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CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Published: September 23, 2021 doi: 10.3791/62304
* These authors contributed equally


Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.


An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.


Transformation is a basic tool to evaluate the expression of exogenous genes1,2. Many biological aspects of resource plants are common to all plants; therefore, functional studies of certain genes canbe carried out in model plants (such as Arabidopsis)3. Yet, many genes in plants are unique in their function and expression patterns, requiring studies in their own or closely related species, especially for resource plants3,4. Plant cells can sense various signals that enable plants to show specific changes in gene expression, metabolism, and physiology in response to different environmental stress conditions5,6,7. Flavonoids are crucial players in the signaling process of plants that is responsive to environmental stresses5,8,9. In addition, the flavonoid content in horticultural and medicinal plants is also an important indicator for quality evaluation10. Identification of genes involved in the regulation of flavonoid synthesis in response to external signals is crucial for understanding the mechanism of flavonoid synthesis in plants. Several studies have revealed that the application of exogenous hormones can promote the accumulation of flavonoids6,11. A stable transformation system and gene function validation method are essential to demonstrate the function of genes and to understand secondary metabolism in plants.

Agrobacterium-mediated transformation is widely used in DNA insertion5,8,9. Agrobacterium tumefacient can transfer ring genes into the chromosomes of plant cells, and exogenous phytohormones induce single or a few host cells that can regenerate plants to obtain stable transformants12,13,14. Agrobacterium tumefacient-mediated transformation methods are more applicable to plant species suitable for in vitro manipulation, while most perennial woody plants limit the application of this method because of their regeneration difficulty4,15. A. rhizogenes is also able to modify the genome of host cells16. In the present study, we have developed an efficient and stable A. rhizogenes-mediated transformation procedure. A. rhizogenes contains a second binary plasmid carrying non-natural gene T-DNA in addition to the Ri plasmid. The host plant is infected, and a composite plant can be obtained with transgenic hairy roots emerging from the wild-type shoot16,17. The A. rhizogenes-mediated transformation systems are suitable for application in woody plant research due to their fast, low cost and non-required plant regeneration. More than 160 kindsof plants have successfully induced hairy roots, and most of which are in Solanaceae, Compositae, Cruciferae, Convolvulaceae, Umbelliferae, Leguminosae, Caryophyllaceae, and Polygonaceae18,19. Compared with A. tumefaciens, A. rhizogenes showed higher efficiency in the mediated transformation of pigeon pea17,20.

In this study, pigeon pea was used as an example to introduce the transformation process mediated by A. rhizogenes. From inoculation to rooting, the experiments lasted for 5 weeks. We identified the transformation of the adventitious root through morphology and the GFP reporter gene, and the transformation efficiency was as high as 75%. Also, we treated the composite plant with JA and ABA, as well as detected transcripts and secondary metabolites by quantitative real-PCR and HPLC (high performance liquid chromatography). It is confirmed that the expression level of CcCIPK14 responds not only to JA and ABA but also affects the biosynthesis of flavonoids. This system is adequate for studying function genes associated with secondary metabolism. It also provides a new approach to studying non-model plants in lack of a sufficient stable transformation system17,21,22.

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NOTE: Pigeon pea is a diploid legume crop that belongs to the family Fabaceae. The pigeon pea seeds used in this experiment are from the Northeast Forestry University of China and are coded 87119. The primary steps of this protocol are illustrated in Figure 1A. The seedling incubation was performed in a high humidity environment at 25 °C under fluorescent lights at 50 µmol photons per m-2s-1 in a 16 h photoperiod. A. rhizogenes strains K599 (NCPPB2659) were preserved in the laboratory. They were stored in yeast mannitol medium (YEP) with 15% glycerol at -80 °C. The protocol described in this work was based on the protocol Meng et al.21.

CAUTION: Deposit all the genetically modified bacteria and plants into the appropriate waste container. Use all hazardous chemicals in a fume hood and dispose of them in the hazardous waste container.

1. Preparation of pigeon pea seedlings

  1. Choose plump and undamaged pigeon pea seeds (Figure 1A) that have been stored for less than 1 year.
  2. Soak the seeds in distilled water for 24 h (Figure 1B). Transfer the swollen seeds to a seed tray and place them in the greenhouse.
  3. Seeds start to germinate after 1-2 days. When the epicotyl reaches 1.5 cm, transplant the seedlings to 10 cm (diameter) x 9 cm (height) pots containing soil and sand in a volume ratio of 3:1.
    NOTE:The soil consists of a mixture of nutritious soil, vermiculite, and perlite at a 2:1:1 ratio.
  4. Grow the seedling in a greenhouse.

