Cardiotonic glycosides from biomass of <Emphasis Type="Italic">Digitalis purpurea</Emphasis> L. cultured in temporary immersion systems

Cardiotonic glycosides are extracted mostly from leaves of Digitalis plants. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process and can be affected by climatic and soil conditions. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research was to obtain biomass of D. purpurea using the temporary immersion system (TIS) and to determine the content of cardiotonic glycosides (digitoxin, digoxin and lanatoside C) as secondary metabolites of commercial value for the pharmaceutical industry. Shoots were cultured in 1,000 ml TIS during 28 days. The effect of four immersion frequencies (once every 2, 4, 6, and 12 h) was studied. Biomass accumulation was influenced by immersion frequency. The maximum biomass accumulation (values in respect of fresh and dry weight) was obtained with immersions every 4 h (six immersions per day). HPLC analysis revealed the presence of digoxin and digitoxin for all immersion frequencies. No lanatoside C was detected in the biomass cultured in TIS. Digoxin concentrations varied depending on the frequencies tested. In contrast, the digitoxin content showed no dependency on the immersion frequency. Net production of digoxin and digitoxin per TIS were found to be higher with immersions every 4 h. The best net production of digitoxin and digoxin per TIS were 167.6 and 119.9 μg, respectively. The development of organ culture based on temporary immersion system can be a reliable method for the steady production of biomass for cardiotonic glycosides production, which is reported for the first time for Digitalis genus in this communication.     

Cycle characteristics in a temporary immersion bioreactor affect regeneration,morphology, water and mineral status of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis>) somatic embryos

Mass regeneration of Coffea arabica L. somatic embryos using a temporary immersion bioreactor was improved by optimizing the immersion cycles, i.e. both the duration and the frequency of immersions. It was demonstrated that increasing the frequency of short immersions (1 min immersions every 24, 12 and 4 h) stimulated embryo production (480, 2,094 and 3,081 embryos/1-l bioreactor, respectively) and improved quality (60, 79 and 85 of torpedo shaped embryos, respectively). On the other hand, an increase in the immersion duration (1, 5 and 15 min) inhibited embryo regeneration (from 2,094 to 428 embryos per 1-l bioreactor) and negatively affected their morphological quality (from 79 to 49 torpedo-shaped embryos) and the conversion of embryos into plants (from 70 to 33). A 15 min immersion duration applied every 4 h produced hyperhydric symptoms in 90 of the embryos. Hyperhydric embryos were characterized by higher fresh weight and water content, more negative values for water potential and higher K⁺ content when compared to normal torpedo-shaped embryos. Micrographs showed structural problems in the globular stage, such as the existence of an irregular epidermis and an absence of reserves. Whatever the immersion cycle used, the somatic embryos exhibited water and mineral characteristics very different from those of their zygotic counterparts. The use of 1 min immersions every 4 h led to the production of the largest quantities of torpedo-shaped embryos without hyperhydricity that succeeded in regenerating plants (75 conversion).     

Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system

Here, we evaluated the efficiency of shoot multiplication of Vanilla planifolia Jacks. ex Andrews using solid medium, partial immersion, and a temporary immersion system (TIS) to improve micropropagation in this species. Clusters of shoots were cultivated in vitro using Murashige and Skoog (MS) medium supplemented with 9.55 μM benzyladenine (BA) and 100 mL L^?1 coconut water. For the TIS, a RITA^® system was used and three immersion frequencies were evaluated (every 4, 8, and 12 h) with an immersion time of 2 min. After 30-d culture, the TIS produced the maximum multiplication rate (14.27 shoots per explant) when using an immersion frequency of 2 min every 4 h, followed by the partial immersion system (8.64 shoots per explant), and solid medium (5.80 shoots per explant). Next, the effect of the volume of culture medium per explant was also evaluated for TIS. The most suitable volume of culture medium for shoot formation was 25 mL per explant, which increased the rate of multiplication to 17.54 shoots per explant. Root initiation was 90% successful in TIS using half-strength MS medium supplemented with 0.44 μM naphthaleneacetic acid (NAA) and an immersion frequency of 2 min every 4 h. With this system, the shoot multiplication rate increased threefold compared to that obtained with solid medium. In addition, this system produced good results for the transplantation and acclimation (90% of survival) of in vitro-derived plants. These results offer new options for large-scale micropropagation of vanilla.     

