The combined impact of pH and activated carbon on the elemental composition of a liquid conifer embryogenic tissue initiation medium

This study considered the elemental composition of plant tissue culture media in response to pH and two different types of activated C (AC; tissue culture and non acid-washed grades) in liquid media. When tissue culture medium is supplemented with AC the method of AC addition and pH adjustment can greatly impact the final medium pH, in turn, altering mineral availability. Over the pH range of 4–7, Cu and Zn adsorbed (95% and 50%) onto the two physically different ACs to the same extent. As the pH exceeded 5.8, precipitation became pronounced, resulting in 50% reductions in Mn and Fe and smaller reductions in Ca (20%), and P (15%), independent of AC. Non acid-washed AC released significant levels of Mg (65% increase) and Ca (10% increase) at pH 5.8 compared to the no-AC control. No adsorption was indicated for inorganic anions. Low levels for Cu and Zn are a concern when using AC, and low levels of Fe and Mn are a concern when the pH of the medium exceeds 5.8. Due to its impurity content and difficulty associated with its neutralization, non-acid-washed AC may be a poor choice for use in tissue culture medium.Communicated by W.A. Parrott     

Levels and distribution of zinc,copper, magnesium,and calcium in rats fed different levels of dietary zinc

Effects of altered dietary zinc on levels of zinc, copper, magnesium, and calcium in organ and peripheral tissues were studied. When rats fed a zinc-deficient diet (1.3 μg Zn/g) for 28 d were compared with rats fed a control diet (37.5 μg Zn/g), levels of zinc were slightly lower in plasma, hair, and skin and 50% lower in femur and pancreas, whereas the levels of copper were higher in all tissue except plasma. Magnesium levels were higher than controls in the heart and lower in the spleen, whereas the calcium levels were lower in plasma, lung, spleen, kidney, and skin and strikingly higher in brain, hair, and femur. When rats fed a zinc-supplemented diet (1.0 mg Zn/g) were compared to the same conrols, levels of zinc in these were higher in all organs and peripheral tissues studied, except heart, lung, and liver; copper levels were higher in liver, kidney, and spleen; magnesium levels were significantly higher in the spleen, but were little affected in other tissues, although calcium levels were higher in pancreas, spleen, kidney, and skin and lower in plasma and hair. These data indicate that overall copper organ and peripheral tissue levels are affected inversely, and zinc and calcium levels directly, by zinc nutriture.     

Improving ethanol tolerance of a self-flocculating yeast by optimization of medium composition

High ethanol tolerance is a desired property of industrial yeast strains for efficient ethanol fermentation. In this study, the impact of medium composition on ethanol tolerance of the self-flocculating yeast SPSC01 was investigated using a chemically defined medium. Single-factor experiments revealed that besides magnesium and calcium, zinc also exhibited significant protective effect against ethanol toxicity; addition of 0.02 g/l zinc sulfate significantly increased cell viability in the ethanol shock treatment. Metal ions of manganese, cobalt, and ferrous failed to promote ethanol tolerance, although addition of 0.02 g/l cobalt increased ethanol production without apparent influence on ethanol tolerance. Furthermore, Uniform Design method was employed to obtain the medium with high cell viability, and the key nutrient factors in the medium composition were revealed to be (NH₄)₂SO₄, K₂HPO₄, vitamin mixtures, and the metal ions of magnesium, calcium and zinc. The optimized combination of metal ions addition was (g/l): MgSO₄ 0.4, CaCl₂ 0.2, ZnSO₄ 0.01. The highest cell viability (90.2%) of SPSC01 against ethanol shock treatment was observed in the optimized medium, which demonstrated significant improvement of ethanol tolerance of the self-flocculating yeast.     

