https://orcid.org/0000-0001-8241-1775
Figures
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.
Citation: Alles KMA, Dilhani PGLT, Chandrasekera CHWMRB, Bandaranayake PCG (2025) An efficient Rhizobium rhizogenes-mediated transformation system for Cuscuta campestris. PLoS ONE 20(2): e0317347. https://doi.org/10.1371/journal.pone.0317347
Editor: Waqas Khan Kayani, University of Kotli, PAKISTAN
Received: July 30, 2024; Accepted: December 27, 2024; Published: February 21, 2025
Copyright: © 2025 Alles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are included within the manuscript and its Supporting Information files. A minimal data set is provided in the Supporting Information files. All the gene sequences are available from the GenBank database (accession number(s) PQ684448, PQ684449, PQ684450, and PQ684451). **AT ACCEPT: Check data available & accession numbers are public.**
Funding: “The main research funder is the Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya (UOP), Sri Lanka. Part of this work was carried out with the aid of a grant from UNESCO and the International Development Research Centre, Ottawa, Canada. The views expressed herein do not necessarily represent those of UNESCO, IDRC, or its Board of Governors”.
Competing interests: The authors have declared that no competing interests exist.
Introduction
With over 4,500 species, parasitic flowering plants rely on other plants to complete their life cycle. Within this group, the genus Cuscuta, commonly known as Dodder, comprises approximately 200 species, all of which are stem parasites. These Cuscuta species are holoparasitic, meaning they must connect with a host plant within days of germination to survive and grow [1]. They can be found in temperate and tropical regions worldwide, with species such as Cuscuta campestris exhibiting a broad host range [1, 2]. Cuscuta campestris infections often result in reduced host biomass, changes in photosynthetic pigments, and even anatomical alterations in host plants [3–5]. Due to their significant impact on crops, Cuscuta species were added to the US Department of Agriculture’s Noxious Weeds List in 2010.
Extensive research has positioned Cuscuta as a model system for studying plant-plant interactions through the haustoria, which facilitates the exchange of molecules, including DNA, RNA, and proteins, between the host and the parasite [6–9]. However, there is still limited understanding of the underlying biology and genetic regulation of Cuscuta parasitism, especially in functional characterizing genes and pathways. Recent advancements in genome and transcriptomic sequencing have identified candidate genes involved in parasitism, setting a foundation for further research [10–13]. High-throughput functional analysis of these genes through methods like CRISPR/Cas-based genome editing and RNA interference (RNAi) requires a stable genetic transformation system.
Rhizobium-mediated transformation is a prominent method for creating transgenic plants, commonly using Rhizobium tumefaciens (formerly Agrobacterium tumefaciens) and Rhizobium rhizogenes (formerly Agrobacterium rhizogenes), which facilitate stable transformation through both direct and indirect regeneration pathways [14, 15]. Existing transformation protocols, including those for hemiparasitic species like Triphysariya versicolor [16] and Phtheirospermum japonicum [17], and holoparasitic species such as Phelipanche aegyptiaca [18] and Phelipanche ramose [19], demonstrate the versatility of Rhizobium-mediated systems. However, previous transformation attempts for Cuscuta species, including C. trifolii [20], C. europaea [21, 22], and C. reflexa [23], have yielded only limited success, with varied outcomes in gene integration. Recently, a protocol by Adhikari et al. (2024) [24] outlined a callus-based transformation system for C. campestris, though the process is lengthy—taking approximately five months—and reported low transformation efficiency. Dependence of that protocol on external plant growth regulators and indirect callus-based regeneration may present limitations, particularly for studies focused on genes associated with plant growth regulation, since Cuscuta parasitism relies significantly on these regulators, and Ti plasmid strains affect auxin and cytokinin metabolism [25, 26]. A recent review of Balios and colleagues discuss the various factors affect the growth and development of Cuscuta and among them, phytohormones directly regulate haustorium development and functions [27]. Indirect organogenesis could also compromise genetic stability and introduce somaclonal variation, reducing the utility of such systems for comprehensive, high-throughput gene characterization [28].
In light of these challenges, this study aimed to develop an efficient transformation system for Cuscuta that would enable high-throughput functional characterization of genes linked to parasitism. Our research sought to establish optimal environmental, nutritional, and procedural conditions to enhance transformation efficiency, focusing on the direct regeneration of transformed tissues while sustaining parasitic growth. This new system offers Cuscuta researchers a reliable method for investigating the genetic basis of parasitism, bypassing limitations tied to growth regulators and indirect regeneration, thus enabling more stable, reproducible results.