In vitro propagation of three <Emphasis Type="Italic">Agave</Emphasis> species used for liquor distillation and three for landscape

In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.     

Growth and development of carnation ‘Dreambyul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods

The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation ‘Dreambyul’ plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H₂O₂ level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation ‘Dreambyul’ plantlets cultured in the TIS.

     

Improved growth and quality of <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> plantlets using a temporary immersion system

The effects of temporary immersion system (TIS) culture on the growth and quality of Siraitia grosvenorii plantlets were investigated. The TIS promoted the growth and quality of S. grosvenorii plantlets. Proliferation rate, shoot length, fresh weight (FW) and dry weight (DW) of shoots, and total biomass production were significantly (P ≤ 0.05) higher in the TIS than in gelled and liquid medium, respectively. The TIS also decreased callus formation at the base of shoots. Callus diameter was significantly (P ≤ 0.05) lower in the TIS (3.30 mm) than in gelled medium (6.31 mm) and liquid medium (6.77 mm), respectively. FW (50.83 mg) and DW (7.08 mg) of callus in the TIS were also significantly (P ≤ 0.05) lower than those in gelled medium (80.00 and 10.56 mg, respectively) and liquid medium (218.75 and 23.75 mg, respectively). During rhizogenesis, minimal callus was evident at the base of shoots in the TIS, with a well-developed root system. However, the plantlets in gelled medium just produced thick, brown and easily broken roots with obvious callus and fewer secondary roots. The natural-like plantlets of S. grosvenorii obtained in the TIS would probably have positive effects on ex vitro rooting and transplanting in large-scale commercial production.     

Growth, morphogenesis, and essential oil production in Mentha spicata L. plantlets in vitro

To date, plantlet culture has not been explored as a means to obtain secondary metabolites in vitro. However, plantlets readily produce desirable secondary metabolites, which may not be produced in cell suspension or callus cultures. To optimize plantlet growth in vitro, the influences of various physical environments on the growth (fresh weight), morphogenesis (leaf, root, and shoot number), and volatile carbon metabolites (i.e. monoterpene, (−)-carvone) of Mentha spicata L. (spearmint) plants were studied. The carvone content in different portions of sterile plantlets was analyzed. Carvone was only produced from the foliar regions of cultured plantlets and was absent in the callus and roots. The influence of physical support (e.g., agar, glass gravel, liquid, platform or sponge), frequency of media replacement, and culture vessel capacity on spearmint plantlets growth and carvone production was tested. A comparative study was conducted testing the growth, morphogenesis, and secondary metabolism occurring with three different spearmint cultivars grown in either culture tubes containing 25 ml agar medium or in an automated plant culture system (APCS; a sterile hydroponics system) employing a 1-l medium reservoir. Increasing the number of media immersions (4, 8, 12 or 16 immersions d⁻¹) of plantlets growing in the APCS increased growth and morphogenesis responses. Generally, higher culture growth rates resulted in lower carvone treatment⁻¹ (mg carvone g-FW⁻¹); however, overall total carvone ((mg carvone g-FW⁻¹) × g culture FW) increased because of the production of greater biomass obtained per vessel.     

Improving secondary embryogenesis in <Emphasis Type="Italic">Quercus robur</Emphasis>: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA^® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.     

How does plant population density affect the biomass of Ravenna grass?

