Growth responses of seminal roots of wheat seedlings to a reduction in the water potential of vermiculite

We examined the elongation rate, water status and solute accumulation in the seminal roots of wheat seedlings (Triticum aestivum L.) that were growing in vermiculite with a water potential (Ψ_w) ranging from −0 03 to −1 10 MPa. The elongation rate of the primary seminal root was similar to that of the first pair of seminal roots but that of the second pair of seminal roots was lower at all values of Ψ_w tested. The elongation rate was highest in vermiculite with a Ψ_w of −0.03 MPa but did not decrease significantly until the Ψ_w was reduced to −0.15 MPa. Further reductions in Ψ_w reduced the elongation rate markedly. The Ψ_w of mature tissues was always similar to that of vermiculite. The osmotic potential (Ψ_o) decreased to the same extent as the decrease in Ψ_w. Thus, the turgor pressure (Ψ_p) remained unchanged even in vermiculite with a low Ψ_w. In elongating tissues, Ψ_w and Ψ_o were far lower than they were in mature tissues and, thus, reductions in turgor were not significant. Even when the Ψ_w of vermiculite changed, there were no consistent changes in terms of a difference in Ψ_w between elongating plus mature tissues and vermiculite. There were also no consistent changes in levels of osmotica, calculated using the van’t Hoff’s law, in the elongating tissues but the levels in mature tissues increased in vermiculite with a low Ψ_w. Our results suggest that (1) reductions in root elongation in vermiculite with a low Ψ_w were caused by reductions in the extensibility and/or increases in the yield threshold of cell walls and by reductions in the hydraulic conductivity of the tissues; and (2) a seminal root regulates its growth to keep turgor pressure unchanged.     

Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes

One-month old calli of two indica rice genotypes, i.e., Basmati-370 and Basmati-Kashmir were subjected to two iso-osmotic concentrations (−0.57 MPa and −0.74 MPa) created with 50 and 100 mol m⁻³ NaCl or 10 and 18% solutions of PEG-8000. Both genotypes tolerated only low levels of stress and showed severe growth suppression at −0.74 MPa. The degree of stress tolerance of both genotypes was greater for PEG induced stress than for NaCl induced stress. The relative growth rate of callus was reduced under both stresses, however, the reverse was true for callus dry weight. Sodium (Na⁺) content of the callus tissue was increased only under NaCl induced stress. Salt induced stress reduced K⁺ and Ca²⁺ contents, but the PEG induced stress increased them. Higher levels of stress increased the proline content many folds with more increase being under PEG stress than that under NaCl. Water and osmotic potentials of the callus tissue decreased, whereas turgor potential increased under both abiotic stresses. Overall, Basmati-370 was more tolerant to both NaCl and PEG induced stresses than Basmati-Kashmir, because of less reduction in growth and more dry weight. Moreover, Basmati-370 accumulated higher amounts of cations, free proline, and maintained maximum turgor as compared to Basmati-Kashmir. In conclusion, at cellular level, mechanism of NaCl induced osmotic stress tolerance was found to be associated with more ionic accumulation of inorganic solutes and that of PEG induced osmotic stress tolerance with the accumulation of free proline, as an important osmolyte in the cytosol.     

Inhibition of photosynthetic processes in foliose lichens induced by temperature and osmotic stress

Negative effects of osmotically-induced dehydration of two foliose lichen species, Lasallia pustulata and Umbilicaria hirsuta, was studied at physiological (22 °C), low (5 °C) and freezing temperature (−10 °C), using chlorophyll (Chl) fluorescence. In both species, exposure to increasing sucrose concentrations led to a pronounced decrease in potential (F_V/F_M), and actual (Φ₂) quantum yields of photochemical processes in photosystem 2. L. pustulata was more sensitive to osmotic stress, because comparable osmotic dehydration inhibited F_V/F_M and Φ₂ more than in U. hirsuta. Critical concentration of sucrose that fully inhibited photochemical processes of photosynthesis was 2.5 M, which represented water potential (Ψ_w) of −18.8 MPa. Decrease in background Chl fluorescence (F₀) and increase in non-photochemical quenching (qN) revealed two phases of osmotic stress in lichens: phase I with no change (Ψ_w 0 to −6.6 MPa) and phase II (Ψ_w −11.3 to −18.8 MPa) typical by substantial change in Chl fluorescence parameters. Effects of thallus anatomy on species-specific response to osmotic dehydration is discussed and attributed to the results obtained by optical microscopy and Chl fluorescence imaging technique.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Partitioning of photosynthetic electron flow between CO<Subscript>2</Subscript> assimilation and O<Subscript>2</Subscript> reduction in sunflower plants under water deficit

