Liquid Shake Cultures Research Articles

Organogenesis and Cormel Production from Callus Culture of Gladiolus cv. `Balady'

Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.

Adventitious shoot formation from carnation stem segments: a comparison of different culture procedures

Adventitious shoot regeneration from stem expiants of carnation ( Dianthus caryophyllus L., cultivar ‘White Sim’) was studied using three different culture procedures: agar-gelled medium, liquid shaken medium, and an interfacial membrane raft floating on liquid medium. In all culture procedures, expiants derived from the first upper internode exhibited higher adventitious shoot formation than those from the second internode. On agar-gelled medium, the number of regenerated shoots per expiant from the first internode increased with increasing thidiazuron (TDZ) concentration (up to 4 mg l −1) in the presence of 1 mg l −1 naphthaleneacetic acid. In the liquid shaken culture, the number of regenerated shoots peaked at 1 mg TDZ l −1. Maximum shoot regeneration on an interfacial membrane raft was obtained in the presence of 0.25 mg TDZ l −1. A comparison of the overall efficiencies of shoot regeneration for each culture procedure showed that maximum shoot regeneration on the raft was twice and three times that obtained with agar-gelled and liquid shaken media, respectively.

Isolation and characterization of non cerato-ulmin producing laboratory induced mutants of Ophiostoma novo-ulmi

Five laboratory mutants of Ophiostoma novo-ulmi EAN isolate H328 were obtained from uv-irradiated and unirradiated blastoconidia. Although the mutants were initially selected on the basis of various characteristics, including failure to produce cerato-ulmin (CU) in the culture filtrate, growth-temperature responses and colony morphology, they all were eventually also found unable to produce CU when grown in liquid shake culture at 23 °C and 33°, or they produced CU only in negligible amounts. This association suggests possible linkages between CU production and other fungal metabolic pathways. Genetic analysis of crosses between the mutants and a wild type isolate of EAN O. novo-ulmi suggested that each mutant involved a change at a single-locus, at least as far as segregations for colony morphology were concerned. Pathogenicity trials using the five mutants were carried out on four-year old Ulmus carpinifola and U. procera . Two out of the five mutants showed a significant reduction in pathogenicity in comparison to the O. novo-ulmi wild type isolate H328.

Propagation and tuberization of potato bud clusters from bioreactor culture

Single node stem segments fromin vitro potato shoots cultured in liquid medium in the presence of ancymidol (23.4 μM) developed into bud clusters in either shaken flasks or bioreactor cultures. Buds on the clusters developed tubers after subculture to a tuber induction medium with 23.2 μM kinetin, 19.5 μM ancymidol, and 6-8% sucrose. The number of tubers per cluster and their size were higher in agar induction medium on top of which a second layer of liquid medium was added, than in liquid shake or bioreactor cultures. The highest increase in tuber size (i.e., 720 mg fresh weight after 7 weeks), was obtained in agar cultures flushed twice with liquid tuber induction medium. The potential of bioreactor cultures for potato bud proliferation and enhanced tuber development in double layer agar-liquid cultures is discussed.

液体振とう培養におけるササユリ球根の生育に及ぼす窒素源，リン酸イオンおよび硫酸イオンの影響

For liquid-shaking culture of bulbs of Liliurn japonicum Thunb., we analyzed the concentration changes in macro-inorganic ions in the culture media to clarify their relationships with bulb growth.1. Inorganic ions, SO42-, NH4+, and especially H2PO4- were consumed in large quantities by bulbs during culture, whereas the consumption of K+, Mg2+, and Ca2+ was very slow.2. The optimum concentration of NH4+ for bulb enlargement was 10-20 mM. The application of NO3- alone or NH4+ higher than 30 mM as nitrogen source retarded the bulb growth. Root elongation was promoted by application of NO3- as the sole nitrogen source.3. Root growth was poor in the medium containing a high ratio of NH4+ /NO3-. Application of glutamine instead of NH4+ promoted root growth.4. The in vitro rooted bulbs sprouted and bolted abundantly after planting in the soil. Based on the above results, bulbs should be cultured first in a medium containing NH4+ for bulb enlargement and then transferred to a NH4+ free medium for root development. These treatments improved shoot growth after transplanting.5. Although the absorption rate of H2PO4- from the medium with the high concen-tration of NaH2PO4 was higher than that from the medium with the low concentration, bulb enlargement was not promoted.6. High concentration of SO42- in the medium inhibited bule growth.

