In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S′, H′, and D′) based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

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Plant regeneration protocols for sugarcane GT54-9(C9) cultivar were developed for direct organogenesis and indirect somatic embryogenesis, using young leaf segments as explants by studying the influence of different concentrations and types of cytokinin and auxin hormones. For the callus formation from young leaves, a medium containing 4mg/l 2,4-D was found very effective. For embryo formation, MS medium supplemented with 1mg/l Kin and 0.5 mg/l 2,4-D was used. While in the case of direct organogenesis protocol, the medium containing 1mg/l BAP and 2mg/l NAA was the best for direct shoot formation. Data showed that the best shoot regeneration and elongation medium for direct organogenesis and indirect somatic embryogenesis was obtained on medium with 2 mg/l Kin and 0.1 mg/l BAP. Root induction was best performed on 2mg/l NAA and complete plantlets were hardened in the greenhouse before transferring to the field for further evaluation. For transformation, young leaf segments of sugarcane from the cultivar GT54-9(C9) were inoculated and co-cultivated with Agrobacterium tumefaciens strain LB4404 harboring the binary vector pISV2678 with the bar and the gus-intron genes. The obtained putative transgenic plantlets were able to grow under bialaphose containing medium. Stable integration of the bar gene into the plant genomes was tested by PCR and Southern blot hybridization. Histochemical assay and leaf painting analysis were carried out to study the expression of the gus and bar genes in transgenic plants, respectively. The results indicated that the direct organogenesis produced a higher yield of regenerated plants (22% more) within shorter time (4 weeks less). Therefore, this method is recommended for sugarcane regeneration and for further use in genetic transformation via A. tumefaciens with desired genes.

Glyphosate is a commonly used organophosphate herbicide that has an adverse impact on humans, mammals and soil microbial ecosystems. The redundant utilize of glyphosate to control weed growth cause the pollution of the soil environment by this chemical. The discharge of glyphosate in the agricultural drainage can also cause serious environmental damage and water pollution problems. Therefore, it is important to develop methods for enhancing glyphosate degradation in the soil through bioremediation. In this study, thirty bacterial isolates were selected from an agro-industrial zone located in Sadat City of Monufia Governorate, Egypt. The isolates were able to grow in LB medium supplemented with 7.2 mg/ml glyphosate. Ten isolates only had the ability to grow in a medium containing different concentrations of glyphosate (50, 100, 150, 200 and 250 mg/ml). The FACU3 bacterial isolate showed the highest CFU in the different concentrations of glyphosate. The FACU3 isolate was Gram-positive, spore-forming and rod-shape bacteria. Based on API 50 CHB/E medium kit, biochemical properties and 16S rRNA gene sequencing, the FACU3 isolate was identified as Bacillus aryabhattai . Different bioinformatics tools, including multiple sequence alignment (MSA), basic local alignment search tool (BLAST) and primer alignment, were used to design specific primers for goxB gene amplification and isolation. The goxB gene encodes FAD-dependent glyphosate oxidase enzyme that responsible for biodegradation process. The selected primers were successfully used to amplify the goxB gene from Bacillus aryabhattai FACU3. The results indicated that the Bacillus aryabhattai FACU3 can be utilized in glyphosate-contaminated environments for bioremediation. According to our knowledge, this is the first time to isolate of FAD-dependent glyphosate oxidase ( goxB ) gene from Bacillus aryabhattai .

strawberry (Fragaria × ananassa) is an economically important soft fruit crop with polyploid genome, which makes the breeding of new cultivars difficult. simple and efficient methods for transformation and regeneration are required for cultivars improvement in strawberries. In the present study, adventitious shoot regeneration has been investigated in three cultivated strawberry plants, i.e., Festival, sweet charlie, and Florida via direct organogenesis using the in vitro juvenile leaves as explants. explants were collected after sub-culturing on a propagation medium composed of Ms supplemented with 0.5 mg/l Ba; 0.1 mg/l Ga3 and 0.1 mg/l IBa. To select the suitable organogenesis, the explants of the three cultivars were cultured on Ms medium supplemented with different concentrations of TDZ (1, 2, 3, and 4 mg/l), then incubated at a temperature of 22 °c ± 2. Medium containing 2 mg/l TDZ revealed the best regeneration efficiency with the three cultivars (72% for Festival and 73% for sweet charlie and Florida). after 4 weeks the produced shoots were cultured on Ms medium with different concentrations of Ba and Kin to enhance shoot elongation. Results showed that the medium containing 1.5 mg/l Ba and 0.5 mg/l Kin revealed highest elongation efficiency (88% and 94%) for Festival and sweet charlie, respectively. On the other hand, medium media containing 1.5 mg/l Ba and 0.1 mg/l Kin showed highest elongation efficiency (90%) in Florida. elongated shoots were successfully rooted on Ms medium containing 1.5 mg/l Naa. Furthermore, transformation of the two cultivars, Festival and sweet charlie, has been established via Agrobacterium strain LBa44404 containing the plasmid pIsV2678 with gus-intron and bar genes. Three days post-cocultivation, GUs activity was screened using the histochemical assay. The results showed 16% and 18% of the tested plant materials had changed into a blue color for Festival and sweet charlie, respectively. Out of 120 explants only 13 shoots were developed on bialaphos medium for each cultivar, representing 10.8% bialaphos-resistant strawberry shoot. The presence of both the genes bar and uid a was detected by PcR and Northern, giving a transformation efficiency of 5%.