Magnesium Supplementation did not Affect the Increasing Bone Zinc Concentration in Rats Given Excess Calcium as Carbonate

We investigated the effect of magnesium supplementation on zinc distribution in rats given excess calcium as carbonate. Rats were given a control diet (5 g/kg calcium and 0.5 g/kg magnesium), a high calcium diet (HC, 25 g/kg calcium and 0.5 g/kg magnesium) or the high calcium diet supplied with magnesium (HCM, 25 g/kg calcium and 2.5 g/kg magnesium) for 4 weeks. Calcium carbonate and magnesium oxide were used for increasing these mineral concentrations in diets. Although feed intake did not differ among the groups, the excess calcium suppressed feed efficiency, irrespective of dietary magnesium concentration. Femoral magnesium concentration was lower in the HC group than in the control and the HCM groups. Femoral zinc concentration was higher in the HC group and the HCM group than in the control group. The zinc concentration in the kidney was lower in the HC group and the HCM group than in the control group. The excess calcium did not affect zinc concentration in plasma and other tissues such as the liver, testis, and spleen, irrespective of dietary magnesium. These results suggest that the increasing bone zinc and the decreasing renal zinc do not result from magnesium insufficiency in rats given excess calcium as carbonate.     

Achieving desired plant growth regulator levels in liquid plant tissue culture media that include activated carbon

This paper is part of a series considering the impact of activated carbon (AC) on the composition of plant tissue culture media. Using liquid culture media for initiation of Norway spruce embryogenic tissue and eight different ACs, we present a method for achieving target plant growth regulator (PGR) levels in AC-containing medium based on sorption isotherms for individual PGRs. Linear relationships were found between PGR adsorption and specific BET (Brunauer, Emmett, Teller theory) surface area and specific total pore volume of AC. When using a new AC, this linear relationship allows one to achieve multiple PGR levels similar to historic levels through adjustment of the mass of AC based on its relative BET surface area or relative total pore volume. Target levels of PGRs and an initiation success similar to that in medium without AC were achieved with several different AC types when AC mass was adjusted on the basis of pore volume.     

Influence of zinc on lotic plants

SUMMARY. The toxicity of zinc to Hormidium rivulare Kütz. in laboratory culture media is decreased by rises in the levels of magnesium, calcium and phosphate, and increased by rises in pH and cadmium. The effects of all these are sufficiently marked that they may be expected to have considerable importance in the field. In contrast, assays with sodium, chloride and sulphate showed no detectable influence of these ions on zinc toxicity.
When applied at higher concentrations, calcium was always more effective than magnesium, but the reverse was sometimes true at lower concentrations. Both magnesium and phosphate were more effective in reducing zinc toxicity with zinc-tolerant populations than with zinc-sensitive ones. Cadmium was highly toxic, either alone or in combination with zinc; however, calcium had a proportionately greater effect in reducing cadmium toxicity than zinc toxicity. The toxic effects of zinc and cadmium were synergistic, and it seems probable that the presence of cadmium at levels of 0.01 mgl⁻¹ and above will usually lead to a significant increase in the toxicity of any zinc also present. Cadmium (in the absence of zinc) was 34 times more toxic than zinc to a zinc + cadmium sensitive population, and 15.5 times more toxic to a zinc + cadmium tolerant population. Because of the synergistic response, cadmium had an even greater effect in the presence of zinc.     

Changed serum trace element profile in Down's syndrome

Being cofactors of important antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), which are significantly modified in Down's syndrome (trisomy 21), serum levels of microtrace elements zinc, copper, and selenium and of macroelement magnesium are reported in 16 subjects with Down's syndrome (DS) and their respective well age- and sex-matched controls. Serum zinc and selenium levels were significantly lowered in DS subjects, whereas copper levels were elevated. Consequently, a marked increase (40%) of the copper/zinc ratio in DS persons was observed. There were no differences in serum levels of magnesium between DS and control subjects.     

Acidocalcisomes of trypanosomatids have species-specific elemental composition

The elemental composition and stoichiometric profile of elements present in acidocalcisomes of different genera of the Trypanosomatidae family (insect, plant, and mammalian parasites) submitted to parallel cultivation conditions were studied. X-ray microanalysis using transmission electron microscopy in conjunction with a morphometric approach was used to investigate the elemental content, number, distribution, and volumetric density of acidocalcisomes of different species. Microanalytical data showed that the different parasites possess the same elemental composition (oxygen, sodium, magnesium, phosphorus, calcium, iron, and zinc) in their acidocalcisomes. However, the relative concentrations of the elements varied among species, but not within acidocalcisomes of individual species. Iron was detected in acidocalcisomes of all species analyzed, characterizing this element as a constituent of these organelles. Taken together, the results strongly indicate a species-specific composition of acidocalcisomes in trypanosomatid parasites.     

