Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture

The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzylaminopurine - GUS -glucuronidase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - EtOH ethanol - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - NOS nopaline synthase - CaMV cauliflower mosaic virus     

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Optimization of biolistic transformation of embryogenic grape cell suspensions

Embryogenic suspensions of Chancellor (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding ß-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 M kanamycin. Factors that affect biolistic transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 m (M10) resulted in more transient gene expression than 0.771 m diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.Abbreviations AC activated charcoal - GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - IAA-L-alanine indole-3 acetic acid L-alanine - MS Murashige and Skoog - CH casein hydrolysate - Km kanamycin - NPTII neomycin phosphotransferase II     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Requirement of protein and RNA synthesis for lambda repressor inactivation by tif-1: effects of chloramphenicol, neomycin and rifampicin.

Summary The inactivation of repressor was followed by the specific DNA binding assay during the course of lysogenic induction provoked by incubation at 42°C of an E. coli tif-1 lysogenic strain. The presence of up to 400 g/ml chloramphenicol during the inducing treatment did not impair the loss of repressor binding activity, whilst concentrations of 200 g/ml neomycin and 100 g/ml rifampicin effectively inhibited the inactivation of repressor.Residual protein synthesis in the presence of chloramphenicol, neomycin and rifampicin was 5%, 5% and 27% respectively of that observed in the drug-free control. This residual synthesis did not appear to involve amplification of the X-protein. These results suggest that tif-mediated inactivation of the repressor requires the activation of some specific gene(s), the translation of which appears to be resistant to chloramphenicol.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

ATPase-dependent energy spilling by the ruminal bacterium,Streptococcus bovis

When the ruminal bacterium Streptococcus bovis was grown in batch culture with glucose as the energy source, the doubling time was approximately 21 min and the rate of bacterial heat production was proportional to the optical density (1.72 W/g protein). If exponentially growing cultures were treated with chloramphenicol, there was a decline in heat production, but the rate was greater than 0.30 W/g protein even after growth ceased. Since there was no heat production after glucose depletion, this growth-independent energy dissipation (spilling) was not simply due to endogenous metabolism. Stationary cells which were washed and incubated in nitrogen-free medium containing an excess of glucose produced heat at a rate of 0.17 W/g protein. Monensin and tetrachlorosalicylanilide (TCS), compounds which facilitate an influx of protons, caused a more than 2-fold increase in heat production. Dicyclohexylcarbodiimide (DCCD) virtually eliminated growth-independent heat production regardless of the mode of growth inhibition. Because DCCD had little effect on the glucose phosphotransferase system, it appeared that the combined action of proton influx and the membrane bound F₁F₀ proton ATPase was responsible for energy spilling.Abbreviations DCCD dicyclohexylcarbodiimide - TCS tetrachlorosalicylanilide     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 M) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50M lanthanum, 50 M lead, 10 M cadmium, or 200 M manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 M Al in the presence of externally applied 50 M citrate or 400 M malate. The secretion of citrate and malate from intact rye roots exposed to 50 M Al corresponded to 31.3 ± 1.7 M and 11.5 ± 2.5 M, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Changes with age in the absorption spectrum of chlorophyll α in a diatom

Summary Absorption spectra of a young and an old culture of the diatom Pheodactylum tricornutum were measured in thin layers between two opal glass sheets. The spectra at 24° and at -196°C were replotted to give equal areas from 730–625 m to allow direct comparison. At 24°C the spectrum for the difference between the two cultures had a negative component of 18 m half width centered at 675 m and a positive region of W_0.5=26 m near 700 m.The spectra at -196°C may be somewhat distorted by clumping of the cells during freezing but nevertheless the 16 day culture clearly showed a smaller proportion of Ca 670 to Ca 680. This older culture has a shoulder due to a 707 m component. The difference curve at -196°C shows the decrease of an unsymmetrical band peaking at 669 m and an increase at 695 m in addition to the 707 m component. Due to the possibility of distortion, the presence of an actual component at 695 is doubtful in these particular cultures.The room temperature spectrum in the chloropyhll a region for the 5 day culture can be closely fitted by a single probability curve at 675 m having a half-width of 31 m. The sum of two components, with widths more reasonable for chlorophylls, also matched the data well enough. These two probability curves, of 22 m half width, centered on 669 and 683.2 m and had a height ratio, h₆₆₉/h₆₈₃ of 1.18. In the 16 day culture the ratio for these bands changed to 1.11 and there was extra absorption around 700 m.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday     

