees C and, after thawing and storage on ice, remained stable for up to 1 h before declining non-linearly. Crude cell extracts bound [(14)C] 2-carboxy-D-arabitinol 1,5-bisphosphate to a high molecular mass fraction separable by gel filtration chromatography. After pre-treatment of Symbiodinium cell cultures in darkness at temperatures above 30 degrees C, the extracted Rubisco activities decreased, with almost complete loss of activity above 36 degrees C. The implications for the sensitivity to elevated temperature of Symbiodinium photosynthesis are assessed.

The effect of cultivation conditions on the mycelial growth of a dark-septate endophytic isolate

The EF-37 isolate, one of DSE fungi, is beneficial to the growth and development of its host plant, Saussurea involucrata Kar. et Kir. The cultivation requirements including basic culture medium, temperature, light, pH, carbon source and nitrogen compounds were studied for their effects on mycelial growth of a dark-septate endophytic (DSE) fungus EF-37 by using one-factor-at-a-time method. Potato dextrose agar (PDA) was the best medium for the growth of endophyte EF-37. Our studies showed that 20°C, 24 h dark cultivation and pH 7 significantly influenced the growth of endophyte EF-37 on PDA medium. Moreover, glucose and calcium nitrate were found to be the best nutrients for EF-37 growth. Under the optimal cultivation conditions, DSE fungus EF-37 isolate could grow actively. This is the first study about the effect of cultivation conditions on the growth of this strain, which provides the preparatory knowledge for the biological characteristics of DSE fungus EF-37. Key words: Cultural conditions, dark-septate endophytic (DSE) fungus EF-37, mycelial growth, optimization, Saussurea involucrata Kar. et Kir.

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR(N143Q) lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR(N143Q) in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.

Synthesis of Coenzyme Q10 and β-carotene by Yeasts Isolated from Antarctic Soil and Lichen in Response to Ultraviolet and Visible Radiations

The effect of different doses of visible (Vis), ultraviolet-capital A, Cyrillic (UVA), and mixed light (UVA + Vis) upon coenzyme Q(10) (CoQ(10)) and beta-carotene synthesis and biomass yield by the Sporobolomyces salmonicolor AL(1), Cryptococcus albidus AS(55), Cryptococcus laurentii AS(56), and C. laurentii AS(58) strains isolated from Antarctic samples was investigated. The beta-carotene concentration in the red strain biomass increased by 52% under irradiation with 11 J/cm(2) Vis, and the CoQ(10) concentration rose by 37% in relation to the control quantity obtained through dark cultivation. Under irradiation with 6 J/cm(2) UVA, the S. salmonicolor AL(1) strain synthesized 15% more beta-carotene; C. albidus AS(55), 22%; C. laurentii AS(56), 44%; and C. laurentii AS(58), 35% in relation to the control quantity. Irradiation with a low UVcapital A, Cyrillic + Vis dose significantly stimulated beta-carotene biosynthesis by the strains of the Cryptococcus genus (87%, 138%, and 100%), whereas S. salmonicolor AL(1) increased the beta-carotene content to a smaller degree (55%). Higher doses of all three irradiation types inhibited beta-carotene accumulation. Vis suppressed CoQ(10) biosynthesis in the Cryptococcus strains, whereas UVcapital A, Cyrillic and UVcapital A, Cyrillic + Vis inhibited it in all four strains. The S. salmonicolor AL(1) strain pre-treated with 0.02 J/cm(2) UVA synthesized twice as much CoQ(10) and beta-carotene when cultivated in the presence of Vis light in an 11-J/cm(2) dose.

Lipid production in Porphyridium cruentum grown under different culture conditions

Autotrophic growth of Porphyridium cruentum under 18:12 h and 12:12 h light:dark cycles showed the maximum cell concentration of 2.1 g-dry wt./L, whereas the specific growth rate, 0.042 (1/h), at 18:6 h is faster than that of 12:12 h, 0.031 (1/h), respectively. The highest lipid accumulation level, 19.3 (%, w/w), was achieved at 12:12 h cycle. Under dark cultivation condition with 10 g/L of glucose, the lipid accumulation in the cell was 10.9 (%, w/w), whereas the heterotrophic growth with glycerol as the carbon resource showed low level of cell concentration and lipid production, compared to that of glucose. The glucose was decided to be a suitable carbon resource for the heterotrophic growth of P. cruentum. The lipids from P. cruentum seemed be feasible for biodiesel production, because over 30% of the lipid was C16-C(18:1). The cultivation time and temperature were important factors to increase the maximum cell concentration. Extending the cultivation time helps maintain the maximum cell concentration, and higher lipid accumulation was achieved at 25 degrees C, compared to 35 degrees C. The fed-batch cultures showed that, under the light condition, the specific production rate was slightly decreased to 0.4% lipid/g-dry wt./day at the later stage, whereas, under the dark condition, the specific production rate was maintained to be a maximum value of 1.1% lipid/g-dry wt./day, even in the later stage of cultivation. The results indicate that the heterotrophic or 12:12 h cyclic mixotrophic growth of P. cruentum could be used for the production of biodiesel in long-term fed-batch cultivation of P. cruentum.

