Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Influences of medium consistency and shoot density on in vitro shoot proliferation of Populus alba × P. grandidentata

The in vitro shoot proliferation of Populus alba × P. grandidentata was affected by the medium consistency and shoot density, but not by three sizes of vessels. After 4 weeks of culture, the fresh weight and number of shoots per explant on liquid medium were significantly greater than those on agar-solidified medium. In particular, 3.2 shoots, 7 mm or longer per explant, were produced on liquid medium compared with 1.6 shoots per explant or agar-solidified medium. The fresh weight per explant after 4 weeks of culture on liquid medium and agar-solidified medium were 0.68 and 0.25 g, respectively. Increasing the number of shoots per vessel slowed the growth of the explants as measured by fresh weight and the number of shoots produced. There was little difference in the number of shoots produced between vessels with 1 or 2 shoots per vessel, but there were many fewer shoots produced when 3 shoots were placed in each vessel.Journal Paper No. J-11977 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2210.     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus L

Summary Comparative studies of carnation micropropagation under four different ventilation rates showed that using gas-permeable filters, with gelled or liquid media and modifying the volume of culture medium, it was possible to establish a suitable hydric state to obtain good proliferation rates with gelled and liquid medium, as well as optimal acclimatization of microcuttings. The following parameters were measured: ventilation rate, gas exchange coefficients, relative water loss, increase of agar concentration, micropropagation rates, percentage of hyperhydricity, and acclimatization rates. Our results confirm that it is possible to avoid hyperhydric plants cultured in liquid medium with the use of ventilated culture vessels through the control of the water relations during the multiplication phase and, at the same time, keeping the micropropagation rate.     

Nutritional and gibberellic acid requirements in kiwifruit vitroponic cultures

Summary Residual macronutrients, carbon source, and gibberellic acid (GA₃) in the culture medium were measured throughout the whole culture period of kiwi (Actinidia deliciosa Chev. cv. Hayward) explants cultured in liquid medium using cellulose plugs as explant support. The objective of this study was to adapt the composition of the culture medium to an automated culture system with the possibility of applying 6-benzyladenine pulses so as to improve the efficiency of kiwifruit micropropagation. Ammonium and phosphate were the most consumed ions at the end of the culture period (35 d). Murashige and Skoog medium might be too rich for the culture of kiwifruit in liquid medium except with respect to phosphate which decreased significantly toward the end of the culture period. Sucrose, and the glucose resulting from the hydrolysis of the former, were taken up by the kiwi explants throughout the whole culture period. GA₃ absorption by the kiwi explants occurred gradually throughout the subculture, although 67% of this initially added plant growth regulator remained in the culture medium at the end of the 35-d culture period.     

Production of fruit-bodies of a mycorrhizal fungus,Lyophyllum shimeji,in pure culture

Cultivation of mycorrhizal fungus,Lyophyllum shimeji, was examined using selected strains capable of forming primordia in pure culture. Mycelia grew fastest on barley grains containing synthetic liquid medium. The primordia readily formed in test-tubes after lowering the incubation temperature from 23°C to 15°C. The co-existence of pine seedlings had no promotive effect on primordium formation. Fruit-bodies formed on a medium consisting of barley, beech sawdust, and liquid synthetic nutrients in 500-ml glass bottles. Mature fruit-bodies produced basidiospores. The spores thus produced could germinate on an agar medium and formed mycelial colonies. Thereby, the life cycle inL. shimeji was accomplished in pure culture without using the host plant.     

Attached Growth of Sphaerotilus natans in Continuous-Flow Apparatus and Its Growth Inhibition by 9-beta-d-Arabinofuranosyladenine

Sphaerotilus natans grew at the maximum specific growth rate (mu(max)) of 0.43/h when cultivated on PGY medium at 25 degrees C. The organism mainly grew attached to inside of the culture vessels when the culture medium was fed to the completely mixed continuous-flow apparatus at a dilution rate above mu(max) and the attached growth was directly related to the dilution rate. When a low concentration of the medium was supplied to the apparatus, almost all of the cells grown were filamentous and attached to the inside of the vessels. When a high concentration of the medium was fed, the organism grew as single cells or short chains and flowed out into the effluent. The attached growth of S. natans in the continuous-flow apparatus was inhibited by the minimal inhibitory concentration of 0.5 to 1.0 mug of 9-beta-d-arabinofuranosyladenine per ml. 9-beta-d-Arabinofuranosyladenine showed bacteriocidal activity against S. natans at a concentration of 50 to 100 mug/ml.     

