Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were inoculated with 1 ml of diluted (OD_600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin^?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin^?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration. Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Enhancement of 17alpha-hydroxyprogesterone production from progesterone by biotransformation using hydroxypropyl-beta-cyclodextrin complexation technique

An enhancement of 17α-hydroxyprogesterone (17α-HP) production from progesterone by biotransformation using hydroxypropyl-β-cyclodextrin (HPβCD) complexation together with aeration and sonication technique was demonstrated. The progesterone–hydroxypropyl-β-cyclodextrin complex was prepared by co-evaporation method. The percentage yield of 17α-HP from P of 11.26 ± 0.64% at 24 h was observed in Curvularia lunata ATCC 12017. In the complex form of P, together with sonication at 40 kHz for 5 s and aeration, the yield of 17α-HP was increased to 72.92 ± 4.28% which was about 6.5 and 1.3 times of that from the uncomplexed (P) and the complexed (PC), respectively without sonication and aeration. The increased aqueous solubility of P by complexation with HPβCD was the main factor which increased the yield of 17α-HP, while aeration had more effect on P than PC. Sonication did not significantly increased the yield of the product from both P and PC. When both aeration and sonication were used in the PC system, the product yield was increased significantly more than that from P. The result from this study can be applied for the biotransformation of other poor aqueous soluble precursors.     

Possible effects of the detachment of stromal lamellae from granal stacks on salt-induced changes in spillover. A study by sonication of chloroplasts

Salt-induced chlorophyll fluorescence and spillover changes in control and briefly sonicated chloroplasts have been studied under conditions where Photosystem II traps are closed. In a low-salt medium containing 10 mM KCl, control envelope-free chloroplasts exhibited good spillover, as measured by low chlorophyll fluorescence yield at room temperature, a high ratio of the fluorescence peaks $\text{F}{}_{735}\text{F}{}_{685}$ at 77 K, and increased Photosystem I activity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and Photosystem II light. In contrast, when stacked chloroplasts were briefly sonicated and subsequently diluted into a low-salt medium, a high fluorescence yield at room temperature and a low ratio of $\text{F}{}_{735}\text{F}{}_{685}$ at 77 K persisted. When unstacked chloroplasts were sonicated and then diluted into a high-salt medium, the room temperature fluorescence yield remained low. The results are interpreted in terms of a model relating the changes in chlorophyll fluoresecence with the lateral diffusion of Photosystem I and Photosystem II chlorophyll-protein complexes in the plane of the thylakoid membrane creating randomized or segregated domains, depending on the degree of electrostatic screening of surface charges (Barber, J. (1980) FEBS Lett. 188, 1–10). It is argued that brief sonication of stacked chloroplasts separates stromal membranes from granal stacks, thus limiting the inter-mixing of the photosystems via lateral diffusion even when the ionic composition of the medium is varied. Consequently energy transfer from Photosystem II to Photosystem I is relatively poor and chlorophyll fluorescence from Photosystem II is enhanced. The loss of the salt effect on sonicated unstacked membranes can also be accommodated by the model. In this case it seems that the generation of small membrane fragments does not allow the normal salt-induced phase separation of the pigment-protein complexes to occur.     

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study

Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.     

Development of sensitive methods for the detection of myobacteria by DNA probes

A sensitive method allowing the detection of mycobacteria by DNA probing has been developed. Besides the choice of a relevant probe encoding for part of the ribosomal RNA genes, the critical step allowing this high sensitivity was the method by which mycobacteria were lysed. Sonication of mycobacteria in the presence of chloroform followed by dot blot by this lysate gave the highest sensitivity in the detection of sequences homologous to the DNA probes.     

Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae

A comparative evaluation of Bligh and Dyer, Folch, and Cequier-Sánchez methods for quantitative determination of total lipids (TLs) and fatty acids (FAs) was accomplished in selective green (Ulva fasciata), red (Gracilaria corticata), and brown algae (Sargassum tenerrimum) using a full factorial categorical design. Applications of sonication and buffer individually on lipid extraction solvent systems were also evaluated. The FA recoveries obtained from the aforementioned methods were compared with those of direct transesterification (DT) methods to identify the best extraction methods. The experimental design showed that macroalgal matrix, extraction method, and buffer were key determinants for TL and FA recoveries (P ? 0.05), exhibiting significant interactions. But sonication gave erratic results with no interaction with any of the factors investigated. The buffered solvent system of Folch rendered the highest TL yield in U. fasciata and G. corticata while the buffered system of Bligh and Dyer gave the highest yield in S. tenerrimum. DT methods were more convenient and accurate for FA quantification and rendered 1.5–2 times higher yields when compared with the best conventional method, minimizing the use of chlorinated solvents, their cost of analysis, and disposal. The buffered solvent system was found to be the most appropriate for lipid research in macroalgae.     

A novel hybrid technology for remediation of molasses-based raw effluents

A novel three-step technology for treatment of four molasses-based raw industrial effluents, varying in their COD, color and turbidity is reported here. Sequential steps involved in this treatment are; (1) sonication of the effluents, (2) whole-fungal treatment of these by a ligninolytic marine fungus and (3) biosorption of the residual color with heat-inactivated biomass of the same fungus. Sonication reduced the foul odor and turbidity of the effluents. It increased biodegradability of the effluents in the second stage of treatment. Laccase production in the presence of all the four effluents was directly correlated with their decolorization. After the third step, a reduction of 60-80% in color, 50-70% in COD and 60-70% in total phenolics were achieved. Comparative mass and nuclear magnetic resonance spectra indicated increasing degradation of the effluent components after each stage. Toxicity (LC₅₀ values) against Artemia larvae was reduced by two to five folds.     

A transgenic tropical maize line generated by the direct transformation of the embryo-scutellum by <Emphasis Type="Italic">A. tumefaciens</Emphasis>

An efficient Agrobacterium-mediated transformation system, from which transgenic tropical maize plants were directly generated without previous crosses with laboratory or temperate lines, was established. Experimental evaluations were focused on two main issues: (i) establishment of appropriate tissue culture conditions, which induced somatic embryogenesis from the scutellum-cells, and (ii) the delivery of T-DNA toward these cells. High rates of embryogenic-calli, mainly generated from the embryo-scutellum, were obtained when 15 mg l⁻¹ AgNO₃ were included into the N6-based induction medium; rates up to 19 plants per gram were regenerated from these induced calli. Regarding the Agrobacterium strains evaluated for their transformation capability on the tropical maize line LPC13 used here, best results were obtained from the EHA105 cells when applied at OD_{550 nm} = 0.5–1.0. Physical microwounds before the Agro-infection proved to be an excellent way to promoting both the T-DNA transferring toward the embryo-scutellum and the increasing of rates of transient GUS expression. The highest frequencies of transient GUS expression corresponding to the scutellum-cells as well as the regeneration of whole transgenic plants emerged from them, were obtained using immature embryos wounded by bombarding at 80 lb/in² followed for vacuum infiltration before and during the Agro-infection, respectively, or using embryos wounded by 5 s-sonication (without vacuum infiltration) before the Agro-infection. Transformation frequencies up to 5.41% and 6.82% were obtained from the Agro-infected embryos wounded by particle-bombardment and sonication, respectively. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.     

超声波辅助农杆菌介导CP基因转化番小瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV 外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV 菌株、美国夏威夷PRSV 菌株、泰国PRSV 菌株及日本PLDMV 菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset 的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS 6%蔗糖 1%葡萄糖,pH 5.7)调整至光密度OD_(600(?)m)=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml 无菌离心管装载胚性愈伤材料进行15s 的超声波处理,在80块被转化的胚性愈伤中获得21个CP 基因G 转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s 的超声波处理48块被转化的胚性愈伤中获得8个CP 基因B 转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP 基因转化上均表现出相似的效果。试验还表明:120mg/L 是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR 和Southern 印迹杂交进行了鉴定。