2. Activation of A. rhizogenes

NOTE: The strain used for A. rhizogenes transformation was the K599 preserved at -80 °C. The binary vector pROK2 (pBIN438;http://www.biovector.net/product/428388.html) contains green fluorescent protein (GFP) as an indicator gene and a kanamycin resistance gene as a selectable marker to transform A. rhizogenes.

  1. Thaw A. rhizogenes on ice.
  2. Dip the bacteria and line them evenly onto yeast mannitol medium (YEP) supplemented with 8 g/L of agar powder, 25 mg/L of rifampicin, and 25 mg/L of kanamycin (YRK, pH 7.0).
  3. Incubate at 28 °C for 16 h.
  4. Select monoclonal colonies. Culture them in a 50 mL tube containing 10 mL of YEB medium supplemented with 25 mg/L of rifampicin and 25 mg/L of kanamycin (YRK, pH 7.0). Place the centrifuge tube on a shaking incubator with a rotational radius of 10 cm, at 28 °C and 200 rpm for 16 h.

3. Plant transformation using A. rhizogenes

NOTE: Select healthy plants to infect A. rhizogenes using the following injection procedure. This procedure results in transformed hairy roots. To analyze the gene function of CcCIPK14,a control is needed. A. rhizogenes solutions with empty vector or CcCIPK14-pROK2 plasmids were injected into seedlings to induce hairy roots.

  1. Inoculate A. rhizogenes
    1. Choose pigeon pea seedlings with the same growth status.
    2. Empty the air from the 1 mL syringe and aspirate 0.3 mL of bacterial liquid. Slowly push the pushrod to fill the syringe needle with bacterial liquid21.
    3. First fix the stem of the pigeon pea seedling with tweezers, and then prick the syringe needle into the stem at 1 cm.
    4. When withdrawing the syringe, completely submerge the needle tip into the stem. Slowly push the pushrod to pump the bacterial liquid out of the penetrating wound.
      ​NOTE: The attachment of residual bacterial liquid to the wound in bacteria droplets can improve infection and transformation efficiency.
  2. Seedling management
    NOTE: High temperature and humidity can improve the infection efficiency of A. rhizogenes.
    1. Place seedlings inoculated with A. rhizogenes in a tray (30 cm x 60 cm x 6 cm) with 1 L of water. Use a transparent plastic lid (30 cm x 60 cm x 30 cm) as a cover to maintain the temperature and humidity of the internal environment.
    2. Periodically remove fallen leaves and flocculent hyphae attached to leaf tips and fallen leaves.
    3. After a week, the callus begins to appear in the wound. Subsequently, keep the the plastic lid half-open.
    4. After 4-5 weeks, most of the callus tissue differentiated into adventitious roots. Remove the plastic lid.
    5. Add 1 L of water to the tray every 3 days to keep the soil moist.
      ​NOTE: Pigeon pea is a drought-tolerant plant; too much water can inhibit plant growth.

4. Identification of transformed hairy roots

NOTE: Transformed hairy roots can be identified based on the morphology and gene level. This procedure primarily focuses on reporter gene (GFP) identification assay.

  1. Collect the root tips of the hairy root and mark the remaining part.
  2. Evaluate whether there is green fluorescence under a confocal laser scanning microscope.
  3. Triturate 0.1 g of hairy roots with strong fluorescence signal into fine powder in liquid nitrogen.
  4. According to the plant genomic DNA kit manufacturer's instructions, prepare the genomic DNA of pigeon pea independent transgenic lines by the modified cetyltrimethylammonium bromide (CTAB) method23.
  5. Use 500 ng of genomic DNA template and primers for PCR. The primers are shown in Table 1.
  6. Perform the following amplification cycle: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing of primers at 55 °C for 30 s, and primer extension at 72 °C for 30 s. After 36 cycles and a final extension of 10 min at 72 °C, analyze the amplified products on a 1% agarose gel.
  7. Stain the gels with nucleic acid staining and visualize them under UV light.

5. Exogenous hormone treatment

NOTE: The positive composite plants were treated with exogenous hormones to study the effect of CcCIPK14 on metabolic. The composited plants induced by A. rhizogenes were divided into three groups: JA treatment group, ABA treatment group, and control group (Figure 3A).

  1. Reduce watering 3 days before exogenous hormone treatment.
  2. Prepare both JA and ABA solutions at a concentration of 5 mg/L.
    NOTE: The JA and ABA powder are first dissolved in ethyl alcohol, and then filled to the target volume with distilled water.
  3. Using a spray bottle, spray JA and ABA solutions uniformly on the leaves of the plants. Treat the control group with water.
    ​NOTE: An average of 10 mL of solution was sprayed on each seedling.
  4. Cover with the plastic lid immediately after spraying treatment. Put it back in the artificial climate greenhouse.

