Quality of micropropagated plants—Vitrification

Summary Vitrification-Hyperhydrous shoot development, effects the survival and quality of several micropropagated plants ex-vitro. The leaves which are the immediate organ to be affected, exhibit abnormal morphology and physiology. Leaf malfunction is apparently a stress response to very rich media and high relative humidity. The understanding of the underlying mechanism of vitrification and its control in vitro can contribute to a more efficient micropropagation. Vitrification was found to be associated with elevated ethylene production which was related to hypolignification and poor cell wall development. Liquid and low agar media induced callose formation along with reduced and disoriented cellulose biosynthesis, manifested also in non-functioning guard cells. Malfunctioning stomata, in addition to defective cuticle contributed to increased transpiration and desiccation of in vitro formed leaves. The activity of various enzymes, associated with cell wall synthesis, was low and total proteins in normal leaves was higher than in vitreous ones. Various measures were found to reduce vitrification; lowered matrix and water potential in the medium, reduction in RH, low NH ₄ ⁺ , changes in Ca⁺⁺ levels and the removal of ethylene. These measures improved leaf morphogenesis, survival and the quality of several micropropagated plant species. Presented in the Session-in-Depth Transition of Plants From Culture to Establishment In Vivo,“ at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Blood plasma concentrations of microelements in endurance trained volunteers during hypokinesia and chronic hyperhydration

The objective of this investigation was to determine the effect of daily intake of fluid and salt supplementation (FSS) on increased urinary losses of microelements that developed during hypokinesia (decreased number of walking steps/d). The studies were performed on 30 endurance-trained male volunteers aged 23–26 yr, with an averaged maximum oxygen uptake of 65 mL/kg/min during 364 d of hypokinesia (HK). All volunteers were divided into three equal groups: Ten volunteers were placed continuously under an average of 10,000 running steps/d (14.2 km/d) (control subjects), ten volunteers subjected continuously to HK without the use of FSS (hypokinetic subjects), and ten volunteers were continuously submitted to HK and consumed daily FSS (hyperhydrated subjects). For the simulation of the hypokinetic effect the hypokinetic and hyperhydrated volunteers were kept under an average of 3,000 walking steps/d (2.7 km/d) for 364 d. Prior to their exposure to HK the volunteers were on an average of 10,000 running steps/d (14.2 km/d). During the prehypokinetic period of 60 d and during the hypokinetic period of 364 d were determined renal excretion of microelements responses of endurance-trained volunteers. In the hyperhydrated volunteers urinary excretion of iron, zinc, copper, manganese, cobalt, nickel, lead, tin, chromium, aluminum, molybdenum, and vanadium decreased, whereas in the hypokinetic volunteers it increased significantly. It was concluded that chronic hyperhydration may be used to attenuate urinary excretion of microelements in endurance-trained volunteers during prolonged restriction of muscular activity.     

Daily hyperhydration effect on electrolyte deficiency of endurance-Trained subjects during prolonged hypokinesia

The aim of this study was to evaluate the effect of a daily intake of fluid and salt supplementation (FSS) on the deficiency of electrolytes, which is characterized by higher rather than lower plasma concentration of electrolytes during prolonged hypokinesia (HK) (decreased number of km taken per day). Forty long distance runners aged 22–25 yr with a peak V0₂ 65.4 mL min^-1 kg^-1 with an average 14.2 km d running distance were selected as subjects. They were equally divided into four groups: 1) unsupplemented control subjects (UCS); 2) unsupplemented hypokinetic subjects (UHS); 3) supplemented hypokinetic subjects (SHS), and 4) supplemented control subjects (SCS). During the investigation of 364 d, groups 2 and 3 maintained an average running distance of less than 4.7 km per day, groups 1 and 4 did not experience any modification in their normal training routines and diets. During the preexperimental period of 60 d and during the experimental period of 364 d urinary excretion of electrolytes and concentrations of sodium, potassium, calcium, and magnesium in plasma were determined. Whole blood hemoglobin, hematocrit index, plasma osmolality, and plasma protein concentration were measured. In the UHS plasma concentration of electrolytes and urinary excretion thereof, fluid elimination, hematocrit, whole blood hemoglobin, plasma osmolality, and plasma protein concentration increased significantly (p < 0.05) when compared with the UCS, SCS, and SHS groups. In the SHS plasma concentration of electrolytes and urinary excretion thereof, fluid excretion, whole blood hemoglobin, hematocrit, plasma osmolality, and plasma protein concentration decreased when compared with the UHS and increased insignificantly when compared with the UCS and SCS groups. It was concluded that FSS may be used to prevent or minimize electrolyte deficiency in endurance-trained volunteers during prolonged restriction of muscular activity.     