Ravenna grass, Tripidium ravennae (L.) H. Scholz, is known to produce an abundance of biomass, but how plant density affects its biomass potential remains unknown. The objectives were to determine the effects of plant density on biomass yield; plant growth traits; biomass?carbon, nitrogen, and ash concentrations; heating value; nitrogen removal; and sucrose concentration in leaves and culms. The treatments consisted of five plant densities (1,250; 2,500; 5,000; 10,000; and 20,000 plants per hectare) in a randomized complete block design with four blocks. Plots were nonirrigated, unfertilized, and harvested once during the dormant season each year. Data were collected from 2015?2019. Dependent variables that varied with plant population density (p < .05) were biomass yield, number of reproductive culms per plant, reproductive culm diameter, reproductive culm sucrose concentration, and nitrogen removal with biomass. Biomass yield ranged from 5.6 to 16.3 Mg/ha for plant densities of 1,250–20,000 plants per hectare, respectively. Combined over years, nonlinear regression of the data showed the equation for biomass yield to plateau at 16.2 Mg/ha at a plant density of 10,640 plants per hectare. As plant density increased, the number of reproductive culms per plant, culm diameter, and culm sucrose concentration significantly decreased. At 1,250 plants per hectare, the number of reproductive culms per plant, culm diameter, and culm sucrose averaged 70, 10.2 mm, and 63.2 g/kg, respectively. Nitrogen removed with biomass significantly increased as biomass yield increased with plant density. At a density of 10,000 and 20,000 plants per hectare, the amount of nitrogen removed annually in the harvested biomass averaged 88 kg/ha. The data suggest that 10,000 plants per hectare would produce the greatest annual biomass yields; however, research is needed to determine the nutrient requirement for Ravenna grass to sustain biomass production at that density.     

Alleviation of hyperhydricity of sugarcane plantlets produced in RITA® vessels and genotypic and phenotypic characterization of acclimated plants

The effect of employing a RITA® system in one or both of somatic embryo induction and germination stages was investigated, and it was deemed far superior to a semi-solid (agar) substrate in terms of in vitro plant yields for sugarcane genotype N41. Approximately 18,000 plants/leaf roll were obtained in vitro in 12 weeks, when both culture stages were undertaken using temporary immersion, compared with approximately 2000 plants/leaf roll produced on semi-solid medium. However, due to hyperhydricity, only ~ 34% of the plants produced in RITA® survived acclimatization. To overcome this, and realize the potential yields of the RITA® system, various culture conditions were investigated, viz. nutrient and sucrose supplies, a rockwool substrate and the immersion regime. Of these, increasing the resting time between immersions from 1 min/12 h to 1 min/72 h, and lowering MS nutrient to 1/2 strength, proved the most beneficial, resulting in 60% acclimation success. Genetic fidelity of these plants was investigated by AFLP analyses where only 0–0.9%, of polymorphic bands were scored compared with the conventionally- propagated N41 control. Phenotypic characterization of plants grown in the field for 6 months showed that, although all in vitro derived plants had a reduced stalk diameter relative to the control, there were no significant differences regarding stalk mass, height and population.     

Temporary immersion systems for the mass propagation of sweet cherry cultivars and cherry rootstocks: development of a micropropagation procedure and effect of culture conditions on plant quality

Sweet cherry (Prunus avium L.) varieties and cherry rootstocks are an important part of the fruit industry, and difficulties associated with mass propagation provide an opportunity for the use of temporary immersion systems (TIS). We show the establishment of culture procedures for four genotypes: the rootstocks Maxma-14 and Colt and the varieties ‘Van’ and ‘Rainier.’ The starting explants were internodal segments from seedlings kept in solid propagation medium (PM) (Driver-Kuniyuki Walnut (DKW) base supplemented with indole butyric acid, benzyl amino purine; ascorbic acid, myo-inositol, and agar). Segments were cultured under TIS for 14 d and led to whole plant generation after 30 d of culturing in solid rooting media, which depended on whether they are varieties or rootstocks. A 15-d acclimatization phase led to establishment in greenhouse. The efficiency of TIS was specifically analyzed for the two best PM-derivative media and compared to cultures using solid medium. A number of shoots (P _x), biomass (Q _x), and sucrose consumption (SC) were evaluated for these purposes. The results showed that Maxma-14, Colt, and ‘Van’ TIS cultures had improved performance in comparison to solid cultures, whereas ‘Rainier’ showed no differences. The number of immersions influenced all of the productive parameters (P _x, Q _x, and SC), whereas genotype affected P _x, and the time of immersion influenced SC. The best Q _x and P _x values were obtained with the rootstocks Maxma-14 and Colt, as well as the variety Van; these showed no hyperhydration. Physiological studies show that 14-d TIS-produced shoots represented an intermediate stage between solid-derived and adult plants, although the photosynthetic efficiencies of these materials revealed a lack of autotrophic ability at this point.     

