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—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^−1 benzyladenine (BA) and 0.2mgl^−1 α-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.     

Different shake flask closures alter gas phase composition and ajmalicine production in Catharanthus roseus cell suspensions

The type of closure chosen for plant cell cultures can significantly alter the headspace gas composition of a culture, leading to major differences in the production of secondary metabolites. In cell suspension cultures of Catharanthus roseus, ethylene accumulated in cultures with limited gas exchange and appeared to inhibit the production of ajmalicine. The variability in product yields between replicates can also be attributed to gas composition differences.     

Carbon dioxide and ethylene evolution in the culture atmosphere of Magnolia cultured in vitro

Subcultured explants of Magnolia soulangeana Soul, were incubated in tissue culture containers fitted with different types of closures. Type of closure affected the CO₂ concentration, with levels as high as 14% CO₂ being detected. The ethylene concentration increased gradually with time, to as much as 2–3 ppm after 9 weeks. There was a large variation in the composition of the atmosphere within the containers of any one type. The way by which a container was closed influenced exchange of the inner gas phase with the surrounding atmosphere and was important in determining the development of the cultured tissues.     

Production of yam microtubers using a temporary immersion system

Yam clones ‚Pacala Duclos’ and ‚Belep’ in temporary immersion system culture showed favourable results on shoot growth stage and in the development of microtubers in comparison with solid culture media. Cultures in temporary immersion systems in both clones obtained a higher microtuber number per plant, with greater fresh weight and diameter in comparison with solid culture media. Besides, 45 and 47% of microtubers greater than 3.0 gFW for ‚Belep’ and ‚Pacala Duclos’ clones respectively, were obtained. Those tubers may be planted without acclimatization and may be stored for a prolonged period of time.     

Measurement of gas exchange rates in plant tissue culture vessels

The aerial microenvironment in culture vessels has a significant effect on the growth and development of plantlets in vitro. Since the gas exchange between outside air and inner air can influence the microenvironment of culture vessel, it is necessary to measure the air exchange rate for various vessels. In this study, water vapor was used as the tracer gas, and the change of absolute humidity inside the vessel was calculated continuously by the measured values of a relative humidity sensing element. The outside environment was maintained at constant humidity level by a saturated salt solution. The RH data were transformed into absolute humidity and the specific humidity ratio. The air exchange rates of several tissue culture vessels were then calculated. The exchange rate was between 0.0145 h^–1 to 0.0376 h^–1. This technique provides an inexpensive, rapid and simple way to determine the air exchange rate of a culture vessel within a short period. The effects of the air current velocities on the exchange rates of vessels were also determined.     

A cultivar comparison of plant regeneration from suspension cells,callus, and cormel slices ofGladiolus

Callus was initiated from either cormel slices or in vitro-grown plants of sixGladiolus cultivars cultured on Murashige and Skoog’s basal salts medium supplemented with either 10 mg/liter (53.8 µM) 1-naphthaleneacetic acid, 2 mg/liter (9.3 µM) dicamba, or 0.5 mg/liter (2.2 µM) 2,4-dichlorophenoxyacetic acid. More plants were regenerated from callus of the cultivar “Peter Pears” as compared to “Jenny Lee,” “Florida Flame,” or “Golden Year.” No plants were regenerated from callus of “Rosa Supreme” or “Purity White.” Plants were regenerated from 2 and 6-mo.-old suspension cells of “Jenny Lee” and “Peter Pears” but not from “Florida Flame.” Cormel slices cultured on Murashige and Skoog’s basal salts medium supplemented with 1 mg/liter (4.4 µM) 6-benzylaminopurine regenerated plants from all six cultivars indicating a cultivar-independent system of plant regeneration.     