Production of ?-glucosidase by Aspergillus terreus

The production of beta-glucosidase by Aspergillus terreus was investigated in liquid shake cultures. Enzyme production was maximum on the 7th day of growth (2.18 U/ml) with the initial pH of the medium in the range of 4.0-5.5. Cellulose (Sigmacell Type 100) at 1.0% (wt/vol) gave maximum beta-glucosidase activity among the various soluble and insoluble carbon sources tested. Potassium nitrate was a suitable nitrogen source for enzyme production. Triton X-100 at 0.15% (vol/vol) increased the enzyme levels of A. terreus. The test fungal strain showed an ability to ferment glucose to ethanol.

Ophiostoma himal-ulmi sp. nov., a new species of Dutch elm disease fungus endemic to the Himalayas

In September–October 1993 a research survey was conducted in northern Himachal Pradesh, western Himalayas, as part of an investigation into the origins of Dutch elm disease. No disease symptoms were observed in the area, but an apparently endemic Ophiostoma similar to the known Dutch elm disease pathogens Ophiostoma ulmi and O. novo-ulmi was isolated from around breeding galleries of scolytid beetles in the bark of Ulmus wallichiana . Over 200 isolations of the fungus were made. These were later resolved into a minimum of 20 different genotypes, which were used to further characterize the fungus. Like O. ulmi and O. novo-ulmi the Himalayan Ophiostoma is outcrossing and heterothallic, with two sexual compatibility types, A and B, occurring in a near 1:1 ratio in nature. In interfertility tests, strong pre- and post-zygotic reproductive isolation was revealed between the Himalayan Ophiostoma and sexually compatible isolates of O. ulmi or the EAN or NAN races of O. novo-ulmi . The Himalayan Ophiostoma also exhibits a unique combination of physiological and morphological characteristics including a distinctive colony type, ability to produce synnemata on malt extract agar, production of perithecia with long necks, a very high level of cerato-ulmin toxin production in liquid shake cultures, and moderate to strong vascular wilt pathogenicity on U. procera . Its reproductive isolation from O. ulmi and O. novo-ulmi and its other biological features demonstrate that it is a distinct sibling species from O. ulmi and O. novo-ulmi , and it is designated as a new species, Ophiostoma himal-ulmi sp. nov. The discovery of O. himal-ulmi should help resolve the problem of the origins of Dutch elm disease, while the occurrence of an apparently endemic Dutch elm disease system in the Himalayas may present new opportunities for the biological control of the disease elsewhere. These possibilities are discussed.

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.

In vitro growth and microcycle conidiation of Idriella bolleyi, a biocontrol agent of cereal pathogens

The minimum nutrient requirements for growth of Idriella (= Microdochium ) bolleyi in shaken liquid culture were mineral salts, nitrate nitrogen, glucose and thiamine. Typical batch culture kinetics were displayed in this medium and were assessed by culture turbidity when sodium alginate was used to induce growth by dispersed mycelia. Conidia were produced during exponential growth and reached maximum numbers ( ca 6 × 10 7 ml −1 ) by the deceleration or early stationary phase. Applying the Monod equation to culture parameters at different glucose concentrations, the minimum doubling time in glucose-nitrate-thiamine medium at 25 °C was calculated as between 5·25 and 6·1 h, and K s (limiting substrate concentration at half maximum specific growth rate) was between 0·049 and 0·077% glucose. Conidial germination was oxygen-dependent but did not require nutrients. On water agar or 0·1% glucose agar most spores germinated by germ-tubes, but up to 15% of spores swelled and produced further conidia (2–5) from one or both poles of the parent spore. This microcycle conidiation was suppressed at higher glucose levels. The findings are discussed in relation to production of biocontrol inocula and efficiency of I. bolleyi in biocontrol on roots.

Effect of prehelminthosporol, a phytotoxin produced by Bipolaris sorokiniana, on barley roots

The effects of prehelminthosporol, a toxin produced by Bipolaris sorokiniana, were investigated on barley roots. A concentration of 30 μg∙mL−1 prehelminthosporol or higher in nutrient solution significantly increased the rate of nuclear disintegration in the root cortex cells. The toxin also increased leakage of ATP from intact roots of barley seedlings grown in nutrient solution, but the growth rate of the seedlings was not significantly affected. However, diluted filtrate from liquid shake cultures of the fungus inhibited the growth of the seedlings by 21 – 73% over a 5-day period. The results indicate that the toxin may play an important role in pathogenesis by killing or weakening plant cells in advance of the growing hyphae and facilitating nutrient uptake and further growth of the fungus in plant tissue. Different cultivars of barley differed in sensitivity to prehelminthosporol, but sensitivities were not correlated with known levels of resistance to B. sorokiniana. Key words: Cochliobolus sativus, Hordeum vulgare, root cortex, toxin, prehelminthosporol, ATP.