In sunflower (Helianthus annuus L.) grown under controlled conditions and subjected to drought by withholding watering, net photosynthetic rate (P _N) and stomatal conductance (g _s) of attached leaves decreased as leaf water potential (Ψ_w) declined from −0.3 to −2.9 MPa. Although g _s decreased over the whole range of Ψ_w, nearly constant values in the intercellular CO₂ concentrations (C _i) were observed as Ψ_w decreased to −1.8 MPa, but C _i increased as Ψ_w decreased further. Relative quantum yield, photochemical quenching, and the apparent quantum yield of photosynthesis decreased with water deficit, whereas non-photochemical quenching (q_NP) increased progressively. A highly significant negative relationship between q_NP and ATP content was observed. Water deficit did not alter the pyridine nucleotide concentration but decreased ATP content suggesting metabolic impairment. At a photon flux density of 550 μmol m⁻² s⁻¹, the allocation of electrons from photosystem (PS) 2 to O₂ reduction was increased by 51 %, while the allocation to CO₂ assimilation was diminished by 32 %, as Ψ_w declined from −0.3 to −2.9 MPa. A significant linear relationship between mean P _N and the rate of total linear electron transport was observed in well watered plants, the correlation becoming curvilinear when water deficit increased. The maximum quantum yield of PS2 was not affected by water deficit, whereas q_P declined only at very severe stress and the excess photon energy was dissipated by increasing q_NP indicating that a greater proportion of the energy was thermally dissipated. This accounted for the apparent down-regulation of PS2 and supported the protective role of q_NP against photoinhibition in sunflower.     

The effect of NaCl on water relations,chlorophyll, and protein and proline contents of two cultivars of blackgram (Vigna mungo L.)

The physiological basis of salt tolerance of two cultivars of blackgram, cv Candhari Mash (relatively salt tolerant) and cv Mash 654 (salt sensitive), was assessed in salinized sand culture at the flowering stage. Increasing NaCl concentration in the rooting medium significantly reduced the chlorophyll a, chlorophyll b, and total chlorophyll, leaf water potential (Ψ_w), leaf solute potential (Ψ_s), and leaf turgor potential (Ψ_p) in both the cultivars. Leaf protein and proline content was increased as a result of increasing salt concentration in both cultivars. High salt concentrations had no significant effect on the seed protein content of both cultivars. At high salinities, cv Candhari Mash had significantly greater chlorophyll a, chlorophyll b and total chlorophyll, leaf water potential, solute potential, and turgor potential than cv Mash 654, but the latter had greater leaf proline content than cv Candhari Mash. Cultivars did not differ significantly for both leaf and seed protein contents. The relatively salt tolerant cv Candhari Mash maintained high leaf water potential and turgor potential to resist salt injury. Leaf proline content had negative correlation with salt tolerance in blackgram.     

Regeneration through organogenesis in rattan

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l⁻¹ glutamine, 100 mg l⁻¹ arginine, 100 mg l⁻¹ asparagine, 60 g l⁻¹ sucrose, 8 g l⁻¹ Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Screening for drought tolerance in Sorghum using cell culture

Summary Callus growth from 10 cultivars ofSorghum bicolor (L.) Moench was measured with increasing levels of polyethylene glycol (PEG) as an osmoticum in the medium to determine whether differences among these cultivars at the cellular level in response to osmotic stress existed. These cellular ratings were compared to field ratings from the 10 tolerant-to-susceptible cultivars when grown under drought conditions to determine whether cellular ratings corresponded to differences in drought tolerance at the plant level. Callus cultures were grown on Murashige and Skoog inorganic salt formulation plus vitamins, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin and sucrose, supplemented with 0 to 25% (wt/vol) PEG corresponding to −0.2 to −1.62 MPa osmotic potential. Results suggest that PEG-induced osmotic stress on callus cultures can be used to screen sorghum cultivars for potential early field (preflowering) drought tolerance. This implies that at least a component of the early field drought tolerance in sorghum may have a cellular basis. This study was supported by U.S. Agency for International Development Grant AID/DSAN/XII/G-0149, and USDA Competitive Grants Program.     

