A RE-EDUCATIVE REGIMEN FOR CHRONIC (FUNCTIONAL) CONSTIPATION.
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This paper studied the chilling tolerance and the activity of protective system against active oxygen of own-rooted and grafted watermelon seedlings. The results showed that under low temperature stress, the chilling tolerance of the watermelon seedlings grafted on figleaf was higher than that of the seedlings grafted on Chaofeng F1, and that of the own-rooted seedlings was the lowest. The decrease of chlorophyll, ascorbate and GSH contents and superoxide dismutase, ascorbate peroxidase and dehydroascorbate reductase activities as well as the increase of MDA content in the leaves of both own-rooted and grafted watermelon seedlings indicated an injury on the protective system against active oxygen, and induced lipid peroxidation under low temperature stress. However, the activity of protective system against active oxygen was higher in grafted watermelon seedlings than in own-rooted watermelon seedlings. The activity of protective system against active oxygen in figleaf gourd-grafted watermelon seedlings with stronger chilling tolerance was higher than that of Chaofeng F1-grafted watermelon seedlings with weaker chilling tolerance. It could be concluded that the enhancement of chilling tolerance in grafted watermelon seedlings was related to the increase of antioxidants and antioxidative enzyme activities.
The 5' flanking region of ADP-Glucose Pyrophosphorylase large subunit gene wml1, which covers 1864 bp, was isolated from watermelon genomic DNA by Uneven PCR. Sequence analysis indicated that there were putative TATA box and CAAT box at position 1237bp and 1284bp of the cloned fragment, respectively. Two transcriptional fusions between 5' regulational sequence of wml1 and beta-glucuronidase gene were constructed. Constructs containing 180bp-1752bp fragment could drive GUS gene transient express specifically in watermelon fruit, while 958bp-1752bp had no promoter activity. Putative fruit-specific cis-acting elements may locate between 180bp and 958bp.
Cucurbitacins are feeding stimulants for diabroticite beetles, including corn rootworms and cucumber beetles, which can be added to a bait containing an insecticide thereby reducing the levels of other insecticide treatments needed to control these pests. One of them, cucurbitacin E-glycoside, is water soluble and easily processed from mutant bitter Hawkesbury watermelons (BHW) that express elevated levels of cucurbitacin. Storage of BHW extract at room temperature resulted in a 92% reduction of cucurbitacin E-glycoside over two months, while refrigeration or freezing resulted in a 60% loss of the active ingredient during this time. The loss of the active ingredient was correlated with an increase in BHW extract pH from 5 to greater than 9. The increase in pH of the BHW extracts at room temperature appeared to be due to the growth of certain bacteria, especially Bacillusspp. In refrigerated extracts, the pH remained relatively constant, and bacterial growth was dominated by bacteria such as Lactobacilli. An alternative to refrigeration is concentration of BHW extract. One means of concentration is spray drying, but the high sugar content of the BHW extract (20mg/ml glucose, 40mg/ml fructose) makes this technique impractical. Fermentation of the BHW extract by the yeast, Saccharomyces boulardii, eliminated the sugars and did not raise the pH nor alter the cucurbitacin E-glycoside content of the extract. Elimination of the sugars by fermentation produced an extract that could be successfully spray dried. BHW extract fermented by S. boulardii produced a higher level of feeding stimulation for spotted cucumber beetles in laboratory choice tests. When applied to cucumbers, there was no difference in control of spotted and striped cucumber beetles between baits of fresh or fermented juices combined with the same insecticide.
The purpose of this study was to enhance shoot regenerative efficiency of watermelon by optimizing culture parameters. Cotyledons at 4-day seedlings were explanted and cultured on MS medium supplemented with different combinations of hormones. In "Yixuan" cultivar, the plantlet regenerative frequency and shoots produced by per explant was as low as 10% and 0.4 respectively at all combinations tested when distal parts of cotyledons were inoculated. Whereas, maximum regenerative frequency of 100 percentage and 10.3 shoots regenerated from per explant were observed on MS medium supplemented with 5 mg/L BA and 0.1 mg/L IAA using basal parts of cotyledons as explants. Likewise, in "Jingxinmuben" cultivar, the highest regenerative frequency of 100 percentage and 6.9 shoots regenerated from per explant were obtained when proximal parts of cotyledons were cultured on MS medium containing 2 mg/L BA and 0.5 mg/L IAA. In the present study, adventitious buds were initiated after only 4 d in culture and intact plantlets were obtained in 40 d culture. This will facilitate quicker gene transformation and polyploidy induction of watermelon in vitro.
