Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

Salt resistance of chickpea genotypes in solutions salinized with NaCl or Na2SO4

Summary To assess the potential for developing a salt resistant cultivar of chickpea (Cicer arietinum L.) 160 genotypes were screened for percent survival after 9 weeks in greenhouse solution cultures, with 50 mM NaCl or 25 mM Na₂SO₄. All plants grew well in the sulfate treatment but only cv. L-550 survived the chloride treatment. Salt damage appeared and developed slowly. To check these apparent effects of cultivar and kind of anion, three genotypes including cv. L-550 were then grown in solutions with isoosmotic NaCl or Na₂SO₄ at three levels (−0.044, −0.088, and −0.132 MPa), and in a separate experiment cv. L-550 was grown with NaCl and Na₂SO₄ at four levels: 10, 20, 30 and 50 mM Na. Salt composition affected shoot weight less than salt level or cultivar did. Shoot dry weight was only slightly less in chloride treatments than in isoosmotic sulfate, and for the least sensitive cultivar (L-550) this held only at the highest salt level, corresponding to that in the screening trial. Further, sensitivity to sulfate and to chloride was equal when sodium concentrations in shoots were equal, regardless of anion compositions of media. Shoot Na concentration was a useful negative indicator of growth under salt stress regardles of cultivar, and may be a useful tolerance indicator also for other species that neither accumulate nor efficiently exclude Na.     

Optimization of nutrient levels in the medium increases the efficiency of callus induction and plant regeneration in recalcitrant indian barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis>

Summary The present study reports that a revised nutrient concentration in the basal medium improved shoot bud induction and subsequent plant regeneration in barley (Hordeum vulgare L. var. BL-2). Cultures were raised from immature embryos on MSB₅ medium supplemented with picloram. Concentrations of five nutrients were varied. The effect of these nutrients was investigated on (1) induction, (2) induction and subculture, and (3) induction, subculture and regeneration stages. The basal MSB₅ medium was not optimal for each phase of barley culture. Decreased ammonium nitrate, increased potassium dihydrogen phosphate, sodium molybdate, cobalt chloride, and addition of glycine enhanced shoot bud induction and plant regeneration. The different media that were optimal for immature embryo culture were: MSB₅ medium supplemented with 20.70 μM picloram, 10.30 mM NH₄NO₃, 6.25 mM KH₂PO₄, 2.06 μM Na₂MoO₄, 0.55 μM CoCl₂, and 26.64 μM glycine (for induction); MSB₅ medium supplemented with 12.47 μM picloram, 10.30 mM NH₄NO₃, and 0.55 μM CoCl₂ (for subculture); and MSB₅ medium supplemented with 0.2 μM picloram and 10.3 mM NH₄NO₃ (for regeneration). Primary cultures required 6wk (without transfer) for morphogenic callus formation. Callus required 4wk of subculture and another 4wk on regeneration medium for optimal plant regeneration. The revised medium could also promote regeneration of the recalcitrant barley genotype RD-2552. Histological analysis showed that the major pathway of differentiation was through shoot bud formation.     

Uptake of inorganic ions and compatible solutes in cultured mangrove cells during salt stress

Summary Callus of the mangrove plant, Sonneratia alba J. Smith, established from pistils of flower buds were cultured on solid Murashige and Skoog medium supplemented with 0 to 500 mM NaCl. Maximum growth was observed with 50 mM NaCl, and net growth of callus occurred for concentrations up to 200 mM NaCl. At 500 mM NaCl, growth of callus was completely inhibited, although a part of the tissue was still alive after 30 d. Cellular levels of Na⁺ and Cl⁻ were greatly increased by the treatment with NaCl. Uptake of K⁺ was also enhanced and was accompanied by increasing levels of Na⁺ and Cl⁻ so that the Na⁺/K⁺ ratio was almost constant (4.1–4.2) in callus grown with 50–200 mM NaCl. Levels of Mg²⁺ and Ca²⁺ were not changed significantly with 50–200 mM NaCl, whereas levels of free NH ₄ ⁺ , NO ₃ ⁻ and SO ₄ ²⁻ ions, which are convertible to organic compounds, were lowest in callus grown with 50 mM NaCl. The rate of conversion of ¹⁵NH ₄ ⁺ into macromolecules during 30 d culture with 0–100 mM NaCl did not vary greatly, but 200 mM NaCl reduced the biosynthesis of macromolecules from this ion. The highest rate of conversion of ¹⁵NO ₃ ⁻ into macromolecules was observed at 50 mM NaCl. Identification of compatible solutes with NMR-spectroscopy indicated that mannitol is the compatible solute for intact plants of Sonneratia alba, but no accumulation of mannitol was found in calluses, not even in those grown at high concentrations of NaCl.     