Multivariate analysis on the distribution of elements in plants

The present review accentuates the elemental composition of plants as investigated by various researchers utilizing sophisticated techniques. Plants contain plethora of elements which extend their benefits to humans both in terms of medicine as well as nutrition. Therefore, there is a need to explore the elemental composition of plants to investigate their nutraceutical importance. In this review, the elemental data was analyzed using different multivariate techniques. The average values of elements used in this review are sodium (Na) (241.1 µg/g dw), magnesium (Mg) (1202.4 µg/g dw), aluminum (Al) (159.6 µg/g dw), silicon (Si) (133.7 µg/g dw), phosphorus (P) (1947 µg/g dw), sulfur (S) (1483.1 µg/g dw), chloride (Cl) (624.5 µg/g dw), potassium (K) (3008.5 µg/g dw), calcium (Ca) (1663.1 µg/g dw) and iron (Fe) (143.6 µg/g dw) respectively. Maximum accumulation of Na, K, Ca, Mg, Fe, P and S occurs in Poaceae, Lamiaceae, Lauraceae, Sapotaceae, Apiaceae, Pedaliaceae and Brassicaceae, respectively.     

Somatic embryogenesis in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.): improving culture initiation with abscisic acid and silver nitrate

Loblolly pine (Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7 µM), silver nitrate (20 µM), and guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt (10 µM) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l -naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4–6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.Abbreviations ABA Abscisic acid - AC Activated carbon - BA 6-Benzylaminopurine - 8-Br-cGMP Guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt - NAA -Naphthaleneacetic acid Communicated by G.C. Phillips     

Statistical optimization of culture media for growth and lipid production of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> LB572

Botryococcus braunii has an outstanding ability to produce lipid; however, it is a slow-growing green microalgae. Statistical optimization of growth media was performed to faster growth and to increase lipid concentration. The effect of media composition on the growth of B. braunii LB572 was examined using fractional factorial design and central composite design. The media components examined include sodium carbonate, potassium phosphate, calcium chloride, magnesium sulfate, ferric citrate, and sodium nitrate. The results indicated that potassium phosphate and magnesium sulfate were major impact factors. The optimum concentrations of potassium phosphate and magnesium sulphate were found to be 0.058 and 0.09 g/L, respectively, for growth and 0.083 and 0.1 g/L, respectively, for lipid production. These values were validated using bubble column photobioreactors. Lipid productivity increased to 0.19 g/L/day in lipid-optimized media, with an average biomass productivity of 0.296 g/L/day and 64.96% w/w. In growth-optimized media, lipid productivity was 0.18 g/L/day, with an average biomass productivity of 0.304 g/L/day and 59.56% w/w.     

Factors influencing regeneration from protoplasts of Asparagus densiflorus cv. Sprengeri

This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7 mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt solution with 7 mm CaCl₂ ^.2H₂O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks. Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997     

Comparison of element levels in minimal and complex yeast media

The cellular functions are strongly influenced by the composition of the environment. In particular, phenotypes of microbial strains are modulated by concentrations of ions in the culture medium, and differences in element levels may be responsible for a phenotypic variability observed when microbial strains are grown on synthetic versus complex media. In this report, we analyzed the levels of nine elements (magnesium, potassium, sodium, calcium, iron, copper, manganese, zinc, and phosphorus) and sulphate ions in commercially available peptone and yeast extract and compared them with those in yeast nitrogen base routinely used for preparation of synthetic minimal media. We observed that whereas some elements are present at similar levels, the levels of others differ by a factor as high as 20. The observed differences should be taken into account when interpreting different phenotypes observed for microbial strains grown on synthetic versus complex media.     