Results
Preparation of explants after seed germination
Sterilized C. campestris seeds were germinated with 3 cm long shoots after three days of incubation at 29°C in a dark environment (Fig 1A). The shoot tip without the apical meristem, the middle part of the shoot, and the root portion of the shoot (Fig 1B) were used for in-vitro growth while the shoot tip without the meristem, the middle part of the shoot of 3-days-old seedling and the root tip of the 1 day-old seedling (Fig 1C) was used for R. rhizogenes-mediated 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.
Having a good regeneration protocol from possible explants for inoculation is a key factor for the success of Rhizobium-mediated transformation. Since there was no reported tissue culture protocol for C. campestris, we checked the possibility of direct or indirect regeneration on artificial media. We tested previously optimized media for other species of Cuscuta in-vitro culture. Also, we examined two explant factors: age and types of explant.
Optimization of in-vitro growth of C. campestris
A three-factor experiment was performed to evaluate the regeneration of C. campestris in different types of explants (shoot tip, middle, root), different culture media (K, MMS), and with different ages of seedlings (3, 5, 7 days old).
Direct regeneration and callus formation commenced 2–3 weeks post-placements on the plates in both K and MMS media (Fig 2A, 2B, 2D and 2E). Over the following weeks, callus proliferation was evident, particularly in K medium where substantial calli were observed after 8 weeks (Fig 2C). Additionally, shoot protuberances emerged as indirect regeneration in MMS medium after the same duration (Fig 2F).
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.
There was a significant interaction among the 3 factors- age, medium, and explant type. Since the interactions had significant effects, the specific effect of each factor on the calli formation and direct regeneration was not evaluated (Table 1). On average, the calli formation or direct regeneration of 7-days-old seedlings was lower in both media compared to 3-days and 5-days-old seedlings suggesting the suitability of young seedlings for in-vitro culture. Root explants also showed lower efficiency than shoot tip or middle explants with both media. Middle explants from both 3 and 5-day-old seedlings grown on K medium showed higher calli formation efficiencies of 96% and 64%, respectively. Interestingly, the shoot tip explants of both 3 and 5-day-old seedlings grown on MMS medium showed better direct regeneration efficiencies of 46% and 60% respectively. For raw data, see S1 Table.
Therefore, we selected the shoot tip and middle explants and both K and MMS media for the transformation experiments. Though there was no significance observed between 3-day and 5-day-old seedlings, and because of the demonstrated preference for younger plant tissues in previous transformation experiments [16, 29] we selected 3-day-old seedlings as the choice of explant age for subsequent experiments.
Optimization of R. rhizogenes-mediated transformation for C. campestris
The R. rhizobium-mediated transformation was done using the streak infection method. First, we tested the explant type, co-incubation period, and culture media on transformation efficiency. To determine the optimal conditions of three explant types; the shoot tip, middle parts of the 3-days-old seedling (Fig 1B), and root tips of the one-day-old seedlings (Fig 1C) were inoculated with two different co-incubation periods one week or two weeks. We used the root tips of 1-day-old seedlings since we utilized R. rhizogenes, known for its typical use in root transformation. Additionally, three different culture media MS, K, and MMS were tested for each method. We also included the widely used MS [30] medium, as it has previously used for Cuscuta transformation [20, 22].
Transformation events were assessed with YFP expression, under the fluorescence microscope. YFP expression was observed in C. campestris cells after the 3–4 weeks of transformation (Fig 3A). Transformation efficiency was defined as the percentage of cuttings with YFP expression. Stable transformation efficiency was defined as the percentage of cuttings with YFP expression that showed consistent expression over 6–8 weeks while the transient expression efficiency was defined as the percentage of cuttings that showed temporary YFP expression in transformed tissues. The stable transformation events were observed with MMS and K media, while the basal MS medium did not exhibit stable expression of the YFP (Table 2). However, transient transformation, was evident across all three media, with only the explant type and culture media playing a significant role. For raw data, see S2 Table.
(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).