Phosphorus-induced micronutrient disorders in hybrid poplar

The effect of calcium on the nodulation of lucerne was studied using EGTA, a specific calcium-chelator. First, the effects of the chelator were tested on hydroponically grown plants at pH 7.0. Optimal numbers of nodules were obtained in nutrient solution containing 0.2 mM CaCl₂. When 0.4 mM EGTA was given additionally, nodulation was completely inhibited. Nodulation was restored specifically with CaCl₂, but not with MgCl₂. For studies in an acid soil (pH-H₂O 5.2), lucerne seedlings were grown in rhizotrons. 67% of the seedlings became nodulated when the soil around the seed was neutralized locally with 1.0 μmol of K₂CO₃ in drops of 12 μL volume. When native calcium was removed with 2 μmol of EGTA, nodulation was reduced to 12%. However, addition of EGTA to soil resulted in a drop of pH from 6.1 to 5.2. A phosphate buffer could also not keep soil-pH sufficiently stable. Such pH-decreases could be avoided by placing agar blocks containing 6 μmol of EGTA for three hours on freshly developed roots. This treatment reduced nodulation from 87% to 32%, with soil-pH lowering only from 6.2 to 6.0. Nodulation could be restored by adding 2 μmol of CaCl₂. The depletion of soil-calcium could depress nodule formation only during the first day after inoculation.     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

Rhizosphere salinity and phytoalexin accumulation in soybean

Summary Rhizosphere salinity decreased the capacity of soybean to accumulate a pterocarpanoid phytoalexin (glyceollin) in the stem in response toPhytophthora megasperma var.sojae. Rapid (48h) accumulation was depressed by NaCl, Na₂SO₄, CaCl₂ and MgSO₄ applications. Time-course accumulations was slowed by applications. Time-course accumulation was slowed by application of 0.131M NaCl. Glyceollin accumulation was also reduced in plants subjected to a period of high salinity stress (0.177M NaCl, 72 h) after a period of nonsalinized growth. Calcium chloride completely suppressed glyceollin accumulation in normally-resistant plants but no susceptibility to the fungus was observed.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

披针叶黄华试管苗适应盐胁迫的渗透调节机制

以披针叶黄华(Thermopsis lanceolata)试管苗为材料,通过组培方法研究其在0、0.2%、0.4%、0.6%、0.8%和1.0%NaCl和Na2SO4胁迫30d后的生长、有机渗透调节物质和无机渗透调节物质(Na+、K+和Ca2+)含量的变化,以探讨其耐盐性机制。结果显示:(1)随NaCl和Na2SO4胁迫浓度的增加,披针叶黄华试管苗叶片脯氨酸和可溶性糖含量均显著持续增加,且NaCl胁迫下脯氨酸上升的幅度均大于相同浓度Na2SO4胁迫下的增幅,而可溶性糖上升的幅度却小于相同浓度Na2SO4胁迫下的幅度;可溶性蛋白含量随NaCl浓度的增大呈先升高后降低的趋势,但随Na2SO4浓度的增加呈持续上升的趋势。(2)随NaCl和Na2SO4浓度的增加,披针叶黄华试管苗Na+含量呈增加趋势且各处理均显著高于对照,Ca2+含量和叶片K+含量却呈逐渐减少趋势且各处理均显著低于对照,而根系K+含量呈先降后升的趋势;Na2SO4胁迫下披针叶黄华试管苗叶片Na+含量上升幅度以及K+和Ca2+含量下降幅度均明显低于相同浓度NaCl胁迫组;而Na+/K+和Na+/Ca2+比值随NaCl和Na2SO4浓度增加而升高;NaCl胁迫下,叶片Na+/K+和Na+/Ca2+高于相同浓度Na2SO4胁迫下的比值,而根系Na+/K+和Na+/Ca2+却低于相同浓度Na2SO4胁迫下的比值。研究表明,盐胁迫下,披针叶黄华试管苗通过抑制叶片中Na+积累并增加可溶性糖和可溶性蛋白含量,在根系中维持较高K+和Ca2+含量以及较低水平Na+/K+和Na+/Ca2+比,以降低披针叶黄华细胞渗透势来适应盐渍环境;披针叶黄华对NaCl胁迫的调节能力弱于Na2SO4。     

