Abdullah A. Alsadon

Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

Background Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. Methodology/Principal Findings To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. Conclusions/Significance We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.

Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics

Background Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. Principal Findings A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. Conclusions We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

Characterisation of alleles of tomato light signalling genes generated by TILLING

Targeting Induced Local Lesions IN Genomes (TILLING) combines chemical mutagenesis with high throughput screening to allow the generation of alleles of selected genes. In this study, TILLING has been applied to produce a series of mutations in genes encoding essential components of the tomato light signal transduction pathway in an attempt to enhance fruit nutritional quality. Point mutations to DEETIOLATED1 (DET1), which is responsible for the high pigment2 (hp2) tomato mutant, resulted in elevated levels of both carotenoid and phenylpropanoid phytonutrients in ripe fruit, whilst immature fruit showed increased chlorophyll content, photosynthetic capacity and altered fruit morphology. Furthermore, genotypes with mutations to the UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), COP1 and COP1like were also characterised. These genotypes largely did not display phenotypes characteristic of mutation to light signalling components but their characterisation has enabled interrogation of structure function relationships of the mutated genes.

Covering Materials Incorporating Radiation-Preventing Techniques to Meet Greenhouse Cooling Challenges in Arid Regions: A Review

Cooling greenhouses is essential to provide a suitable environment for plant growth in arid regions characterized by brackish water resources. However, using conventional cooling methods are facing many challenges. Filtering out near infra-red radiation (NIR) at the greenhouse cover can significantly reduce the heating load and can solve the overheating problem of the greenhouse air. This paper is to review (i) the problems of using conventional cooling methods and (ii) the advantages of greenhouse covers that incorporate NIR reflectors. This survey focuses on how the cover type affects the transmittance of photosynthetically active radiation (PAR), the reflectance or absorptance of NIR and the greenhouse air temperature. NIR-reflecting plastic films seem to be the most suitable, low cost and simple cover for greenhouses under arid conditions. Therefore, this review discusses how various additives should be incorporated in plastic film to increase its mechanical properties, durability and ability to stand up to extremely harsh weather. Presently, NIR-reflecting covers are able to reduce greenhouse air temperature by no more than 5°C. This reduction is not enough in regions where the ambient temperature may exceed 45°C in summer. There is a need to develop improved NIR-reflecting plastic film covers.

Evaluation of PE Films Having NIR-Reflective Additives for Greenhouse Applications in Arid Regions

Linear-low-density-polyethylene- (LLDPE-) based formulations with several near-infrared- (NIR-) reflective pigments were prepared by melt blending technique and their subsequent films were prepared by blown film extrusion process. Thermal properties of these films were evaluated using differential scanning calorimetry (DSC). The results showed that the melting and crystallization temperatures ( and , resp.) of these formulations were almost similar to that of control resin. The melt viscosity was measured by stress-controlled rotational rheometer and melt flow index (MFI) instruments. Rheological measurements indicated that the blend formulations with NIR-reflective additive have similar melt viscoelastic behavior (storage modulus and dynamic viscosity) to the control resin. The mechanical test performed on NIR-reflective films showed similar values of tensile strength for blend samples as that of control resin. The spectral radiometric properties of the blend films were evaluated in the solar wavelength range of 200–1100 nm and found to be improved over the control sample without having NIR-reflective pigment.

In vitro rooting of leguminous plants: Difficulties, alternatives, and strategies for improvement

Leguminous plants include many of the most important food crops and are also cultivated for forage, used as a rich fiber source, and are important in the manufacture of pharmaceutical compounds. Their ability to biologically fix atmospheric nitrogen enhances soil fertility. Beyond conventional propagation methods, modern in vitro technology offers new means for propagating these plants. Significant progress has been achieved in somatic embryogenesis and embryo rescue, despite legumes’ notorious recalcitrance to transformation and regeneration of legumes. The establishment of stable and reproducible regeneration protocols has resulted in commercially successful propagation of some legume species. Still, regeneration protocols for many other legumes have not been successful, primarily due to poor in vitro rooting during regeneration. This review addresses the factors constraining rooting, describes alternative methods to promote in vitro rooting, and provides a summary of in vitro rooting studies performed across a large number of leguminous species.