Plasma trace elements concentrations in trained subjects after exposure to hypokinesia and daily hyperhydration

The objective of this investigation was to evaluate the effect of a daily intake of fluid and salt supplementation (FSS) on blood plasma trace elements concentrations in physically healthy volunteers after exposure to 364 d of hypokinesia (decreased number of steps per day). The studies were performed after exposure to 364 d of Hypokinesia (HK) on 30 long-distance runners of volunteers who had a VO₂ max 67 mL/kg/min and were ranging in the age of 19–24 yrs. Prior to their exposure to HK all volunteers were on an average of 10,000 steps/d. For the simulation of the hypokinetic effect the volunteers were kept under an average of 3000 steps/d. All volunteers were divided into three equal groups. The first group of volunteers subjected to HK and received daily FSS (water 26 mL/kg body wt and sodium chloride 0.16 g/kg body wt.), the second groups of volunteers submitted only to HK, and the third group of volunteers underwent a normal ambulatory life and served as control. The content of manganese, calcium, magnesium, iron, lead, copper, tin, nickel, zinc and cobalamine were determined in blood plasma of volunteers. By the end of the hypokinetic period the blood plasma concentration of microelements increased significantly in the hypokinetic subjects (second group), whereas in the hyperhydrated subjects (first group) decreased. It was concluded that prolonged restriction of motor activity induced significant increases in blood trace elements concentrations whereas daily hyperhydration had a normalizing effect on their concentration in blood plasma. This indicates that daily hyperhydration may be used to normalize blood plasma concentrations of microelements in physically healthy volunteers subjected to prolonged restriction of motor activity.     

Zinc metabolism in endurance-trained volunteers during prolonged restriction of muscular activity and chronic hyperhydration

The objective of this study was to evaluate the effects of hypokinesia (HK) and fluid- and salt supplementation (FSS) on zinc metabolism in endurance-trained volunteers (ETV) for a period of 364 d. Thirty long-distance runners aged 22–25 yr with a peak V_O2 of 67 mL/min/kg with an average 13.8 km/d running distance were chosen as subjects. They were equally divided into three groups:

Cytokinins,auxins and activated charcoal affect organogenesis and anatomical characteristics of shoot-tip cultures of lisianthus [<Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> (RAF.) Shinn]

Summary Variants from seed-propagated Lisianthus [Eustoma grandiflorum (Raf.) Shinn] were shoot-tip cultured to observe the effects of cytokinins, auxins and activated charcoal on organogenesis and anatomical characteristics. N⁶-Benzyladenine (BA) and kinetin at high concentrations (13.32–22.2 and 13.94–23.23 μM) resulted in good shoot formation but high percentages of hyperhydric shoots. Increased indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) concentrations favored root formation, while increased naphthaleneacetic acid concentration adversely affected root formation. Both shoot and root development were suppressed by activated charcoal. The highest percentage of regeneration and the largest number of glaucous shoots with an average of 15 shoots per explant after 4 wk of culture were obtained when the shoot tips were cultured on MS (Murashige and Skoog, 1962) medium supplemented with 4.44 μM BA and 1.47–4.92 μMIAA and IBA. In vitro-grown leaves had a higher number of stomata than field-grown leaves but the length and diameter of stomata showed no significant difference between the two types. Field-grown leaves had well-developed epicuticular wax layers. which were not observed on hyperhydric leaves. Hyperthydric plantlets could not survive when transplanted to soil, whereas glaucous plantlets survived in more than 80% of cases. Variation in soil type resulted in a slight difference in plantlet survival. Based on the results of our experiment, this protocol should be useful for the rapid micropropagation of lisianthus.     

