We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.

Bioregulators have a great effect on vital processes of plant growth and development. Known plant bioregulators include Naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA). Natural or synthetic plant bioregulators are organic compounds that affect the physiological processes in the plant, either to control some of these processes or to modify them. For example these bioregulators can affect the nature of the process, either by accelerating or decelerating plant growth, rates of maturation and also by altering the behavior of the plants or their products. Also, enhancement of important nutrients in human diet could be achieved by bioregulators.   This study uses the model crop plant Tomato (Lycopersicon esculentum). Tomato is affected by a group of bioregulators, this group contains compounds which are powerful antioxidants in vitro. The current study aims to find out the effect of some plant bioregulators (IAA, IBA and NAA) on tomato growth, total protein content and enzyme activities of ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT). This study also investigates the effect of the above mentioned bioregulators on the level of RNA expression for SOD, CAT and TPX1 genes. The analytical quantification of target gene expression showed the induced effect of NAA on SOD expression and reducing effect of the other bioregulators (IAA and IBA) on CAT and TPX1 expression. However, at the protein level, we foundthat IBA and IAA caused a minor effect on total protein content while a significant effect was recorded on the total protein level using NAA. Upon measuring the enzyme activity of ascorbate peroxidase and catalase, we found that both the exogenous NAA and IBA stimulated ascorbate peroxidase activity in tomato while there was no considerable difference detected in IAA treated plants. Also, there was no considerable difference detected in catalase activity of all bioregulator-treated plants compared with the control.

Low transformation efficiency and long generation time for production of transgenic Gerbera jemosonii plants leads to vulnerable gene function studies. Thus, transient expression of genes would be an efficient alternative. In this investigation, a transient expression system for gerbera petals based on the Agrobacterium infiltration protocol was developed using the reporter genes β-glucuronidase (gus) and green florescence protein (gfp). Results revealed the incapability of using the gfp gene as a reporter gene for transient expression study in gerbera flowers due to the detection of green fluorescent color in the non-infiltrated gerbera flower petals. However, the gus reporter gene was successfully utilized for optimizing and obtaining the suitable agroinfiltration system in gerbera flowers. The expression of GUS was detectable after three days of agroinfiltration in gerbera cultivars "Express" and "White Grizzly" with dark pink and white flower colors, respectively. The vacuum agroinfiltration protocol has been applied on the cultivar "Express" for evaluating the transient expression of the two genes involved in the anthocyanin pathway (iris-dfr and petunia-f3' 5'h), which is responsible for the color in flowers. In comparison to the control, transient expression results showed change in the anthocyanin pigment in all infiltrated flowers with color genes. Additionally, blue color was detected in the stigma and pollen grains in the infiltrated flowers. Moreover, blue colors with variant intensities were observed in produced calli during the routine work of stable transformation with f3' 5'h gene.

ABSTRACT. Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium-mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5 th day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at »70 kDa from freshly extracted protein, while two degraded bands of »55 and »45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.

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Egyptian Journal of Botany http://ejbo.journals.ekb.eg/ 22 R EVERSE genetic approach was used to isolate and characterize Medicago truncatula containing "knockout" mutations in gene involved in nodulation process and in many other physiological processes. More than 60 Tnt1-Flanking sequence tags (FSTs) ranging from ~70bp to ~600bp were isolated, sequenced and submitted to Genebank referring for akt1 mutant characterization. The proper Tnt1-insertion was mapped in chromosome number 8 of Medicago truncatula genome. It was precisely identified upstream the base number 141 and downstream of the ATG start codon of Medicago truncatula potassium channel AKT1 gene (MtAKT1). MtAKT1 gene encodes inwardly rectifying potassium channel was isolated, sequenced, and submitted to Genebank with accession number MN649185.1 .M. truncatula akt1 mutant is achieving higher number of deformed root nodules and shorter root length compared to wild type. akt1 nodules exhibited reduced size occupied with un-differentiated cells and abnormalities in symbiotic nodule zones. Non-functional nitrogen fixation zone and compacted infection zone were observed as well. AKT1 null mutant exhibits attenuation in its ability to maintain the proper K + and Na + ions content in akt1 seedlings which is 2-3 fold less than wild type seedlings,. In contrast, akt1 seedlings showed three-fold increase in Ca ++ ion concentration compared to wild type.In conclusion Medicago truncatula mutant, akt1 is Tnt1-retrotransposon mutant impaired in inwardly rectifying potassium channel AKT1. As the first reported AKT1 null mutant was isolated from legume plants, this mutant is displaying abnormalities in nitrogen fixation organ and affecting ions uptake.

Genetic mutagenesis is a very efficient tool in studying genes function. Because of great benefits of legumes as human food and animal feed worldwide, we used a model plant Medicago truncatula for identification gene function related to nitrogen fixation process. Our mutant is a Medicago mutant line contains a tobacco Tnt1 retro-transposon mobile element with the two Long Terminal Repeats (LTR) inserted within the genome. Our mutant is predicted to contain a mutation in gene/s belonging to symbiotic interaction between legume and rhizobia. A novel technique was used based on using fluorescent oligonucleotide primers against oligonucleotide primers for Tnt1-LTRs of our mutant. This novel protocol was very successful in detection the polymorphism between our mutant line and the wild variant R108 using Biosystems 310 Genetic Analyzer. Electropherograms of the mutant line and wild type gave a total of 561 well- resolved AFLP peaks, 357of which were polymorphic peaks and 204 were monomorphic peaks. This novel technique enables the calculation percentage of polymorphism between the mutant line and the wild type. Additionally primers combinations amplified more bands from others to detect polymorphism between the plants

The production of new cultivars via recombinant DNA technology is important in applied agriculture. Promoters play fundamental roles in successful transformation and gene expression. Fragments of the upstream regulatory region of the movement protein gene of the Tomato yellow leaf curl virus (TYLCV; two fragments) and Watermelon chlorotic stunt virus (WmCSV, two fragments) and one fragment of the coat protein putative promoter of TYLCV (CPTY-pro) were isolated to assess their abilities to drive expression in monocot and dicot plants. We used bioinformatic analyses to identify tentative motifs in the fragments. The five promoter fragments were isolated, fused with the GUS reporter gene, and transformed into tomato, watermelon, and rice plantlets via Agrobacterium infiltration. GUS expression driven by each putative promoter was analysed using histochemical and fluorometric analyses. In both dicots and the monocots, the highest level of GUS expression was obtained using a truncated regulatory region from TYLCV (MMPTY-pro) followed by a truncated regulatory region from WmCSV (MMPWm-pro). However, the corresponding full-length fragments from TYLCV and WmCSV showed essentially equivalent expression levels in the fluorometric GUS assay compared with the enhanced Cauliflower mosaic virus e35S-pro. In addition, CPTY-pro showed no expression in either the dicots or the monocot. This study demonstrated that MMPTY-pro and MMPWm-pro may be useful as plant promoters.