Growth and biosynthesis of rugulovasines in Penicillium variabile Sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

Mechanism to combat cobalt toxicity in cobalt resistant mutants of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Characteristics of fungal species tolerant to high levels of metals in natural environment can be amplified by isolation and selection of resistant mutants. Step-by-step culturing led to identification of highly stable Co-resistant (Co^R) mutants of A. nidulans. Based on two distinct morphological features, Co-resistant mutants were categorized as Co^RI and Co^RII. The two mutants varied in their growth behavior and colony morphology that were reflected in supplemented as well as unsupplemented growth media over the generations. As compared to the Co^RI, Co^RII mutant exhibited sparse mycelia and conidiation but secreted higher amount of melanin. Co^R mutants could tolerate up to 2.5mM Co in the medium, however, required a threshold concentration of 0.25mM Co for optimal growth and germination. Absence of Co in the medium caused a stressful situation for the Co^R mutants and led to the secretion of a white extracellular precipitate found to be a glycoprotein. In response to interactions with Co-ions, Co^R mutants produced oxalic acid and bioprecipitated Co as Co-oxalate providing scope for metal reclamation as well as oxalic acid extraction. The mutants could help to recover the insoluble Co-oxalate salt from aqueous solutions by entrapping it in their growing mycelial meshwork.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

High frequency in vitro regeneration of<Emphasis Type="Italic">Kigelia pinnata</Emphasis> L. via organogenesis

An effective and reproducible protocol for the micropropagation ofKigelia pinnata L through high frequency callus regeneration is described. Seeds were surface sterilized before culturing on Gamborgs basal medium (B5 medium). After two weeks the cotyledonary node along with a portion of the hypocotyl were carefully excised from well-developed embryos and subcultured on B5 medium supplemented with different concentrations of 2,4-D and BAR The cultured cotyledonary node expiants showed callus formation at the base of the lower cut end of the hypocotyl. This callus showed shoot initiation after two weeks of subculture on the regeneration medium supplemented with various concentrations of BAP alone or in combination with NAA. The highest number of shoot regeneration occurred on medium containing 5 γM BAP and 0.1 γM NAA. The optimum rooting of the regenerated shoots was observed on 1/2B5 medium supplemented with 4 γM IBA. Micropropagated plants were successfully established in soil in field condition with a survival frequency of 100%.     

The fungus Penicillium variabile Sopp 1912 isolated from permafrost deposits as a producer of rugulovasines

It has been established that relict fungi Penicillium variabile Sopp can synthesize clavine alkaloids, rugulovasines A and B, which are revealed in this species for the first time. Submerged cultivation of the strain-producer revealed several microcycles of conidia formation. The synthesis of alkaloids was also of a cyclic character. The synchronism of cyclic rugulovasine biosynthesis and conidia formation was revealed. Zinc ions stimulated fungal growth but had a negative effect on the biosynthesis of rugulovasines.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

Development of Media and Automated Liquid Fermentation Methods to Produce Desiccation-Tolerant Propagules ofTrichoderma harzianum

A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process.     

Micropropagation of sweet viburnum (<Emphasis Type="Italic">Viburnum odoratissimum</Emphasis>)

A micropropagation protocol for shoot culture of sweet viburnum (Viburnum odoratissimum) is described. Nodal explants, initially established on MS medium, were transferred to WPM supplemented with combinations of BA and GA₃. Maximum shoot multiplication was observed on explants cultured on medium supplemented with BA concentration higher than 1.1 μM, and 14 μM GA₃. Although Stage II medium supplemented with BA concentration higher than 1.1 μM resulted in increased shoot multiplication, it also caused a decrease in shoot length. A negative carry over effect of GA₃ on rooting was observed in subsequent Stage III cultures. The presence of GA₃ in Stage II medium promoted shoot elongation, but it also caused a decrease in microcutting rooting. For this reason, 0.5 μM BA and 14 μM GA₃ were selected for optimum Stage II shoot multiplication. Although 100% microcuttings formed roots when cultured on medium containing 6.0 μM NAA, significant callus formation was observed and ex vitro survival rate was low (49%). Rooting was achieved after 3 weeks with 82% of microcuttings on medium supplemented with 3 μM IBA. The survival rate of plantlets under ex vitro conditions was 100% after 3 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 30 plants.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Development of a submerged-liquid sporulation medium for the johnsongrass bioherbicide<Emphasis Type="Italic"> Gloeocercospora sorghi</Emphasis>

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Gloeocercospora sorghi. In phase I, 18 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, butter bean and lima bean brines (1.5–4.6 mS/cm) provided best conidiation. In phase II, a fractional-factorial design was utilized to screen 76 different medium adjuncts in combination with butter bean brine for improved sporulation. d-Mannitol and carboxymethylcellulose (CMC) were the only acceptable factors that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 2.69 mS/cm butter bean brine +0.043 M d-mannitol +0.37% w/v CMC with an expected titer of 1.51×10⁷ conidia/ml. Actual mean titer attained with the model-derived medium was 1.91×10⁷ conidia/ml. Optimal sporulation occurred at 25.5°C in this medium and conidia remained viable up to 2.71 days when stored at 12°C. No significant difference was observed in virulence of conidia produced on agar vs washed conidia produced in the model-derived (liquid) medium.     

Induction of submerged conidiation of the biocontrol agent Penicillium oxalicum

Induction of submerged conidiation of Penicillium oxalicum has been examined using a range of synthetic and complex media and complex media supplemented with by-products of the brewing industry. Only one method (Morton's method), consisting of growth in a glucose/salts-based medium (C:N ratio 62.5, medium A) for 24 h and then transference to the same medium without a nitrogen source (medium B), induced conidiation. Levels of sporulation were significantly (P = 0.05) increased by addition of calcium or poly(ethylene glycol) 6000 to medium B. The optimum age for transference of the mycelium was 24 h and the optimum pH was 6. Calcium was an induction factor when added to medium A (C:N ratio 62.5) of Morton's method. It was concluded that nitrogen depletion and calcium addition to a medium with high C:N ratio are the factors inducing conidiation of P. oxalicum. Maximum levels of conidiation (35 × 10⁶ spores ml⁻¹) were obtained when the nitrogen level in medium A of Morton's method was further reduced (C:N ratio 142.9) and calcium (20 mM) was added. These results are the essential starting point to investigate liquid fermentation systems for the biocontrol agent P. oxalicum. Received: 19 November 1996 / Received revision: 25 March 1997 / Accepted: 27 March 1997     

The histological analysis of indirect somatic embryogenesis on <Emphasis Type="Italic">Drosera spathulata</Emphasis> Labill

We studied indirect somatic embryogenesis in the callus tissue of Drosera spathulata Labill. originated from isolated leaves. Callogenesis was induced on MS medium (Murashige and Skoog 1962), supplemented with various concentrations of NAA and BA. Somatic embryos regenerated on half-strength MS medium supplemented with 20 μM of NAA or without growth regulators. The highest efficiency of somatic embryo production was achieved on hormone-free medium. Globular, heart-, torpedo- and cotyledonary-shaped embryos were observed in embryogenic clusters. Histological and scanning electron microscopy analysis verifies somatic embryogenesis. Regenerated plants were transferred to soil and were grown to maturity.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.