1.Introduction

Agrobacterium rhizogenes contains a root-inducing(Ri)plasmid that contains root locus(rol)genes in the T-DNA region,including rolA,rolB,rolC,and rolD,and is able to induce hairy roots from the wounded surface of explants after infection[1].The hairy roots can be maintained in culture or hosted in composite plants with untransformed aerial tissue.Agrobacterium rhizogenes-mediated transformation has been widely used in many plants,such as Glycine max[2],Capsicum annuum[3],Lotus corniculatus[4],Prunus[5],Pisum sativum[6],and Catharanthus roseus[7].

2018年中国皮卡市场增势迅猛。1-10月，全国皮卡车企累计销售36万 辆皮卡，累比增长13.20%，其销量增长率已经超越所有车型和整体车市的增长。面对皮卡行业的春天，郑州日产适时推出了“高品质SUV级皮卡”锐骐6。作为一款全新皮卡车型，锐骐6如何能赢得消费者的认可？

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Soybean(Glycine max(L.)Merr.)is one of the most important crops and has high oil and protein contents.With the development of biotechnology,advances in breeding,functional research and targeted genetic modifications have become essential for studying soybean.Thus,efficient transformation systems are required to advance soybean research.At present,Agrobacterium tumefaciens-mediated transformation and biolistic methods are the methods most frequently used for soybean transformation[8,9].However,these techniques are too inefficient and labor-intensive to meet the increased demands of research[2].

Several successful soybean studies have been performed using Agrobacterium rhizogenes and taking advantage of the hairy root system.For gene function studies,overexpression of GmACSL2(long-chain acyl-CoA synthetase 2)in soybean hairy roots was observed to reduce lipid and fatty acid content,suggesting that GmACSL2 is an important enzyme that catalyzes the five fatty acids(C16:0,C18:0,C18:1,C18:2,and C18:3 fatty acids)to form acyl-coenzymes [10].Overexpression of TaNHX2(Na+/H+antiporter 2)in hairy roots improves the salinity tolerance of transgenic roots.Under salt stress,a general growth inhibition in hairy roots was observed,but hairy roots transformed with the control vector without TaNHX2 showed much less growth(on a dry-weight basis)than transgenic hairy roots overexpressing TaNHX2[2].For promoter studies,the specificity of the soybean root promoter could be used to detect expression in hairy roots.Using this method,activities,enhancers,repressors,and the core region of the promoter could be easily observed in the hairy root system[11,12].The soybean hairy root system was also used to test the expression efficiency of an RNAi vector and was successfully applied to the CRISPR/Cas9 system[13–15].

The protocol described in this study is a rapid and efficient approach for inducing soybean hairy roots in vitro.The technology is more practical than other methods,owing to its high productivity and low cost.Transgenic hairy roots canbe obtained in a short period of time(approximately 3–4 weeks)and can be directly used for research.In comparison,the Agrobacterium tumefaciens-mediated transformation approach typically requires up to 3–4 months to obtain a T0 plant and the T1 transgenic plants needs to be harvested for research[2].

The seeds of five soybean cultivars were used in this protocol for transformation.All tested cultivars were able to produce hairy roots,and the proportion of hairy roots generated was between 90%–99%.Zigongdongdou had the highest generation proportion of almost 99%,followed by Jack and Williams 82 with>95%and Heihe 27 and Zhonghuang 30 with>90%(Table 1).The positive proportion of the hairy roots was between 30%–60%.The highest positive proportion forhairy roots was 58%in Zigongdongdou,and the lowest was 35%in Zhonghuang 30(Table 1).

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2.Materials and methods

2.1.Plant materials

According to reporter gene in the designed vector,any reporter gene can be used to detect transgenic hairy roots.GUS can be assessed by GUS staining,whereas GFP and DsRed2 can be assessed by fluorescence.Hairy roots were directly screened using a dissecting fluorescence microscope(Nikon SMZ1500),and transgenic hairy roots showed GFP or DsRed2 fluorescence labeling.Histochemical GUS assays were performed following Jefferson[18].The hairy roots were placed in GUS staining solution(50 mmol L−1 sodium phosphate,pH 7.0,0.5 mmol L−1 potassium ferrocyanide,0.5 mmol L−1 potassium ferricyanide, 0.5 mg mL−1 5-bromo-4-chloro-3-indolyl-β-D-glucuronide(X-Gluc),0.1%Triton X-100,and 20%methanol)and incubated at 37°C overnight.GUS staining was observed under a Nikon SMZ1500 microscope and photographed with a Nikon DS-Fil camera.