Production of dimethyl triselenide and dimethyl diselenenyl sulfide in the headspace of metalloid-resistant Bacillus species grown in the presence of selenium oxyanions

A Bacillus species harvested from the environment is metalloid resistant and, when grown anaerobically in complex growth medium and amended with the selenium oxyanion selenate, selenite, or selenocyanate, produces volatile organoselenium compounds in bacterial culture headspace. Two novel compounds so far undetected in bacterial culture headspace, CH₃Se₂SCH₃ and CH₃SeSeSeCH₃, are produced and can be detected using solid-phase microextraction and gas chromatography with either fluorine-induced chemiluminescence or mass spectrometric detection. Differences in the electron impact fragmentation pattern of the mixed sulfur/selenide compounds allow the tentative differentiation between the symmetric and asymmetric isomers in this bacterium’s headspace in favor of the asymmetric CH₃SeSeSCH₃ isomer.     

Ethylene production by Agrobacterium rhizogenes strains in vitro and in vivo

Research was initiated to determine whether Agrobacteriumrhizogenes strains, used for plant transformation, are a source ofethylene and which compound these bacteria use for its production. A4 and LBAstrains produced ethylene on solid MG medium, used for culture of bacteria, andon MS medium used for plant in vitro culture. The enhancedethylene production in the presence of methionine and2-keto-4-methylthiobutyricacid (KMBA), but not in the presence of glutamic acid and1-aminocyclopropano-1-carboxylic acid (ACC), was observed. The removing ofethylene from the culture atmosphere caused the inhibition of bacterial growthand indicates, that these strains need this gas for their growth. Theinoculation of petunia explants with A. rhizogenes causedincreased ethylene production by the explants. Ethylene present in flasksduringthe growth of hairy roots of Petunia hybrida inhibitedtheir growth. These results indicate that in the flasks where the planttransformation takes place the source of ethylene, affecting the root growth,ispossibly not only the plant explant but also bacteria and pathogenesis.     

Mineral nutrition and plant morphogenesis

Summary Plant morphogenesis in vitro can be achieved via two pathways, somatic embryogenesis or organogenesis. Relationships between the culture medium and explant leading to morphogenesis are complex and, despite extensive study, remain poorly understood. Primarily the composition and ratio of plant growth regulators are manipulated to optimize the quality and numbers of embryos or organs initiated. However, many species and varieties do not respond to this classical approach and require further optimization by the variation of other chemical or physical factors. Mineral nutrients form a significant component of culture media but are often overlooked as possible morphogenic elicitors. The combination of minerals for a particular plant species and developmental pathway are usually determined by the empirical manipulation of one or a combination of existing published formulations. Often only one medium type is used for the duration of culture even though this formulation may not be optimal for the different stages of explant growth and development. Furthermore, mineral studies have often focused on growth rather than morphogenesis with very little known of the relationships between mineral uptake and morphogenesis. This article examines the present knowledge of the main effects that mineral nutrients have on plant morphogenesis in vitro. In particular, the dynamics of nitrogen, phosphorus, and calcium supply during development are discussed.     

Comparison of Soil VOCs measured by soil gas,heated headspace,and methanol extraction techniques

Comparisons of soil volatile organic compound (VOC) measurement techniques and soil properties expected to influence these measurements were performed at two dissimilar sites. A total of 41 soil gas, 52 heated headspace, and 51 methanol extraction/purge‐and‐trap measurements were obtained on collocated samples. Contaminants present at both sites included cis‐1,2‐dichloroethene, 1, 1, 1‐trichloroethane, trichloroethene, and tetrachloroethene. Heated headspace offered the highest sensitivity, as indicated by the greatest percentage of detections per number of analyses. The statistical regression between headspace concentrations and methanol extraction concentrations was highly significant (p < 0.001) with r² = 0.53. Headspace concentrations (range, 7 to 4250 ng/g) ran approximately 20 to 30% of the methanol extraction concentrations (range, 260 to 7300 ng/g), indicating that the methanol was able to extract significantly more of the chlorinated hydrocarbons (CHCs) than the headspace extraction, even in soils with relatively low organic carbon contents (< 0.25%). None of the soil properties (gravimetric moisture content, organic carbon content, percent sand, and percent clay) significantly improved the regression fit. The soil gas responses were unlike either headspace or methanol extraction data. CHC measurements by vapor extraction/soil gas could not be used to predict soil CHC concentrations at these sites.     

