Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration

Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tumefaciens infection and transformation and because transgenic mature events are less competent for regeneration. We have shown that reinvigoration allows manipulation of the vegetative phase to increase the potential for transformation and regeneration without loss of competence for flowering and fruiting. To produce transgenic plants from clementine mandarin (Citrus clementina hort. ex Tanaka), we optimized the conditions of the source material both ex vitro and in vitro. Grafting of mature buds on juvenile rootstocks in the spring and preventing multiple bud sprouting by removing all but one bud permitted selection of vigorous first flushes for in vitro culture. Use of additional virulence genes from A. tumefaciens to increase transformation frequency and optimization of culture media and conditions to enhance explant cell competence for T-DNA integration and organogenesis resulted in efficient and reliable transgenic plant production. Transformed regenerants from explants, cultured in media without antibiotics, were identified by a screenable marker (either beta-glucuronidase or green fluorescent protein (GFP)), creating the possibility of generating transgenic clementine plants without antibiotic resistance marker genes. Stable integration of foreign genes was demonstrated by Southern blot analysis, and expression of these foreign genes was confirmed by detection of GFP fluorescence in leaves, floral organs and fruits of the transgenic plants.     

Antibacterial activity of fresh flower heads of Chrysantemum coronarium

The antibacterial activity of the methylene chloride and methanol extracts of Chrysantemum coronarium fresh flower heads was investigated. The methylene chloride extract showed discrete antimicrobial activity.     

Uleine and demethoxyaspidospermine from the bark of Plumeria lancifolia

The isolation of (+)-uleine (1) and (+)-demethoxyaspidospermine (2) from the bark of Plumeria lancifolia is reported along with (1)H- and (13)C-NMR data.     

Variations in growth, survival and carbon isotope composition (delta(13)C) among Pinus pinaster populations of different geographic origins

To evaluate differences in growth and adaptability of maritime pine (Pinus pinaster Ait.), we studied growth, polycyclism, needle tissue carbon isotope composition (delta(13)C) as an estimate of water-use efficiency (WUE) and survival of seven populations at 10 years of age growing in a performance trial at a provenance test site in Escaroupim, Portugal. Six populations were from relatively high rainfall sites in Portugal and southwestern France (Atlantic group), and one population was from a more arid Mediterranean site in Spain. There were significant differences between some populations in total height, diameter at breast height, delta(13)C of bulk needle tissue, polycyclism and survival. A population from central Portugal (Leiria, on the Atlantic coast) was the tallest and had the lowest delta(13)C. Overall, the variation in delta(13)C was better explained by the mean minimum temperatures of the coldest month than by annual precipitation at the place of origin. Analyses of the relationships between delta(13)C and growth or survival revealed a distinct pattern for the Mediterranean population, with low delta(13)C (and WUE) associated with the lowest growth potential and reduced survival. There were significant negative correlations between delta(13)C and height or survival in the Atlantic group. Variation in polycyclism was correlated with annual precipitation at the place of origin. Some Atlantic populations maintained a high growth potential while experiencing moderate water stress. A detailed knowledge of the relationships between growth, survival and delta(13)C in contrasting environments will enhance our ability to select populations for forestry or conservation.     

Trypanocidal activity of extracts and fractions of Bertholletia excelsa

Crude extracts and fractions of Bertholletia excelsa stem barks were tested for trypanocidal activity. Acetone and methanol extracts showed significant in vitro trypanocidal activity against trypomastigote form of Trypanosoma cruzi since in the concentration of 500 microg/ml, the parasites were reduced in 100% and 90.3% respectively, whereas the triterpene betulinic acid pure isolated from hexane extract presented 75.4%.     

Effects of two iron sources on iron and cadmium allocation in poplar (Populus alba) plants exposed to cadmium

Effects of 10 microM cadmium (supplied as Cd nitrate) on the utilization and allocation of iron (Fe) were investigated in poplar (Populus alba L.) plants grown in nutrient solution with Fe(III)-EDTA or Fe(III)-citrate as the Fe source. The effects of Cd were also compared with those of Fe deprivation. The accumulation of Fe in roots was 10-fold higher in plants grown with Fe-citrate than with Fe-EDTA. Cadmium decreased leaf chlorophyll concentrations and photosynthetic rates, and these decreases were more marked in plants grown with Fe-citrate than with Fe-EDTA. In both Fe treatments, addition of Cd caused large increases in root and shoot apoplasmic and non-apoplasmic Cd contents and increases in root Fe content; however, Cd decreased shoot Fe content, especially in plants grown with Fe-citrate. New leaves of plants grown with Fe-citrate had small cellular (non-apoplasmic) Fe pools, whereas these pools were large in new leaves of plants grown with Fe-EDTA. Non-apoplasmic Cd pools in new leaves were smaller in plants grown with Fe-citrate than with Fe-EDTA, indicating that inactivation of non-apoplasmic Cd pools is facilitated more by Fe-EDTA than by Fe-citrate. In the presence of Cd, Fe-EDTA was also superior to Fe-citrate in maintaining an adequate Fe supply to poplar shoots. Differences in plant responses to Fe-EDTA and Fe-citrate may reflect differences in long-distance transport of Fe rather than in acquisition of Fe by roots.     

