Changes of phenylethanoid and iridoid glycoside distribution in various tissues of shoot cultures and regenerated plants of Harpagophytum procumbens (Burch.) DC. ex Meisn

Harpagophytum procumbens is a medicinal plant containing several compounds with pharmaceutical activity. Previously, we established shoot culture and in vitro regenerated plants of H. procumbens. In this study, HPLC and LC-ESI-MS were used to identify harpagoside, harpagide, verbascoside and isoverbascoside in various tissues (stems, leaves and callus) of shoots multiplied on Schenk and Hildebrandt (SH) solid medium supplemented with 0.57 μM indole-3-acetic acid (IAA) and 8 μM 6-benzylaminopurine (BAP), as well as in stems, leaves and root tubers of in vitro propagated plants grown in the greenhouse for 3, 6 and 12 months. The content of the compounds was also determined by HPLC. For comparison, control H. procumbens plants initiated from seeds were analyzed. H. procumbens shoots grown under in vitro conditions accumulated lower amounts of iridoids and phenylethanoids than the plants derived from them. The levels of analyzed compounds were higher in the organs of 3- or 6-month-old plants than in those of 12-month-old plants. Differences in the distribution of secondary metabolites were also observed between organs. The aerial parts (stems, leaves) of 3-month-old in vitro regenerated plants were characterized by the highest amounts of phenylethanoids, which significantly exceeded those detected in control plants. Total iridoid content, calculated as the sum of harpagoside and harpagide, was highest in the root tubers of 6-month-old plants. In these organs the level of harpagoside was comparable to that in root tubers of 6-month-old seed-propagated plants, but the level of harpagide was much lower.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

Development of an efficient callus proliferation system for Rheum coreanum Nakai,a rare medicinal plant growing in Democratic People’s Republic of Korea

A clonal mass propagation to obtain mountainous sources of Rheum coreanum Nakai, a rare medicinal plant in Democratic People’s Republic of Korea was established by rhizome tissue culture. Whole plants were selected and collected as a vigorous individual free from blights and harmful insects among wild plants of R. coreanum grown on the top of Mt. Langrim (1.540 m above the sea) situated at the northern extremity of Democratic People’s Republic of Korea. Induction of the callus was determined using four organs separated from the whole plant and different plant growth regulators. The callus was successfully induced from rhizome explant on MS medium containing 2.4-D (0.2–0.3 mg/l). In the MS medium supplemented with a combination of BAP (2 mg/l) and NAA (0.2 mg/l), single NAA (0.5 mg/l), or IBA (0.5 mg/l), a higher number of shoot, root and plantlets was achieved. The survival rate on the mountainous region of the plantlets successfully acclimatized (100%) in greenhouse reached 95%, and yields of crude drug and contents of active principles were higher than those obtained by sexual and vegetative propagation. This first report of R. coreanum tissue culture provides an opportunity to control extinction threats and an efficient callus proliferation system for growing resources rapidly on a large scale.     

Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn.—An endangered plant

Leptadenia reticulata (Retz.) Wight. & Arn. is an important medicinal plant, belongs to the family Asclepiadaceae. This plant is known for its medicinal uses since 4500 BC. Presently this is an endangered species (Arya et al., 2003). Six shoots (2–4 cm long) per node differentiated on MS medium + 5.0 mg/l of BAP + additives. Incorporation of additives in the culture medium promoted growth of cultures. The shoots differentiated per explant were repeatedly transferred on to fresh MS + 1.0 mg/l of BAP + 0.1 mg/l of NAA and additives. The regenerated shoots were subcultured for further multiplication on MS + 1.0 mg/l BAP + 0.5 mg/l Kin + 2-iP (0.5 mg/l) and 0.1 mg/l of NAA + additives regularly after an interval of 3 weeks. Addition of ammonium sulphate in the medium resulted in increase in shoot number and promoted elongation also growth of cultures was sustained even if subculturing was delayed (26 ± 2 days). Success was also achieved in defining protocol for in vitro regeneration of shoots from petiole derived callus. Shoots regenerated in vitro by both processes were rooted in vitro on 1/4 strength of MS medium + 3.0 mg/l of IBA after 15–20 days. Cent percent of the shoots rooted ex vitro, if the in vitro regenerated shoots were treated with 200 mg/l of IBA. The in vitro–ex vitro rooted plantlets were hardened under different regimes of temperature and humidity in a greenhouse. The hardened plantlets were transferred to soil in polybags. More than 95% plants survived in field conditions. Total dry biomass harvested per year was 2800 kg/acre.     