2.2.Plasmid construction

In addition to the desired transgenic construct,plasmids for the transformation of soybean require a linked reporter gene marker.We designed a plasmid with three reporter genes(GUS,GFP,and DsRed2)under the constitutive promoter CaMV 35S for rapidly checking for positive hairy roots.

2.3.Strain

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2.4.Preparation of culture medium

The media used in this protocol were(1)solidified YEP medium,composed of 10 g L−1 tryptone,5 g L−1 yeast extract,5 g L−1 NaCl,and 15 g L−1 agar(pH 7.0);(2)liquid YEP medium,composed of 10 g L−1 tryptone,5 g L−1 yeast extract,and 5 g L−1 NaCl(pH 7.0);(3)MS liquid medium,composed of 4.33 g L−1 MURASHIGE&SKOOG BASAL SALT MIXTURE(PhytoTechnology,M524),30 g L−1 sucrose (pH 5.8)and 1 mL L−1 of MURASHIGE&SKOOG VITAMIN SOLUTION(PhytoTechnology,M533);(4)germination culture medium(GCM),composed of 3.1 g L−1 GAMBORGS BASAL SALT MIXTURE(PhytoTechnology,G768),20 g L−1 sucrose,7 g L−1 agar(pH 5.8),and 1 mL L−1 of GAMBORGS VITAMIN SOLUTION(PhytoTechnology,G249);(5)co-cultivation culture medium(CCM),composed of 0.433 g L−1 MURASHIGE&SKOOG BASAL SALT MIXTURE,30 g L−1 sucrose,3.9 g L−1 MES(Sigma,M3671),7 g L−1 agar(pH 5.4),1 mL L−1 of MURASHIGE&SKOOG VITAMIN SOLUTION,150 mg L−1 DTT(Sigma,D5545),and 0.02 g L−1 AS(Sigma,D134406);(6)washing culture medium(WCM),composed of 2.165 g L−1 MURASHIGE&SKOOG BASAL SALT MIXTURE,30 g L−1 sucrose(pH 5.8),1 mL L−1 of MURASHIGE&SKOOG VITAMIN SOLUTION,250 mg L−1 cefotaxime sodium salt(Sigma,C7039),and 250 mg L−1 carbenicillin disodium salt(INALCO,1758–9317);(7)induction culture medium(ICM),composed of 2.165 g L−1 MURASHIGE&SKOOG BASAL SALT MIXTURE,30 g L−1 sucrose,0.6 g L−1 MES,7 g L−1 agar (pH 5.8),1 mL L−1 of MURASHIGE&SKOOG VITAMIN SOLUTION,250 mg L−1 cefotaxime sodium salt,and 250 mg L−1 carbenicillin disodium salt;and(8)callus induction culture medium(CICM),composed of 2.165 g L−1 MURASHIGE&SKOOG BASAL SALT MIXTURE,30 g L−1 sucrose,0.6 g L−1 MES,7 g L−1 agar(pH 5.8),1 mL L−1 of MURASHIGE&SKOOG VITAMIN SOLUTION,1 mg L−1 2,4-D(Sigma,D7299),0.2 mg L−1 6-BA(Sigma,B3408),250 mg L−1 cefotaxime sodium salt,and 250 mg L−1 carbenicillin disodium salt.All media were autoclaved at 121°C for 15 min.

2.5.Detection of transgenic hairy roots

Five soybean cultivars(Williams 82,Jack,Zigongdongdou,Heihe 27,and Zhonghuang 30)were used for Agrobacterium rhizogenes-mediated transformation.

3.Results

An overview of the protocol is shown in Fig.1.The process had five primary working phases:(1)the selected soybean seeds were sterilized with chlorine gas for 16–20 h.(2)The sterilized seeds were planted onto GCM and cultured for 4–5 days,depending on genotype.A higher temperature(30°C)will accelerate the germination process,so that a short germination time is more appropriate.During this time,the Agrobacterium strain was prepared.(3)Cotyledons with the wounded points of attachment of the cotyledon and hypocotyls were immersed in a suspension of the Agrobacterium strain for 30 min.(4)The explants were transferred to CCM and cultured for 5 days.(5)After rinsing in WCM,the explants were transferred to ICM.Hairy roots formed in 10–12 days in ICM.

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Fig.1–Schematic illustration of the protocol for soybean hairy root induction.The phase column includes sterilization,germation,infection,co-cultivation,and induction.GCM,germination culture medium;CCM,co-cultivation culture medium;ICM,induction culture medium.