Fruit ripening is associated with a number of physiological and biochemical changes. They include degradation of chlorophyll, synthesis of flavor compounds, carotenoid biosynthesis, conversion of starch to sugars, cell wall solublisation and fruit softening. These changes are brought about by the expression of specific genes. People are interested in the molecular mechanism involved in the regulation of gene transcription during fruit ripening. Many fruit-specific promoters such as PG, E4, E8, and 2A11 have been characterized and shown to direct ripening-specific expression of reporter genes. AGPase plays the key role in catalyzing the biosynthesis of starch in plants. It is a heterotetrameric enzyme with two small subunits and two large subunits, which are encoded by different genes. In higher plants, small subunits are highly conserved among plant species and expressed in all tissues. And the large subunits are present at multiple isoforms and expressed in a tissue-specific pattern. In fruits, the expression pattern of the large subunits varies with plant species. That made it important to study the transcriptional regulation of the large subunits of AGPase in different plant species. Northern-blot analysis indicates in watermelon, an isoform of the large subunits Wml1 expressed specifically in fruits, not in leaves. The 5' flanking region of Wml1, which covers 1573bp, has been isolated through the method of uneven PCR. And transient expression assay has shown that the 1573bp (named WSP) can direct fruit-specific expression of GUS gene. Our goal in this study was to scan the promoter region for main regulatory regions involved in fruit-specific expression. A chimaeric gene was constructed containing the WSP promoter, the beta-glucuronidase (GUS) structural sequence as a reporter gene and the nopaline synthase polyadenylation site (NOS-ter). The plasmid pSPA was digested with Hind III + Hinc II and promoter fragment of 1573bp (from 180bp to 1752bp) was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-16. The same insert was then cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by HindIII + Bgl II to produce pISPA-16. Three 5'-end deletions of the promoter were obtained and fused to GUS gene in plant transient expression vector pBI426: the 1201bp fragment (from 551bp to 1752bp) was generated by digestion of pBSPA-16 with BamH I + SnaB I, the 898bp fragment (from 854bp to 1752bp) by BamH I + EcoRV. Both fragments were ligated with pBluescript SK(-) digested by BamH I + Sma I, to produce pBSPA-12 and pBS-PA-9. The inserts were cut out with HindmIII + BamH I and ligated with pBI426 digested by Hind III + Bgl II, to produce pISPA-12 and pISPA-9. The 795bp fragment (from 957bp to 1752bp) was generated by digestion of pSPA with Hinc II + EcoR I, promoter fragment was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-8. The same insert were cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by Hind III + Bgl II. The 1573bp fragment and three 5'-end deletions were delivered into watermelon leaf, stem, flower and fruit of different development stages (5, 10, 20 days after pollination) via particle bombardment using a biolistic PDS-1000/He particle gun. Bombardment parameters were as follows: a helium pressure of 1200 psi, vacuum of 91432.23Pa, 7 cm between the stopping screen and the plate. Histochemical assay were done on all the tissues bombarded after incubation for 2 days. The 1573bp fragment had the strongest promoter activity, and can induce GUS expression in fruits of 5 and 20 days after anthesis and flowers, but not in fruits of 10 days after anthesis, leaves and stems. Fragments of 1201bp and 898bp can induce GUS expression only in fruits of 20 days after anthesis, and with lower expression levels than 1573bp. Fragment of 795bp was not able to direct GUS expression in any of the tissues bombarded (data not shown). It can be concluded that of the 1573bp, 1201 bp, 898bp Wml1 5'flanking regions include the necessary information directing fruit-specific expression. Deletion from 180bp to 551bp doesn't affect the fruit-specificity of the promoter, but lowered the expression level. There may be some cis-acting elements located in this region, which can enhance external gene expression in later stages of fruit development. Deletion from 854bp and 958bp led to loss of GUS expression. This region includes the necessary information needed for gene expression as well as the regulatory elements for fruit-specific transcription.
The occurrence of 4 pesticide residues, imidacloprid, carbendazim, methiocarb, and hexythiazox, was assessed in several fruits (oranges, tangerines, watermelons, and date plums) from the Valencian Community (Spain). A total of 227 samples--56 oranges, 134 tangerines, 13 watermelons, and 24 date plums--were taken from an agricultural cooperative representative of the area studied during 2001. The pesticides were determined by liquid chromatography/mass spectrometry after sample extraction with ethyl acetate and anhydrous sodium sulfate. Recoveries of 4 pesticides at 2 fortification levels (0.02 and 0.2 mg/kg), the lower of which was the quantification limit, ranged from 60 to 108%. Concentrations of imidacloprid, carbendazim, methiocarb, and hexythiazox ranged from 0.02 to 0.75 mg/kg in 184 samples (54 oranges, 119 tangerines, 3 watermelons, and 8 date plums). Nineteen samples contained methiocarb or hexythiazox residues that exceeded the maximum residue limits. Calculation of the estimated daily intakes of the 4 pesticides studied and their comparison with the Acceptable Daily Intakes established by the Food and Agriculture Organization of the World Health Organization demonstrated the safety of fruit consumption and showed the importance of monitoring for pesticide residues.
A method using solid-phase extraction for cleanup, followed by high-performance liquid chromatography with ultraviolet detection (HPLC/UV), was developed for the determination of forchlorfenuron residues in watermelon. The pesticide is extracted from the sample with acidic acetonitrile, and the extract is loaded onto a primary-secondary amine (PSA) column. The pesticide is eluted with acetone and determined by HPLC/UV. The PSA column was found to provide effective cleanup, removing the greatest number of sample matrix interferences. The acetonitrile extraction followed by the PSA cleanup provided recoveries of >95%, coefficients of variation (precision) of <10%, and sensitivity of 0.005 mg/kg, in agreement with the directives for method validation in residue analysis. The proposed method was successfully used to determine forchlorfenuron residue levels and dissipation rates in watermelon grown in an experimental greenhouse in Beijing, People's Republic of China.