Genetic Diversity of Peanut Core Collection from US Using Simple Sequence Repeats

Groundnut is a member of genus Arachis and the crop is divided into two subspecies and six botanical varieties based on morphological characteristics （Krapovickas and Gregory, 1994）. The International Crops Research Institute for the Semi-Arid Tropics （ICRISAT） holds more than 14 000 groundnut accessions from which a core collection of 1 704 have been developed （Upadhyaya et al., 2003）. Holdbrook et al. （1993） developed a groundnut core collection of 831 accessions from a total of 7 432 US groundnut accessions based on morphological characteristics. The large number and variability of accessions in GenBanks create problems in knowing which germplasm to select for breeding purpose. Only a few established cultivars and elite breeding lines have been utilised in breeding programmes. A core collection is a fraction of accessions from the entire collection which represent most of the available genetic diversity of the species. A core collection can extensively be evaluated and information derived from them can be applied to the whole collection. Identification of DNA markers associated with the botanical varieties of groundnut would be useful in genotyping, germplasm management and evolutionary studies.     

Microsatellite Marker Based Assessment of Genetic Diversity among Cultivars, Landraces and Wild Relatives in Rice

India being the primary center of origin for rice had a very large treasure of local land races, most of which are out of cultivation today. The exact genetic potential and their differences from commercial varieties and the magnitude of heterogeneity still present in them are not well catalogued. Hence the need to characterize the available land races has become imminent in the modem day concept of crop improvement （Rezai and Frey, 1990）. The present study addresses the utility of SSR markers in divulging the genetic relationships at molecular level among the major component factions office gennplasm viz., local cultivars, land races and wild species collected from a wide range of agro-geographical regions of Indian subcontinent.     

Cloning and Sequence Analysis of Lipoxygenase Gene cDNA from Cucumber Fruit （Cucumis sativus L.）

Lipoxygenases are nonheme-iron-containing dioxygenases that catalyze the hydroperoxidation of unsatrated fatty acids containing a cis, cis-1,4-pentadiene structure producing hydroperoxy acids with conjugated dienes. LOX activity has been found in a wide range of plants. Typical substrates for LOX in plants are linoleic acid and linilenic acid fatty acids. The function of various LOXs in plants is unknown, but their participation in all stages of plant growth and development has been suggested （Hildebrand, 1989; Siedow, 1991）. Some of the physiological processes in whicn lipoxygenses have been implicated include wounding （Saravitz and Siedow, 1996）, pathogen attack （Melan et al., 1993）, seed germination （Kato et al., 1992）, fruit ripening （Ferrie et al., 1994）, plant senescence （Paliyath and Droillard, 1992）. The study on the role of lipoxygenase in ripening and senescence fruit focused on tomato and strawberry. Cloning LOX gene of cucumber fruit will make us further understand the molecularaction mode of this enzyme during fxuit ripening and senescence. In this paper we isolated the partial nucleotide sequences of cucumber fiuit lipoxygenase gene and discuss the characterization of it.     

The Application of TDZ in Enhancing Regeneration of Transgenic Plants

At present, transgenic plants are globally grown. Availability of a reliable regeneration system predominantly from a single transformed cell is the prerequisites for gene transfer, but regeneration is still a key problem （Wenzel, 2006）. The application of genetic modification technology in plants is closely related to the efficiency of the regeneration protocol. Shoot formation is oilen enhanced by the combination of auxins and cytokinins. TDZ （Thidiazuron, N-phenyl-N＇-1,2,3-thiodiazol-5-ylurea）, a non-purine, has been shown to promote differentiation of organized centers of growth in cultured tissues at much lower concentrations, and shoot regeneration occurs with an efficiency comparable to or greater than that of other cytokinins （Fiola et al., 1990）. By addition of TDZ, a series of plants which were difficult to culture in vitro or less sensitive to plants growth regulators obtained somatic embryos and regenerated plants, some of them have become the eminent transformation system for genetic engineering. TDZ has been reported to be very efficient in stimulating adventitious shoot production in several recalcitrant plants. TDZ is considered as a potential growth regulator for in vitro shoot regeneration and somatic embryogenesis of several crops. This review summarized how the new growth regulator TDZ to affect the regeneration of transgenic plants.