In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l^–1 resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l^–1 gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.     

In vitro Post-germination Growth and Development of Embryos of Alectra (Scrophulariaceae)

In vivo, seeds of the obligate root parasite Alectra vogelii Benth. (Scrophulariaceae) germinate only after being soaked in water for a period of time (pretreatment) followed by stimulation by certain factors exuded from a host root. Germinated seedlings do not develop beyond radicle emergence, and finally die, unless their radicles make contact with and penetrate into a host root conductive system. In vitro, germinated embryos obtained by exposing sterilized and pretreated seeds to root exudate of Vigna unguiculata were aseptically cultured on Knop's, White's and Murashige and Skoog's media. The embryos grew into seedlings with shoots and roots on a medium containing mineral salts and sucrose, but not on mineral salts alone. Seedling performance was generally not improved when the mineral salts-sucrose media were supplemented with vitamins. Shoot extension growth was better on Murashige and Skoog's mineral salts-sucrose medium than on Knop's or White's medium. However, seedling development was greatly boosted when cultivated on White's minerals salts-sucrose medium supplemented with coconut milk. Seedlings turned green on transfer to light but did not flower. The successful culture of these embryos and seedlings on a simple, chemically defined medium of mineral salts and sugar suggests that these nutrient components are the minimal external requirements for stimulation and support of normal seedling growth. These may be obtained in vivo by the parasite's tapping of the host root conductive system.     

Growth and anthocyanin production by carrot suspension cultures grown under chemostat conditions with phosphate as the limiting nutrient

Chemostat cultures of carrot suspension cultures, where growth was limited by the concentration of phosphate in the input medium, were achieved by replacing a fixed proportion of the culture with fresh medium at daily intervals. In the range 0.05–0.30mM phosphate in the input medium and at a specific growth rate of 0.357 days^?1, steady-state culture density but not anthocyanin in the cells was strictly proportional to the input phosphate concentration with no intercept. At a phosphate concentration of 0.10mM and growth rates from 0.105 to 0.430 days^?1, the steady-state culture density could not be described by Monod's model of chemostat cultures, but could be described by Nyholm's model. The steady-state levels of anthocyanin were not strictly proportional to the steady-state biomass under all conditions, showing that anthocyanin production is not completely growth associated.     

A Simplified Medium for the Growth of Maize (Zea mays) Endosperm Tissue in Suspension Culture

In vitro cultures of maize (Zea mays L.) endosperm derived from the dent inbred A636 have been maintained in liquid culture using Straus' medium for over six years. We have studied the growth of this tissue in four basic media and various modifications of the organic constituents of these media. Auxins and kinetin did not improve growth rate or degree of cell dispersion and thiamine (0.4 mg/l) was the only vitamin required by this tissue. Growth equal to that in the standard Straus medium and improved cell separation was obtained in a medium containing only inorganic salts, sucrose, and thiamine. Although asparagine was not required when high quantities of NH₄NO₃ and KNO₃ were included, more rapid growth was obtained when 2 g/l of asparagine was added. The simplified medium reported in this paper should facilitate the use of maize endosperm tissue in various studies of metabolism, hormone biosynthesis, etc.     

MORPHOGENETIC STUDIES ON HAWORTHIA: ESTABLISHMENT OF TISSUE CULTURE AND CONTROL OF DIFFERENTIATION

Investigations were carried out on the in vitro morphogenetic responses of inflorescence segments and gynoecia of several species of Haworthia (Liliaceae). Morphogenetic responses of explants were not species specific. It was found that coconut milk was essential for the growth and differentiation of Haworthia tissue if White's basal medium was used. However, growth and differentiation could be supported by a modified Murashige and Skoog's medium, without any supplements. The investigations demonstrated’ the importance of inositol and ammonium nitrogen in the nutrition of Haworthia tissue cultures. A chemical control of callusing and shoot and root differentiation was obtained by providing appropriate amounts of auxin and cytokinin in the culture medium.     

A Procedure for Comparing Growing and Non-growing Tetrahymena Cultures: Ribonucleotide Pools in Tetrahymena Starved for the Essential Amino Acid Histidine1

ABSTRACT A new starvation procedure permitted the study of early events in a protozoon's growth cycle. Growing cultures of Tetrahymena that differed from non-growing cultures by one variable were produced by adding histidine to cells deprived of that amino acid in an otherwise complete medium. Alterations of the nucleotide pools were examined in +His and in -His cultures in the period preceding RNA synthesis by cells in +His medium. High performance liquid chromatographic analysis provided a balance sheet for the difference in purine compounds in the two cultures. The change in rNTP levels occurred only when the cells were resuspended in a fresh medium and was not a function of cell density. These observations point to the presence of a factor(s) in the old medium that inhibits the energy charge increase in rNTP and in purine accumulation.     

