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.

Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

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。

根癌农杆菌介导的香蕉炭疽菌转化

利用农杆菌(Agrobacterium tumefaciens)介导的转化方法,以香蕉炭疽菌(Colletotrichum musae)的分生孢子为受体,pTHPR1为双元载体,成功实现了植物病原真菌香蕉炭疽菌的遗传转化.在诱导培养基pH 5.6、乙酰丁香酮200μg/mL、共培养时间24 h等适宜条件下,最高转化率可达260个转化子/106个孢子.对转化子的PCR检测表明,T-DNA已整合到香蕉炭疽菌的基因组中,转化子的遗传表现稳定.

根癌农杆菌介导水稻成熟胚及抗褐飞虱基因植株的获得

以水稻(Oryza sativa L.)籼型两系恢复系M5274和晚粳稻不育系N55S成熟胚为材料,以携带有双元载体的根癌农杆菌(Agrobacterium tumefaciens)EHA105为载体进行抗飞虱基因Bph14、Bphi008A、Osg1的遗传转化,共获得515棵再生植株,包括198株Bph14转基因植株、72株Bphi008A转基因植株和245株Osg1转基因植株。PCR检测结果表明,获得的515株再生植株中,有244株阳性转基因植株,3个不同抗褐飞虱基因中,转Bph14基因的再生苗阳性率最高,增加筛选次数能有效减少转化所得的假阳性植株。

Characterization of Avirulent TnphoA Mutants in <i>Agrobacterium tumefaciens</i>to Enhance Transformation Efficiency

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.

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.

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.

Overexpression of SOS Genes Enhanced Salt Tolerance in Sweetpotato

The production of transgenic sweetpotato （cv.Xushu 18） plants exhibiting enhanced salt tolerance using salt overly sensitive （SOS） genes was achieved through Agrobacterium tumefaciens-mediated transformation.A.tumefaciens strain EHA105 harbors a binary vector pCAMBIA3301 with SOS genes （SOS1,SOS2 and SOS3） and bar gene.Selection culture was conducted using 0.3 mg L^-1 phosphinothricin （PPT）.A total of 40 plants were produced from the inoculated 170 cell aggregates via somatic embryogenesis.PCR analysis showed that 37 of the 40 regenerated plants were transgenic plants.The in vitro assay demonstrated that superoxide dismutase （SOD） and proline were significantly more accumulated and malonaldehyde （MDA） was significantly less accumulated in 21 transgenic plants than in control plants when they were exposed to 86 mmol L^-1 NaCl.Salt tolerance of these 21 plants was further evaluated with Hoagland solution containing 0,51,86,and 120 mmol L^-1 NaCl in the greenhouse.The results indicated that 6 of them had significantly better growth and rooting ability than the remaining 15 transgenic plants and control plants.Expression of SOS genes in the 6 salt-tolerant transgenic plants was demonstrated by RT-PCR analysis.This study provides an alternative approach for improving salt tolerance of sweetpotato.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.

Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in <i>Artemisia annua</i>L., as Reported by a Promoter-GUS Fusion

Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.

Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud

We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0 - 1.5mm in size, C58cl of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production, shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptll ELISA (nptII enzyme assay), PCR, Southern blot and leaf bleach, 29 positive plants were identified from two genotypes of Bobwhite and Yangmai 10, with an average transformation efficiency of 0.82%. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.

Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation

In order to improve stress tolerances of turf-type tall fescue （Festuca arundinacea Schreb.）, Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses, and that relative electronic conductivity of in vitro leaves treated with low and high temoeratures, dehvdration and high salinity stresses was 25-30% lower in transgenic plants than in control plants.In addition,it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato （cv. Lizixiang） was achieved using Oryzacystatin-I （OCI） gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Low Co-Cultivation Temperature at 20°C Resulted in the Reproducible Maximum Increase in Both the Fresh Weight Yield and Stable Expression of GUS Activity after <i>Agrobacterium tumefaciens</i>-Mediated Transformation of Tobacco Leaf Disks

The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.

Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.