Soil water availability and rooting depth as determinants of hydraulic architecture of Patagonian woody species

Adaptations of species to capture limiting resources is central for understanding structure and function of ecosystems. We studied the water economy of nine woody species differing in rooting depth in a Patagonian shrub steppe from southern Argentina to understand how soil water availability and rooting depth determine their hydraulic architecture. Soil water content and potentials, leaf water potentials (Ψ_Leaf), hydraulic conductivity, wood density (ρ_w), rooting depth, and specific leaf area (SLA) were measured during two summers. Water potentials in the upper soil layers during a summer drought ranged from −2.3 to −3.6 MPa, increasing to −0.05 MPa below 150 cm. Predawn Ψ_Leaf was used as a surrogate of weighted mean soil water potential because no statistical differences in Ψ_Leaf were observed between exposed and covered leaves. Species-specific differences in predawn Ψ_Leaf were consistent with rooting depths. Predawn Ψ_Leaf ranged from −4.0 MPa for shallow rooted shrubs to −1.0 MPa for deep-rooted shrubs, suggesting that the roots of the latter have access to abundant moisture, whereas shallow-rooted shrubs are adapted to use water deposited mainly by small rainfall events. Wood density was a good predictor of hydraulic conductivity and SLA. Overall, we found that shallow rooted species had efficient water transport in terms of high specific and leaf specific hydraulic conductivity, low ρ_w, high SLA and a low minimum Ψ_Leaf that exhibited strong seasonal changes, whereas deeply rooted shrubs maintained similar minimum Ψ_Leaf throughout the year, had stems with high ρ_w and low hydraulic conductivity and leaves with low SLA. These two hydraulic syndromes were the extremes of a continuum with several species occupying different portions of a gradient in hydraulic characteristics. It appears that the marginal cost of having an extensive root system (e.g., high ρ_w and root hydraulic resistance) contributes to low growth rates of the deeply rooted species.     

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.     

Poly(3-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> in fed-batch culture

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l⁻¹). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH₄Cl and K₂HPO₄ on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH₄Cl and 0.22% (w/v) K₂HPO₄ and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l⁻¹, respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH₄Cl, and K₂HPO₄ were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l⁻¹, PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l⁻¹ h⁻¹.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Somatic embryogenesis and plantlet production using rejuvenated tissues from serial grafting of a mature <Emphasis Type="Italic">Kalopanax septemlobus</Emphasis> tree

An effective micropropagation technique via somatic embryogenesis has been developed using tissue from serially grafted shoots generated from a mature Kalopanax septemlobus tree (~40 y old). Callus was induced from leaf segments obtained from the grafts by culturing the explants in Murashige and Skoog (MS) medium supplemented with 2,4-D and 3% w/v sucrose under darkness. The effects of sucrose, coconut water, and polyethylene glycol (PEG-3350) were evaluated as factors to promote development of somatic embryos (SEs) from embryogenic callus. More than 90% of explants formed callus; however, only 2.5%, or 20 leaf segments out of 800 explants, formed embryogenic callus after 8 wk of culture. High sucrose concentrations (3% and 5% w/v) were effective in inducing SEs. Treatment with 2–10% v/v coconut water also had a positive effect on embryo induction. A synergistic effect on SE induction was obtained using sucrose and PEG, with presence of the latter compound resulting in smaller, more uniform SEs. Embryo germination and conversion to plantlets were significantly influenced by the gelling agents. In general, gelrite-gelled medium was superior to agar-gelled medium. In gelrite-gelled medium, gibberillic acid (GA₃) enhanced embryo germination. Converted plantlets in an artificial soil mixture showed a 91% survival rate and displayed no distinct morphological variations. Our results indicate that reliable somatic embryogenesis and plant production can be achieved with rejuvenated tissues after repeated grafting of shoots derived from a mature Kalopanax septemlobus tree.     