Reducing Competitive Suppression of a Rare Annual Forb by Restoring Native California Perennial Grasslands

Populations of the rare annual forb Amsinckia grandiflora may be declining because of competitive suppression by exotic annual grasses, and may perform better in a matrix of native perennial bunchgrasses. We conducted a field competition experiment in which Amsinckia seedlings were transplanted into forty 0.64‐m² experimental plots of exotic annual grassland or restored perennial grassland. The perennial grassland plots were restored using mature 3 cm‐diameter plants of the native perennial bunchgrass Poa secunda planted in three densities. The exotic annual grassland plots were established in four densities through manual removal of existing plants. Both grass types reduced soil water potential with increasing biomass, but this reduction was not significantly different between grass types. Both grass types significantly reduced the production of Amsinckia inflorescences. At low and intermediate densities (dry biomass per unit area of 20–80 g/m²), the exotic annual grasses reduced Amsinckia inflorescence number to a greater extent than did Poa, although at high densities (>90 g/m²) both grass types reduced the number of Amsinckia inflorescences to the same extent. The response of Amsinckia inflorescence number to Poa biomass was linear, whereas the same response to the annual grass biomass is logarithmic, and appeared to be related to graminoid cover. This may be because of the different growth forms exhibited by the two grass types. Results of this research suggest that restored native perennial grasslands at intermediate densities have a high habitat value for the potential establishment of the native annual A. grandiflora.     

Temporary immersion systems (RITA®) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production

Somatic embryogenesis in cork oak (Quercus suber L.) is an efficient tool that allows the production of large number of embryos from selected quality and productive trees. Temporary immersion systems (TIS) are an alternative to semi-solid or liquid culture that combine the advantages of liquid culture and avoid the associated problems. Parameters that affect the TIS multiplication efficiency of Q. suber L. embryogenic cultures were evaluated. Immersion frequencies of 1 min every 6 or 4 h increased the fresh weight 3.7 or 7.5-fold compared with an immersion frequency of 1 min every 12 h or cultures on semi-solid medium, respectively. The cellular fate of embryogenic cultures was also affected by the immersion frequency, 1 min every 6 h was the best for mass propagation of proliferative developmental stages (embryogenic calli and embryo clusters) while 1 min every 4 h promoted the formation of single, fully developed cotyledonary embryos. An initial amount of 1.5 g fresh weight of proliferative tissues produced the best results in RITA^® containers while 0.5 g of embryogenic callus was the best for semi-solid cultures.     

Effect of immersion systems,lighting, and TIS designs on biomass increase in micropropagating banana (Musa spp. cv. 'Grande naine' AAA)

Development of in vitro techniques has enabled rapid clonal propagation, regeneration, and multiplication of genetically manipulated superior clones, production of secondary metabolites, and ex situ conservation of valuable germplasm. This has been possible not only due to the refinements of culture methodologies and applications of cutting-edge areas of molecular biology but also due to the judicious inclusion of engineering principles and methods to improve and refine the system. In the present study, we used engineering principles and methods to transform basic in vitro techniques into commercially viable technologies. We investigated two types of temporary immersion systems (TIS), two types of lighting, and two different gas exchange systems during in vitro banana (Musa spp. cv. 'Grande naine') shoot cultures. After 7 wks, all banana shoots cultured in standard TIS (5-L glass vessels, type 1) showed superior vegetative growth for the evaluated parameters. We also found that illumination provided by light emission diodes (LEDs) was superior to the use of white fluorescent lamps at the same light intensity (40 μmol m^?2 s^?1). Shoots treated with compressed air for immersion and additional gas exchange during the culture period in the glass vessel of TIS systems resulted in higher propagation rates and a larger number of shoots harvested as well as a larger number of roots formed per shoot after the 7-wk culture period.     

