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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.Β 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.

Protocol

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 Β°C under fluorescent lights at 50 Β΅mol 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 Β°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 Β°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 transformΒ 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 Β°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 Β°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 Β°C for 5 min, denaturation at 94 Β°C for 30 s, annealing of primers at 55 Β°C for 30 s, and primer extension at 72 Β°C for 30 s. After 36 cycles and a final extension of 10 min at 72 Β°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 Β°C for further investigation.

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 th...

Discussion

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.Β 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 o...

Disclosures

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.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
0.1 mL qPCR 8-strip tube (with optical caps)KIRGEN, Shanghai, ChinaKJ2541
ABASolarbio Life Science, Beijing, ChinaA8060
Agar powderSolarbio Life Science, Beijing, ChinaA8190
CentrifugeOsterode am Harz, Germanyd37520
CFX Connect TW Optics ModuleBio-rad, US1855200
constant temperature incubatorShanghai Boxun Industry & Commerce Co., Ltd, Shanghai,ChinaBPX-82
Diposable Petri dishCorning, US
Dry BathGingko Bioscience Company/Coyote bioscience, ChinaH2H3-100C
Eastep Total RNA Extraction Kit50Promega, Beijing, ChinaLS1030
Electronic balanceTianjin, ChinaTD50020
Filter papeHangzhou wohua Filter Paper Co., Ltd, China
FiveEasy PlusMettler Toledo, Shanghai, China30254105
Flowerpot 9*9China
JASolarbio Life Science, Beijing, ChinaJ8070
KanSolarbio Life Science, Beijing, ChinaK8020
MagicSYBR MixtureCWBIO, Beijing, ChinaCW3008M
Mini MicrocentrifugeScilogex, Beijing, ChinaS1010E
NaClSolarbio Life Science, Beijing, ChinaS8210
NanPhotometer N50 TouchIMPLEN GMBH, GermanyT51082
Purelab untra
RifampicinSolarbio Life Science, Beijing, ChinaR8010
Seedling box 30*200China
ThermalΒ CyclerΒ PCRBio-rad, UST100
Thermostatic oscillatorBeijing donglian Har lnstrument Manufacture Co.,Ltd,ChinaDLHE-Q200
Tomy AutoclaveTomy, JapanSX-500
TryptoneSolarbio Life Science, Beijing, ChinaLP0042
UEIris II RT-PCR System for First-Strand cDNA Synthesis( with dsDNase)US Everbright INC, Jiangsu, ChinaR2028
Yeast Extract powderSolarbio Life Science, Beijing, ChinaLP0021

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