Role of endogenous purine metabolism in thidiazuron-induced somatic embryogenesis of peanut (Arachis hypogaea L.)

Somatic embryogenesis was induced at the hypocotyledonary notch region of intact peanut (Arachis hypogaea L.) seedings cultured on a medium containing 10 mol·L^–1 thidiazuron (TDZ). Inclusion of the purine analogs 2,6-diaminopurine (DAP), azaadenine or azaguanine to the thidiazuron amended medium inhibited the embryogenic response of the seedlings. DAP-mediated inhibition was not overcome by the addition of adenine sulphate. Inhibition of the embryogenic response by DAP provides evidence that the TDZ-induced accumulation of purine cytokinins is an essential component of TDZ-induced somatic embryogenesis process. Analyses of the endogenous level of purine metabolites indicated that supplementation of the media with TDZ resulted in an overall increase in the endogenous cytokinins while DAP inhibited the purine recycling resulting in decreased levels of endogenous adenine and zeatin.     

Recent advances in Pelargonium in vitro regeneration systems

Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation of regeneration by growth regulators, choice of explant and characterization of induction and expression phases of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium opened up the possibilities for developing genotypes with novel characters, including resistance to some of the major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional crop improvement programs, thereby providing a valuable resource to the horticultural industry.     

Somatic embryogenesis and Agrobacterium-mediated transformation system for scented geraniums (Pelargonium sp. `Frensham')

Plant regeneration via somatic embryogenesis was achieved from leaf petioles of Pelargonium sp. `Frensham' cultured on Murashige and Skoog medium containing 15 μM N⁶-benzyladenine, and 5 μM α-naphthaleneacetic acid (NAA). More than 80% of these somatic embryos converted into plants when isolated and cultured on Murashige and Skoog medium supplemented with 15 μM NAA. Stable transgenic plants were obtained by co-cultivation of the petioles (prior to culture) with Agrobacterium tumefaciens strains LBA4404 (harbouring a binary vector pBI121 carrying the nptII and gus genes) and LBG66 (harbouring a binary plasmid pJQ418 carrying the gus/int:nptII fusion gene). Transformants were selected using kanamycin and transformation was verified by β-glucuronidase histochemical assay and polymerase chain reaction. Southern analysis further confirmed the integration of these genes into the genome of transgenic plants. We report here for the first time, an Agrobacterium-mediated model transformation system coupled with regeneration via somatic embryogenesis for production of transgenics in Pelargonium sp. Received: 20 September 1996 / Accepted: 13 November 1996     

Characterization of somatic embryogenesis in Pelargonium××hortorum mediated by a bacterium

Several cultivars of hybrid seed geranium (Pelargonium×hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos at high frequency when explants were co-cultivated with a morphogenesis promoting bacterium. This bacterium was isolated as an in vitro contaminant from cultures of geranium seedling explants and identified as belonging to the genus Bacillus and species circulans. Co-cultivation of hypocotyl explants with the bacterium promoted somatic embryo formation and improved both the frequency and quality of somatic embryos. In the cultivar Ringo Rose, the least responsive among the cultivars screened, the embryogenic response was more than four times that of axenic cultures. Nearly 70% of these embryos converted into plantlets, while the somatic embryos induced under axenic conditions developed poorly and plantlet formation was inconsistent. Among the different treatments of bacterial culture tested (autoclaved culture, culture filtrate, sonicated bacterial culture, sonication of bacterial culture followed by filtration, HPLC fractionation of crude bacterial lysate), only two HPLC fractions promoted embryogenesis to a marginal degree. Co-cultivation of the explants with bacterium during the first week of induction was crucial for obtaining high-frequency embryogenesis, indicating the role of bacterial stimuli during the induction process. Received: 23 June 1998 / Revision received: 20 August 1998 / Accepted: 27 October 1998     

Tryptophan is a precursor for melatonin and serotonin biosynthesis in in vitro regenerated St. John's wort (Hypericum perforatum L. cv. Anthos) plants

Hypericum perforatum cv. Anthos) is presented. Isotope tracer experiments were performed on plantlets regenerated from thidiazuron-induced stem explants and grown on MS basal medium for 2 months. Radiolabel from ¹⁴C-tryptophan was recovered as ¹⁴C-indoleacetic acid, ¹⁴C-tryptamine, ¹⁴C-5-hydroxytryptophan, ¹⁴C-serotonin and ¹⁴C-melatonin in the treated St. John's wort plantlets. Chromatographic peak identity was confirmed by high performance liquid chromatography-mass spectrometry-mass spectrometry and quantification of melatonin by radioimmunoassay. Significantly more radiolabel was recovered in serotonin relative to melatonin under low light conditions with this ratio being reversed under increased lighting, indicating that the rate of flow through this biosynthetic pathway is regulated, at least in part, by light. Received: 9 November 1999 / Revision received: 16 December 1999 / Accepted: 21 December 1999     

Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium x hortorum Bailey)

Summary Root outgrowths formed on the root tissue of geranium (Pelargonium x hortorum Bailey cv. Kim and cv. Shone Helena) plants in response to treatment with the phenylurea derivative, thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea; TDZ). Treatment with the cytokinin N⁶-benzylaminopurine (BAP) or the auxin -naphthaleneacetic acid (NAA) did not result in stimulation of similar abnormal structures on the root tissue. Significantly more outgrowths developed on roots of plants treated with 10 M and 20 M TDZ than on control plants or those treated with 1 M TDZ for the eight-week treatment period. Some outgrowths produced shoots and plantlets while still attached to roots, and regenerants were easily separated from the root tissue and transferred to soil in the greenhouse where they grew to maturity. Histological observations suggested these outgrowths originated from the vascular cambium region of the root.     

Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos')

 St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N′-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 μmol·l^–1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders. Received: 19 March 1999 / Revision received: 5 July 1999 · Accepted: 17 August 1999     

Resistance to Botrytis cinerea in scented geranium transformed with a gene encoding the antimicrobial protein Ace-AMP1

Scented geranium (Pelargonium sp. `Fren-sham') was transformed with a gene encoding an antimicrobial protein (Ace-AMP1) from onion through an Agrobacterium-mediated transformation system. The binary vector pFAJ3033 contained the coding region of the Ace-AMP1 preproprotein-encoding cDNA. Transformants were verified by polymerase chain reaction and Southern blot analysis. Transgenic plants expressing high levels of Ace-AMP1 were identified by immunoblots and those plants were shown to have increased resistance to Botrytis cinerea leaf infection. Received: 23 July 1998 / Revision received: 24 November 1998 / Accepted: 5 December 1998     

Metal Tolerance of Scented Geranium (Pelargonium sp. ‘Frensham’): Effects of Cadmium and Nickel on Chlorophyll Fluorescence Kinetics

The ability of scented geraniums (Pelargonium sp. Frensham) to tolerate metal stress was assessed using chlorophyll a fluorescence kinetics. The effects of various concentrations of cadmium and nickel in the culture solution on photosynthetic efficiency in scented geranium was evaluated in comparison to two well-established metal accumulators, the Indian mustard (Brassica juncea), and the sunflower (Helianthus annuus), under greenhouse conditions. The efficiency of the photosynthetic apparatus was affected to varying degrees at all metal concentrations for the plants tested. High concentrations of cadmium (1000 mg L^-1) did not significantly affect the efficiency of photosystem II activity, expressed as the ratio of variable fluorescence to maximal fluorescence (Fv/Fm), which remained high (0.738) in scented geraniums, but decreased significantly (P < 0.05) in Indian mustard (0.089) and sunflower (0.026) plants following 4 days of metal exposure. Similar trends were observed for nickel treatments. Also, the number and size of active photosynthetic reaction centers, as measured by the Fv/Fo ratio, was not significantly affected by metal exposure in scented geranium plants, while the ratio significantly decreased in Indian mustard and sunflower seedlings. The results suggest that scented geranium plants were able to overcome metal stress through (1) maintaining an efficient photosystem II activity, which is required for plant metabolism and physiological functions, as well as to overcome metal ion mediated stress, and (2) restricting damage to the photosynthetic apparatus (reaction centers) by metal ions.     

Inhibitory effect of GA3 on the development of thidiazuron-induced somatic embryogenesis in geranium (Pelargonium xhortorum Bailey) hypocotyl cultures

Somatic embryogenesis in geranium (Pelargonium xhortorum Bailey cv Scarlet Orbit Improved) can be achieved by incubating hypocotyl explants on MS medium supplemented with thidiazuron (TDZ; 10 M for 3 days followed by subculture on medium devoid of any plant growth regulators. The presence of gibberellins (GAs) during both the induction and expression phases of embryogenesis was significantly detrimental to somatic embryo formation on the hypocotyl explants. The addition of the GA-synthesis inhibitors paclobutrazol, uniconazole or ancymidol during the period of growth and differentiation of somatic embryos increased the number of somatic embryos formed on each explant. However, paclobutrazol added during the period of induction had no significant influence on somatic embryo formation. Results suggest that both exogenously supplied as well as endogenous GAs play a role, albeit a negative one, on somatic embryogenesis of geranium.Abbreviations MS Murashige and Skoog (1962) medium - MSO basal medium devoid of any plant growth regulator - TDZ N-phenyl-N1,2,3-thidiazol-5-ylurea (thidiazuron)