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Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To establish a system for rapid identification of millet gene functions, in this study, we compared the effects of explants from different foxtail millet cultivars, the concentrations of acetyleugenone, bacterial solution, and co-culture time on the hairy root induction mediated by Agrobacteria rhizogenes strain K599. When the shoot tips were infected with bacterial solution 0.5 optical density (OD) and containing 100 µmol L-1 acetyleugenone, and co-cultured for three days, the induction efficiency of hairy roots reached to 80.24%. Using this system, we analyzed the transformation efficiency of the hairy roots, the subcellular localization of SiDVL1and SiDVL3, and gene functions of SiNHX2, SiCBL4, and SiCBL7. The PCR results of GFP gene and GFP fluorescence microscopic observation indicated that the transformation efficiency of the hairy roots was over 70%. The subcellular localization of SiDVL1 and SiDVL3 was similar in millet hairy-root and tobacco epidermal cells. Simultaneously, the survival rates of SiNHX2, SiCBL4, and SiCBL7 transgenic millet were significantly higher than that of empty vector transformed millet. Therefore, our study established an efficient and rapid method to analyze the subcellular location and to identify the function of genes from foxtail millet. [ABSTRACT FROM AUTHOR]

Bifidobacterium can be used as probiotic that confers various health benefits to its host. The functional analysis of specific genes can help elucidate the probiotic mechanism of Bifidobacterium. However, the low transformation efficiency has hindered the in-depth study of its function by genetic manipulation. In this study, we optimized transformation methods for Bifidobacterium animalis AR668 to improve the transformation efficiency up to 2.15 ×105 CFU/μg DNA. Additionally, the expression of competence gene ssb2 could enhance the transformability which was the first attempt to apply it to B. animalis. Moreover, the transformation efficiency was increased after the plasmids were modified by gene0033 and gene1509 which limited the interference of the restriction-modification system in the uptake of foreign DNA by cells. Based on the above optimization, the transformation efficiency of 7.2 ×105 CFU/μg DNA for AR668 was obtained, which was more than 100 times higher than that of the original condition. Together, we successfully improved the transformation efficiency of B. animalis AR668, contributing to its genetic manipulation and modification. [ABSTRACT FROM AUTHOR]

One of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli ( E. coli ) in advance using adopted methods of M1 (MgCl 2 + CaCl 2 ) and M2 (CaCl 2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT- hisC . Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD 600 = 4), incubated for 3 h. Pellets were harvested by centrifugation ( 4000 rpm; 4 °C; 15 min ) . Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at λ 595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 10 6 ) than M1 (3.10 × 10 5 ). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; P < 0.05) than 600 µM IPTG (0.896 ± 0.052 mg/ml) and not different to 400 µM IPTG (1.298 ± 0.080 mg/ml). This research indicated that transformation efficiency needs to be taken account in prior of optimization of the protein expression. [ABSTRACT FROM AUTHOR]

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog (MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD 600 = 0.8–1.0 and the co-cultivation at 25 °C for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gusA gene by PCR using the gusA specific primers in the T 0 lines, there were 4 transgenic lines from 286 T 0 lines (1.4% transformation efficiency). However, histochemical glucuronidase (GUS) assay demonstrated that only three of four transgenic lines exhibited gusA expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium -mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l−1 in combination with 0.1 mg l−1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA 3) (1.0 mg l−1), IAA (0.5 mg l−1), and ZEA (0.5 mg l−1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l−1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium -mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes. [ABSTRACT FROM AUTHOR]

The preparation of competent cell is the central step of bacteria transformation and has a great impact on transformation efficiency of nucleic acid. The aim of the research was to study the factors such as ionic species, strain types, plasmid concentration, ice-bath time and incubation time. The result shows that ionic species is fatal to the transformation efficiency, preparation of competent cells by monovalent ions (Li+, Na+, K+) and tervalent ion (Al3+) do not have capacity of transformation, preparation of competent cells by bivalent ion (Ba2+, Ca2+, Mn2+, Mg2+, Sr2+) has capacity of transformation. On the whole, the efficiency of Ca2+ was found to be the optimum bivalent ion, following by Sr2+>Mn2+>Mg2+>Ba2+. On the other factors that affecting transformation efficiency, the transformation efficiency was the best when plasmid concentration was 100ng/mL, and ice-bath time should be controlled at about 30 min and incubate time selected as 60 min. Experiment of natural transformation later proves the existence of the phenomenon of natural transformation and it's also related to factors such as calcium chloride concentration, plasmid concentration and others. [ABSTRACT FROM AUTHOR]

Abstract: Chlamydomonas offers several potential advantages as a single-celled autotrophic recombinant protein production system including: facile transformation systems for all three genomes, low production costs, and the ability to secrete proteins. However, transgene expression levels from the nuclear genome of Chlamydomonas are often inadequate for industrial applications. With the objective of optimizing nuclear transgene expression, we surveyed transgene (luciferase) expression driven by seven different nuclear gene promoters and three different transcription terminators. The results demonstrate that in addition to the choice of nuclear gene promoters used, transcriptional terminators can have strong influence on transgene expression. We show that the psaD terminator improved transgene expression when paired with a variety of different gene promoters. Among those tested, the psaD–psaD promoter–terminator expression cassette gave the highest expression levels. This expression cassette was then used to express a human protein of pharmaceutical value, human butyrylcholinesterase (huBuChE). Chlamydomonas cells were able to accumulate the luciferase–huBuChE fusion protein to 0.4% of total soluble protein levels, which is comparable to the expression levels of the same protein obtained in plants. In addition, this study found that the form (linear or supercoiled) of the transforming DNA used for Chlamydomonas transformation had significant impact on the transformation efficiency and the level of transgene expression and stability. These results demonstrate that transgene expression in Chlamydomonas can potentially be scaled for commercial production of recombinant proteins. [Copyright &y& Elsevier]

Pseudomonas, due to its diversity in habitat and metabolic type, makes it have broad prospects applying in bioremediation, bioconversion, and biocontrol, while the introduction of exog- enous gene is the key link to genetically modified Pseudomonas. The preparation and transformation of competent cells are the important methodological basis of the introduction of exogenous gene. In this paper, three Pseudomonas strains (P. putida TS11, P. stutzeri DNB, and P. mendocina JJ12) isolated from a petroleum-contaminated soil were taken as the recipient strains, and a three-factor and four-level orthogonal experiment was conducted to investigate the effects of CaCl2 concentration, heat shock duration, and recovery duration on the preparation and transformation efficiency of the strains competent cells. The results showed that CaCl2 concentration was the most important factor affecting the transformation efficiency (P<0. 05), and the transformation efficiency was improved markedly when the Pseudomonas cells were repeatedly washed with sterile distilled water before the preparation of competent cells. When the P. putida TS11 cells were treated with 100 mmol0·L-1 of CaCl2, heat-shocked for 3 minutes at 42 ° and incubated for 1.5 hours at 30 °C, the P. stutzeri DNB cells were treated with 50 mmol·L-1 of CaCl2, heat-shocked for 6 minutes, and incubated for 1.5 hours, and the P. mendocina JJ12 cells were treated with 75 mmol·L-1 of CaCl2, heat- shocked for 4. 5 minutes, and incubated for 0. 5 hours, the transformation efficiency of exogenous plasmids in the three strains all achieved 105 cells.μg-1 DNA. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to the establishment of a new system for Plant Transformants. It states that system utilizes the toxin, toxoflavin as selection agent and the toxoflavin lyase (tflA) as selectable marker gene. The result of the study validates the use of tflA gene as a non antibiotic selectable marker for the transformed plants.