6. Sample collection and preservation

NOTE: After 3 h of exogenous hormone treatment, plant materials from different treatment groups were collected.

  1. Remove tawny and contaminated hairy roots and select those with a white appearance. Collect these hairy roots and dry them out with absorbent paper.
  2. Divide the hairy roots collected from each seedling into two parts. Put one part into a numbered test tube and wrap it using marked tinfoil.
  3. Lyophilize the tinfoil in liquid nitrogen, and then store all harvested tin foil at -80 °C for further investigation.

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Representative Results

A. rhizogenes -mediated hairy root transformation on pigeon pea
This study described the step-by-step protocols for the genetic transformation of hairy roots mediated by A. rhizogenes, which has significance in the field of plant molecules. It took about 5 weeks to get hairy roots from the roots of pigeon pea infected by A. rhizogenes. Figure 1A showed an overview of the entire transformation process, from the injection of A. rhizogenes to obtain composite plants with hairy roots. Proliferating tissueswere observed around 1 week after infection, and its differentiation to adventitious roots was observed around 2 weeks, and a large number of hairy roots were produced at 35 days (Figure 1B). This GFP made it possible to distinguish genetically modified hairy roots from non-transgenic hairy roots through fluorescence. In Figure 2, the transformed hairy roots with GFP-pROK2 were visualized under the confocal green fluorescent protein channel. Induction of hairy roots by A. rhizogenes with the empty vector did not show green fluorescence (Figure 2A). The generated adventitious roots were identified by morphology and GFP reporter gene; the results confirmed that K599 could successfully infect and transform pigeon pea (Figure 2). The plant genomic DNA was prepared (CTAB method) for PCR analysis. The GFP was present only in hairy roots induced by A. rhizogenes carrying the GFP-pROK2 plasmid; neither the untransformed plant tissues nor the roots induced by A. rhizogenes with empty vectors were present (Figure 2B,C). These results indicated that the binary vector had been successfully transformed into the adventitious root genome16,21.

To improve the transformation efficiency of A. rhizogenes,the protocol was optimized. Infection withA. rhizogenes caused abnormal growth of normal roots and leaves of the host plant and even led to death. There are several possible explanations for this result. In the early vegetative growth stage, the spontaneous propagation and transformation of host cells consumed large amounts of nutrients, which maybe the main reason for inhibiting the development of seedling and even causing plant death24,25.Besides, the mechanical damage caused by inoculation may have also affected the seedlings24,26,27. Meanwhile, the growth status of seedlings determines the differentiation efficiency of callus. Inoculation is best done when the seedlings are 7 days old and the stems of seedlings are incompletely lignified (Figure 1A). Under this condition, the efficiency of A. rhizogenes-mediated callus production was 76%. The findings from these studies suggest that relative humidity can affect the proliferation of callus. The proliferation and differentiation of proliferating tissues and the growth of adventitious roots both need to maintain a high relative air humidity. Many roots were created within 4 to 5 weeks of inoculation and have begun to penetrate the soil. The overall transformation efficiency of this experiment can be as high as 72%.Transformation efficiency (%) = (number of positive compound plants / number of infected plants) x 100.

Evaluation of the CcCIPK14 gene function in pigeon pea using the transgenic hairy root system
Hairy roots of CcCIPK14-OE lines were obtained using the above-mentioned hairy root induction protocol. The hairy roots could attach to the infection points on the stems to form a composite plant. As only the hairy roots are genetically modified in composite plants, they can also be used to study signal transduction between the roots and the above-ground part. The phytohormone solutions (JA and ABA) were sprayed uniformly on the leaves of composite plants, and the hairy roots were collected after 3 h (Figure 3A).The function of CcCIPK14 in transgenic roots was verified by gene expression analysis and metabolite determination. The results showed that the expression level of CcCIPK14 was up-regulated after JA and ABA treatment. Besides, the expression levels of two key enzymes for Genistein synthesis in the CcCIPK14-OE line, CcHIDH1, and CcHIDH2 were up-regulated. Compared with the control group, the Genistein content in transgenic roots increased to 0.060 mg/g FW. We compared the phenotypes before and after the transgenic plants were treated with JA/ABA, and there was little difference in morphology through comparison (Figure 3B). CcCIPK14, a key functional gene in the flavonoid metabolic pathway, can respond to hormone signals and participate in genistein biosynthesis in pigeon pea.

