Mutation in the Agrobacterium his I gene enhances transient expression in pepper

Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation.In this work,Agrobacterium was found to induce a strong immune response in pepper,which might be the reason for T-DNA being difficult to express in pepper.An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves.Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death,presumably due to reduced bacterial growth.However,mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response.The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed.

Enemies atpeace:Recentprogressin Agrobacterium-mediated cereal transformation

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Agrobacterium _mediated Transformation of Rice with Help of Bombardment

Oryza sativa L. ssp. japonica cv. Zhonghua 8, which is recalcitrant to infection of Agrobacterium tumefaciens (Smith et Townsend) Conn strain EHA105 with ordinary binary vector pCambia 1301, was transformed through Agrobacterium-mediated transformation with help of bombardment. The transformation efficiency can be raised greatly. Single copy of gene insertion in the genome of transgenic rice plants was proved by Southern analysis and the expression of GUS gene was observed. GUS gene and hygromycin-resistant gene show 3:1 segregation in progenies of the transgenic rice plants.

Researches of Agrobacterium rhizogenes Ri Plasmid rol Genes

Expression of rol genes from Ri plasmid of Agrobacterium rhizogenes not only leads to the excessive formation of adventitious roots, but also exhibits various genetically modified characteristics that have broad prospects for the application of plant genetic improvement. Since the 1980s of the last century, much progress has been made in the studies of A. rhizogenes, in particular the agropine type Ri plasmid rol genes and their applications for plant genetic improvement, which involves the structure and function of Ri plasmid, the characters of rol genes, the influence of rol genes expression on plants growth and development, and the applications of rol genes for genetic improvement of forest tree. In this paper, the advances in this field are reviewed and the existing problems about the application of rol genes for genetic improvement of forest tree are also discussed.

Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens

Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe （Aloe barbadensis Miller） by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride （25 rain） for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride （10 min）. Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium （30 rain） in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24℃. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHAI05 and C58CI was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHAI05 than to C58C1. Infected tissues were selected using G418 （10.0-25.0 mg/L） to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe.

异养硝化好氧反硝化菌株Agrobacterium tumefaciens LAD9羟胺氧化酶的分离纯化

羟氨氧化酶(Hydroxylamine oxidase,HAO)的作用方式直接决定了异养硝化好氧反硝化细菌的代谢途径,分离得到纯度较高的HAO也就成为研究这类细菌脱氮机制的重要环节。以异养硝化好氧反硝化细菌Agrobacterium tumefaciens LAD9为代表,建立了该菌株HAO的分离纯化方法:首先采用渗透压休克法提取细胞周质液,然后采用DEAE Sepharose CL-6B离子交换层析和Sephacryl S-100凝胶过滤层析对细胞周质液进行分离纯化。结果表明,经过离子交换层析可得到分子量分别为55.3、35.7和19.2kD的杂蛋白,进一步经过凝胶过滤层析即可得到电泳纯的HAO,纯化倍数为5.79,产率为39.71%。对其酶学性质的初步研究表明,该菌株HAO的分子量为18.8 kD,能够将羟胺氧化为亚硝酸盐氮,且Fe2+的加入可显著增强其酶活。

Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium

[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.

Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF

[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

一株放射性土壤杆菌(Agrobacterium cradiabacter)产胞外多糖性质的鉴定及其发酵工艺条件初探

通过对本实验室筛选得到的一株放射性土壤杆菌LQC-99产生的胞外多糖性质的鉴定,得出该菌株产生的胞外多糖为可得然胶。经过摇瓶发酵实验,初步确定其最佳发酵培养基配方及最适摇瓶发酵条件。摇瓶实验确定的最佳培养基组成为(g/L):蔗糖50.0,(NH_4)_2HPO_4 2.0,KH_2PO_4 2.0,MgSO_4·7H_2O 1.0,CaCO_3 2.0,玉米浆2.5(v/v)。最佳发酵条件为:初始pH 7.0,装液量为80ml/500ml摇瓶.摇床转速为180 rpm,温度为30℃。在此条件下,发酵产胶量可达32g/L。16L自动机械搅拌罐放大试验结果表明,初糖投量为50g/L,在发酵过程中不改变操作参数的情况下,当转速为350 rpm,通气量1:1(v/v)时,经90～96h发酵,可得然胶产量可达35g/L,转化率达到70%;将蔗糖投料量提高至60g/L,(NH_4)_2HPO_4则相应提高至2.4g/L时,胶产量可达到40g/L,转化率仍旧高达66.7%;进一步提高投糖量则可得然胶产量几乎不变,残糖升高,延长发酵时间无明显作用。

Optimization of Agrobacterium Mediated Genetic Transformation System of Tomato Meifen No.1

[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used as the explant,and the Agrobacterium mediation method was used to optimize its genetic transformation efficiency so as to establish the efficient Agrobacterium mediated genetic transformation system of tomato cotyledon.[Result] The highest transformation efficiency was obtained when the explants were cultivated for 2 d on MS ＋ 2.0 mg/L 6-BA＋ 0.5 mg/L IAA medium and then infected with Agrobacterium EHA105（OD = 0.4）for 5 min;it was proved by PCR analysis that the target nptII gene had been integrated into the genome of regenerated plants.[Conclusion] The result in this study had provided basis for the transfer of valuable genes into tomato Meifen No.1.