Multi‐symbiotic systems: functional implications of the coexistence of grass–endophyte and legume–rhizobia symbioses

The coexistence of symbionts with different functional roles in co‐occurring plants is highly probable in terrestrial ecosystems. Analyses of how plants and microbes interact above‐ and belowground in multi‐symbiotic systems are key to understand community structure and ecosystem functioning. We performed an outdoor experiment in mesocosms to investigate the consequences of the interaction of a provider belowground symbiont of legumes (nitrogen‐fixing bacteria) and a protector aerial fungal symbiont of grasses (Epichloё endophyte) on nitrogen dynamics and aboveground net primary productivity. Four plants of Trifolium repens (Trifolium, a perennial legume) either inoculated or not with Rhizobium leguminosarum, grew surrounded by 16 plants of Lolium multiflorum (Lolium, an annual grass), with either low or high levels of the endophyte Neotyphodium occultans. After five months, we quantified the number of nodules in Trifolium roots, shoot biomass of both plant species, and the contribution of atmospheric nitrogen fixation vs. soil nitrogen uptake to above ground nitrogen in each plant species. The endophyte increased grass biomass production (+ 16%), and nitrogen uptake from the soil – the main source for the grass. Further, it reduced the nodulation of neighbour Trifolium plants (?50%). Notably, due to a compensatory increase in nitrogen fixation per nodule, this reduced neither its atmospheric nitrogen fixation – the main source of nitrogen for the legume – nor its biomass production, both of which were doubled by rhizobial inoculation. In consequence, the total amount of nitrogen in aboveground biomass and aboveground productivity were greatest in mesocosms with both symbionts (i.e. high rhizobia + high endophyte). These results show that, in spite of the deleterious effect of the endophyte on the establishment of the rhizobia–legume symbiosis, the coexistence of these symbionts, leading to additive effects on nitrogen capture and aboveground productivity, can generate complementarity on the functioning of multi‐symbiotic systems.     

Effects of mycorrhizal infection, soil phosphorus availability and fruit production on the male function in two cultivars of Lycopersicon esculentum

The effects of mycorrhizal infection, soil P availability and fruit production on the male function of reproduction were examined in two cultivars of tomato (Lycopersicon esculentum Mill.). Tomato plants were grown in a greenhouse under three treatment combinations: non‐mycorrhizal, low P (NMPO); non‐mycorrhizal, high P (NMP3); and mycorrhizal, low P (MPO). In addition, all treatment combinations were grown both with and without fruit. Fruit production decreased final leaf biomass, flower production and in vitro pollen tube growth rates, often reducing the beneficial effects of increased P uptake. Thus, fruit production diverted resources from subsequent vegetative growth, flower production and pollen development. As the growing season progressed, mean pollen production per flower and in vitro germination and tube growth decreased. Mycorrhizal infection and high soil P conditions increased final leaf biomass, flower production, mean pollen production per flower (in one cultivar) and in vitro pollen tube growth rates. Thus, mycorrhizal infection and high soil P conditions increased pollen quantity and quality, thereby enhancing fitness through the male function. Similar trends in these treatments suggested that mycorrhizal effects on the male function were largely the result of improved P acquisition.     

