Micropropagation of Endangered Species Daphne cneorum

A new protocol for micropropagation of endangered Daphne cneorum through multiple shoot formation has been developed. Two different types of explants (dormant apical buds and in vitro seed-derived young seedlings) from plants in two different localities were used for the initiation of multiple shoots on agar woody plant medium (WPM) with 0.2 mg dm^–3 benzylaminopurine (BAP), 0.1 mg dm^–3-indolebutyric acid (IBA), 200 mg dm^–3 glutamine, and 200 mg dm^–3 casein hydrolysate. From 10 seeds only one germinated and the multi-apex culture bearing 12 shoots sprouted out from in vitro seed-derived young seedling. After 6-month cultivation 35 multi-apex cultures were achieved from in vitro seed-derived young seedling. On 1/3 strength WPM medium supplemented with 2.83 mg dm^–3 IBA 50 % of cultures (clusters of 3 – 5 shoots) rooted but no rooting occurred in the presence of -naphthaleneacetic acid (NAA). The rooted plantlets were acclimatized for 4 weeks in the greenhouse and then transferred into natural conditions. The plants successfully survived the winter and flowered.     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.     

Micropropagation of an Endangered Orchid Anoectochilus formosanus

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^–3 benzyladenine or 1 – 2 mg dm^–3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^–3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented with 2 % sucrose and 0.5 g dm^–3 activated charcoal. Rooting was induced in 100 % of the regenerated shoots in the same media. The plantlets were acclimatized and established in greenhouse.     

Efficient regeneration of <Emphasis Type="Italic">Brassica napus</Emphasis> L. hypocotyls and genetic transformation by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm^–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm^–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm^–3 benzyladenine and 0.3 mg dm^–3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm^–3 of indolebutyric acid and 10 mg dm^–3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.     

Nickel hyperaccumulation in shoot cultures of Alyssum markgrafii

Shoot cultures of Alyssum markgrafii O.E. Shulz, endemic nickel hyperaccumulating species of central Balkan, were established and maintained on Murashige and Skoog medium supplemented with 0.2 mg dm^–3 benzyladenine (BA). Nickel in form of NiCl₂ . 6 H₂O was supplemented at 22 different concentrations ranging from 0.0001 to 15 mM but none of them was lethal to cultures. High Ni²⁺ concentrations (10 mM or more) arrested shoot growth which, upon transfer to Ni-free medium, commenced via axillary bud proliferation. Shoots that developed from axillary buds through the subculture manifested increased tolerance to Ni²⁺ expressed as shoot elongation. Shoot multiplication and dry biomass production decreased with increase of Ni²⁺ in medium. Only the accumulation of Ni²⁺ in tissues increased with Ni²⁺ content of the medium. Apart from shoot cultures, high Ni²⁺ accumulation was registered in undifferentiated callus cultured on medium with 0.5 mg dm^–3 BA and 0.5 mg dm^–3 naphthylacetic acid. Highest content of accumulated Ni was 2.37 g g^–1 (d.m.) in shoots and 2.65 g g^–1 (d.m.) in callus, both measured on medium with 15 mM Ni²⁺.     

Nitrate and phosphate removal by<Emphasis Type="Italic"> Spirulina platensis</Emphasis>

The cyanobacterium Spirulina platensis was used to verify the possibility of employing microalgal biomass to reduce the contents of nitrate and phosphate in wastewaters. Batch tests were carried out in 0.5 dm³ Erlenmeyer flasks under conditions of light limitation (40 mol quanta m^–2 s^–1) at a starting biomass level of 0.50 g/dm³ and varying temperature in the range 23–40°C. In this way, the best temperature for the growth of this microalga (30°C) was determined and the related thermodynamic parameters were estimated. All removed nitrate was used for biomass growth (biotic removal), whereas phosphate appeared to be removed mainly by chemical precipitation (abiotic removal). The best results in terms of specific and volumetric growth rates ( =0.044 day^–1, Q _x =33.2 mg dm^–3 day^–1) as well as volumetric rate and final yield of nitrogen removal ( =3.26 mg dm^–3 day^–1, =0.739) were obtained at 30°C, whereas phosphorus was more effectively removed at a lower temperature. In order to simulate full-scale studies, batch tests of nitrate and phosphate removal were also performed in 5.0 dm³ vessels (mini-ponds) at the optimum temperature (30°C) but increasing the photon fluence rate to 80 mol quanta m^–2 s^–1 and varying the initial biomass concentration from 0.25 to 0.86 g/dm³. These additional tests demonstrated that an increase in the inoculum level up to 0.75 g/dm³ enhanced both NO₃ ^– and PO₄ ^3– removal, confirming a strict dependence of these processes on biomass activity. In addition, the larger surface area of the ponds and the higher light intensity improved removal yields and kinetics compared to the flasks, particularly concerning phosphorus removal ( =0.032–0.050 day^–1, Q _x =34.7–42.4 mg dm^–3 day^–1, =3.24–4.06 mg dm^–3 day^–1, =0.750–0.879, =0.312–0.623 mg dm^–3 day^–1, and =0.224–0.440).     