Selection of Maize Inbred Lines with High Regeneration and Susceptibility to Agrobacterium tumifacien

Ten-maize inbred lines of maize （Zea mays L.） with high-induction rate and proliferation ability of embryonic calli were selected from 70-maize inbred lines by immature embryo culturing. Some of the embryonic calli were transferred onto regeneration medium to examine the ability of regeneration, some were transformed via Agrobacterium tumifaciens C58 carrying intron-β-glucuronidase （gus） gene, and GV3301 carrying the green fluorescent protein （gfp） gene to study the susceptibility of different genotypes in maize to A. tumifaciens. All embryonic calli initiated from 10-maize inbred lines were able to regenerate into plantlets, and the regeneration frequencies of inbred lines 6010, 6038, 6015, 6051, and 6060 were 61.11%, 31.94%, 45%, 33.33%, and 56.94%, respectively, which were higher than that of other lines. Analysis of variance indicated that the susceptibility of the various genotypes in maize to A. tumifacien C58 showed a significant difference among each other, and the inbred lines 6010, 6015, 6051, 6050, 6058, 6060, 6069, 6077 were susceptible to A. tumifacien C58, of which frequency of gus expression were over 70%. Expression of GFP was observed in six-inbred lines （6050, 6015, 6051, 6058, 6069, 6077）. The inbred lines 6051, 6010, 6015, 6060, and 6050 had the high regeneration and the susceptibility to A. tumifaciens C58; and the inbred lines 6051, 6015, and 6060 had the high regeneration and the susceptibility to Agrobacterium tumifaciens GV3301.

石油生物催化脱硫菌Agrobacterium tumefaciens UP3的分离筛选

从胜利油田被原油污染的土壤中筛选到一株能有效降解模型化合物二苯并噻吩 (DBT)的菌株。根据常规的形态分析、生理生化性状及 16SrDNA序列分析 ,将其鉴定为根癌土壤杆菌 (AgrobacteriumtumefaciensUP3)。该菌不能以十二烷、十六烷、液体石蜡和萘作为唯一碳源和能源生长 ,具有工业应用的潜力。对该菌株DBT降解能力的初步研究表明 ,5 4h内可将 5 0 0mg L的DBT降解至 15 0mg L。对降解产物的分析表明 ,根癌土壤杆菌降解DBT的途径与Kodama路线及 4_S路线不同。

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.

Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

Factors Affecting Agrobacterium-Mediated Transformation Efficiency in Rice

Several important factors affecting the efficiency of Agrobacterium-mediated rice transformation were studied with several predominant commercial indica and japonica rice cultivars. As far as indica rice callus was concerned, CC medium was the best and the quality of callus was improved with the addition of 1.0 to 2.0 mg/L ABA. It decreased the percentage of browning calli and improved the callus growing state by addition of a certain amount of sorbitol to the subculture medium. NB medium was the best for callus initiation of japonica rice, but the improvement in the quality of callus of japonica subspecies was not obvious by adding ABA. During the period of subculture, to a certain degree, increasing the sucrose concentration could improve the proportion of hygromycin resistant calli. Furthermore, the transformation efficiency would be higher by applying selection pressure in the selection stage, removing selection pressure during the plantlet differentiation period and applying selection pressure again during seedling hardening period. Besides, suitable combination of plant hormones was beneficial for callus differentiation. An efficient /Agrobacterium-mediated rice transformation system had been established for several rice cultivars and a lot of transgenic rice plants had been obtained.

石油生物脱硫菌Agrobacterium tumefaciens UP-3

对能降解二苯并噻吩(DBT)的根癌土壤杆菌AgrobacteriumtumefaciensUP3菌株进行了固定化研究,以聚乙烯醇(PVA)和海藻酸钠(SA)混合物为包埋法固定化载体,固定化最佳操作条件为4℃交联,PVA和SA混合物总浓度7%,两者最佳浓度比为6,细胞浓度为0.05g/mL。当DBT加入量为2.7mmol/L时,UP-3的静息细胞最高脱硫率为13%,而固定化细胞的脱硫效率超过了60%;固定化细胞的最佳使用条件为降解5d,温度28℃～32℃。

Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.