Effect of temperature on growth and cerato-ulmin production of Ophiostoma novo-ulmi and O. ulmi

Ophiostoma novo-ulmi and O. ulmi , previously named ‘aggressive’ and ‘non-aggressive’ subgroups of the old species O. ulmi , are distinguished by a wide range of morphological, molecular, genetical and physiological characters, observed both in vivo and in vitro . They show different virulence on elms of moderate resistance, that seems to be correlated with the ability to synthesize phytotoxic compounds, of which the most important is cerato-ulmin (CU). To date, O. novo-ulmi isolates have been considered the greatest producers of CU in contrast to O. ulmi isolates, that synthesize very little or no toxin. We demonstrate that the synthesis of CU is temperature-dependent and that the two species seem to have different temperature optima for its production. However, while the difference in CU production is linked to temperature, it is not a consequence of differences in fungus growth in liquid shake culture caused by these temperature differences.

大型培養槽を用いた多芽体の大量増殖に関する研究

The shoot primordium is a suitable inoculant for large scale culture of shoots comparing with the shoot. itself. A culture method of multiple shoots starting from the shoot primordium was established. The influence of scale up conditions on the growth of multiple shoots originating from the primordia was also investigated.Shoot primordium of Stevia rebaudiana has been induced and maintained in modified MS liquid medium containing NAA and BA. The primordia grew constantly by about 7 times in 1 month in the liquid shake culture. The multiple shoots were then induced efficiently from the primordia by eliminating the growth hormones from the MS medium. They grew constantly by about 20 times in 1 month in the liquid shake culture. The growth of shoots in culture vessels of different sizes, 0.3 l (flask), 6 l (jar fermentor) and 500 l (bioreactor), were compared by inoculating the primordia at a fixed density of about 10 gl-1. The shoots grew actively to fill up culture vessels within a month, and the final weight of shoots per unit volume of medium was similar in each culture vessel. This result strongly suggests that multiple shoots can be mass propagated in a large scale without affecting the culture efficiency.

Growth and formation of true conidia by Metarhizium flavoviride in a simple liquid medium

In shaken liquid culture on media containing sucrose and brewers' yeast or peptone, 5 isolates of Metarhizium flavoviride from acridoid hosts produced submerged sporogenous cells and spores morphologically indistinguishable from aerial phialides and conidia. Submerged conidia produced by IMI 330189 were 4·6 × 2·6 m, within the size range described for M. flavoviride var. minus aerial conidia. Submerged hyphae and conidia contained green pigment. One of four isolates of M. anisopliae from acridids and other insects produced non-pigmented hyphal bodies (‘blastospores’) by budding under similar conditions. Production of vegetative biomass in M. flavoviride was enhanced by increasing the concentration of either the carbohydrate or the organic nitrogen component. The onset of conidiation was controlled both by the ratio of yeast to sucrose and the quantity of yeast in the medium. Increasing the yeast concentration delayed the onset of conidiation, suggesting that this was triggered when the nitrogen component was depleted. After initiation, conidiation was limited by carbohydrate availability. More than 1·5 × 109 conidia ml−1 were formed after 7 d in media containing 20 g yeast and 20 g sucrose or 30 g yeast and 30 g sucrose l−1.

Deoxyscytalidin and Lignicol: New Metabolites from Scytalidium Species

As part of a chemosystematic study of the genus Scytalidium several strains belonging to the closely related species Scytalidium album, Scytalidium lignicola, Scytalidium aurantiacum and Scytalidium circinatum have been grown in liquid shake culture. The various strains examined did not show a consistent pattern of metabolites. Scytalidin [1], the major metabolite of Sc. album was produced by two of three tested strains but not demonstrated by any strain of Sc. lignicola, Sc. aurantiacum or Sc. circinatum. The scytalidin-producing strains also produced a closely related but undescribed metabolite, deoxyscytalidin [2]. One strain of Sc. lignicola produced a new compound which we call lignicol [3] [...]

Assessment of liquid culture procedures for in vitro propagation of Rheum emodi

Micropropagation of an endangered Indian medicinal plant, Rheum emodi Wall., was achieved on Murashige and Skoog's medium using different liquid culture procedures. Liquid static (submerged, semi-submerged and with filter paper bridge) and shake (80 and 120 rpm) culture procedures were assessed for their effects on growth and multiplication rates. Best results were obtained using liquid shake cultures, which resulted in 50% reduction in medium requirement, 37.5% reduction in time and 1.5–2.2 fold increase in growth and multiplication rate. Liquid culture-raised plantlets facilitated easy transplantation and 90–95% survived transfer to potting mix in glasshouse.