Determination of dimethyl sulphide in blood and adipose tissue by headspace gas analysis

A method for the headspace analysis of dimethyl sulphide in blood and adipose tissue has been established. Blood (0.2 ml) or adipose tissue (0.5 g) with added dimethyl sulphide was sealed in a 10-ml vial using PTFE sheet to prevent escape of dimethyl sulphide from the headspace. Equilibration was performed at 60°C for 4 h, and 20 μl of gaseous phase sampled from the headspace was subjected to gas chromatography (with flame photometric detection). Calibration curves were prepared for the two samples. Linearity was observed in the range from 5–10 μg to 2 mg.     

Tissue culture in synthetic atmospheres: diffusion rate effects on cytokinin-induced callus growth and isoflavonoid production in soybean [Glycine max (L.) Merr. cv. Acme]

Concentration is one factor that is known to determine how metabolic gases influence the growth and secondary metabolism of plant tissues in culture. How actual gas bioavailability influences these processes has not been studied despite its potential importance in specialized applications. A simple model system, soybean [Glycine max (L.) Merr. cv. Acme] callus culture, was selected for experiments because exogenous cytokinin (6-benzylaminopurine; BAP) elicits two types of responses: (1) enhanced callus proliferation, and (2) rapid induction of the isoflavonoid daidzein (7,4′-dihydroxyisoflavone). Synthetic atmospheres supplying metabolic gases with higher or lower bioavailability than in air were created by replacing the nitrogen moiety in standard air with either helium or argon, respectively. Callus was cultured on agar or in liquid shake cultures according to standard procedures. At an optimal cytokinin concentration for stimulation of callus proliferation, 4.4 × 10⁻⁷ M BAP, increased diffusion rates for the metabolic gases resulted in greater weight gain in agar cultures. Weight gain was 11% higher for He-treated and 39% lower for Ar-treated cultures than for the nitrogen control. In contrast, there was no significant effect of metabolic gas diffusion rate on daidzein production in either agar or liquid cultures. Apart from the potential application of these synthetic atmospheres for enhancing plant tissue culture growth, they may have unique value for the space program as an effective way of replicating the gas exchange limitations posed for plants by microgravity (Ar atmosphere), and as a countermeasure for this limitation (He atmosphere).     

Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Summary Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog&#x2019;s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog&#x2019;s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg &#x00B7; liter^&#x2212;1), and BAP (1 mg &#x00B7; liter^&#x2212;1) plus NAA (0.1 mg &#x00B7; liter^&#x2212;1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.     

Assessing the effects of chamber placement, manual sampling and headspace mixing on CH4 fluxes in a laboratory experiment

A laboratory experiment was conducted with two types of closed static chambers to estimate the effects of chamber placement, manual headspace sampling and headspace mixing on methane (CH₄) fluxes. Chamber fluxes were compared to a known reference flux in a chamber calibration system. The measurements were conducted with three types of soils (coarse dry, fine dry and fine wet quarts sand) at five flux levels ranging from 60 to 2000 ??g CH₄ m^?2 h^?1. We found that the placement of a non-vented chamber disturbed the initial CH₄ concentration development within the chamber headspace for 10 to 30 s. Excluding this short period from the flux calculation resulted in a lower flux estimate (mean±SE) of 126?±?26 ??g CH₄ m^?2 h^?1 compared to 134?±?26 ??g CH₄ m^?2 h^?1 if data from time zero of the enclosure were included. We also found that in non-mixed chambers (no fan mixing) the gas sampling by syringes or gas bottles disturbed the development of CH₄ concentration during the enclosure. Furthermore, flux estimates in non-mixed chambers were significantly underestimated (on average 36%) compared to the measured reference fluxes. However, the use of fans to constantly mix the chamber headspace during enclosure significantly improved the goodness-of-fit of the regression analysis used to calculate the flux and further eliminated the disturbance of the manual sampling on the concentration development. We recommend that chambers should be vented during the placement of the chamber, and that fans are used as an integrated part of static chambers while headspace mixing with syringes should be avoided.     