DEVELOPMENT OF PLANTS FROM PROTOPLASTS OF SOLANUM (SOLANACEAE)

Experiments were performed to determine conditions critical to the isolation and culture of protoplasts from leaf mesophyll cells of a Solanum Section Tuberarium diploid clone, a S. phureja × S. chacoense F₁ hybrid. The optimum concentration of cellulase (Cellulysin) was 0.4%, while pectinase (Macerase) was inhibitory, even at a low concentration (0.075%). Maximum yields of protoplasts were achieved when the enzyme solution was not changed during incubation, and slow oscillation (60 rpm) on a shaker was used. When detached leaves were held under low light intensities or in the darkness for 3–5 days prior to protoplast production, results were more consistent than when the leaves were used directly from the greenhouse. Following dark or low light treatment the optimum osmolarity of the isolation and growth media was approximately 0.3–0.4 M. Of nine growth media tested only those of Nitsch and Ohyama and of Upadhya supported callus development, but the callus was often loose and did not differentiate roots or shoots. Callus from protoplasts cultured in the Upadhya medium modified by addition of glycine, vitamins, and casein hydrolysate, and subsequently transferred to the medium of Lam, formed roots and shoots when cultures were maintained in light. Mature plants were obtained following transfer to modified White's medium and later transplantation to soil.     

Propagation of lettuce (Lactuca sativa) breeding material by tissue culture

A tissue culture method using Murashige and Skoog's (MS) medium was devised to propagate healthy plants from field grown lettuce plants selected for seed production. Explants (2–3 mm long) from axillary buds were successfully grown on MS + 1.0 or 2.0 mg litre^-1 kinetin and 6.4 mg litre^-1 IAA to promote shoot growth. Concentrations of 0.5 and 4.0 mg litre^-1 kinetin gave poor shoot growth. The cultures were successfully rooted after 3–4 wk on MS + 6.4 mg litres^-1 IAA after transfer from MS + 1.0 mg litre^-1 kinetin and on MS + 4.8 mg litre^-1 IAA after transfer from MS + 2.0 mg litre^-1 kinetin. Concentrations of 3.2 and 8.0 mg litre^-1 IAA gave poor root initiation. Root initiation was more successful when cultures were grown at 40 Wm^-2 than in cultures grown at 5 Wm^-2. Rooted cultures were established in compost with a 90–95% success rate and the regenerated plants flowered c. 18 wk after the cultures were initiated.     

GROWTH AND HABITUATION IN TISSUE CULTURES OF ENGLISH IVY,HEDERA HELIX

Tissue cultures from both juvenile and adult stems of English ivy, Hedera helix L., were established in White's medium supplemented by coconut water and auxin (usually naphthalene acetic acid). With repeated transfers, cultures were habituated in less than a year to grow well without coconut water by using an auxin and kinetin. Cultures from juvenile seedlings were less demanding in requirements for growth. In all types of cultures occasionally small areas of rapidly growing cells were noticed. These when isolated gave rise to rapidly growing cultures with many cells of unusual appearance. Abnormally long cells and chain-type cells were abundant. When 0.1 mg/ liter of kinetin was added to the medium, these cells grew well without auxin and coconut water.     

Fungi and Gram-negative bacteria as soilborne minor pathogens of goat's rue (Galega orientalis Lam.)

Fungi and Gram-negative bacteria were isolated from inside the roots of field-grown goat's rue (Galega orientalis). Fungi were isolated from three plants out of a total of 45 tested. Two multinuclear Rhizoctonia solani isolates were identified to the anastomosis group 5 (R. solani AG-5-Gal) using pairings with known AG test cultures. One fungal isolate was identified to Phoma chrysanthemicola. Gram-negative bacteria were isolated from three plants out of 25 tested. They were identified using classical methods, the BIOLOG identification system based on the utilisation of 95 different carbon sources, and the MIDI system for the analysis of whole cell fatty acids. The two latter systems were computer-associated and utilised an extensive reference library of isolates. One bacterial isolate was identified as Enterobacter agglomerans and two isolates as Pseudomonas marginalis. R. solani AG-5-Gal reduced the emergence of Lupinus luteus, L. polyphyllus and french bean (Phaseolus vulgaris) and the growth of broad bean (Viciafaba), L. luteus and french bean, but did not cause obvious damage in goat's rue and pea (Pisum sativum). However, R. solani AG-5-Gal was re-isolated from the roots of all the test plant species following inoculation. P. chrysanthemicola reduced the emergence of L. polyphyllus and the growth of goat's rue, french bean and broad bean, and it was re-isolated from all of the test plant species (except for french bean) following inoculation. All the bacteria reduced the emergence of french bean, but not that of goat's rue and pea, when applied to the soil. When the roots were dipped into bacterial suspension, all the bacteria damaged french bean and L. polyphyllus. Additionally, P. marginalis JV3 damaged goat's rue and red clover. The pathogenicity of the fungi and bacteria were not changed when they were double-inoculated in pairs, except for R. solani AG-5-Gal and P. marginalis JV2 which reduced the emergence of goat's rue when inoculated together but not when inoculated separately.     