Figure 1
Figure 1: Induction of the A. rhizogenes -mediated compound plant. (A) Flow chart of the hairy roots transformation system, including activating A. rhizogenes, seedling cultivation, Agrobacterium inoculation, seedling management, and obtaining compound plants. A representative image showing the key stages. (B) The three main stages of adventitious root formation at the inoculation site: callus appears, callus proliferates, and differentiates. Scale bars are 1 cm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Transgenic hairy root analysis. (A) GFP signal in transgenic hairy roots, with empty vector-containing Agrobacterium solution control. Scale bars are 50 µm. (B,C) PCR amplification of genes (GFP) from genomic DNA isolated from wild-type roots and stems, leaves, and transgenic roots of compound plants. CK: Empty Vector #1-4: plasmid (binary vector pROK2 carrying GFP) as the positive control. Please click here to view a larger version of this figure.

Figure 3
Figure 3: CIPK14 function analysis by hairy root method and hormone treatment. (A) Process flow chart for verification and analysis of gene function through compound plants. (B) Phenotypic changes of compound plants before and after treatment with JA, ABA, and H2O. Scale bars are 1 cm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Gene expression level and secondary metabolite content of hairy roots with empty vector and plasmid CcCIPK14-pROK2 (binary vector pROK2 with CcCIPK14). CcCIPK14 expression level (A), secondary metabolite content in transgenic hairy roots (B), and expression levels of CcHIDH1 and CcHIDH2 in hairy roots after hormone treatment (D). The relative expression level was normalized to that of the actin control. Data are presented as mean ± standard deviation (n = 3),* indicates a significant difference (P < 0.05) between CcCIPK14-OE and the controls (empty vector lines) using Student's t-test. (C) The biosynthesis pathway of Genistein. The enzyme catalyzing reaction HIDH, 2-hydroxyiso flavanone dehydratase, CIPK14 (Calcineurin B-like protein-interacting protein kinases). Treatments with the same letters were not significantly different based on the mean comparison by Duncan's test at p < 0.05. Please click here to view a larger version of this figure.

Primer Sequence(5'-3')

Table 1: Primers used in this study.

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The rapid characterization of gene function is the common goal in the study of most species, and it is particularly important for the development of resource plants. The A. rhizogenes-mediated transformation has been widely used in the hairy root culture. The hairy root culture (HRC), as a unique source of metabolite production, plays a pivotal role in metabolic engineering18,28. The application of this technology is mainly limited to the function of genes in vivo21. Here, we provide a basic method for studying gene function based on the previous A. rhizogenes -mediated transformation system. This method can be used to verify a variety of gene functions, such as response to environmental stress and exogenous hormones. The CcCIPK14-OE line showed that CcCIPK14 could promote the accumulation of flavonoid metabolites and respond to exogenous hormones JA and ABA. CcHIDH1 and CcHIDH2 are two essential flavonoid synthase genes, which were significantly up-regulated in the CcCIPK14-OE line. These results indicate that the method can be used as an effective tool for evaluating gene function and secondary metabolites.

An effective genetic transformation system is a prerequisite for the verification and analysis of gene function29,30. A. rhizogenes is a bacterium with a natural evolutionary mechanism31. It contains a root-inducing (Ri) plasmid that contains root locus (rol) genes in the T-DNA region, including rolA, rolB, rolC, and rolD, which can induce hairy roots from the wound surface of the explants27,32. The A. rhizogenes -mediated transformation system has many advantages33. First, in most species, Ri-transformed cells of the host plant can spontaneously differentiate into roots with normal phenotypes33. When additional binary vectors are used to integrate exogenous DNA, Ri plasmids can provide the possibility of obtaining transformed hairy roots without using exogenously applied plant hormones for organogenesis34. Second, Ri-transformed roots are genetically stable, because it is speculated that these transformed roots are developed from a single transformed cell17,33. Moreover, these hairy roots can maintain attachment to wild-type branches, resulting in composite plants16,20. Ri-transformed roots can simulate normal roots, providing experimental materials for verification and analysis of gene functions35. This transformation system is suitable for plants that are difficult to regenerate, such as pigeon pea, which has low regeneration frequency36,37. However, since only the hairy roots are transgenic, the function of genes on the entire plant level cannot be assessed, and transgenic traits also cannot be passed on to the offspring through sexual reproduction.

