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.

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.

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.

Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi

The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials. Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites. The regenerated plants from hairy root were characterized by normal leaf morphology and stem growth but a shallow and more extensive root system than normal plants. Opine synthesis, PCR and Southern blot confirmed that T- DNA had been integrated into the A. pseudoalhagi genome. Acetosyringone (AS) was found to be vital for successful transformation of A. pseudoalhagi.

Hairy Root Cultures and Plant Regeneration in <i>Solidago nemoralis</i>Transformed with <i>Agrobacterium rhizogenes</i>

By screening a native plant extract library we identified Solidago nemoralis as a novel source of agonists for alpha7 nicotinic receptors for acetylcholine with therapeutic potential. The next phase of our drug discovery strategy is to increase the yields of active compounds in the plant species by gain of function mutations in hairy root cultures [1]. Here we report a protocol for Agrobacterium rhizogenes-mediated genetic transformation of hairy root cultures of Solidago nemoralis which will enable this. Leaf explants of this species were successfully transformed with a frequency of 30%-35% using A. rhizogenes strain R1000 harboring the binary vector pCambia 1301. Transformation was confirmed using the β-glucuronidase (GUS) histochemical assay. Transformed hairy roots showed spontaneous regeneration of adventitious shoots in media without the addition of cytokines, albeit at very low frequency. However, media supplementation with auxin (α-naphthaleneacetic acid, NAA) increased shoot regeneration frequency to 35% and resulted in viable adventitious shoots. Transformation was confirmed at all phases of plant regeneration by GUS staining. Hairy root transformation of Solidago altissima has been previously reported, but this is the first report of genetic transformation of S. nemoralis. The protocol will allow for a large population of activation tagged mutants of S. nemoralis to be generated which will be then screened for the presence of stable mutants which are over-producing metabolites with activity at alpha7 nicotinic receptors. These over-producing mutant cultures will then be regenerated into intact mutant plants.

Rapid,Efficient and High-Performance Protocol for Agrobacterium rhizogenes-Mediated Hairy Root Transformation of the Common Bean Phaseolus vulgaris

A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizogenes was developed for the common bean Phaseolus vulgaris. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly efficient procedure for achieving hairy root transformation frequencies as high as 100% as visualized by GUS reporter gene expression system. Transgenic hairy roots in these young composite plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput genomic analysis to study root biology and endosymbiosis in common bean.

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.

Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae

Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection.Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease.The traditional way of R gene functional validation requires stable transformation that is both time-and labor-consuming.In this study,a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed.The transformation positive rate was over 80%in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation.The system was applicable to different B.napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea.In particular,two known CR genes,CRA3.7.1 and CRA8.2.4 were used respectively,as example to show that the system works well for CR gene study combined with subsequent P.brassicae infection in B.napus.Most importantly,it works both in over-expression that led to disease resistance,as well as in RNAi which led to disease susceptible phenotype.Therefore,this system can be used in batch-wise identification of CR genes,and also offered the possibility of manipulating key genes within the P.brassicae genome that could improve our knowledge on host-pathogen interaction.

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth; typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT; 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.

黄芩毛状根的诱导及响应面法优化培养条件

[目的]应用响应面法优化黄芩毛状根的培养条件。[方法]利用发根农杆菌1.2556感染黄芩获得毛状根,在单因素试验的基础上,利用Box-Benhnkcn设计和响应面分析法对影响黄芩毛状根生长的培养条件(培养时间、温度、摇床转速和接种量)进行分析,优选最佳培养条件。[结果]毛状根在无外源激素的MS培养基上自主生长,表现出典型的发根特征。毛状根的最优培养条件为:培养时间24.95 d、温度27.4℃、摇床转速123.69 r/min、接种量0.500 g;在此条件下,黄芩毛状根的生长量为(fm)9.271 28 g/瓶。[结论]该研究优化了黄芩毛状根的培养条件,为黄芩的进一步开发利用提供了依据。

