An efficient Rhizobium rhizogenes-mediated transformation system for Cuscuta campestris

Abstract

Parasitism has evolved independently in various plant families, with Cuscuta campestris (field dodder) being an economically significant example. Despite advances in genomics and transcriptomics, functional studies in C. campestris are limited by the lack of an efficient genetic transformation system. This study introduces a highly effective Rhizobium rhizogenes-mediated transformation system for C. campestris using a pBIN plasmid harboring a Yellow Fluorescence Protein reporter gene. We optimized transformation and regeneration by assessing explant type, media composition, and plant growth regulators. Notably, host plant contact was essential for transgenic shoot regeneration. Over 70% transformation efficiency was achieved using cuttings co-incubated with modified Murashige and Skoog medium and 5 mg/L Benzylaminopurine, followed by transfer to tomato hosts. Additionally, we developed a complete in-vivo protocol over 30% regeneration efficiency. Transgenic shoots were confirmed for rol gene expression and haustoria formation, advancing functional studies in C. campestris.

Fig 1. Germinated seedlings used for in-vitro culture and transformation.

(A) Germinated seedling plate. (B) 3 days old seedling. 1: cut the meristem. 2: cut the tip part. (C) 1-day-old seedling (right after germination). Scale bar B– 1 cm, C– 2 mm.

Fig 2. In-vitro culture of C. campestris on artificial media.

In-vitro growth (A, B, C) on K medium and (D, E, F) on MMS medium. (A, D) Direct regeneration (B, E) callus growth (C) callus proliferation (after 8 weeks) on K medium. (F) Emerging of shoot protuberances—Indirect regeneration (after 8 weeks) on MMS medium. Scale bar A, C, D, F = 2 mm B, E = 5 mm.

Fig 3. Transformation events reported on artificial media under the fluorescence microscope.

(A) YFP expression in C. campestris cells after 3–4 weeks of the transformation. (B) YFP expressing callus growth. (C-D) YFP expression with regeneration initiation from cut end (Arrow). Initiated shoots did not elongate to make a full plant. (E-F) Explants growing on the same media without Rhizobium inoculation did not show any YFP expression. (scale bar A = 0.5 mm, B = 1 mm, C-F = 2 mm).

Fig 4. Growth and development of C. campestris cuttings harvested from outside.

(A) Introduction of the cuttings to tomato plants. (B) Development of a new tip from the cut surface (arrow). (C) Development of haustoria. (D) Normal growth on the host was maintained on a plate. (Scale bar A, B, C– 1 mm, D– 1 cm).

Fig 5. Elongation of transgenic Cuscuta shoots expressing YFP that directly transferred to the tomato host after Rhizobium inoculation.

(A, B, C, D) Elongated YFP expressing Cuscuta shoots after one week of transferring to the host. (D) The transgenic Cuscuta stem damaged the host. The host stem has turned brown (square). (E) Transient phenotypes and (F) Chimeric phenotypes showing on elongated shoots. C = Cuscuta, C+ = YFP positive Cuscuta, C- = YFP negative Cuscuta, T = Tomato. Scale bar A, B, E, F = 1 mm, C = 0.2 mm, D = 0.5 mm.

Fig 6. Elongation of transgenic Cuscuta stems expressing YFP that co-incubated with Rhizobium on MMS media.

(A, C, E, G, I, K) images under YFP fluorescence. (B, D, F, H, J, L) images under white light. (A, B, C, D) Elongation of YFP expressing stems which were transferred to the host directly after co-incubation on MMS media and (E, F, G, H) transferred to the host after 5 days on MMS or ½ MS media. (I, J) Explants transferred to the host without Rhizobium inoculation. C = Cuscuta, C+ = YFP positive Cuscuta, C- = YFP negative Cuscuta, T = Tomato. Scale bar A, B = 0.2 mm, C, D = 5 mm, E, F = 1 mm (zoomed E, F = 0.2 mm), G, H, I, J = 1 cm.

Fig 7. Elongation of YFP expressing shoots transferred to the tomato host at different time points.

Treatment 1. Cuscuta explants were directly transferred to the host after Rhizobium inoculation. Treatment 2. Cuscuta explants were co-incubated with Rhizobium on MMS medium. After co-incubation, they were divided into two sets. Set 1. Transferred to the host. Set 2. Transferred to MMS or ½MS medium and cultured for 5 days and then transferred to the host. Values represented by different letters are significantly different (Pr>F = <0.0001).

Fig 8. Regeneration efficiency of YFP expressing shoots with different media and plant growth regulator combinations.

T1. 0 NAA and 0 BAP, T2. 0 NAA and 10 BAP, T3. 0.5 NAA and 0 BAP, T4. 0.5 NAA and 10 BAP. Each hormone concentration is represented by mg/L. Each value represents mean ± SD of three experiments each with 3 technical replicates.

Fig 9. Branching of YFP elongated shoots.

(A) Branching with different media and plant growth regulators in treatment 2—set 2. (B) Difference in branching in treatment 2 between set 1 and set 2. (Treatment 2. Cuscuta explants were co-incubated with Rhizobium on MMS medium. After co-incubation, they were divided into two sets. Set 1. Transferred to the host. Set 2. Transferred to MMS or ½MS medium and cultured for 5 days and then transferred to the host). T1. 0 NAA and 0 BAP, T2. 0 NAA and 10 BAP, T3. 0.5 NAA and 0 BAP, T4. 0.5 NAA and 10 BAP. Each growth regulator concentration is represented by mg/L. Each value represents mean ± SD of three experiments each with 3 technical replicates.

Fig 10. Confirmation of transgene integration via PCR.

1,2: YFP positive C. campestris, 3: R. rhizogenes, 4: negative control (non-transformed C. campestris), M-100 bp molecular weight marker (Promega G210A).

Fig 11. Rhizobium rhizogenes mediated transformation procedure for C. campestris.

(A) Three days old germinated seedlings. (B) Cutting the shoot tip. (C) Rhizobium inoculation. (D) Placing the cuttings on the respective medium. (E) Cuttings directly placed on host and kept in 15°C for 7–10 days. (F-G) Cuttings transferred onto host after co-incubation on MMS for 10 days and taken picture after 5 days (Scale bar = 1 cm).