High-frequency shoot regeneration from cotyledon explants of watermelon cv. sugar baby

Summary A protocol for in vitro shoot regeneration from cotyledon explants of Citrullus lanatus (Thunb.) Matsum. & Nakai cv. Sugar Baby is described. The cotyledons excised from 7-d-old aseptic seedlings showed the highest percentage of shoots on Murashige and Skoog (MS) + N⁶-benzyladenine (BA; 3.0 μM) + N⁶-[2-isopentenyl] adenine (2iP; 3.0 μM) and MS + BA (3.0 μM) + indole-3-acetic acid (IAA; 3.0 μM). Whereas the latter medium induced shoot regeneration after the callusing of the explant, the former stimulated direct shoot formation. The regenerated shoots were rooted and the resulting plants were established in earthen pots with 55% success.     

Light and the maintenance of photosynthetic competence in leaves of Populus balsamifera L. during short-term exposures to high concentrations of sulfur dioxide

Leaves of Populus balsamifera grown under full natural sunlight were treated with 0, 1, or 2 l SO₂·1^-1 air under one of four different photon flux densities (PFD). When the SO₂ exposures took place in darkness or at 300 mol photons·m^-2·s^-1, sulfate accumulated to the levels predicted by measurements of stomatal conductance during SO₂ exposure. Under conditions of higher PFD (750 and 1550 mol·m^-2·s^-1), however, the predicted levels of accumulated sulfate were substantially higher than those obtained from anion chromatography of the leaf extracts. Light-and CO₂-saturated capacity as well as the photon yield of photosynthetic O₂ evolution were reduced with increasing concentration of SO₂. At 2 l SO₂·1^-1 air, the greatest reductions in both photosynthetic, capacity and photon yield occurred when the leaves were exposed to SO₂ in the dark, and increasingly smaller reductions in each occurred with increasing PFD during SO₂ exposure. This indicates that the inhibition of photosynthesis resulting from SO₂ exposure was reduced when the exposure occurred under conditions of higher light. The ratio F _v/F _M (variable/maximum fluorescence emission) for photosyntem II (PSII), a measure of the photochemical efficiency of PSII, remained unaffected by exposure of leaves to SO₂ in the dark and exhibited only moderate reductions with increasing PFD during the exposure, indicating that PSII was not a primary site of damage by SO₂. Pretreatment of leaves with SO₂ in the dark, however, increased the susceptibility of PSII to photoinhibition, as such pretreated leaves exhibited much greater reductions inF _V/F _M when transferred to moderate or high light in air than comparable control leaves.Abbreviations and symbols A₁₂₀₀ photosynthetic capacity (CO₂-saturated rate of O₂ evolution at 1200 mol photons·m^-2·s^-1) - F_o instantaneous fluorescence emission - F_M maximum fluorescence emission - F_V variable fluorescence emission - PFD photon flux density (400–700 nm) - PSII photosystem II     

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.     

Salt tolerance inNitellopsis obtusa

Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyteNitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl₂), no cell survived for more than one day. Within the first 30 minutes, membrane potential (E_m) depolarized and membrane resistance (R_m) decreased markedly. Simultaneously, cytoplasmic Na⁺ increased and K⁺ decreased greatly. At steady state the increase in Na⁺ content was roughly equal to the decrease in K⁺ content. The Cl^– content of the cytoplasm did not change. These results suggest that Na⁺ enters the cytoplasm by exchange with cytoplasmic K⁺. Both the entry of Na⁺ and the exit of K⁺ are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K⁺, Na⁺, and Cl^– remained virtually constant, suggesting that rapid influx of Na⁺ from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl₂, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na⁺] and [K⁺] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl₂, Em decreased, Rm increased, cytoplasmic Na⁺ and K⁺ returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca²⁺ in salt tolerance inNitellopsis are discussed; one a reduction in plasmalemma permeability to Na⁺ and the other a stimulation of active Na⁺-extrusion.     

Callus formation and plant regeneration from tubercles of Coryphantha elephantidens (Lem.) Lem.

Summary Callus cultures were established from pith tissue of Coryphantha elephantidens (Lem.) Lem. on Murashige and Skoog (MS) basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin. Highest shoot regeneration frequency was observed on a medium containing 6.9 μM kinetin and 2.3 μM 2,4-D under 30 μE m⁻² s⁻¹ light intensity with a 16-h photoperiod. Calluses retained organogenic potential throughout several passages of subculture (18 mo.). Shoots were rooted on MS medium without plant growth regulators. All (100%) plantlets transplanted to soil survived acclimatization. Regenerated plants showed good overall growth and were morphologically similar to the mother plants.     

