Morphological characteristics of pollen from triploid watermelon and its fate on stigmas in a hybrid crop production system.

Hybrid crop production is more reliant on pollinators compared to open-pollinated crops because they require cross-pollination between a male-fertile and a male-sterile line. Little is known about how stigma receipt of pollen from male-sterile genotypes affects reproduction in hybrids. Non-viable and non-compatible pollen cannot fertilise plant ovules, but may still interfere with pollination success. Here we used seedless watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) as a model hybrid plant, to evaluate the morphology, physiology, and movement of pollen from inter-planted genotypes (diploids and triploids). We found that pollen from triploids ('Exclamation' and 'Royal Armada') and diploids ('SP-6', 'Summer Flavor 800', and 'Tiger') was visually distinguishable. Pollen in triploids had more deformities (42.4-46%), tetrads (43-44%), and abnormal growth of callose plugs in pollen tubes. The amount of pollen in triploids to germinate on stigmas was low (8 ± 3%), and few pollen grains produced pollen tubes (6.5 ± 2%). Still, contrary to previous reports our results suggest that some viable pollen grains are produced by triploid watermelons. However, whilst honey bees can collect and deposit pollen from triploids onto stigmas, its effect on hybrid watermelon reproduction is likely to be minimal due to its low germination rate.

Dietary watermelon residue influencing the nonspecific immunity of juvenile Pseudorasbora parva.

The study explored the improvement of disease resistance, non-specific immunity and anti-oxidation reactions for Pseudorasbora parva (PP) using dietary watermelon residue. The cumulative PP mortality and the pathogenic bacteria number in 15-45% groups reduced relative to those in control group (CK). Under 15-45% groups, AKP, ACP activities and akp, acp genes expression levels were increased markedly in nonspecific immunity system. Similarly, antioxidant response (SOD, CAT activities) and their genes was promoted also at 15-45% groups. Organic matter (vitamin and polyphenols) in watermelon residue improved AKP, ACP, SOD, CAT activities by increasing corresponding gene expressions. Theoretically, they could also function as stimulus signal, active center or composition to modulate enzyme activities and gene expressions. Besides, watermelon residue ameliorated NF-kB, mTOR responses pathway, and consequently suppressed Aeromonas hydrophila which augmented disease resistance.

Doubled Haploid Production in Watermelon.

Doubled haploid (DH) technology is very advantageous in plant breeding. This technique is beneficial for reducing the time required to obtain pure lines and contributes to the selection efficiency. Using this technique, 100% homozygosity can be achieved in a single generation, while the development of stable lines using the traditional self-pollination method takes from 6 to 8 years. It has long been used in diverse crops including cucurbits. DHs can be obtained via parthenogenesis (pollination mostly with irradiated pollen), gynogenesis (in vitro culture of ovules and ovaries), and androgenesis (in vitro culture of microspores and anthers). All these methods have been used for over 30 years to develop haploid and DH lines in cucurbit crops. Nowadays, many researchers benefit from these techniques routinely. However, there are still many limits for using DH technology in watermelon breeding programmes. The number of developed DH lines is still very low.In this chapter, we present a protocol based on the different studies on haploids and DHs induced in watermelon through irradiated pollen technique, unfertilized ovule/ovary culture and anther/microspore culture. According to the results of all these studies, it is crucial to develop an efficient protocol for haploid embryo induction to enhance the frequency of obtaining haploid embryos in watermelon.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants.

Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 µg (in 100-µL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.

Purification and partial characterization of low molecular weight vicilin-like glycoprotein from the seeds of Citrullus lanatus.

The watermelon (Citrullus lanatus) seeds are highly nutritive and contain large amount of proteins and many beneficial minerals such as magnesium, calcium, potassium, iron, phosphorous, zinc etc. In various parts of the world, C. lanatus seed extracts are used to cure cancer, cardiovascular diseases, hypertension, and blood pressure. C. lanatus seed extracts are also used as home remedy for edema and urinary tract problems. In this study, we isolated protein fraction of C. lanatus seeds using various protein separation methods. We successfully purified a low molecular weight vicilin-like glycoprotein using chromatographic methods followed by SDS-PAGE and MALDI-TOF/MS identification. This is the first report of purification of a vicilin like polypeptide from C. lanatus seeds. In next step, we extracted mRNA from immature seeds and reverse transcribed it using suitable forward and reverse primers for purified glycoprotein. The PCR product was analysed on 1% agarose gel and was subsequently sequenced by Dideoxy DNA sequencing method. An amino acid translation of the gene is in agreement with amino acid sequences of the identified peptides.

ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin.

The flowering pattern of watermelon species (Citrullus spp.) is either monoecious or andromonoecious. Ethylene is known to play a critical role in floral sex determination of cucurbit species. In contrast to its feminizing effect in cucumber and melon, in watermelon ethylene promotes male flower development. In cucumber, the rate-limiting enzyme of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), regulates unisexual flower development. To investigate the role of ethylene in flower development, we isolated four genomic sequences of ACS from watermelon (CitACS1-4). Both CitACS1 and CitACS3 are expressed in floral tissue. CitACS1 is also expressed in vegetative tissue and it may be involved in cell growth processes. Expression of CitACS1 is up-regulated by exogenous treatment with auxin, gibberellin or ACC, the immediate precursor of ethylene. No discernible differential floral sex-dependent expression pattern was observed for this gene. The CitACS3 gene is expressed in open flowers and in young staminate floral buds (male or hermaphrodite), but not in female flowers. CitACS3 is also up-regulated by ACC, and is likely to be involved in ethylene-regulated anther development. The expression of CitACS2 was not detected in vegetative or reproductive organs but was up-regulated by auxin. CitACS4 transcript was not detected under our experimental conditions. Restriction fragment length polymorphism (RFLP) and sequence tagged site (STS) marker analyses of the CitACS genes showed polymorphism among and within the different Citrullus groups, including watermelon cultivars, Citrullus lanatus var. lanatus, the central subspecies Citrullus lanatus var. citroides, and the desert species Citrullus colocynthis (L).

Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon.

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.