Dietary zinc effects on zinc, calcium, and magnesium content in bones of growing rats

The aim of the present study was to assess dietary zinc effects on femur weight and mineral content in growing rats. For this purpose, 70 weanling Sprague-Dawley rats were divided into four groups. Each group was subject to a diet containing 2 (BZ), 5 (DZ), 10 (MZ), and 30 (CZ) ppm zinc. The calcium and magnesium content in all diets was 5 g/kg and 507 mg/kg, respectively. The animals were kept on this regime for 28 d and then sacrificed and their femurs were removed for analysis using atomic absorption spectrophotometry. The weights of the BZ and DZ groups were significantly different from the MZ and CZ groups (38.5±10.5, 89.9±13.7, 118.6±13.6 and 134±19.9 g, p<0.01) respectively. There were no differences between the MZ and CZ groups. Femur weight also varied with dietary zinc, as it was significantly different among all groups (BZ, 265±49 mg; DZ, 380±40 mg; MZ, 452±54 mg; CZ, 735±66 mg; p<0.01). The femur zinc content varied with diets, following a different pattern than the above parameters. Femur zinc from the BZ group (51.5±5.4 ppm) was significantly different from the MZ and CZ groups (115.9±14.2 and 175.0±13.5 ppm, respectively), whereas the DZ group (62.5±11.3 ppm) did not differ from the other three groups. The femur content of calcium (BZ, 83.2±9.8 mg/g; DZ, 88.0±9.2 mg/g; MZ, 90.2±13.6 mg/g; CZ, 83.1±14.7 mg/g) and magnesium (BZ, 1.82±0.13 mg/g; DZ, 1.98±0.09 mg/g; MZ, 1.93±14 mg/g; CZ, 1.83±0.19 mg/g) were not significantly different among the groups, nor was the calcium-magnesium ratio. These results suggest that although dietary zinc deficiency retards growth and causes bone fragility, bone deposition of calcium and magnesium and its ratio are not affected.     

Plant regeneration from protoplasts of peppermint (Mentha piperita L.)

Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - KIN kinetin - ZEA zeatin - CPW cell and protoplast wash solution - B5 Gamborg et al. (1968) mineral elements - MS Murashige and Skoog (1962) mineral elements     

Elemental composition of anatomically distinct regions of rat liver

Experiments were conducted to test the commonly held assumption that analysis of a portion of rat liver is representative of the elemental concentration of the whole organ. Male Sprague-Dawley rats (initial body weight approximately 250 g) fed a chow diet or weanling male Long-Evans rats (initial body weight approximately 50 g) fed a semipurified diet with or without copper in the mineral premix were sacrificed after 4 wk on their respective diets and livers were dissected into seven portions representing major anatomical divisions of this organ. Elemental analyses by atomic absorption spectroscopy (calcium, magnesium, iron, zinc, copper, manganese), atomic emission spectroscopy (sodium, potassium), or colorimetric assay (phosphorus) demonstrated no statistically significant differences in composition of these nine elements among anatomical regions of liver. Dietary copper deficiency led to equivalently reduced copper concentration in all portions of rat liver and did not cause any other significant alterations in liver composition of these nine elements within the 4 wk of these studies. These results confirm the validity of the common assumption that analysis of a portion of rat liver can be representative of the elemental composition of the whole organ. This conclusion will allow more analyses to be performed on fewer animals, thereby reducing animal use and reagent costs without sacrificing analytical accuracy. Publication No. 517 of the Bureau of Nutritional Sciences.     

Effect of rigidity of gel medium on benzyladenine-induced adventitious bud formation and vitrification in vitro in Picea abies

Observations on the effects of different degrees of rigidity of both an agar (Tayio) and a non-agar (Gelrite) gel on the uptake of radiolabelled N⁶-benzyladenine (¹⁴C-BA) were also extended to mode of application and positioning of the explant. Regression analysis showed a highly significant inverse correlation between ¹⁴C-BA accumulation and degree of gel stiffness. Significantly greater numbers of adventitious buds per explant were induced at low to medium levels of rigidity (2.5–10 g Tayio 1⁻¹, 1–5 g Gelrite 1⁻¹); this advantage was almost completely nullified at the lower levels (2.5 and 5.0 g Tayio 1⁻¹, 1 and 1.5 g Gelrite 1⁻¹) as a result of the high incidence of vitrification. In addition to turgor distension, vitrified buds displayed cellular damage. Explants with their cotyledons flattened onto the agar surface accumulated less ¹⁴C-BA after 96 h than upright explants, but produced greater numbers of adventitious buds, pseudobuds and phylloids. It was suggested that BA was taken up only by "target" cells, presumably the differentiating subsidiary cells of those stomatal complexes in surface contact with the medium. Pulse treatments of relatively short durations (2 h) with optimal concentrations of BA (ca 125 μ M ), followed by subculturing on hormone-free media gelled with 10 g agar 1⁻¹, produced a satisfactory balance between yield and competence of adventitiously-induced buds.     