Novel approaches for regulating gas supply to plant systems <Emphasis Type="Italic">in vitro</Emphasis>: Application and benefits of artificial gas carriers

Summary The composition of the gaseous environment within tissue culture vessels is a critical factor in determining in vitro plant growth and morphogenic responsiveness. Consequently, the provision of an adequate and sustainable oxygen supply for cultured plant cells (including isolated protoplasts), tissues and organs is a crucial prerequisite for optimization and regulation of such cultural responses. During the past decade, research has focused on improving growth and development using artificial gas carriers based on inert perfluorocarbon (PFC) liquids and hemoglobin (Hb) solution. Supplementation of culture media with such artificial oxygen carriers has demonstrated beneficial effects of increased and sustainable cellular mitotic division and subsequent biomass production in a diverse range of plant species, during both short- and longer-term culture. Studies have targeted systems where oxygen availability is actually or potentially a major growth-limiting factor. Undoubtedly, gas carrier-facilitated improvements in regulating the supply of respiratory gases to cultured cells, tissues and organs will have increasingly important biotechnological and practical implications in the context of plant micropropagation, somatic hybridization, transgenic plant production, and secondary product biosynthesis.     

Bioreactor headspace measurement by inverted siphon

This paper descibes a new way to measure the headspace, and hence broth volume, of a closed reactor producing gas, or into which some gas can be introduced. The measuring element is an inverted siphon through which reactor gas flows in discrete volumes or boli. Experiments have shown that the gas boli volumes are directly proportional to the headspace volume of the reactor and theoretical considerations confirm this observation.     

In vitro shoot regeneration from cotyledons of immature ovules ofImpatiens platypetala Lindl.

Summary An efficient and reproducible protocol has been developed for in vitro shoot regeneration from cotyledonary explants derived by germinating immature ovules ofImpatiens platypetala Lindl. &#x2018;TR6-27-2&#x2019;. Cotyledonary explants were cultured on a modified Murashige and Skoog (MS) agar-solidified medium containing 7.5g &#x00B7; liter^&#x2212;1 sucrose, 22.2&#x00B5; M N⁶-benzyladenine (BA), and 0.54&#x00B5;M &#x03B1;-naphthaleneacetic acid (NAA). The induction of organogenic tissues occurred after 6 to 8 wk in culture. Exogenous auxin and cytokinin were essential for the induction of organogenic tissues and survival of explants, and BA was most effective for the induction of organogenic tissues, compared with other cytokinins tested. The addition of glutamine (500 mg &#x00B7; liter^&#x2212;1) was also important for growth of organogenic tissues after induction and for reducing explant death during culture. The induction of organogenic tissue was also influenced by the type of cotyledon cultured and the age of the donor seedlings. On average, eight shoots per explant were induced from organogenic tissues larger than 0.5 cm in diameter 6 to 8 wk after transfer to a modified MS agar-solidified medium without NAA and BA reduced to 4.44&#x00B5;M. Shoots longer than 0.5 cm in length were successfully rooted 2 to 4 wk after transfer to a basal MS medium containing 30g &#x00B7; liter^&#x2212;1 sucrose.     