Production of rhoifolin and tiliroside from callus cultures of Chorisia chodatii and Chorisia speciosa

The current work aims to stimulate the production of rhoifolin and tiliroside as two valuable phytochemicals from Chorisia chodatii Hassl. and Chorisia speciosa A. St.-Hil. callus cultures. A comparison between three explants from the in vitro germinated seedlings of both species for callus induction and accumulation of both flavonoids was carried out. Highly efficient calluses were induced from the leaves, stems and roots of C. chodatii seedlings on Gamborg’s B5 (B5) and Murashige and Skoog (MS) media containing 2.0 mg/l β-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenin (BA) or kinetin (Kn), while those of C. speciosa seedlings efficiently produced calluses on both media supplemented with 0.5 or 1.0 mg/l NAA and 0.5 mg/l BA. Besides, the highest contents of rhoifolin (1.927 mg/g DW) and tiliroside (1.776 mg/g DW) from C. speciosa cultures were obtained from the calluses of seedlings’ roots and stems maintained on B5 medium containing 1.0 mg/l NAA and 0.5 mg/l BA, respectively. On the other hand, the maximum rhoifolin content (0.555 mg/g DW) from C. chodatii cultures was obtained from the calluses of seedlings’ stems grown on B5 medium supplemented with 2.0 mg/l NAA and 0.5 mg/l BA, whereas the highest tiliroside content (0.547 mg/g DW) was provided by the root explants on B5 medium containing 2.0 mg/l NAA and 0.5 mg/l Kn. Both flavonoids were bioaccumulated in greater amounts than the wild and cultivated intact plants, which provides a promising tool for their future commercial production under a controlled environment, independent of climate and soil conditions.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

Effects of nutrient media,different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond × peach) vegetative rootstock

The objective of this research was to assess the effects of different media i.e. Murashige and Skoog (MS) and Quoirin and Lepoivre (QL), cytokinin type i.e. 6-Benzyladenin (BA) and 6-Benzylaminopurine (BAP) and cytokinin concentration on in vitro proliferation of the G × N15 rootstock. To evaluate the effects of different media and cytokinin type, two separate experiments were conducted as factorial based on completely randomized design, and single nodes were used as explants. The results showed that MS nutrient medium was found to be superior to QL nutrient medium. Regarding the interaction between media and growth regulators, the best interaction was found in MS medium supplemented with 1 mg l⁻¹ BAP resulting in 8.5 new micro shoots/explant while 7.75 shoots were observed in MS medium containing 1.25 mg l⁻¹ BA. The longest length of new micro-shoots (2.10 cm) was obtained in hormone-free MS medium. Findings of this study showed that there is a significant correlation between the hormone level and plantlet height and formed callus weight so that an increase in BAP and BA levels in both of MS and QL media resulted significantly in height decrease and callus weight increase. The results also suggest that the best and the worst plantlets in terms of quality were observed in hormone-free QL medium and MS medium supplemented with 1.25 mg l⁻¹, respectively. These results reflect the fact that the presence of high amounts of NH₄NO₃ and cytokinin especially BAP in culture medium triggered inhibitory effect on shoot growth.     

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.     

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.     

Transformation of tef (Eragrostis tef) by Agrobacterium through immature embryo regeneration system for inducing semi-dwarfism

Successful application of genetic transformation for integration of a transgene is much dependent upon availability of an efficient in vitro plant regeneration procedure and detection of transgene insertion and expression. Isolated immature embryos (IEs) of Eragrostis tef cultivar DZ-01-196 were used for embryogenic callus formation and the callus was transformed with GA inactivating gene PcGA2ox under the control of a triple CaMV 35S promoter using Agrobacterium transformation procedure. Embryogenic callus was induced from immature embryos in a medium containing KBP minerals in the presence of 2,4-dichlorophenoxiyacetic acid. The embryogenic calli were further inoculated with Agrobacterium and the calli were grown in co-cultivation medium (CCM) followed by selection in KBP and regeneration (K4NB) media. Putatively transformed E. tef embryogenic calli were tolerant to treatment with the selectable marker kanamycin, while 75 mg l^− 1 geneticin inhibited growth of non-transformed shoots derived from matured embryos completely after 12 days. A total of 55 plants were regenerated from all the embryogenic calli to fully viable plants setting seeds at maturity. Eight putatively transformed T₀ plants were produced carrying the transgene in their genome which was detected by PCR. Sequence analysis confirmed amplified PCR products to have 97.2 and 99.8% sequence identity to PcGA2ox and nptII, respectively. However, detection of the transgene, PcGA2ox or nptII, in T₁ plants was inconsistent although phenotypic analysis of T₁ plants showed changes in pheno-morphic and agronomic characters such as plant height, number of internodes, tillering, panicle length, biomass, yield as well as GA content. Culm reduction was due to absence of elongation of the upper-most internodes. Panicle length in semi-dwarfed plants showed no relation with culm length. GA analysis showed plants with semi-dwarf phenotype to be associated with a low level of bioactive GA₁ and its immediate precursors. Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.     

Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems

An efficient micropropagation protocol was developed for Ruta graveolens Linn. using shoot tip meristems derived from a 4-month-old field grown plant. In vitro shoot regeneration and proliferation was accomplished on Murashige and Skoogs (MS) semi-solid medium in addition to different doses of cytokinins viz.6- benzyl adenine (BA), Kinetin (Kn) or 2-isopetynyl adenine (2iP), singly or in combination with auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Highest regeneration frequency (27.6%) was obtained on (MS) medium composed of BA (10 µM) with maximum number (9.4) of shoots and 4.3 cm shoot length after 4 weeks of incubation. Among various combinations tried best regeneration frequency (71%) of multiple shoot formation with highest number (12.6) of shoots per shoot tip explants were achieved in MS medium augmented with a combination BA (10.0 µM) and NAA (2.5 µM) after 4 weeks of incubation. The optimum frequency (97%) of rhizogenesis was achieved on half-strength MS medium having 0.5 µM IBA after 4 weeks of incubation. Tissue culture raised plantlets with 5–7 fully opened leaves with healthy root system were successfully acclimatized off in Soilrite? with 80% survival rate followed by transportation to normal soil under natural light. Genetic stability among in vitro raised progeny was evaluated by ISSR and RAPD markers. The entire banding pattern revealed from in vitro regenerated plants was monomorphic to the donor. The present protocol provides an alternative option for commercial propagation and fruitful setting up of genetically uniform progeny for sustainable utilization and germplasm preservation.     

Indirect organogenesis from various explants of Hildegardia populifolia (Roxb.) Schott & Endl. – A threatened tree species from Eastern Ghats of Tamil Nadu,India

Hildegardia species are an important resource for fiber industry. This investigation was conducted to develop a plant regeneration protocol for Hildegardia populifolia (Roxb.) Schott & Endl. via indirect organogenesis Callus was obtained from leaf, internode and petiole explants, among these explants internode explant gave best result on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). The highest percentage (100%) of regeneration was obtained with benzyladenine (BA) (2.0 mg/l) + indole-3-acetic acid (IAA) (0.1 mg/l) + glutamine (25 mg/l) + thidiazuron (TDZ) (0.5 mg/l) from internode explants. Shootlets were highly rooted on MS medium supplemented with 3.0 mg/l indole-3-butyric acid (IBA). In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facilitate further development of reliable procedures for this genus.     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

Pharmacological properties and in vitro shoot production of Barleria argillicola – A critically endangered South African species

Barleria argillicola Oberm. is a critically endangered species, endemic to a small area in the KwaZulu-Natal Province, South Africa. Animals are known to forage on this plant species, suggesting its therapeutic or nutraceutical potential. This study investigated the antibacterial, acetylcholinesterase inhibition, antioxidant and phytochemical properties of this species with a view to exploring its medicinal potential. The possibility of in vitro propagation as a conservation strategy was also examined. Dichloromethane extract showed a good antibacterial activity (with minimum inhibitory concentration less than 1 mg/ml) against all the tested micro-organisms. Methanol extract exhibited a stronger antibacterial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa than the Gram-positive bacterium Staphylococcus aureus. Extracts obtained from the aerial parts and roots demonstrated a dose-dependent acetylcholinesterase inhibition and antioxidant activities. Higher iridoid, flavonoid and condensed tannin contents were recorded in the aerial parts compared to the roots although the total phenolic content was higher in the roots. The highest in vitro shoot proliferation of 4.60 ± 0.51 and 4.0 ± 0.47 shoots per explant was achieved using shoot-tip and single nodal explants respectively, after four weeks of culture in Murashige and Skoog medium supplemented with 5 μM benzyladenine riboside (BAR). Further supplementation of the medium with naphthalene acetic acid (NAA) or indole butyric acid (IBA) concentrations did not significantly increase shoot proliferation.     

The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57 μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8 μM) or TDZ (thidiazuron) (0.2, 0.5, 1 μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1 μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35 mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55 mg/g dry wt). Verbascoside accumulation was also stimulated by 1 μM TDZ; its level was about six times higher than that found on the control medium (6.2 mg/g dry wt vs. 1.03 mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8 μM BAP.     