Micropropagation of Aloe arborescens Mill: A step towards efficient production of its valuable leaf extracts showing antiproliferative activity on murine myeloma cells

A micropropagation protocol for Aloe arborescens has been developed in order to maximize the multiplication index and to minimize the cycle length. Explants were sterilized in NaOCl and subcultured weekly to overcome the effects of released polyphenol that, otherwise, caused browning of the cut surface, tissue damage and death. In order to identify the combination of plant growth regulators giving a suitable multiplication index, five substrates having Murashige and Skoog salts as a basis and differing in the type and concentration of auxins and cytokinins were tested. Explants cultured on the substrate containing 1.0 mg l−1 α‐Naphthaleneacetic acid and 2.0 mg l−1 6‐benzylaminopurine showed abundant sprouting from their base and from axillary meristems. The mean multiplication index obtained with this substrate was about 3.5. For callus induction, only substrates combining an auxin and a cytokinin were effective; percentages of explants producing callus in dark culture conditions were higher than those obtained in the light. Biological effects of leaf extracts from micropropagated plants of A. arborescens on proliferation of the murine myeloma cell line P3X were evaluated. Preliminary results confirmed the presence of bioactive molecules and their activity in inhibiting cell proliferation; such activity was lower than that of analogous leaf extracts from three‐year‐old plants.

EMPLOYMENT OF ORGANIC ACIDS TO ENHANCE ASTAXANTHIN FORMATION IN HETEROTROPHICCHLORELLA ZOFINGIENSIS

The green alga Chlorella zofingiensis produces significant amounts of the valuable ketocarotenoid astaxanthin under heterotrophic growth conditions. In this study, pyruvate, citrate and malic acid were investigated, for the first time, to promote the formation of secondary carotenoids including astaxanthin in C. zofingiensis in the dark. The addition of pyruvate, citrate and malic acid into the culture medium at a concentration above 10 mM stimulated biosynthesis of astaxanthin and other secondary carotenoids. The addition of 100 mM pyruvate enhanced the yield of astaxanthin from 8.36 to 10.72 mg/L, representing an increase of 28.2%. Citrate and malic acid also had stimulatory effects on the formation of astaxanthin. The results showed that organic acid supplementation was potentially useful for commercial production of astaxanthin by C. zofingiensis in dark heterotrophic culture.

Gynogenesis in the vine cacti Hylocereus and Selenicereus (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.

Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.)

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.

Adventitious shoot regeneration from the leaves of in vitro grown ‘Zhongli 1’ pear (Pyrus spp.)

The pear (Pyrus spp.) is one of the most important temperate fruit crops. The technique of adventitious shoot regeneration from leaves is considered to be one of the shortcuts in the research on pear genetic modification and cellular engineering, which, however, has not been widely used. As the regeneration frequency of pear leaves is usually very low, the research on adventitious shoot regeneration from pear leaves is eagerly needed. In this experiment, the factors affecting shoot and bud regeneration from the leaves of ‘Zhongli 1’ pear were studied, and an efficient protocol for shoot regeneration was established. The results showed that different types of basic media, different combinations of plant growth regulators, leaf placement on medium, periods of dark culture and the use of silver nitrate (AgNO3) on culture media all significantly affected the adventitious shoot regeneration frequency of ‘Zhongli 1’ pear. The details are as follows: (1) Among three kinds of basic media, NN69 was better for ‘Zhongli 1’ shoot regeneration, followed by half (½) MS, while full MS had no effect on shoot regeneration; (2) Thidiazuron (TDZ) was better than 6-benzylaminopurine (6-BA) for ‘Zhongli 1’ regeneration, with an optimal concentration of 1.5 mg · L−1, and the regeneration rate under this concentration could reach 85%, with 2.72 buds per leaf. 0.5 mg · L−1 indole-3-butyric acid (IBA), which induced a higher regeneration frequency, was a better choice for pear regeneration compared with 0.3 mg · L−1 naphthaleneacetic acid (NAA). Among the different combinations of plant growth regulators, TDZ + IBA was better for inducing high regeneration frequency; (3) The abaxial surface of leaves touching the medium was beneficial for leaves to uptake nutrients from the medium, and because of that, the regeneration frequency of leaves was significantly higher than that of leaves touching the medium with their adaxial surfaces (obverse side of leaf); (4) Dark culture was necessary for bud regeneration, and the best duration for dark culture of ‘Zhongli 1’ pear was 21 days; (5) The addition of 1.0 mg · L−1 AgNO3 into the culture medium could promote adventitious shoot regeneration significantly. A high adventitious shoot regeneration frequency was obtained in this research, which will be beneficial for further research on efficient and stable in vitro plant regeneration systems and genetic modification of pear.