3.1.Sterilization of soybean seeds and germination

Five-day-old seedlings were used for infection experiments.Cotyledons with 0.5-cm hypocotyls were cut from seedlings(Fig.4-a,b).Two methods may be used to divide the pair of cotyledons into individual ones.One is to cut from cotyledon to hypocotyls(Fig.4-c)and the other is to cut from hypocotyls to cotyledon(Fig.4-d).The pair of cotyledons is divided into single ones(Fig.4-e),and the points of attachment of the cotyledon and hypocotyls are wounded with a blade(Fig.4-f)previously dipped into the A.rhizogenes K599 strain prepared earlier,such that one cotyledon has seven or eight scars(Fig.4-g).The cotyledons are then immersed in an A.rhizogenes culture and shaken at 50 r min−1 at 28 °C for 30 min(Fig.4-h).During seed germination,CCM is prepared.

3.2.Preparation of the Agrobacterium strain for infection

During seed germination,the YEP plates and YEP liquid medium were prepared.Bacteria(from glycerol stock)were streaked onto the surface of the YEP plates containing appropriate antibiotics(Fig.3-a)and the plates were incubated at 28 °C for 2 days(Fig.3-b–d).Next,a single colony was placed into a 20 mL of fresh YEP liquid containing appropriate antibiotics,and incubated at 28°C overnight with shaking at 200 r min−1.The Agrobacterium strain used for infection can be prepared using either of the two methods described below.One method is to use the YEP liquid directly for immersion after incubation.When the OD600 of the Agrobacterium culture reaches 0.6,the YEP liquid can be used directly for immersion(Fig.3-e).An alternative method is to resuspend the bacteria in MS liquid medium.When the OD600 of the Agrobacterium culture reaches 1.2,the bacteria are collected by centrifugation at 5000 r min−1 for 10 min and resuspended in MS liquid medium at an OD600 of 0.6 for the immersion step(Fig.3-f).

3.3.Preparation of explants and infection

Healthy seeds,typically with smooth surfaces and without disease lesions,were carefully selected and placed side by side in Petri dishes.The open Petri dishes were placed in a vacuum desiccator in a fume hood(Fig.2-a,b).A 100 mL volume of 10%sodium hypochlorite was placed in a 100-mL beaker in the desiccator,after which 4 mL of 12 mol L−1 HCl was slowly added and the desiccator was immediately covered with a lid and sealed with Vaseline.The seeds were exposed to the chlorine gas for 16–20 h(Fig.2-c,d).The time for seed sterilization should not exceed 20 h,as a long sterilization time will result in poor germination.After sterilization,the Petri dishes were removed.During sterilization,the germination culture medium was prepared.The sterilized seeds were cultivated in GCM,with each dish typically containing 20 seeds(Fig.2-e).The seeds were grown in a growth chamber at 28°C for 5 days(Fig.2-f).The entire cotyledons from 5-day-old seedlings were then harvested as explants.

Fig.2–Sterilization of soybean seeds and germination.Healthy seeds were carefully selected and arranged side by side in Petri dishes(a),the open Petri dishes were placed in a vacuum desiccator in the fume hood(b),a 100-mL beaker was placed in the desiccator,and 100 mL of 10%sodium hypochlorite and 4 mL of 12 mol L−1HCl were added(c),the seeds were kept in the chlorine gas for 16–20 h(d),the sterilized seeds were cultivated in GCM(e),and the 5-day-old seedlings were harvested for the explants(f).GCM:germination culture medium.Scale bar,10 mm.

Fig.3 –Preparation of the Agrobacterium strain for infection.Bacteria stored in glycerol at−80 °C(a),inoculating loop soaked bacteria(b),streaking culture on the YEP plates containing the appropriate antibiotics(c),incubation at 28°C for 2 days(d),direct use of YEP liquid for immersion when the Agrobacterium strain reaches an OD600of 0.6(e),and resuspension of the bacterial strain in MS liquid medium at an OD600of 0.6(f).

3.4.Co-cultivation

After infection,the explants were dried on sterile filter paper and then transferred to CCM covered with sterile filter paper and incubated under 16 h light/8 h dark at 22°C(Fig.5-a).During 5 days of co-cultivation,the cotyledons expanded and the Agrobacterium adhered to the surface of the cotyledons(Fig.5-b).It can be seen that the cotyledon is markedly larger than before co-cultivation(Fig.5-c,d).If the cotyledons have not swelled after 5 days of co-cultivation,hairy roots will not emerge.This result may indicate that the Agrobacterium strain is incompetent and that the Agrobacterium will need to be reprepared for successful infection.During the co-cultivation,WCM and ICM were prepared.