The effect of culture growth phase on induction of the heat shock response in Yersinia enterocolitica and Listeria monocytogenes

The effect of culture growth phase on induction of the heat shock response in Yersinia enterocolitica and Listeria monocytogenes, was examined. Exponential or stationary preconditioned cultures were heat shocked and survivor numbers estimated using selective and overlay/resuscitation recovery techniques. The results indicate that prior heat shock induced increased heat resistance in both micro-organisms to higher heat treatments. Heat-shocked cells of each micro-organism were able to survive much longer than non-heat-shocked cells when heated at 55 degrees C. The size of the change in heat resistance between heat-shocked and non-heat-shocked cells was greatest for exponential cultures (X:X). Results indicate that the overall relative thermal resistance of each pathogen was dependent on cell growth phase. Stationary cultures (S:S) were significantly (P < 0.01) more thermotolerant than exponential cultures (X:X) under identical processing conditions. Under most conditions, the use of an overlay/resuscitation recovery medium resulted in higher D-values (P < 0.05) compared with a selective recovery medium.     

Nutritional Requirement for Production of Antifungal Substance by Enterobacter aerogenes and Bacillus subtilis Antagonists of Phytophthora cactorum1

Anker's medium with glucose and Thornton's medium were most suitable for growing Enterobacter aerogenes and Bacillus subtilis respectively, antagonists of P. cactorum, the causal agent of apple crown rot. Calcium nitrate was thebest source of nitrogen for growing cultures of E. aerogenes and B. subtilis. E. aerogenes produced the maximum amount of antifungal substance at 200 and 400 mg/l of nitrogen in the medium. Phosphate supplied either in the potassium or calcium form did not change the growth of either antagonist. An addition of 200 mg/l of N and 400 mg/l of P significantly enhanced the production of antifungal substance by E. aerogenes on Anker's medium with glucose. Thornton's medium supplemented with 200 mg/l of N and 100 mg/l of P produced the maximum amount of antifungal substance from B. subtilis. Generally, soil extracts without enrichment did not support the growth of either antagonist; E. aerogenes required at least 400 mg/lof, both N and P while B. subtilis required 200 mg/l of N and 800 mg/l of P for the maximum production of antifungal substance. When ammonium phosphate was added to soil extracts, only a small amount of antifungal substance was produced by E. aerogenes and none by B. subtilis. These results indicate that E. aerogenes and B. subtilis need N and P fertilization of the sterilized soil for the maximum production of the antifungal substance that inhibits the growth of P. cactorum.     

CYCLOMORPHOSIS IN SCENEDESMUS ARMATUS (CHLOROPHYTA): AN ORDERED SEQUENCE OF ECOMORPH DEVELOPMENT1

Scenedesmus armatus (Chod.) Chod, growth and morphology were monitored in medium 7 (oligotrophic) and Bristol's medium (eutrophic); cultures in both media were incubated at 10 and 22° C. Growth rate at 10° C was reduced, i.e. only one doubling in 7 days in medium 7 and 2.3 doublings in Bristol's, compared to 4.3 and 6 doublings at 22° C over the same period. Unicells as well as cells of colonies were larger at the cold temperature. The lengths of cells were not significantly different regardless of temperature or medium, but cell width was markedly increased at the lower temperature. Additionally, an arcuate, eight-celled, multispined ecomorph, which resembled several previously described taxa, was produced at 10° C. It becomes a component of a previously published ordered sequence of ecomorph development for this species. Based on data now accumulated in both the laboratory and the field, these temporal changes are interpreted to be a cyclomorphosis, driven by a coupling of nutrient availability and temperature. Within the addition of new cold temperature (spring) ecomorphs, the ordered sequence of ecomorphs for S. armatus is a succession from unicells to multispined eight-celled colonies to quadricaudate colonies, ending with acaudate four-celled ecomorphs.     