Micropropagation of Spilanthes acmella Murr.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^–3 BA in combination with 0.1 mg dm^–3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^–3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.     

Micropropagation in Peanut (Arachis hypogaea L.)

Multiple shoots in Arachis hypogaea L. could be induced from the de-embryonated cotyledons (DC), embryo-axes (EA) and mature whole seeds (MWS) in MS medium supplemented with different levels of benzylaminopurine (BAP). DC was the most suitable explant with 57.9 % induction and more than 40 shoots per explant in 31.6 % of cases. Though EA and MWS had high percent induction at or above 30 mg dm^–3 BAP, only 10 – 14 shoots per explant were observed. In DC, multiple shoots were confined to the proximal end and in EA they originated from the axillary bud region. Histological studies on DC confirmed the origin of shoots from the region of attachment with the embryo. Shoots could be rooted in MS medium containing 2 g dm^–3 charcoal and 200 mg dm^–3 casein hydrolysate. Sixty percent of the rooted plantlets could be established in the field.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

High Frequency Multiple Shoot Regeneration from Decapitated Embryo Axes of Chickpea and Establishment of Plantlets in the Open Environment

Multiple shoot regeneration from the cut plumular ends of embryo axes of chickpea (Cicer arietinum L.) was evaluated on Murashige and Skoog medium having different concentrations of thidiazuron (TDZ) (0.1 to 10.0 mg dm^–3) 6-benzylaminopurine (BAP) (0.5 and 1.0 mg dm^–3), kinetin (0.5 and 1.0 mg dm^–3) or zeatin (2.0 and 4.0 mg dm^–3). TDZ (0.2 mg dm^–3) was found to be the most effective cytokinin as it produced multiple shoots in 100 % of the explants from genotypes C235, ICC5166, ICC12269, ICC4951, ICC11531, BG256 and a local cultivar. Shoots were elongated on growth regulator-free medium, and rooted on growth regulator-free medium containing 1/4 MS salts + full vitamins + 3 % sucrose. Plantlets formed were acclimatized for 12 – 15 d in MS medium with a gradual reduction in sucrose concentration and transferred into pots filled with soil and kept in the field; this resulted in more than 70 % survival. The plants developed normally and produced fertile flowers and set seeds. Low temperatures, maximum 19.0 °C, and minimum 8.2 °C, during the first 15 d of transfer favoured survival on transfer to pots.     

Parallel plastic tank experiments with cultures of marine diatoms

The reproductibility of tank experiments concerning unicellular marine algal development was analyzed by means of parallel experiments with cultures ofThalassiosira rotula andSkeletonema costatum, using large flexible plastic tanks under semi-natural conditions. The tanks (3–4 m³, 4–5 m deep) were exposed in the German Bight at a station in the outer harbour of helgoland. The water was obtained from the open North Sea in towable tanks; it was filtered (plate filter), enriched with nitrate (20–30 gat dm^–3), phosphate (1.3–2.3 gat dm^–3) and silicate (15–23 gat dm^–3)-nearly natural springtime concentrations in this area-and inoculated with 10³–10⁵ cells dm^–3. The water was mixed with non-metal stirring equipment. Within 5 days, concentrations of 10⁶–10⁷ cells dm^–3 in an exponential growth phase were obtained. In experiments withT. rotula a parallel development was achieved in spite of some contamination by surrounding water. This is the case for nearly all parameters analyzed (nutrient salts, phytoplankton, bacteria, C, N and particulate carbohydrates). The heterotrophic bacteria, which were determined by means of the plate method, reached concentrations of up to 10⁶ (T. rotula) and 10⁵ (S. costatum) CFU cm^–3, respectively. They showed a consistent retrograde development at diatom concentrations above a certain level. The crop did not increase again until the diatoms had reached the stationary phase. During exponential growth ofT. rotula (G=8.9–11.7 h) a partially synchronous cell division was observed. There were also rhythms with respect to cell size (pervalvar axes) and chain length (number of cells). For the experiments withS. costatum (G=10–11.4 h) diurnal variations of cell size and chain length occurred. The present results indicate acceptable reproducibility of algal development and related phenomena in enclosed water bodies.     