Our primary focus was to assess the factors contributing to stable transformation, as the generation of complete transgenic plants relies on the establishment of stable transformed cells. We identified that MMS and K media which contain lower levels of nutrient conditions than normal MS medium play a critical role in the formation of stable transgenic cells.
During the prolonged culture, a YFP-expressing callus was also formed in MMS medium (Fig 3B). Furthermore, stable YFP expression with regeneration initiation from the cut surface was observed in MMS medium (Fig 3C and 3D). The control experiment carried out by dipping the explant in sterilized distilled water did not show any YFP expression (Fig 3E and 3F).
Culture media and explant type collectively contributed to the transformation events significantly while the duration of co-incubation at 15°C or any other interactions did not. Shoot tip explants grown on the MMS medium resulted in the highest stable transformation efficiency of 22% with one-week and 14% with two-week co-incubation periods, which were not statistically significant. According to the results we used shoot tip explants for further experiments.
There were no significant differences in either transient or stable transformation efficiency between the two co-incubation periods, one week or two weeks. As such, an additional one-week incubation period at 15°C did not further increase the transformation rate.
Optimization of different plant growth regulators for transformation efficiency and initiation
We optimized the suitable parameters to get a better transformation efficiency and elongation of transgenic shoots defined as regeneration. Here we aimed to investigate the impact of different types and concentrations of Auxins and Cytokinins for co-incubation and initiation media on the regeneration of YFP-expressing tissues. In previous experiments, we used MMS medium with 1 mg/L BAP and 3 mg/L NAA for co-incubation. This was named, the regular MMS medium.
We analyzed the data to see the best growth regulator combination for the highest YFP expression in transformation events. When the BAP was added as cytokinin, 5 mg/L of BAP with no NAA in both co-incubation and initiation resulted as the best. When TDZ was cytokinin, the regular MMS medium for co-incubation and 0.05 mg/L TDZ with 0.5 mg/L NAA for initiation worked best (Table 3). We followed up the explants growing on the same media combinations for 6 months with subculturing in 6 weeks and we could see YFP calli and the initiation of YFP regeneration, but we did not see elongation of YFP expressing shoots. For raw data, see S3 Table.
Nevertheless, there was no significant difference between using BAP or TDZ as cytokinin. Therefore, the subsequent experiments focused on the elongation of YFP-expressing shoots, we optimized for a media combination solely with BAP as the cytokinin. This choice was influenced by the cost-effectiveness of BAP compared to TDZ. Additionally, using TDZ for optimal efficiency would require the inclusion of NAA, and our objective was to minimize the use of plant growth regulators in the transformation process. Hence, we chose to proceed with 5 mg/L BAP for growth of transgenic shoots. We observed the tip development initiation in Cuscuta cuttings from both cut surfaces. Although 5 mg/L BAP resulted in high transformation efficiency and initiation of transgenic shoots, there was no elongation of these shoot tips.
Furthermore, we examined the orientation of meristem initiation to determine whether the cuttings have the capacity to generate new shoots from both cut surfaces or if there is a directional specificity. This investigation aimed to ascertain whether shoot development is influenced by the concentration of growth regulators and proximity to either the meristem or the root on one side. Interestingly, lower hormone levels prompted tip development on both sides. Our findings suggest that there is no influence from the proximity of the shoot meristem of cuttings while lower hormonal levels may play a key role in inducing new shoot development (Table 4). For raw data, see S4 Table.
Involvement of host plant in elongation of transgenic stems
Since there was no elongation of transgenic shoots in any of the growth regulators tested, we checked the need for the presence of a host plant and different media for YFP expressing shoot elongation. We used a local tomato variety susceptible to C. campestris. When we introduced shoot tip parts of Cuscuta collected from the wild without meristem directly on the tomato host with no bacterial infection, they developed tips and continued the growth. We checked the difference between cuttings with or without meristems and both of them showed normal elongation with no significant difference. Therefore, we introduced the shoot tip explants from seedlings (used for previous experiments) with bacteria infection to the same susceptible host plant and they also developed a tip and continued the growth (Fig 4A–4D). The growth rate was higher in Rhizobium-infected seedling explants than in the wild-collected tips from mature C. campestris (Table 5). For raw data, see S5 Table.
(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).