NUTRITIONAL AND MORPHOGENETIC INVESTIGATIONS ON CALLUS CULTURES OF NEOMAMMILLARIA PROFILERA MILLER (CACTACEAE)

Neomammillaria prolifera (Cactaceae), when grown on Murashige and Skoog's medium supplemented with fresh coconut milk, showed very little growth. Various concentrations and combinations of growth regulators which did not cause callusing had no apparent effect on the normal growth rates of intact plants. Healthy green calli obtained on a 2,4-D and kinetin-containing medium exhibited extremely fast growth and very specific growth requirements. Relatively high amounts of 2,4-D (10–20 mg/liter), kinetin (1–2 mg/liter), and coconut milk (20–60%) were required at all times for continued proliferation of callus on subculturing. Moreover, the callus was very tolerant to extremely high concentrations of other growth regulators (IAA, NAA, IBA, and GA up to 100 mg/liter) in the presence of 2,4-D and coconut milk. These substances could not replace 2,4-D for callusing or continued growth of callus. It was not possible to establish root cultures or to induce callusing of roots. Attempts to induce differentiation in callus were unsuccessful, except for sporadic root initiation in some cultures. A comparison of these results with similar studies on other succulents demonstrates some basic physiological similarities among this group of plants.     

Somatic embryogenesis from cell suspension and protoplast cultures ofCapsella bursa-pastoris (L.) Medic

Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium. These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization.     

Soybean [Glycine max (L.) merrill] embryogenic cultures: The role of sucrose and total nitrogen content on proliferation

Summary To improve proliferation of soybean cultures in liquid medium, the effects of sucrose; total inorganic nitrogen; content of No₃ ⁻, NH₄ ⁺, Ca²⁺, PO₄ ³⁻, K⁺; NH₄ ⁺/NO₃ ⁻ ratio; and medium osmotic pressure were studied using cv. Jack. Sucrose concentration, osmotic pressure, total nitrogen content, and ammonium to nitrate ratio were found to be the major factors controlling proliferation of soybean embryogenic cultures. Growth decreased linearly as sucrose concentration increased from 29.7 mM to 175.3 mM. A sucrose concentration of 29.2 mM, a nitrogen content of 34.9 mM, at 1 to 4 ammonium to nitrate ratio were found to be optimal for the fastest proliferation of soybean embryogenic cultures. There was no significant effect on proliferation of cultures when concentrations of NH₄ ⁺, Ca²⁺, PO₄ ³⁻, and K⁺ were tested in the range of 3.50 to 10.50, 1.02 to 3.06, 0.68 to 2.04, and 22.30 to 36.70 mM, respectively. The relative proliferation of embryogenic cultures of four soybean genotypes was evaluated in Finer and Nagasawa medium and in the new medium formulation. Despite genotype-specific differences in growth, the genotypes tested showed a biomass increase in the new formulation equal to 278, 269, 170, and 251% for Chapman, F138, Jack, and Williams 82, respectively, relative to their growth on standard FN medium. Due to its lowered sucrose and nitrogen content, we are referring to the new medium as FN Lite.     

Production of <Emphasis Type="Italic">Cocconeis neothumensis</Emphasis> (Bacillariophyceae) biomass in batch cultures and bioreactors for biotechnological applications: light and nutrient requirements

Cocconeis neothumensis, a benthic diatom living as epiphyte on Posidonia oceanica leaves, was hypothesised to synthesize secondary metabolites inducing apoptosis in the androgenic gland of the protandric shrimp Hippolyte inermis. The optimization of C. neothumensis cultures is a primary aim in order to identify and characterise the active compounds produced by this diatom. Light intensity and concentration of nutrients, such as silicates and selenium, are of major importance in determining the growth rate of diatoms and the maximum produced biomass. Thus, we evaluated the growth performances of C. neothumensis cultures at different light intensities, corresponding to those recorded in Posidonia oceanica meadows in April, when the induction of sexual reversal in Hippolyte inermis occurs; the growth performances were also determined in relation to different selenium and silicate availability in the culture medium and the possible interactions between these two micronutrients were evaluated. Two methods of cultivations were compared: batch cultures in Petri dishes and cultures in a bioreactor with a continuous medium flow in the system. C. neothumensis showed a faster growth at low light intensities, although with a good acclimation capacity in the range from 60 to 140 μmol photons m⁻² s⁻¹. The presence of selenium in the medium improved both the exponential growth rate and the maximum cell density. The same results were evidenced for a silicate concentration double that in Guillard f/2 medium. On the other hand, a simultaneous increase of silicate concentration and the presence of selenium led to a loss of the positive effects detected with single nutrients. This result suggests either a possible antagonism in selenium and silicate uptake or a negative interaction between these two micronutrients in Cocconeis. The yields of the two tested cultivation methods calculated in terms of diethyl ether extract dry weight per unit of substrate area were highest for the bioreactor.     