Induction Technique of Picea koraiensis Nakai Embryogenic Callus

Culture conditions for embryogenic callus induction from immature zygotic embryos of Picea koraiensis were studied.The effect of basal medium,light conditions,collection period of immature seeds on induction rate of embryogenic callus were compared through L16(42×2)mixed level orthogonal experiment,subsequently culture temperature optimization experiment were also performed.The results were as follows:the optimal basal medium and culture conditions were improved RJW medium,dark culture,zygotic embryo collection date of July 20,culture temperature of 22 ℃。The highest embryogenic callus induction rate(81.3%)was achieved on the improved RJW medium supplemented with 1.0 mg·L-1 BA,5.0 mg·L-1 NAA,20 g·L-1 sucrose,450 mg·L-1 L-glutamine,750 mg·L-1 casein hydrolysate,2.0 g·L-1 gelrite,dark culture under 22℃,the average induction frequency was 81.3%.

Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of Phalaenopsis orchids

The influence of light regime, explant position and orientation on direct embryo formation from leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were investigated to optimize the protocol for regenerating of this orchid. When explants were cultured in light, direct embryogenesis was retarded in both species. Embryos showed whitish to pale green in color and larger size than those cultured in darkness. Furthermore, light regime induced explant browning, embryo necrosis and eventually low plantlet conversion rate. Sixty days of culture in darkness is the most suitable duration for direct embryo induction. Explant orientation also significantly affected direct embryo formation, and explants placed adaxial-side-up on culture medium had higher embryogenic response than abaxial-side-up orientation. In both species, the cut end had highest embryogenic competence than other parts of the explant. Moreover, when the leaf explant was cut transversely into two segments, the leaf basal segment had higher embryogenic competence than the leaf tip segment.

A novel three‐stage light irradiation strategy in the submerged fermentation of medicinal Mushroom Ganoderma lucidum for the efficient production of ganoderic acid and Ganoderma polysaccharides

A novel three-stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of bioactive metabolites ganoderic acid (GA) and Ganoderma polysaccharides was developed. Significance of light quality, i.e., blue light (390-500 nm, lambda(max) = 470 nm), red light (560-700 nm, lambda(max) = 625 nm), and white light (400-740 nm, lambda(max) = 550 nm), was studied at first. Interestingly, there was a gradual decrease trend of GA content after the culture of day 2 when the maximal GA content was obtained, while GA content decreased slowly under white light irradiation after day 6. The dark environment was favorable to the specific GA biosynthesis (i.e., GA content) before day 6, and after that the optimum was white light irradiation. A relatively lower irradiation density of white light (i.e., 0.94 and 2.82 W/m(2)) was beneficial for the specific GA biosynthesis before day 6, while GA content was higher under higher irradiation density of white light (i.e., 4.70 and 9.40 W/m(2)) at the later-stage of cultivation. 4.70 W/m(2) white light irradiation culture was the best from the viewpoint of GA accumulation. Therefore, a two-stage light irradiation strategy by combing the first 2 days dark culture with the following 4.70 W/m(2) white light irradiation culture was developed. The highest GA production in the two-stage culture was 276.0 +/- 12.5 mg/L, which was increased by 19% compared to 4.70 W/m(2) white light irradiation culture (i.e., 232.4 +/- 15.8 mg/L) and by 178% compared to the dark culture (i.e., 99.4 +/- 1.0 mg/L). Although there still existed a gradual decrease trend of GA content after day 2 when the maximal GA content was obtained in the two-stage culture. Following three-stage light irradiation strategy was further demonstrated in order to turn around the sharp decrease of GA content after day 2. The first-stage was the 2-day dark culture; the second-stage was the following six-day 0.94 W/m(2) white light irradiation culture, and the third-stage was 4.70 W/m(2) white light irradiation culture until the end of fermentation. During the three-stage culture of G. lucidum, the gradual decrease trend of GA content after day 2 was turned around, which suggested that 0.94 W/m(2) white light irradiation was beneficial for the metabolic flux towards the GA biosynthesis. The maximal GA content of 3.1 +/- 0.1 mg/100 mg DW was obtained, which was higher by 41% compared to the two-stage culture. The maximal GA production (i.e., 466.3 +/- 24.1 mg/L) and productivity (i.e., 38.9 mg/L per day) in the three-stage culture were 69 and 101% higher than those obtained in the two-stage culture. This is the first report investigating the significance of light irradiation on the medicinal mushroom submerged fermentation. Such work is very helpful to other mushroom fermentations for useful metabolite production.

