<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Anti-cyanobacterial activity of <Emphasis Type="Italic">Moringa oleifera</Emphasis> seeds

Filtrates from crushed Moringa oleifera seeds were tested for their effects on growth and Photosystem II efficiency of the common bloom-forming cyanobacterium Microcystis aeruginosa. M. aeruginosa populations exhibited good growth in controls and treatments with 4- and 8-mg crushed Moringa seeds per liter, having similar growth rates of 0.50 (±0.01) per day. In exposures of 20- to 160-mg crushed Moringa seeds L⁻¹, growth rates were negative and on average −0.23 (±0.05) .day⁻¹. Presumably, in the higher doses of 20- to 160-mg crushed seeds per liter, the cyanobacteria died, which was supported by a rapid drop in the Photosystem II efficiency (Φ_PSII), while the Φ_PSII was high and unaffected in 0, 4, and 8 mg L⁻¹. High-density populations of M. aeruginosa (chlorophyll-a concentrations of ∼270 μg L⁻¹) were reduced to very low levels within 2 weeks of exposure to ≥80-mg crushed seeds per liter. At the highest dosage of 160 mg L⁻¹, the Φ_PSII dropped to zero rapidly and remained nil during the course of the experiment (14 days). Hence, under laboratory conditions, a complete wipeout of the bloom could be achieved. This is the first study that yielded evidence for cyanobactericidal activity of filtrate from crushed Moringa seeds, suggesting that Moringa seed extracts might have a potential as an effect-oriented measure lessening cyanobacterial nuisance.     

Cadmium and copper toxicity to tench <Emphasis Type="Italic">Tinca</Emphasis><Emphasis Type="Italic">tinca</Emphasis> (L.) larvae after a short-term exposure

At the onset of swimbladder inflation, Tinca tinca larvae were exposed for 24 h to cadmium or copper at 0.0 (control concentration), 0.1, 0.2 and 0.3 mg dm⁻³ at 22°C. From then larvae were reared at 25°C for 9 days in un-supplemented water. Both metals resulted in a significantly reduced growth, survival, and retarded swimbladder inflation in a dose-response manner. The highest Cd and Cu concentration delayed the onset of exogenous feeding (live artemia nauplii) for 2 or 1 days, respectively, comparing to the control concentration. Our results demonstrate a highly toxic effect of Cd and Cu in the studied period of larval ontogeny, when fish seem especially sensitive. Although, at low concentrations and long exposure period, Cu is considered more toxic to fish than Cd, our study revealed the reverse effect for first-feeding larvae of both metals at high concentrations and short exposure.     

Effect of phosphorus and temperature on chlorophyll <Emphasis Type="Italic">a</Emphasis> contents and cell sizes of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> and <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

An experiment was carried out to evaluate the effects of phosphorus concentration (1, 4 and 10 mg l⁻¹) and temperature (15 and 25°C) on chlorophyll a (chl a) contents and cell size/volume of green alga Scenedesmus obliquus and blue green alga Microcystis aeruginosa. Long-term field data from Lake Taihu, a large, shallow eutrophic lake between Jiangsu and Zhejiang Provinces, China, was also used to evaluate the effect of temperature on the model between chl a and total phosphorus (TP). The chl a content of both algae increased with an increase in phosphorus concentration and temperature. Temperatures showed a significantly different effect on chl a content of S. obliquus at a phosphorus concentration of 10 mg l⁻¹, whereas there was no significant difference at the two lower phosphorus levels. For M. aeruginosa, temperatures presented significantly different effects on the chl a contents at three phosphorus concentrations. Chl a content of neither alga presented an interaction between the nutrient and the temperature. Long-term field data from Lake Taihu also indicated that the addition of temperature to the model increased predictability of chl a by TP. The length/diameter and volume of both algae were greater at the lower temperature and phosphorus concentration. Moderate negative correlations were observed between algal size, volume, and chl a content. Our results suggest that phosphorus concentration and temperature could change chl a contents and size in species-specific algal cells and that temperature should be considered when building the model of TP and chl a concentration.     

Energy Demands of Nitrogen Supply in Mass Cultivation of Two Commercially Important Microalgal Species, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

Mass culture of microalgae is a potential alternative to cultivation of terrestrial crops for bioenergy production. However, microalgae require nitrogen fertiliser in quantities much higher than plants, and this has important consequences for the energy balance of these systems. The effect of nitrogen fertiliser supplied to microalgal bubble-column photobioreactor cultures was investigated using different nitrogen sources (nitrate, urea, ammonium) and culture conditions (air, 12% CO₂). In 20 L cultivations, maximum biomass productivity for Chlorella vulgaris cultivated using nitrate and urea was 0.046 and 0.053 g L⁻¹ day⁻¹, respectively. Maximum biomass productivity for Dunaliella tertiolecta cultivated using nitrate, urea and ammonium was 0.033, 0.038 and 0.038 g L⁻¹ day⁻¹, respectively. In intensive bubble-column photobioreactors using 12% CO₂, maximum productivity reached 0.60 and 0.83 g L⁻¹ day⁻¹ for C. vulgaris and D. tertiolecta, respectively. Recycling of nitrogen within the photobioreactor system via algal exudation of nitrogenous compounds and bacterial activity was identified as a potentially important process. The energetic penalty incurred by supply of artificial nitrogen fertilisers, phosphorus, power and CO₂ to microalgal photobioreactors was investigated, although analysis of all energy burdens from biomass production to usable energy carriers was not conducted. After subtraction of the power, nitrogen and phosphorus energy burdens, maximum net energy ratios for C. vulgaris and D. tertiolecta cultivated in bubble columns were 1.82 and 2.10. Assuming CO₂ was also required from a manufactured source, the net energy ratio decreased to 0.09 and 0.11 for C. vulgaris and D. tertiolecta, so that biomass production in this scenario was unsustainable. Although supply of nitrogen is unlikely to be the most energetically costly factor in sparged photobioreactor designs, it is still a very significant penalty. There is a need to optimise both cultivation strategies and recycling of nitrogen in order to improve performance. Data are supported by measurements including biochemical properties (lipid, protein, heating value) and bacterial number by epifluorescence microscopy.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