土壤杆菌固氮、溶磷活性及对植物的促生作用

通过选择性培养基,定性和定量检测了26株土壤杆菌的固氮和溶磷活性,通过盆栽检测了澳洲生防菌株K1026对黄瓜和小麦的促生长作用。结果显示,包括致病性和无致病性的26株土壤杆菌均可在无氮培养基上正常生长;其中19株具有溶磷活性,K1026对磷酸钙液体培养基的溶磷量为109.11μg/m L;对黄瓜幼苗根系生长具有显著促进作用,但对小麦生长没有明显影响。

发根农杆菌介导的杜梨毛状根遗传转化方法

以杜梨(Pyrus betulaefolia)为研究材料,利用发根农杆菌(Agrobacterium rhizogenes)开发了一种简单、快速、高效的杜梨毛状根遗传转化方法。将含有mCherry基因的表达载体pROK2导入发根农杆菌K599,侵染杜梨幼苗,通过注射烟草叶片、常规PCR及体视显微镜观察,检测转化效率。结果表明,利用侵染烟草叶片瞬时表达的方法,证明mCherry可以正常表达;发根农杆菌侵染40株杜梨后,再生毛状根植株为34株,再生率达85%,通过PCR鉴定,转基因毛状根长势良好且鉴定为阳性的植株有6株,阳性率达到17.6%;经体视显微镜观察,可以在转基因根中直接观察到mCherry的红色荧光。通过发根农杆菌直接侵染杜梨苗的方法,成功实现了外源基因在杜梨新生不定根中的表达,操作简便,转化率高,周期较短,为梨树基因功能研究和遗传改良提供了新型工具,为提高梨树遗传转化成功率奠定了基础。

发根土壤杆菌介导的大豆发根转化体系改进

鉴于大豆难以获得转基因植株的现实,借鉴Kereszt等建立的发根土壤杆菌(Agrobacterium rhizogenes)K599介导的大豆转化体系,并针对实验中遇到的具体问题,对原体系进行了适当改进。结果表明:改进的体系操作更简单,对发根培养条件、人员操作技能要求更低,所形成的植株更具生命力。同时,应用改进体系以及RNAi技术,构建了表达线虫特异基因dsRNA的转基因发根植株,可在60 d内在转基因大豆发根上对植物线虫基因的RNAi效果完成验证。改进的体系为培育抗大豆胞囊线虫品种奠定了基础。

Introduction of rol Genes into Cotton (Gossypium hirsutum L.) Genome and Effects of Transgene Expression on the Plant Development

The rol genes cloned from Agrobacterium rhizogenes were transferred to the cotton genomevia Agrobacterium-mediated transformation. Molecular analyses and developmentalidentification of the putative transgenic plants were carried out by means of PCR,Southern blotting and field characterization. The results showed that the expression ofrol genes greatly increased the rooting ability of the transgenic plants, and changed theplant development. Highly male-sterile plants with strong apical dominance and fertileplants with short internodes, stunted growth and improved economic characteristics weresegregated from the T1 transgenic lines of wild rol B gene and the rol B gene driven by35S promoter. The transgenic lines of rol ABC construct usually had normal boll settingand slow growth. Therefore we concluded that the rol genes, modified in suitable ways,could be used to create new cotton varieties with some highly valuable characteristics.

Transformation of Ginkgo Hairy Root and Establishment of Its Suspension Culture Clone

Tender leaves,buds and stems of ginkgo biloba L .were transformed successfully by Agrobacterium rhizogenes with Ri plasmid,and its suspension cultured clone was eastablished.The result of detection of opines suggested that A.rhizogenes had integrated the T DNA of the Ri plasmid into genomic DNA of ginkgo cells.A new way is offered to exploit ginkgo resources with biological technology.