We conducted a series of experiments to identify a suitable time of host infection, the effect of co-incubation, and culture conditions. First, we introduced Cuscuta explants to the tomato host plant soon after the inoculation of bacteria for co-incubation and subsequent growth (Treatment 1). Many explants made new tips on both sides and continued to grow on tomato plants after one week of co-incubation at 15°C, and about 36% of them showed stable YFP expression. The shoots continued elongation with YFP expression (Fig 5A–5C) and made connections to the host (Fig 5D). About 10.5% showed transient YFP expression (Fig 5E) and few explants showed chimeric YFP expression (Fig 5F).
(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.
Furthermore, we explored an alternative medium with different nutrient levels to assess its potential for regenerating YFP-expressing shoots. We included ½ MS medium in our investigations, especially considering that full MS medium initially yielded low transformation efficiency. Concurrently, we optimized hormone levels based on previous tests to enhance the transformation and regeneration process. Therefore, we tested two media, ½ MS and MMS medium with B5 vitamins, and compared them with two different BAP and NAA concentrations and no hormones. For these experiments, we selected the best co-incubation medium among the studied, MMS medium with 5 mg/L of BAP. We initiated three experiments in parallel to test several factors together.
Another set of explants, co-incubated on MMS medium at 15°C for 10 days, was divided into two groups (Treatment 2). The first group (Set 1), directly introduced to the host plant after co-incubation, exhibited elongation of YFP expressing shoots clearly with tip formation at an efficiency of around 71% (Fig 6A–6D). The other group (Set 2) was transferred to MMS or ½ MS medium with cefotaxime, with or without plant growth regulators, after co-incubation. Those were also introduced to the tomato host after 5 days of culture and resulted in complete transgenic shoots expressing YFP at an efficiency of around 61% (S1 Fig). Regenerated Cuscuta shoots displayed stable YFP expression on tomato plants one week after the transfer to the host (Fig 6E–6H). There was no any fluorescence in explants which were transferred to the host without R. rhyzogenes inoculation (Fig 6I and 6J). Explants that continued growing on the culture media with no host did not elongate YFP expressing shoots during the considered period.
(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.
As described, one set was transferred to the host after 5 days of incubation on MMS or ½ MS media, with or without plant growth regulators, across four treatments: T1 (0 NAA & 0 BAP), T2 (0 NAA & 10 BAP), T3 (0.5 NAA & 0 BAP), and T4 (0.5 NAA & 10 BAP), with hormone concentrations in mg/L. All explants generated transgenic Cuscuta shoots with YFP expression, even in the absence of growth regulators (T1). This indicates that growth regulators are not essential for the elongation of transgenic stems after co-incubation. However, explants transferred to the host after co-incubation with bacteria on MMS medium with 5 mg/L BAP (Fig 7 - Treatment 2 –set 1) exhibited a higher efficiency of YFP expressing shoots, suggesting that co-incubation on MMS medium containing 5 mg/L BAP significantly enhances elongation of transgenic stems expressing YFP efficiently (Fig 7). For raw data, see S6–S8 Tables.
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).
Culturing the cuttings in different growth regulators and media combinations for five days did not significantly affect the efficiency of elongation of transgenic shoots (Fig 8).
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.
Further, we monitored the growth of randomly selected shoots growing on the host and growing on the media after co-incubation. The explants introduced to the host made a new tip and showed significantly higher growth than the explants which continued the growth on any MMS or ½ MS medium (Table 5).
The YFP-expressing elongated shoots made branches about 10 days after being introduced to the susceptible host. About eighty-six (86%) of explants that were introduced to the host after co-incubation developed branches (Set 1). The explants were grown on different media and growth regulators for an additional 5 days before being introduced to the host also developed branches with 51% efficiency (Set 2). There was no significance of media or growth regulators for the branching (Fig 9A). Interestingly, the branching of YFP-expressing shoots was higher in set 1 which was transferred to the host after co-incubation (Fig 9B). For raw data, see S9 and S10 Tables.
(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.
Similar results were observed in in vivo conditions, where the germinated seedlings were cut and dipped on the lab bench and grown on tomato seedlings germinated under unsterile conditions, maintained at 15°C for 7 days followed by growing in the lab. However, the fungi infection was observed after 10–14 days, though Cuscuta continued its active growth (S2 Fig).
It is interesting to note that all the cuttings, including wildtype, stable transgenic, chimeric, and transient, developed multiple haustoria when they were attached to the selected tomato host. However, no haustoria development was observed on cuttings maintained on artificial media. Furthermore, the addition of cefotaxime was not necessary in our system as bacterial growth was not observed after introduction onto the host.