Some morphogenic effects of sodium sulfate on tobacco callus

Callus cultures of Nicotiana tabacum L cv. Wisconsin 38 were initiated and grown on shoot-forming (SF) and callus proliferation (CP) medium with or without Na₂SO₄. Two cultures were maintained on SF medium with 0, 0.75, 1 or 1.5% Na₂SO₄ for 2.5 and 3.5 years. In the older culture only callus grown on salt formed shoots throughout the maintenance period, while in the younger culture the control responded best and Na₂SO₄ was inhibitory. Callus from the older culture which had been grown on salt continued to form shoots in the absence of salt. Na₂SO₄ caused adventitious shoot formation in three cultures on CP medium. These shoots were present for 7 subcultures after removal of Na₂SO₄; but established, control callus, did not form shoots when transferred to Na₂SO₄. Callus initiated and maintained on NaCl or mannitol showed a slight increase in shoot initiation. On NaCl, Na₂SO₄ or mannitol, the tissue osmotic potential became more negative and proline concentration increased.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method

Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l⁻¹ of Km for 6 wk and then transfer to 30 mg l⁻¹ of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl⁻¹ cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m⁻²s⁻¹. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.     

Different sealing materials for petri dishes strongly affect shoot regeneration and development from leaf explants of quince ‘BA 29’

Summary The effect of different sealing materials [i.e., polyvinyl chloride (PVC) transparent film, and Parafilm (PARA) for Petri dishes was investigated on shoot regeneration from quince (Cydonia oblonga L.) ‘BA 29’ leaf explants. Leaves were excised from proliferating shoot cultures, transversally scored, and placed with the abaxial side down in 60-mm Petri dishes containing 10 ml of Murashige and Skoog modified medium, with 5.4 μM α-naphthaleneacetic acid, 4.5 μM thidiazuron, 200 mg l⁻¹ cefotaxime, and 0.25% (w/v) Phytagel (IM medium) for shoot bud induction, and cultured in darkness at 22±2°C for 28 d. Then the explants were transferred to standard conditions (16-h photoperiod at 30 μmol m⁻² s⁻¹ photosynthetically active radiation) on a medium similar to IM, except for lack of NAA, and with 0.65% (w/v) agar instead of Phytagel, for an additional 15–28 d. The sealing combinations PARA-PARA, PARA-PVC, PVC-PARA, and PVC-PVC (in the induction-expression phases) were compared during regeneration and for their carry-over effect on shoot development after transfer of explants to an elongation medium (0.9 μM 6-benzyladenine). Carbon dioxide accumulated at 27.2 mmol mol⁻¹ at the end of induction, and gradually decreased from 35.4 mmol mol⁻¹ on day 9 to 22.5 mmol mol⁻¹ on day 28 of the expression phase in PARA-sealed Petri dishes, being always much higher than after sealing with PVC (1–2 mmol mol⁻¹). Ethylene concentration was 0.1 and 0.04 μmol mol⁻¹ in the first part of the induction and expression phase, respectively, in PARA-sealed Petri dishes, and slightly decreased with duration of exposure to light during expression; while it was absent in most PVC-sealed dishes. The PARA-PARA and PVC-PVC (induction-expression) combinations gave, respectively, the worst and best results of regeneration and successive shoot development.     

Shoot regeneration from petioles of coral bells (Heuchera sanguinea Engelm.) cultured in vitro, and subsequent planting and flowering ex vitro

Summary Successful shoot regeneration from petioles, leaves, and petioles with leaves cultured in vitro is reported in Heuchera sanguinea. Petioles or petioles with leaves regenerated more shoots than leaves alone. For culture, the optimum hormonal concentrations were 0.19 μM α-naphthaleneacetic acid combined with 0.44 or 4.4 μM benzyladenine in Murashige and Skoog-based (MS) medium: the regenerating rate and the number of shoots per explant were 60% and 8.6–9.7, respectively. Histological study on petiole culture showed dividing cell clusters including vascular tissues after 1 wk, callus including several dividing cell clusters at the periphery after 3 wk and then apical meristems with immature leaves after 5 wk. Rooting from the regenerated shoots was highest (95%) on MS medium containing 4.9 μM indole-3-butyric acid. Seventy-three percent of rooted plants were successfully acclimatized in pots. When they were cultured in the field, the plants grew and most flowered the following year over winter.