The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary Callus induction and later plant regeneration were studied in four widely grown garlic (Allium sativum L.) cultivars from Europe. Root segments from in vitro plantlets were used as starting material. In addition to cultivar effects, the effects of auxin and cytokinin levels and the position of the segments on the root were studied. There were no statistically significant differences among cultivars for the number of root segments that induced callus in the two series of experiments. The average induction frequency was 34.7% in the first series of experiments. Callus induction on apical root segments was significantly higher compared to callus induction on non-apical root segments in the second series of experiments. Two months after callus induction, callus lines were transferred to a regeneration medium consisting of Murashige and Skoog basal medium supplemented with 30gl⁻¹ sucrose and 1 mgl⁻¹ (4.6μM) kinetin. Calluses derived from different experiments were quite uniform with respect to their regeneration potential. Also it was found that our regeneration system was cultivar-independent. The average shoot regeneration frequency was 17.9% in the first series of experiments. Highly significant differences were found in the frequency of shoot regeneration among different callus induction treatments. When the cytokinin 6-(γ,γ-dimethylallylamino)purine (0.1mgl⁻¹∶0.5 μM) was present during callus induction, shoot regeneration ranged from 30.10 to 47.60%. Shoot regeneration from callus induced on non-apical segments was higher, although not significant, compared to callus induction from apical root segments in the second series of experiments. All in all, an efficient callus induction and plant regeneration system was developed from both apical and non-apical segments taken along the entire length of the roots. This system has potential to be used for garlic transformation.     

Fruit ripening in Vitis vinifera: apoplastic solute accumulation accounts for pre-veraison turgor loss in berries

In Vitis vinifera L. berries, the onset of ripening (known as “veraison”) involves loss of turgor (P) in the mesocarp cells. We hypothesized that P loss was associated with an accumulation of apoplastic solutes in mesocarp tissue prior to veraison. Apoplastic sap was extracted from the mesocarp by centrifugation at the appropriate gravity to measure the apoplast solute potential (Ψ_s^A) and assay the sap composition. The Ψ_s^A was about −0.2 MPa early in development, decreased about 1.0 MPa by veraison, and continued to decrease during ripening to almost −4.0 MPa by the end of berry development. Potassium, malate, tartrate, proline, glucose, fructose, and sucrose were quantified in apoplastic sap. The calculated contribution of these solutes was about 50% of the total Ψ_s^A preveraison, but increased to about 75% as fructose and glucose accumulated during ripening. The contribution of the estimated matric potential to apoplast water potential decreased during development and was only 1.5% postveraison. We conclude that high concentrations of solutes accumulated in the mesocarp apoplast prior to veraison, and that P loss was a direct result of decreased Ψ_s^A. Because Ψ_s^A decreased before veraison, our findings suggest that apoplast solutes play an important role in the events of cellular metabolism that lead to the onset of ripening.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Summary Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 μM 2,4-dichlorophenoxyacetic acid, 17.8 μM 6-benzyladenine, 18.6 μM kinetin, 500 mg l⁻¹ casein hydrolysate, and 500 mg l⁻¹ l-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radieles of mature zygotic embryos. Callus was subcultured on the callus proliferation medium, the same as the induction medium but with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO₃, Ca(NO₃)₂·4H₂O, NH₄NO₃, KCl, ZnSO₄·7H₂O, and MnSO₄·H₂O, 720, 1900, 400, 250, 25.8, and 25.35 mg l⁻¹, respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating the production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4–12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l⁻¹). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1∶1∶1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.     

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (<Emphasis Type="Italic">Petiveria alliacea</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) leaf explants

A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l⁻¹. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7 ± 26.18 mg) and mean root number (23.7 ± 1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both extracts. UV spectral analysis and the use of standard quantitation procedures showed that the quantity of DTS in the somatic embryo extract, at 0.16% (w/v), was approximately 30-fold higher than in rhizogenic/embryogenic callus (0.0055% w/v) of similar fresh weight. These results indicate that it is possible to biosynthesize approximately 6 mg of natural DTS from 3,808 mg of fresh somatic embryos within 10 wk from less than three leaf explants.     