Influence of plant growth regulators on volatiles produced by in vitro grown shoots of Agastache rugosa (Fischer & C.A.Meyer) O. Kuntze

The composition of volatile organic compounds emitted by in vitro shoots of Agastache rugosa (Fischer & C.A. Meyer) O. Kuntze (Lamiaceae) was studied using headspace solid-phase microextraction–gas chromatography–mass spectrometry and compared to the those emitted by adult plants and in vitro-germinated seedlings. Shoot-tip explants were cultured on a solid MS medium supplemented with either 4.4 μM 6-benzyladenine (BA), 9.3 μM kinetin, or 0.45 μM thidiazuron and with either 0.57 μM indole-3-acetic acid (IAA) or 0.41 μM picloram. Shoot proliferation was observed in all these treatments. The presence of these plant growth regulators in the culture medium significantly influenced the composition of volatiles as well as morphogenetic responses observed. The number and quality of regenerating shoots and frequency of axillary bud break were highest in medium containing the BA + IAA combination. Sixty-five compounds were identified in the headspace of the in vitro-produced material and plants cultivated in the field. The in vitro shoots emitted both hydrocarbon (limonene, α-pinene) and oxidized (menthone, isomenthone, pulegone) monoterpenes. The composition of monoterpenes differed depending on the type of auxin—rather than cytokinin—in the medium. The emission of phenylallyl compounds, such as estragole, a major compound in field-grown plants, was markedly lower in shoot cultures.     

The mechanical environment of the chondrocyte: a biphasic finite element model of cell-matrix interactions in articular cartilage

Mechanical compression of the cartilage extracellular matrix has a significant effect on the metabolic activity of the chondrocytes. However, the relationship between the stress–strain and fluid-flow fields at the macroscopic “tissue” level and those at the microscopic “cellular” level are not fully understood. Based on the existing experimental data on the deformation behavior and biomechanical properties of articular cartilage and chondrocytes, a multi-scale biphasic finite element model was developed of the chondrocyte as a spheroidal inclusion embedded within the extracellular matrix of a cartilage explant. The mechanical environment at the cellular level was found to be time-varying and inhomogeneous, and the large difference (3 orders of magnitude) in the elastic properties of the chondrocyte and those of the extracellular matrix results in stress concentrations at the cell–matrix border and a nearly two-fold increase in strain and dilatation (volume change) at the cellular level, as compared to the macroscopic level. The presence of a narrow “pericellular matrix” with different properties than that of the chondrocyte or extracellular matrix significantly altered the principal stress and strain magnitudes within the chondrocyte, suggesting a functional biomechanical role for the pericellular matrix. These findings suggest that even under simple compressive loading conditions, chondrocytes are subjected to a complex local mechanical environment consisting of tension, compression, shear, and fluid pressure. Knowledge of the local stress and strain fields in the extracellular matrix is an important step in the interpretation of studies of mechanical signal transduction in cartilage explant culture models.     

A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

In vitro propagated plants under conditions of low gas exchange generally show morphological and physiological anomalies that lead to high mortality rates during ex vitro acclimatization. The use of gas-permeable membranes increases natural ventilation in culture vessels, photosynthesis and growth rates. However, commercial membranes are expensive, which limits their application. In this study, low-cost, simple to manufacture, alternative membranes were developed to promote gas exchange in jars used for in vitro plant tissue culture. The membranes were developed using polytetrafluoroethylene film and two or three layers of microporous tape (Missner & Missner^?), and were designed to increase the growth of nodal cultures of Pfaffia glomerata (Brazilian ginseng). Conditions that provided higher gas exchange led to an increase in plant growth and content of photosynthetic pigments compared to a closed system without a gas-permeable membrane. The alternative membranes showed similar results for water vapor loss rate and photosynthetic pigments when compared to a commercial membrane. The alternative membranes were also an efficient barrier against contamination and remained intact after being autoclaved multiple times. Among the membranes tested, the traits of the P. glomerata in vitro-derived plants were similar when propagated using the alternative membrane with three layers of microporous tape or the commercial membrane. However, the alternative membrane has a unit cost that is ten times lower than the commercial membrane.