Confirmation of bacterial gene integration in transgenic C. campestris
We conducted the standard assessment of the integration of two genes, rolB and rolC1, as well as virD2, through endpoint PCR using cDNA as the template (Fig 10, S3 Fig). We ensured that any contaminating genomic DNA was removed by treating the RNA with DNAase. In YFP-positive C. campestris, the rolB and rolC1 genes were amplified, while the virD2 was not present in the same samples. This suggests that the plant genome has successfully incorporated rolB and rolC1. The virD2, which is present on the Ti plasmid and outside of the T-DNA region, does not integrate into the plant genome. It was not amplified in plant samples, but it was amplified in R. rhizogenes. Non-transformed C. campestris or the negative control resulted in a product only for the plant endogenous control gene, rbcL. Moreover, multiple sequence alignment of rolB and rolC1 genes also revealed the integration of rolB and rolC1 genes with the correct size product in the YFP-positive C. campestris samples (S4 and S5 Figs).
1,2: YFP positive C. campestris, 3: R. rhizogenes, 4: negative control (non-transformed C. campestris), M-100 bp molecular weight marker (Promega G210A).
Discussion
This study established a highly efficient Rhizobium rhizogenes-mediated transformation system for Cuscuta campestris, enabling rapid production of transgenic materials suitable for various molecular assays. By optimizing explant selection, media composition, and environmental conditions, our system demonstrated transformation efficiencies of up to 70%, with transgenic shoots ready for analysis within 3–4 weeks. The most effective protocol involved dipping shoot tip explants (with sharp cuts on the tip and proximal end) in a creamy bacterial culture (<24 h old), placing them on MMS medium with 5 mg/L BAP in a 15°C growth chamber with a 16-hour light cycle for 5–10 days, and then transferring them to a host environment at 24°C with 12 hours of light. This study also identified a hormone-free option with 36% efficiency, offering researchers flexibility when studying genes sensitive to growth regulators.
Previous transformation attempts in Cuscuta species encountered varied results, with limited gene integration success, lower transformation and subsequent regeneration efficiency, and dependence on external plant growth regulators for a long time, limiting its use in studies investigating genes linked to growth pathways [20–24]. In contrast, our protocol provides a streamlined approach with faster regeneration times and greater efficiency, particularly advantageous for studies where the application of external hormones may interfere with gene functionality related to parasitism, as Cuscuta relies heavily on auxin and cytokinin regulation [26, 27, 31].
In developing this transformation protocol, we optimized conditions across multiple variables, such as using fresh, creamy bacterial cultures less than 24 hours old, making precise, sharp cuts on the explants (shoot tip), the flexibility of 15°C co-incubation duration, and nutrient combinations. Investigating explants of different ages and types revealed that tissues at various growth stages, influenced by active metabolism, affect transformation efficiency. These findings align with previous studies considering explant age and orientation [32]. K and MMS media, adapted from other Cuscuta species, effectively support callus formation and direct regeneration in C. campestris. Unlike standard MS medium, these media have reduced macronutrients, and lack Fe.EDTA salts, and include specific growth regulators (S11 Table). This composition shows potential for broader in vitro cultivation of Cuscuta species. Moreover, the MMS medium with only BAP enhanced the initiation of regeneration of transgenic YFP expression while reducing cost and increasing efficiency compared to TDZ, particularly when minimizing hormonal influence was desired. This protocol was optimized by adjusting these factors, which significantly influenced C. campestris transformation outcomes. The direct regeneration approach for generating transgenic plants not only reduces the overall cultivation time but also minimizes somaclonal variation and complications associated with plant growth regulators, unlike the indirect regeneration methods.
The host’s presence was crucial for the elongation of C. campestris transgenic shoots, highlighting the obligate parasitic nature of this plant. In nature, Cuscuta seedlings cannot continue growth without host attachment shortly after germination [1]. Host dependence for sustained growth may offer insights into the parasitism mechanisms of Cuscuta, particularly regarding hormone signaling pathways necessary for attachment and growth. In our experiments, explants with or without meristems exhibited similar growth when placed on the host, suggesting that host attachment alone is sufficient to support Cuscuta growth, thus opening opportunities for deeper exploration of host-plant interactions and potential parasitism-involved genes.