3.5.Induction of hairy roots and detection

After co-cultivation,the Agrobacterium adhered to the surface of the cotyledons,so that the cotyledons needed to wash in WCM before being transferred to ICM.The cotyledons can be picked up with a triangular flask(Fig.6-a).Typically,the cotyledons were washed three times until WCM became clear(Fig.6-b),after which explants were dried on sterile filter paper.This step can prevent the Agrobacterium from regrowing during the inducing phase.The cleaned cotyledons were then transferred to ICM.The cotyledon was inserted into the culture medium at a 45°angle,with the abaxial surface downward.Each dish contained five cotyledons(Fig.6-c),and was incubated under a 16 h light/8 h dark at 28°C.The hypocotyls formed calluses during the inducing process(Fig.6-d,e).If a callus did not form,positive hairy roots were not produced.In the present study,hairy roots were produced within approximately 10–12 days and grew very quickly.Within 12–15 days,the hairy roots could spread throughout the culture medium(Fig.6-f).

Fig.4–Preparation of explants and infection.Cotyledons with long hypocotyls were cut from 5-day-old seedlings in the medium(a),cotyledons with 0.5 cm hypocotyls for explants(b),the pair of cotyledons were separated from cotyledon to hypocotyls(c),the pair of cotyledons were separated from hypocotyls to cotyledon(d),the pair of cotyledons was divided into two individual cotyledons(e),the attachment points of the cotyledon and hypocotyls were wounded with a blade(f),one cotyledon has seven or eight scars(g),and the cotyledons were immersed into an A.rhizogenes culture and shaken at 50 r min−1at 28 °C for 30 min(h).Scale bar,10 mm.

Fig.5–Explants at the co-cultivation stage.After infection,the explants were dried on sterile filter paper and transferred to CCM covered with sterile filter paper,then incubated in a 16 h light/8 h dark cycle at 22°C(a),5 days after co-cultivation,the cotyledons were expanded and the Agrobacterium adhered to the surface of the cotyledons(b),the cotyledon was co-cultivated for 1 day(c),and the cotyledon was co-cultivated for 5 days(d).CCM:co-cultivation culture medium.Scale bar,10 mm.

Typically,positive roots may be selected by GUS staining,GFP,and DsRed2(Fig.6-g–i).The positive roots can be used for analyzing the activity of reporter genes,assessing phenotypes,and/or conducting a preliminary study of gene function.

Fig.6–Induction of hairy root formation and detection.The cotyledons were washed in WCM(a),the cotyledons were washed three times until WCM became clear(b),the entire,washed cotyledons were transferred to induction culture medium,with each dish containing five cotyledons(c),the hypocotyls formed a callus in the induction process(d,e),the hairy roots were produced and spread onto the whole culture medium(f),positive roots were selected by GUS staining or GFP or DsRed2(g–i).WCM:washing culture medium.Scale bar,10 mm.

3.6.Analysis of transgenic efficiency

In this report,the process for producing soybean hairy roots is described and illustrated.Using this method,90%–99% of the infected explants of five different cultivars produced hairy roots within one month,and 30%–60%of the hairy roots induced were transformed.In addition,the formation of calluses from hairy roots can be successfully induced.An efficient in vitro hairy root system was established,it would be an efficient and rapid platform for study of soybean gene function.

The plasmid vector with the three reporter genes(GUS,GFP,and DsRed2)was mobilized into Agrobacterium rhizogenes K599 via electroporation for use in later soybean infection.

3.7.Calluses induced by hairy roots

The positive hairy roots were used for callus induction by transfer to CICM.Hairy roots need to be transferred to new callus-inducing medium every 2 weeks,and the callus will produce for approximately 30 days.The callus can be used to test the integration of transgenes.Three reporter genes were detected in callus from positive hairy roots(Fig.7).Callus can be continuously conserved in the culture medium for reproduction and shoot induction research.

4.Discussion

Compared with A.tumefaciens-mediated transformation of soybean,the A.rhizogenes-mediated hairy root transformation system has a high transformation efficiency and short transformation period.The procedure can be completed within one month.Hairy roots are usually non-chimeric,because they are derived from single cells and each hairy root consists of uniformly transformed cells[16].Furthermore,hairy roots can grow without exogenous hormones[17].

Table 1–The generation rate of hairy roots and the positive rate of hairy roots.

For each cultivar,60 seeds were used for germination and 80–100 explants from the germinating seeds were excised for transformation.Experiments were repeated in triplicate. 1 Number of explants producing hairy roots/total number of explants×100%. 2 Number of positive hairy roots/total number of hairy roots examined×100%.

