The effects of Zn2+ on guinea pig isolated heart preparations

Isolated guinea pig hearts were perfused, by the Langendorff technique, with 30, 15, 7.5, and 1.5 μM Zn²⁺ in Chenoweth solution. Contractile force, coronary flow, and heart rate were recorded by means of Narco IV Physiograph. Calcium inhibitor (Verapamil 1 μM) and anion inhibitor (DIDS: 0.1, 1, and 5 μM) were used subsequently in the perfusing solutions in order to distinguish some of the possible mechanisms that Zn²⁺ uses to exert its action on cardiac myocytes. Isomolar to zinc concentration of Pb (II) and Co (II) were used to elucidate whether zinc effects on heart are specific for this metal. All hearts were used to estimate their zinc and calcium content by means of AAS (Atomic Absorption Spectrometry). Our findings suggest that the higher the Zn²⁺ concentration, the more toxic effects on heart are expressed by rapid reversible contractile force reduction and reversible specific changes of heart rate and flow. Zinc 1.5 μM in the perfusing solution benefits heart performance, but not significantly. Furthermore, the metal exerts specific effects on guinea pig heart, and it is rather possible that these effects on cardiac myocytes are held through cell membrane receptors.     

Plant regeneration of creeping bluestem (Schizachyrium scoparium (Michx.) Nash var. Stoloniferum (Nash) J. Wipff) via somatic embryogenesis

Summary Creeping bluestem (Schizachyrium scoparium (Michx.) Nash var. stoloniferum (Nash) J. Wipff) embryogenic callus growing on solid medium was used to establish a cell suspension culture in Murashige and Skoog (MS) basal medium supplemented with 1.5 mg l⁻¹ (6.8 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg l⁻¹ (0.88 μM) 6-benzylaminopurine (BA), 0.5 mg l⁻¹ (1.4 μM) zeatin, 0.2 mg l⁻¹ (0.58 μM) gibberellic acid (GA₃), and 10% (v/v) of coconut water (CW). Pro-embryos from suspension culture matured on semi-solid MS medium in about 18 wk, and were then cultured on semi-solid MS medium without growth regulators for 2–3 wk. Shoots were regenerated on MS basal medium supplemented with 3.0 mg L⁻¹ (13.6 μM) 2,4-D, 1.0 mg l⁻¹ (4.4 μM) BA, 1.0 mg l⁻¹ (2.9 μM) GA₃, 0.5 mg l⁻¹ (2.7 μM) 1-naphthaleneacetic acid (NAA), 500 mg l⁻¹ easein hydrolysate, and 10% (v/v) CW. Rooted plantlets were successfully accelimatized to greenhouse and outdoor conditions. Using this protocol, it would be possible to produce at least 1300 fully acclimatized plantlets annually.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

Direct shoot regeneration from leaf segments of mature plants of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> (L.) moench

Summary This is the first communication of direct shoot regeneration from fully developed leaves of potted mature Echinacea purpurea plants. Shoot buds were induced directly on the adaxial surface of mature leaf tissues of E. purpurea 30 d after culture initiation on Woody Plant Medium (WPM) supplemented with various levels of 6-benzyladenine (BA). Maximum shoot organogenesis, with 12–20 shoots per leaf segment, was obtained with 5% coconut milk and 2.5 mg l⁻¹ (6μM) BA in 30 d. Callus was induced using 0.5 mgl⁻¹ (1μM) α-naphthaleneacetic acid and 2.5 mgl⁻¹ (6μM) BA. The regenerated shoots were rooted on WPM supplemented with 1.5 mgl⁻¹ (3μM) of indole-3-butyric acid, 3% sucrose, and 0.85% agarose. Rooted plants were successfully transferred to soil in pots and appeared morphologically normal and flowered in a growth chamber.     