Genetic transformation and overexpression of a rice <Emphasis Type="Italic">Hd3a</Emphasis> induces early flowering in <Emphasis Type="Italic">Saussurea involucrata</Emphasis> Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l⁻¹ benzyladenine (BA), 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l⁻¹ hygromycin, and 500 mg l⁻¹ cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 0.25 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l⁻¹ NAA, 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.     

Combination effect of lactoalbumin hydrolysate and methyl jasmonate on ginsenoside and polysaccharide production in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

Lactoalbumin hydrolysate (LH) at 100 mg L⁻¹ with methyl jasmonate (MJ) at 2 mg L⁻¹ synergistically stimulated ginsenoside accumulation in Panax quinquefolium cells compared with 100 mg L⁻¹ LH. Combination elicitors led to higher ginsenoside productivity (45.93 mg L⁻¹) than single treatment of 100 mg L⁻¹ LH (31.37 mg L⁻¹). This present result will be helpful in providing a tool for enhancing the productivity of ginsenoside by Panax quinquefolium cell cultures on a commercial scale.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.     

Harvesting <Emphasis Type="Italic">Chlorella minutissima</Emphasis> using cell coagulants

Twelve salts were tested for their ability to coagulate microalgae cells in cultures of Chlorella minutissima. The final aim was to develop an easy and efficient approach for harvesting microalgae biomass in dense cultures. Aluminum, ferric, and zinc salts coagulated C. minutissima cultures, while optimum concentration was 0.75 and 0.5 g L⁻¹ for sulfate and chloride salts, respectively. Aluminum salts were most efficient, but caused some cell lysis, which may render this approach inappropriate in some cases. Ferric and zinc salts were ranked second and third, respectively, according to their culture cell-coagulation efficiency. Ferric salts caused a change in the color of the cells, mainly at concentrations higher than 1 g L⁻¹. Zinc salts were less harmful for the microalgal cells, but an additional problem was observed with cell aggregates adhering to the walls of the glass test tubes. Selection of the appropriate coagulant is related to the purpose of the coagulation process.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.     

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. cv. Poinsett76) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L⁻¹ benzyladenine (BA), 20 mg L⁻¹ l-glutamine and 2 mg L⁻¹ phosphinothricin (PPT) or 100 mg L⁻¹ kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L⁻¹) containing MS media with 0.5 mg L⁻¹ gibberellic acid (GA₃) and 0.6 mg L⁻¹ indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T₀ and the progenies. T₁ segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.     

Xanthone compounds in shoot cultures of <Emphasis Type="Italic">Gentianella bulgarica</Emphasis>

Shoot cultures of Gentianella bulgarica established from seedling epicotyls were maintained on MS medium supplemented with BA 0.2 mg l⁻¹ + NAA 0.1 mg l⁻¹. Cultures were prone to precocious flowering requiring the use of small shoot buds for multiplication purposes. The contents of three xanthone compounds identified as DGL, BGL, and DMB, in different plant material were determined by HPLC. The analysis revealed that the production of xanthones was affected by different concentrations of BA in medium. Shoot cultures grown at higher BA concentrations contained more DGL than material grown in nature. The concentrations of other two xanthones were lower in shoot cultures than in plants from nature. The radical scavenging activity of plant extracts and xanthones was investigated by DPPH test. Samples from plants grown in nature showed the highest activity (IC₅₀ = 0.26 mg ml⁻¹), while the extracts of shoot cultures grown in media with higher concentrations of BA showed moderate activities (IC₅₀ from 1.6 to 4.4 mg ml⁻¹).     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.     

Sulphur protects mustard (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.) from cadmium toxicity by improving leaf ascorbate and glutathione

In a pot-soil culture ameliorative effect of sulphur (S) (0 or 40 mg S kg⁻¹ soil) on cadmium (Cd) (0, 25, 50 and 100 mg Cd kg⁻¹ soil)-induced growth inhibition and oxidative stress in mustard (Brassica campestris L.) cultivar Pusa Gold was studied. Cadmium at 100 mg kg⁻¹ soil caused maximum increase in the contents of Cd and thiobarbituric acid reactive substances (TBARS) in leaves. Maximum reductions in growth (plant dry mass, leaf area), chlorophyll content, net photosynthetic rate (P_N) and the contents of ascorbate (AsA), glutathione (GSH) were observed with 100 mg Cd kg⁻¹ soil compared to control. The application of S helped in reducing Cd toxicity, which was greater for 25 and 50 mg Cd kg⁻¹ soil) compared to 100 mg Cd kg⁻¹ soil. Addition of S to Cd-treated plants showed decrease in Cd and TBARS content in leaves and restoration of growth and photosynthesis through increase in the contents of AsA and GSH. Net photosynthetic rate and plant dry mass were strongly and positively correlated with the contents of AsA and GSH. It is suggested that S may ameliorate Cd toxicity and protects growth and photosynthesis of mustard involving AsA and GSH.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.