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated,Thin-Film Liquid System for Micropropagation

Summary Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.     

Biotransformation of ferulic acid and vanillylamine to capsaicin and vanillin in immobilized cell cultures of Capsicum frutescens

Plant tissue culture medium which contained FeEDTA as sole iron source was incubated aseptically in light (16-h photoperiod, 100 mol m^-2 s^-1 PAR) at 20°C without plant tissue. Soluble iron dropped from an initial concentration of 4 mg 1^-1 to less than 0.1 mg 1^-1 in 4 weeks. This occurred in both glass and plastic culture vessels. No loss occurred when medium was incubated at 20°C in darkness. A further experiment showed that soluble iron concentration fell to <0.2 mg 1^-1 in only 4 days but the loss was slower at lower irradiances.Effects of the loss of soluble iron on plantlet growth were assessed by culturing single node stem segments of in vitro potato (Solanum tuberosum L. cv. Arran Banner) plantlets on medium previously exposed to light. Pre-exposure sufficient to reduce soluble iron concentration to <0.1 mg 1^-1 had no inhibitory effect on plantlet development in solidified medium or in liquid medium, except when the liquid medium had been centrifuged before inoculation to remove iron precipitated during pre-exposure to light. The plantlets then became chlorotic.     

Influence of ventilation closure, gelling agent and explant type on shoot bud proliferation and hyperhydricity in Scrophularia yoshimurae—A medicinal plant

Summary We report an improved procedure of in vitro propagation of Scrophularia yoshimurae&#x2014;a medicinally important plant species indigenous to Taiwan. Induction of maximum shoot buds (22.75 per explant) was obtained with shoot tip explant cultured on Murashige and Skoog medium supplemented with 1.0mgl^&#x2212;1 benzyladenine (BA) and 0.2mgl^&#x2212;1 &#x03B1;-naphthaleneacetic acid and gelrite using dispense paper (DP) for ventilation closure of culture vessels. The type of gelling agents (agar and Gelrite) affected both quantity and quality of the shoots induced. Using aluminum foil for ventilation closure resulted in a higher number of hyperhydric shoots. Hyperhydricity was reduced by culturing shoots on a medium devoid of plant growth regulators in conjunction with the use of DP. Plantlet growth in vessels using DP was healthier and all plantlets survived after being transplanted to soil.     

Micropropagation of endangered endemic Brazilian bromeliad Cryptanthus sinuosus (L.B. Smith) for in vitro preservation

An efficient liquid culture system for plant regeneration from leaflessstem–root axes of Cryptanthus sinuosus L. B. Smith(Bromeliaceae) was established. High regeneration rates (93%) were achieved inMurashige and Skoog's medium without growth regulators. Whole plants wereobtained in a single-step procedure, resulting in the production of 25.3± 3.6 plants/explant after 6 months of culture. Incubationof plant material at 35 ± 3 °C resulted in an increaseof 60% in the regeneration efficiency compared with tissues incubated at 28± 2 °C. Moreover, after 5–6 sub-cultures in thesame medium, the axes originated bud clusters that could be continuouslymultiplied and gave rise to 19.4 ± 3.2 whole plants per gram of freshmatter. It was estimated that the liquid culture system described is potentiallyable to produce about 4500 plants/explant/year. Rates of 98% acclimatizationwere achieved. The use of plants produced following this method for populationreinforcement and for in vitro preservation programs ofendangered populations is suggested.     