Effect of growth conditions on electrophysical properties of Rhodobacter capsulatus PG cells

Electrophysical characteristics of cells of the phototrophic bacterium Rhodobacter capsulatus PG grown in complete Hutner medium in light or dark were found to differ depending on the composition of their lipopolysaccharides (LPS). Under dark cultivation, the cells synthesized LPS with a shortened structure that determined the electrophoretic properties of cell surfaces. The observed decrease in the effective high-frequency electroconductivity of the dark-grown cells is assumed to be due to a decrease in the intracellular K+ concentration resulting from increased permeability of cytoplasmic membranes of the cells grown under these conditions.

Physiological Alteration of the Marine Bacterium Vibrio angustum S14 Exposed to Simulated Sunlight During Growth

Growth experiments on the marine bacterium Vibrio angustum S14 were conducted under four light conditions using a solar simulator: visible light (V), V + ultraviolet A (UV-A), V + UV-A + UV-B radiation, and dark. Growth was inhibited mainly by UV-B and slightly by UV-A. UV-B radiation induced filaments containing multiple genome copies with low cyclobutane pyrimidine dimers. These cells did not show modifications in cellular fatty acid composition in comparison with dark control cultures and decreased in size by division after subsequent incubation in the dark. A large portion of the bacterial population grown under visible light showed an alteration in cellular DNA fluorescence as measured by flow cytometry after SYBR-Green I staining. This alteration was not aggravated by UV-A and was certainly due to a change in DNA topology rather than DNA deterioration because all the cells remained viable and their growth was not impaired. Ecological consequences of these observations are discussed.

Dark Earths and manioc cultivation in Central Amazonia: a window on pre-Columbian agricultural systems?

Many commentators highlight the fertility of Anthropogenic Dark Earths (ADE), emphasizing their potential for sustainable agriculture. Some scholars believe that terra mulata (the less fertile, more extensive form of ADE) was created by means of agricultural practices used by large settled populations of pre-Columbian farmers. But what was it that these Amerindian farmers were growing? Until recently, scholarly consensus held that manioc does not perform well on ADE. New research on the middle Madeira River is showing, however, that this consensus was premature. In this region, the most common crop in ADE fields is bitter manioc. Farmers there have various landraces of manioc that they believe yield particularly well on ADE, and logically plant more of these varieties on ADE. Aspects of the behaviour and perception of manioc cultivation among 52 farmers at the community of Barro Alto were measured quantitatively on four terra firme soil types (Terra Preta, Terra Mulata, Oxisols and Ultisols). These farmers plant different configurations of landraces in different soils, according to their perception of the suitability of particular landraces and their characteristics to certain soil types and successional processes. This, in turn, shapes selective pressures on these varieties, as new genetic material incorporated from volunteer seedlings is more likely to contain traits present in the most prevalent landrace(s) in each soil type. Owing to localized population pressure at Barro Alto, manioc is under more intensive cultivation systems, with shorter cropping periods (5-10 months) and shorter fallow periods (1-2 years). The outcome of these processes is different co-evolutionary dynamics on ADE as opposed to non-anthropogenic soils. Further anthropological study of manioc swiddening in one of the richest agricultural environments in Amazonia can fill a gap in the literature, thus opening an additional window on the pre-Columbian period.

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash ( Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.