Plasma volume changes during and after acute variations of body hydration level in humans

This study examined plasma volume changes (deltaPV) in humans during periods with or without changes in body hydration: exercise-induced dehydration, heat-induced dehydration and glycerol hyperhydration. Repeated measurements of plasma volume were made after two injections of Evans blue. Results were compared to deltaPV calculated from haematocrit (Hct) and blood haemoglobin concentration ([Hb]). Eight well-trained men completed four trials in randomized order: euhydration (control test C), 2.8% dehydration of body mass by passive controlled hyperthermia (D) and by treadmill exercise (60% of their maximal oxygen uptake, VO2max) (E), and hyperhydration (H) by glycerol ingestion. The Hct, [Hb], plasma protein concentrations and plasma osmolality were measured before, during and after the changes in body hydration. Different Hct and [Hb] reference values were obtained to allow for posture-induced variations between and during trials. The deltaPV values calculated after two Evans blue injections were in good agreement with deltaPV calculated from Hct and [Hb]. Compared to the control test, mean plasma volume declined markedly during heat-induced dehydration [-11.4 (SEM 1.7)%] and slightly during exercise-induced dehydration [-4.2 (SEM 0.9)%] (P < 0.001 compared to D), although hyperosmolality was similar in these two trials. Conversely, glycerol hyperhydration induced an increase in plasma volume [+7.5 (SEM 1.0)%]. These results would indicate that, for a given level of dehydration, plasma volume is dramatically decreased during and after heat exposure, while it is better maintained during and after exercise.     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Shoot regeneration from nodules of <Emphasis Type="Italic">Charybdis</Emphasis> sp.: a comparison of semisolid,liquid and temporary immersion culture systems

Nodules of Charybdis numidica maintained in liquid Murashige and Skoog (MS) medium with 20 mol BA in the dark were subjected to different treatments under continuous light for shoot regeneration. A high regeneration rate without hyperhydration of the shoots was observed on semisolid basal MS medium with 1% sucrose. The use of liquid MS medium (1% sucrose, no growth regulators) resulted in a significantly lower amount of shoots per gramme of nodules under both submerged and temporary immersion (TI) conditions. Shoot hyperhydration was lowest in a TI system with one 5 min immersion every 24 h. When compared on a per container base, large amounts of shoots could be produced in the TI system with less labour input than in the system with semisolid medium.     

Potassium changes in trained subjects after potassium loading and during restriction of muscular activity and chronic hyperhydration

The objective of this investigation was to determine whether urinary and plasma potassium changes developed during prolonged hypokinesia (HK) (decreased number of km/d) in endurance-trained subjects could be minimized or reversed with a daily intake of fluid and salt supplementation (FSS). The studies were performed on 30 endurance-trained male volunteers aged 23–26 yr with an average peak oxygen uptake of 65 mL/kg min during 364 d of HK. All volunteers were on an average of 13.8 km/d prior to their exposure to HK. All volunteers were randomly divided into three groups: 10 volunteers were placed continuously under an average of 14.0 km/d (control subjects), 10 volunteers were subjected continuously to an average of 2.7 km/d (unsupplemented hypokinetic subjects), and 10 volunteers were submitted continuously to an average of 2.7 km/d, and consumed daily an additional amount of 0.1 g sodium chloride (NaCl)/kg body wt and 30 mL water/kg body wt (supplemented hypokinetic subjects). During the prehypokinetic period of 60 d and during the hypokinetic period of 364 d, potassium loading tests were performed with 1.5–1.7 mEq potassium chloride/kg body wt, and potassium, sodium, and chloride excretion in urine and potassium, sodium, and chloride in plasma were determined. In the unsupplemented hypokinetic volunteers, urinary excretion of electrolytes and concentrations of electrolytes in plasma increased significantly as compared to the control and supplemented hypokinetic groups of volunteers. It was concluded that daily intake of fluid and salt supplementation had a favorable effect on regulation of urinary and plasma potassium changes in trained subjects during prolonged HK.