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l^–1) induced high frequency (88%) development of axillary shoot buds (3.2) in 4–5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26–35 shoots) was recorded when using culture initiation media with 0.5 mg.l^–1 each of BAP and NAA followed by subculture in 0.2 mg.l^–1 BAP. The shoot multiplication rate was further accelerated by reculturing 0.4–0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l^–1 BAP. Shoot cuttings (3.5–7.0 cm) were rooted in 0.2 mg.l^–1 IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - SH Schenk and Hildebrandt (1972) medium     

Micropropagation of Morus laevigata Wall. from mature trees

Multiple shoots were obtained from nodal explants of 10-year-old tree of Morus laevigata on Murashige and Skoog's medium supplemented with different concentrations (0.5–5.0 mg.l^–1) of benzyladenine (BA). Nodal segments taken from in vitro proliferated shoots gave further multiple shoots when cultured on the same basal medium containing 2.5 mg.l^–1 BA. Repeated subculture resulted in rapid shoot multiplication at the average rate of 6-fold per subculture. In vitro raised shoots rooted on MS medium containing 0.1 mg. l^–1 each of 3-indolebutyric acid (ISA) and -naphthaleneacetic acid (NAA). The regenerated plantlets were successfully established in soil under field conditions after a few days of indoor acclimatization.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and development of herbicide-resistant sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> species hybrids) using axillary buds

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing -glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l^–1) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l^–1 6-benzyladenine (BA) and 5.0 mg l^–1 PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l^–1 BA, 1.0 mg l^–1 kinetin (Kin), 0.5 mg l^–1 -napthaleneacetic acid (NAA) and 5.0 mg l^–1 PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l^–1 NAA and 5.0 mg l^–1 PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical -glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50–60% of the transgenic plants sprayed with BASTA (60 g l^–1 glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.Abbreviations BA 6-Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kin Kinetin - NAA -Naphthaleneacetic acid - Nos Nopaline synthase - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PPT Phosphinothricin - YEP Yeast extract and peptone     

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B₅ vitamins, 0.1 mg dm^–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm^–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm^–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.     

Factors Affecting In Vitro Multiplication of Date Palm

Rapid method of in vitro multiplication of date palm was developed. Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm^–3 dimethylaminopurine (2iP) + 1 mg dm^–3 naphthalene acetic acid (NAA). Shoot buds were proliferated from white nodular cultures on hormone free medium. Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm^–3 2iP + 0.5 mg dm^–3 NAA. Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium. Among four concentrations of sucrose used, 30 g dm^–3 speeded up the bud proliferation more than 10, 20 and 40 g dm^–3. However, the largest shoot buds were observed with 40 g dm^–3 sucrose. Solidification of culture media by 1.75 g dm^–3 Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm^–3 Phytagel.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Impact of picolinic acid on the chromium accumulation in fodder radish and komatsuna

Effects of picolinic acid (2-pyridinecarboxylic acid) and chromium(III) picolinate was studied on the chromium (Cr) accumulation of fodder radish (Raphanus sativus L. convar. oleiformis Pers., cv. Leveles olajretek) and komatsuna (Brassica campestris L. subsp. napus f. et Thoms. var. komatsuna Makino, cv. Kuromaru ) grown in a pot experiment. Control cultures, grown in an uncontaminated soil (UCS; humous sand with pH_KCl 7.48, sand texture with 12.4% clay+silt content, organic carbon 0.56%, CaCO₃ 2.2%, CEC 6.2 cmol_c kg^–1, Cr 10.6 mg kg^–1), accumulated low amounts of chromium (less than 5.4 g g^–1) in their roots or shoots. When this UCS was artificially contaminated with 100 mg kg^–1 Cr (CrCl₃) later picolinic acid treatment promoted the translocation of chromium into the shoots of both species. In fodder radish shoots Cr concentration reached 30.4 g g^–1 and in komatsuna shoots 44.5 g g^–1. Application of ethylene diamine tetra-acetic acid (EDTA) to this Cr contaminated soil had similar effect to picolinic acid. When the UCS was amended with leather factory sewage sediment (which resulted in 853 mg kg^–1 Cr in soil), Cr mobilization was observed only after repeated soil picolinic acid applications. From a galvanic mud contaminated soil (brown forest soil with pH_KCl 6.77, loamy sand texture with 26.6% clay+silt content, organic carbon 1.23%, CaCO₃ 0.7%, CEC 24.5 cmol_c kg^–1, Cd 5.0 mg kg^–1, Cr 135 mg kg^–1, and Zn 360 mg kg^–1) the rate of Cr mobilization was negligible, only a slight increase was observed in Cr concentration of fodder radish shoots after repeated picolinic acid treatments of soil. Presumably picolinic acid forms a water soluble complex (chromium(III) picolinate) with Cr in the soil, which promotes translocation of this element (and also Cu) into the shoots of plants. The rate of complex formation may be related to the binding forms and/or concentration of Cr in soil and also to soil characteristics (i.e. pH, CEC), since the rate of Cr translocation was the following: artificially contaminated soil > leather factory sewage sediment amended soil > galvanic mud contaminated soil. Four times repeated 10 mg kg^–1 chromium(III) picolinate application to UCS multiplied the transport of chromium to shoots, as compared to single 10 mg kg^–1 CrCl₃ treatment. This also suggests that chromium(III) picolinate is forming in the picolinic acid treated Cr-contaminated soils, and plants more readily accumulates and translocates organically bound Cr than ionic Cr. Picolinic acid promotes Cr translocation in soil-plant system. This could be useful in phytoextraction (phytoremediation) of Cr contaminated soils or in the production of Cr enriched foodstuffs.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.