Callus induction and somatic embryogenesis in Stevia rebaudiana Bertoni as a medicinal plant.

Stevia rebaudiana (Bert.) from Asteraceae family is a useful medicinal plant that prevents and cures diabetes, blood pressure, weight gain and tooth decay. Due to self-incompatibility in stevia, somatic embryo investigation for artificial seed production is valuable in this plant. In order to evaluate the callus induction characteristics in stevia, a factorial experiment was laid out based on a completely randomized design with three replications. The factors included ten hormone combinations and control, two kinds of media (MS and B5) and two types of explants (leaf and internode). Callus induction characters including the percentage of callus formation, days to callus induction, fresh and dry callus weight were recorded. Analysis of variance showed significant differences (p<0.01) among hormone combinations, media and explant types as well as their interactions. The best treatment for callus induction with minimum time to callus formation was 1 mg/l NAA+1 mg/l BAP. The highest fresh and dry callus weight were obtained on B5 medium supplemented by 1 mg/l 2,4-D+1 mg/l BAP (in leaf explant) and 0.25 mg/l 2,4-D+ 0.1 mg/l BAP (in internode explant). These results can be used in suspension culture. To induce somatic embryogenesis in suspension culture, six hormone treatments were investigated. The highest somatic embryogenesis percentage was obtained in MS medium supplemented by 2 mg/l 2,4-D+ 0.5 mg/l NAA+0.5 mg/l BAP.

Remediation of nitrate-contaminated wastewater using denitrification biofilters with straws of ornamental flowers added as carbon source.

Straws of four ornamental flowers (carnation, rose, lily, and violet) were added into denitrification biofilters using gravel as matrix through vertically installed perforated polyvinylchloride pipes to provide organic carbon for the treatment of nitrate-contaminated wastewater operating in batch mode. Removal efficiencies of nitrate and phosphate, as well as temporal variations of nitrogen and carbon during batches 10 and 19, were investigated and assessed. Nitrate removal was efficiently enhanced by the addition of flower straws, but decreased gradually as the organic substances were consumed. Phosphate removal was also improved, although this very limited. High nitrate removal rates were achieved during the initial 12 h in the two batches each lasting for 3 days, along with the depletion of influent dissolved oxygen due to aerobic degradation of the organic compounds. NO2(-)-N of 0.01-2.83 mg/L and NH4(+)-N of 0.02-1.69 mg/L were formed and both positively correlated to the nitrate reduced. Inorganic carbon (IC) concentrations increased during the batches and varied conversely with the nitrate contents, and could be indicative of nitrate removal due to the highly significant positive correlation between NO3(-)-N removed and IC concentration (r(2) = 0.881, p < 0.0001). It is feasible and economical to use the denitrification biofilter to treat nitrate-contaminated wastewater, although further optimization of carbon source addition is still required.

A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

Direct somatic embryogenesis and plant regeneration from leaf, petiole, and stem explants of Golden Pothos.

Somatic embryos directly formed at cut edges or on the surface of leaf explants, around cut ends or along side surfaces of petiole and stem explants of 'Golden Pothos' [Epipremnum aureum (Linden & Andre) Bunt.] on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) or N-phenyl-N'-1, 2, 3-thiadiazol-5-ylurea (TDZ) with alpha-naphthalene acetic acid (NAA) and a medium called MK containing MS salts with Kao's vitamins, supplemented with 2.0 mg/l TDZ and 0.2 mg/l NAA. Somatic embryos were also produced on MS medium containing 2.0 mg/l kinetin (KN) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) from leaf and petiole explants, MS medium supplemented with 2.0 mg/l CPPU and 0.5 mg/l 2,4-D from petiole and stem explants, and 2.0 mg/l TDZ and 0.2 mg/l or 0.5 mg/l 2,4-D from stem explants. In addition, somatic embryos occurred from stem explants on Chu's N6 medium containing 2.0 mg/l CPPU and 0.2 mg/l NAA. Somatic embryos matured and grew into multiple buds, shoots, or even plantlets after 2-3 months on the initial culture medium. Germination was optimal on MS medium containing either 2 mg/l 6-benzylaminopurine (BA) and 0.2 mg/l NAA or 2 mg/l zeatin and 0.2 mg/l NAA. Shoots elongated better and roots developed well on MS medium with no growth regulators. Approximately 30-100 plantlets were regenerated from each explant. The regenerated plants grew vigorously after transplanting to a soil-less container substrate in a shaded greenhouse.

Induction of direct somatic embryogenesis in garlic (Allium sativum).

An efficient novel method of direct somatic embryogenesis from basal tissue of garlic clove was developed. The influence of plant growth regulators, basal medium and explant type on somatic embryo induction was examined. The best plant growth regulator combination was, 2,4-D and kinetin at 1.0 mg/L and 0.5 mg/L respectively, inducing direct somatic embryogenesis in 60% of explants. White's medium was used as basal medium and somatic embryos developed on explants after six weeks. The technique has potential applicability for problems associated with plant regeneration and virus elimination in garlic.