Somatic embryogenesis from embryogenic cell suspension cultures of california poppy, Eschscholzia californica Cham

Summary A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented with 0.5 mg 1⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition, gyratory shaker speed, various carbon sources, different cytokinins, and AgNO₃ on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog (MS) salts and vitamins supplemented with 40 g 1⁻¹ (117 mM) sucrose, 0.05 mg 1⁻¹ (0.22 μM) 6-benzylaminopurine, and 10 mg l⁻¹ (58.8 μM) AgNO₃. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development of somatic embryos was promoted by AgNO₃ at concentrations below 10 mg l⁻¹ (58.8 μM). A semi-solid medium containing 1.5 g l⁻¹ Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture.     

Plant regeneration through somatic embryogenesis and rapd analysis of regenerated plants in Tylophora indica (Burm. F. Merrill.)

Summary A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l⁻¹; 0.0–13.56 μM) and kinetin (Kn; 0.01 mg l⁻¹; 0.05 μM). The best response for callus induction was obtained on MS medium containing 2 mg l⁻¹ (9.04 μM) 2.4-D and 0.01 mg l⁻¹ (0.05 μM) Kn. After two subeultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5–3 mg l⁻¹; 2.22–13.32 μM) and 2-isopentenyladenine (2ip; 0.53 mg l⁻¹; 2.46–14.76 μM) along with 0.01 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l⁻¹ (9.84 μM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic, DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Clonal micropropagation of <Emphasis Type="Italic">Crataeva adansonii</Emphasis> (DC.) Prodr.: A multipurpose tree

Summary A method of micropropagation through multiple shoot formation from axillary buds of mature tree and rootstock growths of Crataeva adansonii (DC.) Prodr. (a multipurpose tree) has been developed. Factors affecting multiplication rate included season, age of explant source, explant type, type of bud, position of bud on the foliage twig, type of medium, various additives, and explant implantation on the medium. The maximum number of buds was produced from the sixth to 10th axillary buds taken from foliage twigs of 40–50-d-old rootstock growth in the months of October to December. At this time of the year the contamination was minimum. Optimum response was recorded on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 3 mgl⁻¹, 13.3μM) and 1-naphthaleneacetic acid (NAA; 0.05 mgl⁻¹, 0.27 μM) after 21 d of culture. Shoot buds were further multiplied and maintained on BA (1–1.5 mgl⁻¹, 4.4–6.7 μM) while shoots elongated on BA (0.1–0.5 mgl⁻¹, 0.44–2.2 μM) supplemented medium. The number of shoots was further multiplied by using nodal segments of in vitro-regenerated shoots as microcuttings and repeated subculturing of stumps after excising the microshoots. In vitro rooting on growth regulator-free MS medium was possible with 70% of microshoots after 4 wk. From one nodal segment 150 plantlets were produced within 14 wk.     

Highly efficient embryogenesis and plant regeneration of tall fescue (Festuca arundinacea Schreb.) from mature seed-derived calli

Summary We report a protocol for efficient plant regeneration of four tall fescue (Festuca arundinacea Schreb.) cultivars (‘Surpro’, ‘Coronado’, ‘Summer Lawn’, and ‘Fawn’) via somatic embryogenesis. Calli were initiated from mature seeds grown on modified Murashige and Skoog (MMS) medium supplemented with 7.0mgl⁻¹ (31.7μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mgl⁻¹ (0.23 μM) kinetin (Kin). Calli were maintained and proliferated by subculture at monthly intervals on MMS medium containing 4.5 mgl⁻¹ (20.4 μM) 2,4-D and 0.2mgl⁻¹ (0.9 μM) Kin. Somatic embryos (SE) were induced from seed-derived calli on SE-induction medium (MMS supplemented with 2.0 mgl⁻¹ 2,4-D and 0.2mgl⁻¹ Kin). Plantlets were regenerated from somatic embryogenic calli grown on modified SH medium supplemented with 2 mgl⁻¹ Kin. Using this optimized protocol, 78.6–82.3% of mature seeds of all four cultivars produced SE clusters, of which 93.5–95.3% regenerated into plants within 10 wk. The regenerants showed no phenotypic abnormalities.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Micropropagation of Cymbidium ensifolium var. Misericors through callus-derived rhizomes