Influence of isolation technique of half-anthers and of initiation culture medium on callus induction and regeneration in Anthurium andreanum

Optimization of the isolation technique and initiation culture medium are two critical aspects that can determine the success of anthurium half-anther culture. Both aspects in half-anther culture of Anthurium andreanum Linden ex André cv. ??Tropical?? were studied and successfully improved. Untreated half-anthers, when cultured abaxial side down on medium, was the most suitable means of inducing callus. Callus formation was further improved by culturing half-anthers adaxial side down on Winarto-Teixeira (WT) medium (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) supplemented with 0.01?mg/l ??-naphthaleneacetic acid (NAA), 1.0?mg/l 6-benzyladenine (BA) and 0.5?mg/l thidiazuron (TDZ). Gelrite enhanced callus formation (compared to agar) when the concentration was reduced from 2.0 to 1.5?g/l on WT medium. Application of 0.5?mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in WT medium increased callus formation. Most callus formed when half-anthers were cultured adaxial-side down on WT medium supplemented with 0.5?mg/l 2,4-D in combination with 0.01?mg/l NAA, 1.0?mg/l BA and 0.5?mg/l TDZ using 1.5?g/l Gelrite. This ideal medium induced the growth of half-anthers, with as much as 38?% of half-anthers producing callus, or, on average, 3.4 half-anthers/treatment. Callus derived from this optimized protocol regenerated easily and could be multiplied on New Winarto-Teixeira medium (NWT) (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) containing 0.25?mg/l 2,4-D, 0.02?mg/l NAA, 0.75?mg/l BA, 1.5?mg/l TDZ and 2.0?g/l Gelrite. Shoots rooted well on hormone-free NWT medium with 2.0?g/l Gelrite. The plantlets could be easily acclimatized in a substrate of raw rice husk, burned-rice husk and organic manure (1:1:1, v/v/v) with a high survival (100?%) ex vitro in a greenhouse. The results of this study would benefit half-anther culture of other Anthurium cultivars, particularly at the initial stage of callus induction.     

Calcium, magnesium, and zinc status in experimental hypothyroidism

In this study, experimental hypothyroidism was established and used to investigate possible alterations in the calcium, magnesium, and zinc homeostasis by assessing their concentration in plasma and erythrocytes. Hypothyroidism was induced by administration of methimazole an iodine blocker at a dose of 75 mg/100 g food for 3 wk. In the methimazole-induced hypothyroid state, the experimental animals showed a significant decrease in plasma zinc concentration, whereas a significant increase in plasma magnesium concentration occurred. No change was observed in plasma calcium concentration. The erythrocyte zinc and calcium concentrations were found to be increased, whereas magnesium concentration decreased. Erythrocyte magnesium concentration showed a significant positive correlation with T₄ values. The study provides evidence for marked alterations in homeostatis of zinc, magnesium, and calcium.     

Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.)

Summary Suspension cultures which maintained embryogenic potency for more than 18 months were established from excised immature embryos of rice (Oryza sativa L. cv. Konansou). The cultures were subcultured every three days in N6 medium supplemented with proline (10 mM), casein hydrolysate (300 mg/l), sucrose (30 g/l) and 2,4-D (1 mg/l). The frequency of embryogenesis from the embryogenetic suspension cultures reached about 90% when cell clusters (about 1 mm in diameter) were transferred to a solid medium which consisted of N6 medium, NAA (1 mg/l), kinetin (5 mg/l), sucrose (30 g/l) and Gelrite (2 g/l). When smaller clusters of cells (approximately 200–400 m in diameter) were transferred to a liquid medium which consisted of salts of N6 medium diluted with an equal volume of water plus sucrose (45 g/l), NAA (0.01 mg/l) and 4-PU (0.1 mg/l) at a cell density of 13 clusters/ml in 2 ml of medium, somatic embryogenesis was initated at high frequency (about 50%). Morphological evidence is provided to demonstrate that the regeneration occurred via embryogenesis. This is the first report of high-frequency embryogenesis in suspension cultures of rice cells.