In vitro screening for increased drought tolerance in rice

Summary In vitro screening at the cellular level was performed with mature seed-derived callus from five rice varieties, viz. IR 18351-229-3, IR 3185-6-3-3-2, SR 26-B, Nona Bokra, and C 14-8 of diverse geographical origin and with differential drought resistance at the in planta level. Callus was induced from mature seeds on Murashige and Skoog medium supplemented with 2.0 mgl⁻¹ (9 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0, 10.0, and 15.0 gl⁻¹ of high molecular weight polyethylene glycol (PEG, 6000) as stressing agent to create chemical drought. Simultaneous efforts were also made to assess the effects of chemical drought in altering morphogenetic response in different varieties under in vitro culture. Seed germination was almost unaffected in SR 26-B and C 14-8, unlike in other varieties where germination was seriously affected. In general, seed germination was found to be decreased in three genotypes, viz. IR 18351-229-3, IR 3185-6-3-3-2, and Nona Bokra, with increased PEG concentrations. All genotypes displayed callus induction percentage in decreasing order with increased PEG concentrations supplemented in the callus induction medium (CIM), except SR 26-B and Nona Bokra. Callus induction was found to be more on CIM fortified with 5.0 gl⁻¹ PEG. In general, embryogenic callus induction and plantlet regeneration was found to be indirectly proportional to increased PEG concentrations used in CIM. Considering all characters, C 14-8 was found to be most appropriate in developing drought-tolerant lines under in vitro culture conditions followed by SR 26-B and Nona Bokra. A number of putative drought-tolerant plants were developed in C 14-8, SR 26-B, Nona Bokra, and IR 18351-29-3, and forwarded for field evaluation. In the majority of the progenies, a monogenic inheritance pattern for the drought tolerance character was observed.     

Control of leaf cell elongation in barley. Generation rates of osmotic pressure and turgor, and growth-associated water potential gradients

In a previous study on the effects of N-supply on leaf cell elongation, the spatial distribution of relative cell elongation rates (RCER), epidermal cell turgor, osmotic pressure (OP) and water potential (Ψ) along the elongation zone of the third leaf of barley was determined (W. Fricke et al. 1997, Planta 202: 522–530). The results suggested that in plants receiving N at fixed relative addition rates (N-supply limitation of growth), cell elongation was rate-limited by the rate of solute provision, whereas in plants growing on complete nutrient solution containing excessive amounts of N (N-demand limitation), cell elongation was rate-limited by the rate of water supply or wall yielding. In the present paper, these suggestions were tested further. The generation rates of cell OP, turgor and Ψ along the elongation zone were calculated by applying the continuity equation of fluid dynamics to the previous data. To allow a more conclusive interpretation of results, anatomical data were collected and bulk solute concentrations determined. The rate of OP generation generally exceeded the rate of turgor generation. As a result, negative values of cell Ψ were created, particularly in demand-limited plants. These plants showed highest RCER along the elongation zone and a Ψ gradient of at least −0.15 MPa between water source (xylem) and expanding epidermal cells. The latter was similar to a theoretically predicted value (−0.18 MPa). Highest rates of OP generation were observed in demand-limited plants, with a maximum rate of 0.112 MPa · h⁻¹ at 16–20 mm from the leaf base. This was almost twice the rate in N-supply-limited plants and implied that the cells in the leaf elongation zone were capable of importing (or synthesising) every minute almost 1 mM of osmolytes. Potassium, Cl⁻ and NO₃ ⁻ were the main inorganic osmolytes (only determined for demand-limited plants). Their concentrations suggest that, unlike the situation in fully expanded epidermal cells, sugars are used to generate OP and turgor. Anatomical data revealed that the zone of lateral cell expansion extended distally beyond the zone of cell elongation. It is concluded that leaf cell expansion in barley relies on high rates of water and solute supply, rates that may not be sustainable during periods of sufficient N-supply (limitation by water supply: Ψ gradients) or limiting N-supply (limitation by solute provision: reduced OP-generation rates). To minimise the possibility of growth limitation by water and osmolyte provision, longitudinal and lateral cell expansion peak at different locations along the growth zone. Received: 15 October 1997 / Accepted: 12 March 1998