In this scheme, A. rhizogenes carrying binary vectors can induce transgenic hairy roots by an efficient and stable protocol. The basic steps of the transformation method have been outlined in the form of a flowchart (Figure 1A). After 1 week of inoculation with A. rhizogenes, calluses were observed on the wounds of the stem. After 3 weeks, the calluses differentiated to hairy roots (Figure 1B). This method is of significance because it does not require in vitro culture, hence hairy roots were obtained within 5 weeks (Figure 1). Meng et al. conducted a series of similar experiments with this method on different plants, and the results showed that most plants could obtain transgenic roots with the target gene within 2 months21. Pigeon pea, a protein-rich orphan, is growing in semi-arid tropics regions37. The hot and humid culture environment affects the growth of pigeon pea, and can occur deciduous phenotype21; the phenotype can be restored by removing the plastic cover.

During these experiments, many factors must be carefully considered to rapidly obtain transformed roots. The health status of the seedlings after inoculation is the key to the success of this experiment21. Previous studies have shown that A. rhizogenes can induce hairy roots at the inoculation point during multiple stages of seedling growth38. However, the growth status of seedings directly affects the transformation efficiency26. Premature inoculation of A. rhizogenes can inhibit the growth of seedlings and even causes seedling death39,40. To obtain maximum transformation efficiency, A. rhizogene is usually inoculated on 7-day-old seedlings. Besides, air humidity is also a major factor affecting callus proliferation. Premature removal of plastic after inoculation with A .rhizogenes can lead to browning of the callus and dieback of adventitious roots. This study used two different confirmation methods to identify positive transgenic roots: PCR amplification of the inserted gene and GFP fluorescence detection. The test results confirmed the successful conversion. The conversion efficiency is as high as 75%.

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The authors gratefully acknowledge the financial support by National Natural Science Foundation of China (31800509, 31922058), Outstanding Young Talent Fund in Beijing Forestry University" (2019JQ03009), the Fundamental Research Funds for the Central Universities (2021ZY16), Beijing Municipal Natural Science Foundation (6212023), and National Key R&D Program of China (2018YFD1000602,2019YFD1000605-1) and Beijing Advanced Innovation Center for Tree Breeding by Molecular Design. I wish to thank Zhengyang Hou for his guidance in writing the article and to Professor Meng Dong for his guidance on the article idea.


Name Company Catalog Number Comments
0.1 mL qPCR 8-strip tube (with optical caps) KIRGEN, Shanghai, China KJ2541
ABA Solarbio Life Science, Beijing, China A8060
Agar powder Solarbio Life Science, Beijing, China A8190
Centrifuge Osterode am Harz, Germany d37520
CFX Connect TW Optics Module Bio-rad, US 1855200
constant temperature incubator Shanghai Boxun Industry & Commerce Co., Ltd, Shanghai,China BPX-82
Diposable Petri dish Corning, US
Dry Bath Gingko Bioscience Company/Coyote bioscience, China H2H3-100C
Eastep Total RNA Extraction Kit50 Promega, Beijing, China LS1030
Electronic balance Tianjin, China TD50020
Filter pape Hangzhou wohua Filter Paper Co., Ltd, China
FiveEasy Plus Mettler Toledo, Shanghai, China 30254105
Flowerpot 9*9 China
JA Solarbio Life Science, Beijing, China J8070
Kan Solarbio Life Science, Beijing, China K8020
MagicSYBR Mixture CWBIO, Beijing, China CW3008M
Mini Microcentrifuge Scilogex, Beijing, China S1010E
NaCl Solarbio Life Science, Beijing, China S8210
NanPhotometer N50 Touch IMPLEN GMBH, Germany T51082
Purelab untra
Rifampicin Solarbio Life Science, Beijing, China R8010
Seedling box 30*200 China
Thermal Cycler PCR Bio-rad, US T100
Thermostatic oscillator Beijing donglian Har lnstrument Manufacture Co.,Ltd,China DLHE-Q200
Tomy Autoclave Tomy, Japan SX-500
Tryptone Solarbio Life Science, Beijing, China LP0042
UEIris II RT-PCR System for First-Strand cDNA Synthesis( with dsDNase) US Everbright INC, Jiangsu, China R2028
Yeast Extract powder Solarbio Life Science, Beijing, China LP0021



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Liu, T., Song, Z., Du, T., Yang, W., Chen, T., Han, Y., Dong, B., Cao, H., Niu, L., Amin, R., jin, H., Yang, Q., Meng, D., Fu, Y. CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System. J. Vis. Exp. (175), e62304, doi:10.3791/62304 (2021).More

Liu, T., Song, Z., Du, T., Yang, W., Chen, T., Han, Y., Dong, B., Cao, H., Niu, L., Amin, R., jin, H., Yang, Q., Meng, D., Fu, Y. CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System. J. Vis. Exp. (175), e62304, doi:10.3791/62304 (2021).

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