Summary The effects were studied of light intensity, culture method and cytokinins on plant formation from callus-derived rhizomes of Cymbidium ensifolium var. misericors. The results demonstrated that one piece of rhizome produced seven shoot buds in 45 d when cultured in 1/2 MS basal liquid medium supplemented with 0.17 μM N⁶-(2-isopentenyl) adenine, 0.17 μM thidiazuron, 33 μM 6-aminopurine adenine and 1.5 μM naphthaleneacetic acid under 10 μmol m⁻² s⁻¹ artificial light and agitated at 60 rpm on a rotary shaker. These shoot-cm plantlets in 5 mo. when transferred to the same Gelrite-gel basal medium supplemented with 50 g l⁻¹ banana pulp. Plantlets were acclimated and grew well when potted in the greenhouse.     

Micropropagation studies inCeropegia SPP

Summary The purpose of this study was to developin vitro techniques for conserving wild and endemic species ofCeropegia by mass multiplication for subsequent reintroduction in their natural habitat. Micropropagation involving a combination of axillary bud culture, shoot multiplication, somatic embryogenesis andin vitro tuber formation forCeropegia jainii, a rare plant of the Indian sub continent,C. bulbosa var.bulbosa andC. bulbosa var.lushii, common species, was developed. Nodal explants from all species were cultured on 0.5 MS medium with 8.8 μM (2 mg·l⁻¹) N⁶-benzyl aminopurine (BA) to regenerate the axillary buds. These produced multiple shoots when transferred to multiplication medium consisting of 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA, or microtubers when transferred to 0.5 MS medium with 22.2 μM (5 mg·l⁻¹) BA and 23.2 μM (5 mg·l⁻¹) kinetin.In vitro flowering occurred inC. jainii and not in the other two varieties when the plants were cultured on multiplication media with spermine at 0.25 μM (50 μg·l⁻¹) as an additive. Shoot pieces produced callus on MS medium with 9.05 μM (2 mg·l⁻¹) 2,4-dichlorophenoxy acetic acid. Regeneration of the calli by somatic embryogenesis was achieved when they were transferred to 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA. Rooting of the shoots was possible both byin vitro andex vitro means.     

Propagation of rose speciesin vitro

Summary Several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata) and interspecific hybrids were cultured to determine the appropriate concentrations of nutrients and growth regulators for shoot proliferation and root initiation. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal medium [Murashige and Skoog (MS) salts, vitamins, glycine, sucrose, and agar] supplemented with 0 μM to 17.8 μM (4 mg·l⁻¹) 6-benzyladenine (BA) and 0 μM to 0.54 μM (0.1 mg·l⁻¹) naphthalene, acetic acid (NAA). The ability of the explants to proliferate shoots and initiate roots was affected by the genotype, the nodal position of the explant, the strength of the MS basal salts, and the growth regulators used. The buds nearest the apex exhibited the slowest rate of development. Most species had the highest shoot proliferation when cultured on basal MS medium supplemented with 8.9 μM (2 mg·l⁻¹) BA, but the degree varied by species. Root development was enhanced by lowering the concentration of MS salts. With difficult-to-root species, rooting was improved by supplementing the media with 11.4 μM (2 mg·l⁻¹) indole-3-acetic acid (IAA) or by giving them a 7-d dark treatment at 10°C.     