Influence of iturin A on mycelial weight and aflatoxin production byAspergillus flavus andAspergillus parasiticus in shake culture

Iturin A, a peptidolipid produced byBacillus subtilis, inhibits growth of a large number of fungi. In this study, the effects of iturin A were evaluated on nine isolates ofA. flavus and seven isolates ofA. parasiticus in liquid shake culture. The mycelial dry weight of theA. flavus isolates was not significantly influenced by iturin A, however, there was a significant reduction in mycelial dry weight for two of theA. parasiticus isolates. Aflatoxin production was significantly reduced in five of theA. flavus isolates and three of the six aflatoxigenicA. parasiticus isolates. For the other seven isolates, aflatoxin levels were either unchanged or significantly increased in the presence of iturin A. These results indicate that iturin A does not consistently reduce growth or aflatoxin production of these fungi in pure culture.

Regulated expression of the nor-1 and ver-1 genes associated with aflatoxin biosynthesis

RNA transcript accumulation for the ver-1 and nor-1 genes, which are associated with aflatoxin biosynthesis in the fungus Aspergillus parasiticus, was measured before and during aflatoxin production in liquid shake culture. Transcripts were not detected until near the end of trophophase (growth phase) and could still be observed well into stationary phase during batch fermentation in an aflatoxin-supporting growth medium. Maximum accumulation of both transcripts occurred just prior to the onset of stationary phase. Aflatoxin B1 was first detected approximately 8 h after the appearance of the ver-1 and nor-1 transcripts. In contrast, maximum transcript accumulation for the pyrG gene (encoding orotidine monophosphate decarboxylase), which is involved in primary metabolism, was observed at the onset of trophophase when the ver-1 and nor-1 transcripts could not be detected. Accumulation of the ver-1 and nor-1 transcripts was also studied following a nutritional shift from a non-aflatoxin-supporting medium (peptone mineral salts [PMS]) to a glucose-containing medium (glucose mineral salts [GMS]), which does support aflatoxin biosynthesis. Transcripts from ver-1 and nor-1 could not be detected on PMS medium but did accumulate approximately 4 to 7 h following transfer to GMS medium. Additionally, aflatoxins were not detected in PMS medium but were observed to accumulate within 24 h after the shift from PMS to GMS. These data suggest that aflatoxin biosynthesis is in part regulated by the accumulation of the ver-1 and nor-1 transcripts.

Degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Degradation of the BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes) group of organopollutants by the white-rot fungus Phanerochaete chrysosporium was studied. Our results show that the organism efficiently degrades all the BTEX components when these compounds are added either individually or as a composite mixture. Degradation was favored under nonligninolytic culture conditions in malt extract medium, in which extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) are not produced. The noninvolvement of LIPs and MNPs in BTEX degradation was also evident from in vitro studies using concentrated extracellular fluid containing LIPs and MNPs and from a comparison of the extents of BTEX degradation by the wild type and the per mutant, which lacks LIPs and MNPs. A substantially greater extent of degradation of all the BTEX compounds was observed in static than in shaken liquid cultures. Furthermore, the level of degradation was relatively higher at 25 than at 37 degrees C, but pH variations between 4.5 and 7.0 had little effect on the extent of degradation. Studies with uniformly ring-labeled [14C]benzene and [14C]toluene showed substantial mineralization of these compounds to 14CO2.

液体振とう培養によるイチゴ（Ｆｒａｇａｒｉａ ａｎａｎａｓｓａ）の大量増殖に関する研究 シュートの成育におよぼすサイトカイニンとＡＢＡの影響

The effects of different cytokinin levels on multiplication of axillary shoots during culture on agar medium and subsequent growth of shoots in liquid shake culture were examined. The number of axillary shoot buds was highest(about 100) in culture medium containing 1mg/l of 4PU (one kind of cytokinin).Tissue segments with multiple shoot buds thus obtained were further grown in liquid medium by shake culture. The growth of shoot buds into plantlets in shake culture was examined for liquid media containing 0, 0.1 and 1mg/l 4PU in combination with 0, 0.1, 0.3 and 1mg/l ABA. The growth was most faborable in the medium containing 1.0mg/l 4PU in combination with 0.1mg/l ABA. Individual shoots grown under this condition were easily separated by hands, transplanted, and established satisfactorily in soil.

Germination of mcm microconidia of Neurospora crassa

Uninucleate microconidia of high germinability are desirable for measurement of nuclear ratios, recovery of homokaryons following transformation by DNA and for isolation of mutants. The mcm genotype of Neurospora crassa (RM 39-8A; FGSC 7091, a sixth generation progeny from a cross of 74-ORS-6a x Vickraman A) produces abundant microconidia by microcycle microconidiogenesis in liquid shake cultures at 22oC (Maheshwari 1991 Exp. Mycol. 15:346-350).