Trace amounts of nickel in belowground rhizomes of Vaccinium myrtillus L. decrease anthocyanin concentrations in aerial shoots without water stress

Nickel (Ni) may impair plant water balance through detrimental effects on the belowground level. Bilberry (Vaccinium myrtillus L.) plants were grown in a mesic heath forest-type soil and subjected to Ni sulphate (NiSO₄·6H₂O) concentrations of 0, 10, 50, 100 and 500 mg m⁻² during an entire growing season in northern Finland (65°N). Biomass of belowground rhizomes, and tissue water content (TWC) and anthocyanin concentrations of aerial shoots were determined from mature plants in order to study rhizospheric Ni stress, and its possible long-distance effects on aerial shoots. As the major proportion of biomass of bilberry is invested in belowground parts, it was hypothesised that Ni-induced rhizospheric disturbance causes water stress in aerial shoots and increases their anthocyanin concentrations for osmotic regulation. Uptake of Ni from the soil to the rhizome and aerial shoots was measured with X-ray fluorescence spectrometry. Ni concentrations in the soil and rhizome exhibited a dose–response relationship, but the concentrations in the rhizome were about 10-fold lower (<3 mg Ni kg⁻¹) than those in the soil (<30 mg Ni kg⁻¹). Translocation of Ni from the rhizome to aerial shoots did not occur, as Ni concentrations in shoots remained at 1 mg Ni kg⁻¹. Although Ni concentrations in the rhizome were below the threshold values of Ni toxicity (i.e. 10–50 mg Ni kg⁻¹), Ni decreased the rhizome biomass. Anthocyanins decreased in aerial shoots along with the Ni accumulation in the rhizome, while TWC was unaffected. The result suggests that anthocyanins are not involved in osmotic regulation under Ni stress, since anthocyanins in aerial shoots responded to the Ni concentrations in the rhizome despite the lack of water stress.     

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.     

Silicon improves salinity tolerance in wheat plants

Durum wheat (Triticum durum cv. Gediz-75) and bread wheat (Triticum aestivum cv. Izmir-85) were grown in a complete nutrient solution in a growth room to investigate effect of silicone supplied to the nutrient solution on plants grown at salt stress. The experiment was a 2 × 2 factorial arrangement with two levels of NaCl in nutrient solution, 0 and 100 mM, and two levels of silicone (Si) in nutrient solution, 0.25 and 0.50 mM, as Na₂SiO₃. The plants grown at 100 mM NaCl produced less dry matter and chlorophyll content than those without NaCl. Supplementary Si at both 0.25 and 0.5 mM ameliorated the negative effects of salinity on plant dry matter and chlorophyll content. Membrane permeability and proline content in leaves increased with addition of 100 mM NaCl and these increases were decreased with Si treatments. Sodium (Na) concentration in plant tissues increased in both leaves and roots of plants in the high NaCl treatment and Si treatments lowered significantly the concentrations of Na in both leaves and roots. Bread wheat was more tolerant to salinity than durum wheat. The accumulation of Na in roots indicates a possible mechanism whereby bread wheat copes with salinity in the rooting medium and/or may indicate the existence of an inhibition mechanism of Na transport to leaves. Concentrations of both Ca and K were lower in the plants grown at high NaCl than in those in the control treatment and these two element concentrations were increased by Si treatments in both shoots and roots but remained lower than control values in most cases.     

Interactive effects of salinity and water depth on the growth of Melaleuca ericifolia Sm. (Swamp paperbark) seedlings

Melaleuca ericifolia Sm. (Swamp paperbark) is a common tree species in freshwater and brackish wetlands in southern and eastern Australia. The survival of this species in many wetlands is now threatened by increased salinity and inappropriate water regimes. We examined the response of 5-month-old M. ericifolia seedlings to three water depths (exposed, waterlogged and submerged) at three salinities (2, 49 and 60 dS m⁻¹). Increasing water depth at the lowest salinity did not affect survival, but strongly inhibited seedling growth. Total biomass, leaf area and maximum root length were highest in exposed plants, intermediate in waterlogged plants and lowest in submerged plants. Although completely submerged plants survived for 10 weeks at the lowest salinity, they demonstrated negative growth rates and were unable to extend their shoots above the water surface. At the higher salinities, M. ericifolia seedlings were intolerant of waterlogging and submergence: all plants died after 9 weeks at 60 dS m⁻¹. Soil salinities increased over time, and by Week 10, exceeded external water column salinities in both the exposed and waterlogged treatments. In exposed sediment, ∼90% of plants survived for 10 weeks at 60 dS m⁻¹ even though soil salinities reached ∼76 dS m⁻¹. No mortality occurred in the exposed plants at 49 dS m⁻¹, and small but positive relative growth rates were recorded at Week 10. We conclude that at low salinities M. ericifolia seedlings are highly tolerant of sediment waterlogging, but are unlikely to tolerate prolonged submergence. However, at the higher salinities, M. ericifolia seedlings are intolerant of waterlogging and submergence and died rapidly after 5 weeks exposure to this combination of environmental stressors. This research demonstrates that salinity may restrict the range of water regimes tolerated by aquatic plants.     

In vitro study on the effect of cytokines and auxins addition to growth medium on the micropropagation and rooting of Paulownia species (Paulownia hybrid and Paulownia tomentosa)

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.