PHOSPHATE-LIMITED GROWTH KINETICS OF SELENASTRUM CAPRICORNUTUM (CHLOROPHYCEAE)1

Most theoretical studies of phytoplankton growth in aquatic environments assume that relative nutrient utilization abilities regulate species composition. The steady-state phosphate-limited growth kinetics of Selenastrum capricornutum Printz were examined using continuous cultures to characterize the green alga's ability to compete for orthophosphate (Pi) when Pi limits growth. The maximal specific growth rate for Selenastrum at 20 C was 1.20 day^?1, and the concentration where half maximal growth rate occurs was 40 nM Pi. There was an apparent threshold of 10 nM Pi. Cell yields varied inversely with growth rate; thus ability to utilize Pi could not be characterized in terms of the Monod half-saturation constant and maximal growth rate. Instead, we computed the Pi affinity from steady-state flux vs. external Pi concentrations. This affinity was 2.8 l·mg dry wt^?1· day^?1 for Selenastrum. Kinetic evidence from this study suggests that Selenastrum will not be growth competitive with some other common aquatic heterotrophs and autotrophs when Pi limits microbial growth in lakes.     

Carbohydrate supplements to the callus culture medium modify the growth of potato tuber moth larvae feeding on Lycopersicon chmielewskii callus

Lycopersicon esculentum and L. chmielewskii are respectively susceptible and resistant to the potato tuber moth (Phthorimaea operculella Zeller) in the field. Feeding bioassays were conducted with the herbivore caterpillars reared on callus derived from both tomato species and grown in vitro, and the influence of carbohydrate supplements to the callus culture medium, on the insect's feeding behavior was investigated. Newly-hatched larvae fed with L. esculentum or L. chmielewskii callus raised on a medium with 88 mM sucrose, reached a weight of 12–15 mg and 1.5–3.0 mg, respectively, within 9 days. Restriction of larval weight increase in insects reared on L. chmielewskii callus, disappeared when the host tissue was transferred 24 h prior to the callus-insect assay to a medium supplemented with 264 mM of either sucrose, glucose, fructose or mannose. The capability of L. chmielewskii callus to restrict growth of larvae was restored in host tissue retransferred from a medium with 264 mM sucrose to a 24-h incubation on one supplemented with 264 mM of either mannitol, sorbitol, glycerol or myo-inositol, before the callus-insect bioassay. The larval growth response remained unaltered by callus incubated on a medium with 264 mM xylose. The ameliorating effect on insect growth of high sucrose in the callus medium was not due to sucrose as an ingredient of the insect's diet. The diverse response of L. chmielewskii callus, and its dependence on the type of carbohydrate in the medium, rule out effects of these substances as nonspecific medium osmotica. The swift callus responses to carbohydrates (within hours of a change in medium composition), as reflected in the insect's growth, were not accompanied by visible morphological variations in the host tissue. We suggest that suppression by high levels of exogenously applied saccharides and derepression by exogenous polyols and myo-inositol of the impedement to growth of the potato tuber moth larva, reflect the existence in L. chmielewskii of a carbon metabolic control mechanism of gene expression whose products affect insect growth.     

Effect of perfluorodecalin as an oxygen carrier on actinorhodin production by Streptomyces coelicolor A3(2)

Perfluorodecalin, a perfluorocarbon (PFC), was used in this investigation as a dissolved oxygen carrier in the media of Streptomyces coelicolor cultures. The effects of different concentrations of PFC, PFC emulsified with pluronic F-68 and pluronic alone were investigated in the shake-flask cultures using both defined and complex media. In the defined medium with PFC alone, the maximum biomass and actinorhodin concentrations and the volumetric substrate consumption rates increased with increasing PFC concentration. They decreased dramatically, however, when the PFC concentration exceeded 50% (v/v). Emulsifying the PFC with pluronic F-68 resulted in a significant increase in antibiotic concentration while growth was unaffected. The inclusion of more than 4 g/l pluronic alone in the fermentation medium inhibited the growth. In the complex medium with 40% (v/v) PFC, although the final antibiotic concentration was unaffected, the onset of actinorhodin accumulation was 2 days earlier than that in the control. It was demonstrated that PFC and emulsified PFC did not have any deleterious effects on S. coelicolor cultures.