Feeding in the rotifer Brachionus calyciflorus

Summary The laboratory feeding behavior of Brachionus calyciflorus varies depending upon the type of food cell available in suspension. When feeding on the yeast Rhodotorula glutinis, rotifers show a continuous increase in ingestion with increased cell density between 0.01 and 1000 g dry weight ml^-1. Effective clearance rates drop from ca. 50 l animal^-1 h^-1 to less than 0.5 l animal^-1 h^-1 over this food density range. When feeding on Englena gracilis, B. calyciflorus ingestion rates are constant between 1.0 and 100 g ml^-1 of available food, averaging close to 25 ng animal^-1 h^-1. The decrease in clearance rate is more striking than with R. glutinis, dropping from 45 l animal^-1 h^-1 at 0.1 g ml^-1 to 0.13 l animal^-1 h^-1 at 100 g ml^-1. Differences between the patterns obtained with the two food types indicate fundamental dissimilarities in the feeding behavior of this rotifer species when presented with these different foods.     

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica

Summary InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 M 4-PU and 1 M NAA at a cell density of 5 × 10⁴/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1–1 M 4-PU and 1 M NAA, and 10 M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 M 4-PU or 10 M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 M IBA and 0.1 M NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IBA indole-3-butyric acid - 2ip N ⁶-(2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - 4-PU N-(2-chloro-4-pyridyl)-N–phenylurea - TDZ thidiazuron     

Whole plant regeneration of Cicer arietinum from callus cultures via organogenesis

A protocol for whole plant regeneration of Cicer arietinum L. cv. C-235 via organogenesis from callus has been developed. Callus initiation was best when immature leaflets were cultured on MS medium containing 5 or 25 M 2,4-D or NAA in combination with 10 M BA, or 25 M 2,4-D alone. The callus grew most vigorously on MS medum supplemented with 10 M NAA and 5 MBA. Best shoot differentiation was obtained from calli derived from the basal portion of shoot tips on MS medium supplemented with 10 M BA and 0.1 M IBA. The shoot forming ability of calli was enhanced by adding 5 mM potassium phosphate to the medium. Shoots were rooted on a MS medium containing l M IBA. The regenerated plants were grown to maturity and produced viable seed.Abbreviations 2,4-D 2,4-dichlorophen-oxyacetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - Kn kinetin - 2-ip 6-(,-dimethylallylamino)-purine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium     

Influence of plant growth regulators,basal media and carbohydrate levels on the in vitro development of Pinus ponderosa (Dougl. ex Law.) cotyledon explants

Applications of in vitro screening techniques for Pinus ponderosa resistance to Peridermium harknessii could be beneficial in a tree breeding program. Plant growth regulators, basal media formula and carbohydrate levels were examined to determine the various effects each would have on excised cotyledon growth and development. Proliferating green callus was initiated from cotyledon explants on SH basal medium containing 4.4 M BA:5.4 M NAA and 1% sucrose. Subsequent subculturing onto LS medium supplemented with 44.0 M BA: 5.4 M NAA and 2% sucrose improved callus maintenance. The highest frequency of caulogenesis from cotyledon explants occurred on a modified GD medium containing 44.0 M BA: 0.054 M NAA and 4% glucose. The influence of nitrogen source, osmoticum and medium salt concentrations are discussed relative to callus initiation and caulogenesis.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - CD Campbell & Durzan - GD Gresshoff & Doy - LP Le Poivre - LS Linsmaier & Skoog - MC McCown - SH Schenk & Hildebrandt     

The detection and characterization of bacteria-sized protists in “Protist-free” filtrates and their potential impact on experimental marine ecology

Nuclepore filters of 0.6–1.0m pore size have been used to prepare protist-free water for a number of studies in microbial ecology. This procedure has been called into question by a recent study claiming that a significant portion of bacterial loss in filtrates could be due to uncharacterized predators passing through 0.6m filters. We were unable to directly observe protists in 0.6m filtrates using phase contrast, epifluorescence, or transmission electron microscopy. Using the culture techniques of rice grain enrichment and most probable number, however, we were able to observe and quantify several species of bacterivorous nanoflagellates that developed not only in 0.6m, but also in 0.4m seawater filtrates. The ability of predacious nanoflagellates to squeeze through bacteria-sized pores questions studies of bacterial production and chemical cycling that have assumed protist-free filtrates.