Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Effects of sex hormones and gender on attraction thresholds for volatile anal scent gland odors in ferrets

Body odors contribute to mate recognition and sexual partner preference in many mammals, including ferrets. We used a habituation/dishabituation procedure to test whether sex steroid hormones influence whether ferrets will approach and investigate different concentrations of volatile anal scent gland odors from male and female conspecifics. When tested with high concentrations of anal scent gland secretions in oil vehicle, gonadectomized male and female ferrets that received no sex steroids reliably discriminated anal scents from male and female conspecifics. This discrimination most likely reflects gender recognition rather than individual recognition because gonadectomized, sex steroid-treated ferrets discriminated between anal scents of males and females but not between anal scents of individual males or females. Treatment with either the estrogen receptor agonist, estradiol benzoate (EB), or the androgen receptor agonist, 5-alpha dihydrotestosterone proprionate (DHTP), increased investigation of low concentrations of anal scent by gonadectomized ferrets. These data suggest that ferrets could use anal scent gland secretions in mate recognition and that seasonal increases in circulating sex steroid hormones increase ferrets' responsiveness to low concentrations of these odors.     

The Endocannabinoid System and Extinction Learning

The endocannabinoid system has emerged as a versatile neuromodulatory system, implicated in a plethora of physiological and pathophysiological processes. Cannabinoid receptor type 1 (CB1 receptor) and endocannabinoids are widely distributed in the brain. Their roles in learning and memory have been well documented, using rodents in various memory tests. Depending on the test, the endocannabinoid system is required in the acquisition and/or extinction of memory. In particular, the activation of CB1 receptor-mediated signaling is centrally involved in the facilitation of behavioral adaptation after the acquisition of aversive memories. As several human psychiatric disorders, such as phobia, generalized anxiety disorders, and posttraumatic stress disorder (PTSD) appear to involve aberrant memory processing and impaired adaptation to changed environmental conditions, the hope has been fuelled that the endocannabinoid system might be a valuable therapeutic target for the treatment of these disorders. This review summarizes the current data on the role of the endocannabinoid system in the modulation of extinction learning.     

Stimulus change dis-habituates operant responding supported by water reinforcers

The present experiment examined whether habituation contributes to within-session decreases in operant responding for water reinforcers. The experiment asked if this responding can be dishabituated, a fundamental property of habituated behavior. During baseline, rats’ lever pressing was reinforced by water on a variable interval 15-s schedule. During experimental conditions, rats responded on the same schedule and a new stimulus was introduced for 5 min at 15, 30 or 45 min into the 60-min session. The new stimulus was extinction, continuous reinforcement or flashing lights in different conditions. Rate of responding primarily decreased within the session during baseline. Introducing a new stimulus sometimes suppressed (extinction, continuous reinforcement) and sometimes increased (flashing lights) responding while it was in effect. The new stimulus increased responding after it ended and before it was presented in the session. The results are incompatible with the idea that non-habituation satiety factors (e.g., cellular hydration and blood volume) contributed to within-session changes in responding. These satiety factors should increase with increases in consumption, decrease with decreases in consumption and remain constant with constant consumption of water. Nevertheless, all stimulus changes increased operant responding for water. These results support the idea that habituation contributes to within-session decreases in responding for water reinforcers.     

Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 10²–10⁵ colony forming units g⁻¹ fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR^+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 10⁵ – 4.0 × 10⁷ colony forming units g⁻¹ tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.     

Auditory stimulation dishabituates anti-predator escape behavior in hermit crabs (Coenobita clypeatus)

Responses to innocuous stimuli often habituate with repeated stimulation, but the mechanisms involved in dishabituation are less well studied. Chan et al. (2010b) found that hermit crabs were quicker to perform an anti-predator withdrawal response in the presence of a short-duration white noise relative to a longer noise stimulus. In two experiments, we examined whether this effect could be explicable in terms of a non-associative learning process. We delivered repeated presentations of a simulated visual predator to hermit crabs, which initially caused the crabs to withdraw into their shells. After a number of trials, the visual stimulus lost the ability to elicit the withdrawal response. We then presented the crabs with an auditory stimulus prior to an additional presentation of the visual predator. In Experiment 1, the presentation of a 10-s, 89-dB SPL noise produced no significant dishabituation of the response. In Experiment 2 we increased the duration (50 s) and intensity (95 dB) of the noise, and found that the crabs recovered their withdrawal response to the visual predator. This finding illustrates dishabituation of an antipredator response and suggests two distinct processes—distraction and sensitization—are influenced by the same stimulus parameters, and interact to modulate the strength of the anti-predator response.     

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. Havana 425 becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10^–3 per cell generation, was 100–1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10^–10 M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.     

Growth Conditions in In Vitro Culture Can Induce Oxidative Stress in <Emphasis Type="Italic">Mammillaria gracilis</Emphasis> Tissues

In vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) develop calli without any exogenous growth regulators. This habituated tissue spontaneously regenerates morphologically normal as well as hyperhydric shoots. In this study, a possible involvement of activated oxygen metabolism in habituation and hyperhydricity in in vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) was investigated. Significantly higher malondialdehyde (MDA) and carbonyl contents as well as hydrogen peroxide (H₂O₂) production were observed in habituated callus (HC), hyperhydric regenerated shoots (HS), and tumors (TT) in comparison to normal regenerated shoots (NS). Lipoxygenase (LOX) activity showed a similar trend, with a clear increase in activity in HC and HS. The activities of antioxidative enzymes, namely, peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT), were also higher in HC, HS, and TT, whereas an increase in superoxide dismutase (SOD) activity was observed in HC and HS. The majority of antioxidative isoenzymes were common to all cactus tissues, although a few tissue-specific bands were noticed. Significant decreases in phenylalanine ammonia lyase (PAL) activity, total phenolic content, and lignification were found in HS, HC, and TT in comparison to NS. Our results showed the appearance of a prominent oxidative stress in HC, HS, and TT as well as a strong induction of the antioxidant system indicating that activated oxygen metabolism could be involved in habituation and hyperhydricity and linked to the loss of tissue organization in M. gracilis. B. Balen and M. Tkalec contributed equally to this work.     

Operant behavior in dwarf hamsters (Phodopus campbelli): Effects of rate of reinforcement and reinforcer flavor variety

We investigated operant behavior in a novel species, the dwarf hamster (Phodopus campbelli). In two experiments, hamsters were trained to lever-press for food reinforcement. In Experiment 1, rate of reinforcement was manipulated across conditions using four variable-interval schedules of reinforcement (delivering one to eight reinforcers per min). As predicted, within-session decreases in responding were steepest on the richest schedule. In Experiment 2, lever-pressing was reinforced by either a constant or a variety of flavored food pellets. Within-session decreases in responding were steeper when the reinforcer flavor remained constant than when it was varied within the session. In both experiments, subjects hoarded most reinforcers in their cheek pouches rather than consuming them in the operant chambers. These results are incompatible with post-ingestive satiety variables as explanations for within-session decreases in operant responding and suggest that habituation to repeatedly presented reinforcers best accounts for subjects’ response patterns. Additionally, a mathematical model that describes behavior undergoing habituation also described the present results, thus strengthening the conclusion that habituation mediates the reinforcing efficacy of food.