Genotype Williams 82 Jack Zigongdongdou Heihe 27 Zhonghuang 30 Proportion of explants producing hairy roots(%)1 97±1 95±1 99±1 92±1 91±1 Proportion of positive hairy roots(%)2 55±2 46±4 58±2 43±3 35±2

The hairy root system could be an excellent system for molecular studies and genetic Engineering in which the production of transgenic plants is not necessary[19],such as the production of recombinant proteins and secondary metabolites[20–22],metabolic engineering and quick functional analyses of genes[23–25],rhizosphere physiology,and biochemistry[26].Recombinant protein production in the hairy root system has proved to be a rapid,low-cost,and reliable method for the production of valuable proteins.Metabolic engineering can elucidate the biosynthetic pathway for phytochemicals in hairy root cultures.Genetic studies can be performed with the hair root system,including foreign gene expression and gene function analyses.Root physiology studies ranging from nitrogen fixation,iron-deficiency,aluminum toxicity,and host-pathogen interactions can be conducted in hairy root cultures[27,28].

Fig.7 –Reporter gene detection in callus from hairy roots.GUS detection(a–d),callus from hairy roots with the GUS reporter gene(a)and control(b).(c)and(d)show GUS staining of(a)and(b),respectively;GFP detection(e–h),callus from hairy roots with the GFP reporter gene(e)and control(f).Calluses in(g)and(h)were placed under fluorescent light to detect GFP for(e)and(f),respectively;DsRed2 detection(i–l),callus from hairy roots with the DsRed2 reporter gene(i)and control(j).Calluses in(k)and(l)were placed under red fluorescent light to detect DsRed2 for(i)and(j),respectively.Scale bar,5 mm.

Kereszt et al.[29]reported a protocol for Agrobacterium rhizogenes-mediated transformation of soybean that primarily focused on the formation of composite plants by A.rhizogenes in field cultivation with hairy roots produced by hypocotyls.In our protocol,the hairy roots are generated from cotyledons and are completely grown in vitro,which can be controlled more easily than in an open condition.Thus,the experiments can be conducted in a relatively stable environment.Mohammadi-Dehcheshmeh et al.[30]have modified Kereszt's protocol by combining both in vitro and in vivo strategies for hairy root transformation,and the transformation frequency is greatly improved compared with Kereszt's protocol.Our protocol requires only preparation of media using common reagents.

The transgenic hairy roots derived from cotyledons in our protocol can be used in rootbiology and gene function studies but are notsuitable for rootnodule studies.The transgenic hairy roots derived from hypocotyls with aerial shoots represent a better system for root nodule and nitrogen fixation studies.

The transgenic hairy roots can be successfully induced to form calluses,and three reporter genes were detected in calluses induced from soybean transgenic hairy root.The callus generated using our method can support a continuous subculture for long-term maintainance of the materials.Shoots can be generated from calluses in some plants,such as Codonopsis lanceolata[31],Rehmannia glutinosa[32],and Medicago truncatula[33].Unlike for other plant species,there has been no report to date of successful plant regeneration from soybean hairy roots,although the recovery of stable soybean transgenic plants from primary-node explants infected by a disarmed A.rhizogenes strain has been reported[34].If this difficulty is solved,the hairy root system will represent a fast and efficient technology for obtaining transgenic soybean plants.

5.Conclusions

This report describes a detailed protocol for A.rhizogenes transformation of soybean.The present protocol has a high transformation efficiency.On average,90%–99% of A.rhizogenes-infected cotyledons generated hairy roots,and they were produced in all soybean cultivars tested.The positive proportion of hairy root formation,as assessed by detecting the reporter gene,reached 30%–60%.The procedure is rapid and simple.Using this method,hairy roots can be obtained within one month and could be used for gene function studies.Several independent transformation events can be obtained by this method,given that every transgenic root originates from a single cell and represents an independent transformation event.The hairy roots can be used to successfully induce calluses.This protocol may become a powerful tool for soybean genetic engineering.

一辆东风卡车开着近射灯徐徐驶过拐弯处，柏河和徐北风兔子一般窜出来，奔向停在拐弯公路一侧支路上的三轮摩托，迅速发动，打开闪烁的警报灯，朝大卡追去。

This work was supported by the Major Science and Technology Projects of China(2016ZX08010-004),the Ministry of Science and Technology of China(2016YFD0100504)and the CAAS(Chinese Academy of Agriculture Sciences)Innovation Project.

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