Optimizing Culture System of Ri T-DNA Transformed Roots for Citrus grandis cv. Changshou Shatian You

Genetic transformation experiments of the different explants from Citrus grandis cv. Changshou Shatian You infected with Agrobacterium rhizogenes were carried out in darkness or in light. The optimizing culture system of Ri T-DNA transformed roots for C. grandis cv. Changshou Shatian You was constructed as follows： After the ventral wounded striations on the single activation cotyledon were inoculated by A. rhizogenes A4 （logarithmic period）, they were cocultured at （25 ±2）℃ in darkness for 25-30 days; some transformed roots were generated from wounded striations of most cotyledons. The genetically transformed ratio is （83 ± 11）%. Axenic Ri T-DNA transformed roots （hairy roots） were harvested after five subcultures. Explants were activated on MT medium. The MS medium was used for subculture of transformed roots. Mass Ri T-DNA transformed roots in which the hormone was produced independently were harvested from this optimizing culture system. White, fresh Ri T-DNA transformed roots were （1.14 ±0.07） cm long, （0.73 ±0.04） mm wide, and the growth direction of transformed roots was negative geotropism.

Hairy Root and Its Application in Plant Genetic Engineering

Agrobacterium rhizogenes Conn. causes hairy root disease In plants. Hairy root-Infected A. rhizogenes Is characterlzed by a high growth rate and genetic stability. Hairy root cultures have been proven to be an efficient means of producing secondary metabolites that are normally biosyntheslzed In roots of differentiated plants. Furthermore, a transgenlc root system offers tremendous potential for introducing additional genes along with the RI plasmld, especially with modified genes, into medicinal plant cells with A. rhizogenes vector systems. The cultures have turned out to be a valuable tool with which to study the biochemical properties and the gene expression profile of metabolic pathways. Moreover, the cultures can be used to elucidate the Intermediates and key enzymes Involved In the biosynthesis of secondary metabolites. The present article discusses various appllcations of hairy root cultures in plant genetic engineering and potential problems aseoclsted with them.

Establishment of Withania somnifera Hairy Root Cultures for the Production of Withanolide A

Withania sominifera （Indian ginseng） was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy （transformed） roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptll and rolB specific primers and, subsequently, by Southern analysis for the presence of nptll and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated （SH, LS and N6）, with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots （157.4 μg/g dry weight） was 2.7-fold more than in non-transformed cultured roots （57.9 μg/g dry weight）.

INFECTION OF Ri T-DNA-TRANSFORMED ROOTS BY Plasmodiophora brassicae IN Brassica CROPS

Many Ri T-DNA-transformed roots were incited on seedling hypocotyl segments of cabbage, cauliflower and oilseed rape following genetic transformation with Agribacterium rhi-zogencs. The transformed roots were in vitro inoculated with the resting spores of Plasmodi-ophora brassicae on solid medium, in liquid medium and semi-solid medium respectively.On solid medium, the resting spores penetrated root hair cells, but the root cortex was not invaded so that no symptom of clubroot galls was developed. In liquid medium, the root cortex was invaded, giving rise to an abnormal division of root cortical tissues and gall formation. In semi-solid medium, the resting spores penetrated the cells of the cut surfaces of root segments and caused the cut surfaces to swell.

Optimizing hairy root production from explants of Phyllanthus hainanensis,a shrub used for traditional herbal medicine

Phyllanthus hainanensis is a shrub that has been used in traditional herbal medicine.It has great pharmaceutical potential for treating diseases such as cancer and diabetes.As a prerequisite for propagation of this species on a large scale,hairy roots in P.hainanensis were induced using Rhizobium rhizogenes and various factors affecting hairy root induction and growth evaluated.Seven factors were tested:(1)type of explant,(2)type of culture medium,(3)duration of pre-culture,(4)R.rhizogenes inoculum cell density,(5)duration of infection,(6)acetosyringone concentration in the culture medium,and(7)duration of incubation.The optimal protocol for hairy root induction and growth was:young shoots,pre-cultured in Y1 for 2 d,inoculated with R.rhizogenes broth with an OD600 of 0.6 for 20 min,and incubated for 3 d.Putative transgenic hairy roots were initially identified by morphology and then confirmed by polymerase chain reaction.Successful and optimal production of hairy roots is a critical prerequisite for industrial scale clonal propagation of P.hainanensis.Being able to cultivate the plant on a large scale will provide rapid and ready supply of the plant materials that can be used in herbal medicine and in scientific and industrial exploitation.