Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

Abstract

Background Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi. Results In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots. Conclusion We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

Hairy Root Cultures as a Source of Polyphenolic Antioxidants: Flavonoids, Stilbenoids and Hydrolyzable Tannins

“…(Sapindaceae), overexpressing LcMYB1 , contained over 3 mg/g FW of anthocyanidins and nearly 15 mg/g FW of PAs. Wild-type lychee HRs accumulated about 5 mg/g FW PAs and minute amounts of anthocyanidins [ 49 ]. Regulatory functions of PpMYB10.1 and MYB 182 transcription factors in anthocyanidin biosynthesis were also investigated using peach and poplar HR cultures, respectively [ 50 , 51 ].…”

“…Overexpression of LcMYB1 in lychee HRs led to the twofold increase in total flavonol content, in reference to the control roots [ 49 ]. The HRs of Linum album Kotschy ex Boiss.…”

Genome-Wide Characterization of the Aquaporin Gene Family in Radish and Functional Analysis of RsPIP2-6 Involved in Salt Stress

“…The growing tip and 0.5-1 cm elongated hypocotyl (composite plants that contained the transformed hairy roots with a wild-type shoot) were retained for A. rhizogenes infection. Agrobacterium rhizogene harboring RsPIP2-6-GFP (OE) or the empty vector (pCambia1300-GFP: EV) in 50 mL LB liquid medium plus 50 mg/L streptomycin and 100 mg/L kanamycin were incubated overnight at 28 • C on a rotary shaker at 200 rpm until the OD 600 reached 0.8-1.0 (Qin et al, 2021). Bacterial cells were centrifuged at 5000 rpm for 5 min and resuspended in MS liquid medium (OD 600 = 0.8-1.0) containing 100 µM acetosyringone (AS) and infected in the dark for 40-60 min (Huang et al, 2022).…”

“…Therefore, the high-throughput production of transgenic plants in the short run is important for gene function research, especially for plants with a "bottleneck" to plant regeneration (Jian et al, 2009). To date, a fast and efficient transformation technique with A. rhizogenes has been widely used for functional genomics in plants (An et al, 2017;Che et al, 2019;Qin et al, 2021). In radish, only two reports have been successful in developing transgenic plants using the A. rhizogenes-mediated method (Tanaka et al, 1985;Balasubramanian et al, 2018).…”

Integrated metabolome and transcriptome analysis reveals the cause of anthocyanin biosynthesis deficiency in litchi aril

“…In our previous studies, nine structural anthocyanin biosynthesis‐related genes ( LcPAL , LcCHS , LcCHI , LcF3H , LcF3 ′ H , LcDFR , LcANS , LcUFGT , LcGST4 ) and six TFs ( LcMYB1/5 , LcbHLH1/3 , LcABF3 , LcHY5 ) were identified to regulate anthocyanin biosynthesis in litchi pericarps (B. Hu et al, 2016; B. Hu et al, 2019; Lai et al, 2014; Lai et al, 2016; Lai et al, 2019; X. J. Li et al, 2016; Ma et al, 2021; Wei et al, 2011). Furthermore, LcUFGT1 and LcMYB1 have been identified as critical genes in the biosynthesis of anthocyanins in litchi pericarps (X. J. Li et al, 2016; Qin et al, 2021). However, the transcription levels of the aforementioned genes in arils, as well as the reason for the deficiency of anthocyanins in arils, need to be investigated further.…”