Somatic embryogenesis and plant regeneration in yakutanegoyou, <Emphasis Type="Italic">Pinus armandii</Emphasis> Franch. var. <Emphasis Type="Italic">amamiana</Emphasis> (Koidz.) Hatusima,an endemic and endangered species in Japan

Induction of somatic embryogenesis in Pinus armandii var. amamiana, an endemic and endangered species in Japan, was initiated from megagametophytes containing immature zygotic embryos on both media with and without plant growth regulators. Across nine open-pollinated families initiation frequency ranged from 0 to 20%, with an average of 1.5%. Embryogenic cultures were maintained and proliferated on a medium supplemented with 2,4-dichlorophenoxyacetic acid (3 μM) and 6-benzylaminopurine (1 μM). Maturation of somatic embryos occurred on medium containing maltose (50 g l⁻¹), activated charcoal (2 g l⁻¹), abscisic acid (100 μM), and polyethylene glycol (100 g l⁻¹). The frequencies of germination and plant conversion of somatic embryos differed among the embryogenic lines from 16 to 51% and from 12 to 40%, respectively. Growth of regenerated somatic plants has been monitored in the field.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume, Dalbergia sissoo Roxb

Summary A complete and efficient protocol is presented for plant regeneration from cell-suspension cultures of Dalbergia sissoo Roxb., an economically important leguminous tree. Factors influencing callus initiation, establishment of cell-suspension culture, callus formation from embredded microcolonies, and shoot organogenesis from suspension-derived callus were identified. Of the two different auxins tested, callus induction was better on a medium containing naphthalene acetic acid (NAA). The percentage of callus induction increased considerably when NAA at 2.0 mg l⁻¹ (10.8 μM) was added in conjunction with 0.5 mg l⁻¹ (2.2 μM) N⁶-benzyladenine (BA). Of the three different explants evaluated for callus induction, hypocotyl segments were most responsive. Friable hypocotyl-derived callus from the second subculture passage was used to initiate the cell-suspension culture. Optimum growth of the cell suspension was observed in MS medium supplemented with the same growth regulators as described above for callus induction, with an initial inoculum cell density of 1%. The plating efficiency of the microcolonies was greatly influenced by harvesting time and the gelling agent used for plating. Efficiency was highest (93%) with cells harvested at their exponential growth phase and plated in 1.2 g l⁻¹ Phytagel. Shoot organogenesis from callus cultures was higher on a medium supplemented with a combination of BA and NAA than on BA alone. Seventy-one per cent of cultures exhibited shoot-bud differentiation on a medium containing 3.0 mg l⁻¹ (13.3 μM) BA and 0.5 mg l⁻¹ (2.7 μM) NAA. Regenerated shoots were rooted on half-strength MS medium containing 1 mg l⁻¹ each of indole-3-acetic acid (5.7 μM), indole-3-butyric acid (4.9 μM) and indole-3-propionic acid (5.3 μM). Plantlets were acclimated and established in soil.     

Callus induction and plant regeneration of U.S. rice genotypes as affected by medium constituents

Summary This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorbitol or 4% sucrose alone, and 0.5 to 4 mg·L⁻¹ (2.26 to 18.10 μM) 2,4-D with or without 0.5mg·L⁻¹) (2.32 μM) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg·L⁻¹ (9.29 μM) kinetin combined with 0 or 0.1 mg·L⁻¹ (0.54 μM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration.     

Effects of cadmium and copper on zinc transport kinetics by isolated renal proximal cells

Zinc, cadmium, and copper are known to interact in many transport processes, but the mechanism of inhibition is widely debated, being either competitive or noncompetitive according to the experimental model employed. We investigated the mechanisms of inhibition of zinc transport by cadmium and copper using renal proximal cells isolated from rabbit kidney. Initial rates of⁶⁵Zn uptake were assessed after 0.5 min of incubation. The kinetics parameters of zinc uptake obtained at 20°C were a J_max of 208.0±8.4 pmol· min⁻¹·(mg protein)⁻¹, aK _m of 15.0±1.5 μM and an unsaturable constant of 0.259±0.104 (n=8). Cadmium at 15 μM competitively inhibited zinc uptake. In the presence of 50 μM cadmium, or copper at both 15 and 50 μM, there was evidence of noncompetitive inhibition. These data suggest that zinc and cadmium enter renal proximal cells via a common, saturable, carrier-mediated process. The mechanisms of the noncompetitive inhibition observed at higher concentrations of cadmium or with copper require further investigation, but may involve a toxic effect on the cytoskeleton.     

Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.