Factors influencing the production of hardened glaucous carnation plantlets in vitro

Medium type, its water status and the relative humidity in the culture vessel modified carnation leaf development in vitro. Carnation shoot apices cultured on liquid or on 0.8% agar solidified media developed into plantlets having succulent and translucent leaves which are not transplantable to non-aseptic conditions. Increasing the agar and/or sucrose concentration in the medium as well as decreasing the relative humidity in the culture vessel by a desiccant promoted glaucous leaf production. Increased water status (H₂O and relative humidity) increased shoot proliferation and translucency of leaves. Decreased water status reduced shoot proliferation but induced the formation of glaucous leaves. The culture of apices for 5–6 days on liquid medium prior to their sub-culture to 1.5% agar medium improved shoot proliferation and normal leaf development. An agar slant prevented the submergence of apices in water accumulating on the medium and thus reduced leaf translucency. Survival was further increased by the transfer of plantlets in uncapped culture vessels to a desiccator for 1–2 weeks prior to transplanting to soil.     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Growth of Suspension-cultured Acer pseudoplatanus L. Cells in Automatic Culture Units of Large Volume

A phytostat to mass culture higher plant cells in liquid medium is described. This apparatus allowed the culture in batch, turbidostat and chemostat of 20 liters of cells. Automatic control of cell suspension growth was based on culture turbidity. Changes with time of certain cell characteristics, particularly cell respiration and phospholipid content, indicated that the test time to harvest large amounts of sycamore cells (Acer pseudoplatanus L.) in good physiological state was about 2 days before the end of the exponential phase of growth, when the cell density reached one million cells per milliliter of culture.     

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Elicitation is a possible aid to overcome various difficulties associated with the large‐scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator‐free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor‐specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.     

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T_L-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l^–1.     

Plant cell factories as a source for anti-cancer lignans

The review places podophyllotoxin, a powerful anti-cancer material used in clinical treatment of small cell cancers, in focus. The economical synthesis of podophyllotoxin is not feasible and demand for this material outstrips supply. At present, Podophyllum hexandrum (Indian May apple) is the commercial source but it grows in an inhospitable region (the Himalayas) where it is collected from wild stands. Furthermore, the plant is now an endangered species. Alternative sources of podophyllotoxin are considered, e.g., the supply of podophyllotoxin and related lignans by establishing plant cell cultures that can be grown in fermentation vessels. Increase of product yields, by variation of medium and culture conditions or by varying the channelling of precursors into side-branches of the biosynthetic pathway by molecular approaches, are discussed.     

Effect of sucrose concentration on photosynthetic activity of in vitro cultures <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle) germlings

In this work, the effect of sucrose on photosynthetic activity during in vitro culture was studied. Experiments were carried out using uniform somatic embryo-derived germlings of Gentiana kurroo (Royle) confirmed by chromosome counting and flow cytometry technique. Photosynthetic activity was measured by chlorophyll a fluorescence and gas exchange method. The efficiency of photosynthetic apparatus as measured by the ratio F _v/F _m, Yield and qP (light phase of photosynthesis) was the highest when the medium was supplemented with 0.3% sucrose which well corresponded with plant gas exchange. Taking all data into consideration for the best development of photosynthetic apparatus and the most efficient of net photosynthesis of studied germlings would be medium supplemented with 0.2–0.4% of sucrose.     

An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay

A gas exposure system using rotating vessels was improved for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay. This system was composed of 12 square culture vessels, a device for preparation of air containing test gas, and positive and negative control gases at target concentrations and for supplying these gases to the culture vessels, and a roller apparatus in an incubator. Chinese hamster lung cells (CHL/IU) were grown on one side of the inner surface of the square culture vessel in the MEM medium. Immediately prior to exposure, the medium was changed to the modified MEM. Air in the culture vessel was replaced with air containing test gas, positive or negative control gas. Then, the culture vessels were rotated at 1.0 rpm. The monolayered culture cells were exposed to test gas during about 3/4 rotation at upper positions and alternatively immersed into the culture medium during about 1/4 rotation at lower positions. This system allowed the chromosomal aberration assay simultaneously at least at three different concentrations of a test gas together with positive and negative control gases with and without metabolic activations, and duplicate culture at each exposure concentration. Seven gaseous compounds, 1,3-butadiene, chlorodifluoromethane, ethyl chloride, methyl bromide, methyl chloride, propyne, and vinyl chloride, none of which has been tested to date, were tested on CHL/IU for the chromosomal aberration assay using this gas exposure system. All the compounds except chlorodifluoromethane showed positive responses of the structural chromosomal aberrations, whereas polyploidy was not induced by any of these gases. This improved gas exposure system proved to be useful for detecting chromosomal aberrations of gaseous compounds.