Antifungal and growth-promoting activity of Azospirillum brasilense in Zea mays L. ssp. mexicana

Zea mays L. ssp. mexicana (teosinte) is a naturally occurring grass related to maize. The two plants have developed foliar fungal diseases that can be controlled with beneficial bacterial antagonists. While the beneficial effects on stem and root development of the application of bacteria of the genus Azospirillum is widely known, the effects of the bacteria on the control of the phytopathogenic fungi associated with teosinte leaves and seeds are unknown. Bacterium of the species A. brasilense that present acetylene reducing activity, siderophore production and the ability to antagonise pathogenic fungi in vitro and in vivo in teosinte plants were selected for this study, in which the incidence of fungal diseases caused by Alternaria, Bipolaris and Fusarium were reduced in plants. Furthermore, biomass (root and stem) production increased, improving teosinte plant health in greenhouse and field conditions.     

Induction of a dwarf phenotype with IBH1 may enable increased production of plant‐made pharmaceuticals in plant factory conditions

Year‐round production in a contained, environmentally controlled ‘plant factory’ may provide a cost‐effective method to produce pharmaceuticals and other high‐value products. However, cost‐effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild‐type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild‐type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti‐hepatitis B virus antibodies (anti‐HBs) in tobacco plants and found that the production of anti‐HBs per unit fresh weight did not significantly differ between wild‐type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost‐effective production of high‐value products by stably transformed plants in plant factory conditions.     

EFFECTS OF FLOODING AND NUTRIENT ENRICHMENT ON BIOMASS ALLOCATION IN ACER RUBRUM SEEDLINGS

A greenhouse experiment was conducted on Acer rubrum seedlings to evaluate the effects of flood frequency on production and allocation of biomass and to test the effects of N and P fertilization on production and allocation. Seedlings from the Dismal Swamp were subjected to three flood treatments (no flooding, intermittent flooding, and continuous flooding) and four enrichment treatments (no enrichment, N additions, P additions, and N + P additions). More continuous flooding resulted in less biomass production. Biomass increased during the study in all treatments except for root mass in the continuously flooded treatment. However, production of abundant adventitious roots compensated for the lack of normal root growth. Root/shoot ratios exhibited the greatest decreases in the continuously flooded plants. Plants with N + P added had significantly more leaf, stem, and total mass than the nonenriched plants four months into the study. The N + P additions had apparently compensated for the effects of flood stress in the continuously flooded plants by the end of the study. The fertilized seedlings accumulated higher concentrations of N and P, but their nutrient use efficiency (biomass production per unit nutrient absorbed) was lower than in the nonenriched plants. Acer rubrum seedlings survive flooded conditions through several adaptations; however, theirgrowth is slowed by continuous flooding.     

Development of a temporary immersion system (RITA®) for mass production of sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. interspecific hybrids)

Commercial micropropagation of sugarcane is largely determined by the clonal fidelity and the cost of plants produced. Rapid production of plants in vitro reduces the frequency of offtypes in many species. By exploiting the concept of transverse thin cell layer culture, we have developed a rapid, high frequency direct plant regeneration system, called SmartSett®, for commercial sugarcane cultivars grown in Australia. Similar to conventional micropropagation, labour remains the major cost of this plant production system. Hence, to reduce the labour component, we have integrated the SmartSett® system with the RITA® temporary immersion bioreactor. Thin transverse leaf sections or fragmented leaves cultured on agar-based SmartSett® shoot induction medium were used as the starting material for RITA®. Shoot initiation on semi-solid medium prior to transferring to RITA®, culture immersion frequency, explant size and genotype determined the productivity (number of plants produced per unit culture) of the system. Results obtained with cultivar Q165 indicate that explants cultured for 45 d on SmartSett® shoot induction medium were the most prolific, producing on average 275 shoots per vessel after 45 d of culture in RITA with 1 min immersion every 12 or 24 h. Using the fragmented tissue, 14-d-old explants and 3-mm leaf tissue fragments were the most productive. Experiments with three cultivars (Q117, Q165 and Q205) showed that RITA® culture conditions need to be optimised for each cultivar for maximum plant production.