Our first transformation experiment showed that lower nutrient conditions than in MS medium are required for transformation initiation. Incorporating external nutrients or additional plant growth regulators post-co-incubation did not affect the elongation of YFP-expressing shoots, indicating that host-dependent conditions are more relevant for elongation than nutrient availability. Additionally, using sharp cuts, younger seedlings, and maintaining low temperatures were identified as crucial factors in achieving high transformation efficiency, highlighting the specific requirements needed for consistent outcomes in Cuscuta transformation. In these experiments, we used YFP as a reporter gene since it has been previously used as an efficient reporter in parasitic plant roots with high amount of secondary metabolites [33, 34]. Nevertheless, the researchers can select any other reporter gene with appropriate microscopic settings and visual reporters such as RUBY, Luciferase, Beta-glucuronidase (GUS), Chloramphenicol Acetyltransferase (CAT), and Antibiotic resistance genes.
Although our system achieves transgenic materials within 6 weeks, we recommend testing it in other Cuscuta species, particularly those with economic and ecological importance, to verify its broader applicability. Reports indicate that Cuscuta can establish and make haustoria on non-living surfaces such as metal rods or wooden sticks [35]. Adapting the method for use with different non-living surfaces or other host plants, particularly those less tolerant to low temperatures, may be necessary. The current protocol’s 15°C co-incubation period could be incompatible with cold-sensitive hosts, so modifying the duration may be required. Our recent experiments indicate that even a shortened co-incubation time of 5 days at 15°C did not impact transformation efficiency, suggesting room for further adjustments in host-parasite studies.
Our transformation system, adaptable across Cuscuta and potentially other parasitic plants, represents an advancement for functional genomics research. While the hormone-free method offers an effective approach for exploring genes sensitive to growth regulators, more research into other Cuscuta species is recommended to fully validate this system. Additionally, expanding studies to include a broader range of host plants will strengthen its applications, especially with cold-sensitive hosts. This transformation system thus provides a valuable tool for advancing parasitic plant research, supporting gene function analysis, and offering insights for agricultural management and ecological conservation through a more profound understanding of plant parasitism.
Conclusions
This study successfully establishes an efficient Rhizobium rhizogenes-mediated transformation system for Cuscuta campestris, a significant parasitic plant. By optimizing the explant type, technical procedures, and culture conditions, we achieved stable transformation, rapid initiation of direct regeneration, and continuation of parasitic growth. Using BAP in co-cultivation and initiation media, the transformation efficiency reached 70%. Alternatively, for studies where plant growth regulators may interfere with gene or pathway functions, a hormone-free method achieving 36% efficiency is available. Notably, the discovery of host contact as a key factor for transgenic shoot regeneration provides new insights into plant parasitism mechanisms. This transformation system enables functional characterization of C. campestris genes and broadens the potential for in vivo transformation and regeneration directly on the host. Overall, this work offers a valuable tool for understanding the biology and genetics of C. campestris and contributes to the development of effective management strategies for parasitic plants.
Materials and methods
All the media were prepared in the lab following the standard laboratory procedures and the composition of the media is given in the Supplementary file (S11 Table). While the bacteria medium was solidified with Bacto Agar (Meron), the plant growth medium was solidified with 0.4% Phytagel (Sigma). The cultures were maintained under uniform light conditions at 15°C growth chamber and 24°C culture room; a 16 h light and 8 h dark cycle with a light intensity of 85 μmolm-2s-1.
The sterilization, micropropagation, and transformation protocols were optimized separately. The yellow fluorescence protein (YFP) expression was tested using an Olympus (SZX10 Japan) Stereo Zoom research florescence microscope equipped with a YFP filter set with excitation HQ490-500, DM505, and emission HQ515-610. The images were captured with a C-mount CCD camera.
Seed sterilization and germination
Cuscuta campestris seeds were collected from an open-pollinated population growing in Peradeniya, Sri Lanka, and the species was confirmed with the National Herbarium of Sri Lanka (PDA). Seeds were surface sterilized using the method previously described by Furuhashi (1991) [36] with some modifications. The seeds were treated with 95% (v/v) H22SO44 for 1 hour and washed three (3) times with sterile distilled water. Then the seeds were rinsed in 3% (v/v) NaOCl solution, supplemented with Tween-20 for 30 minutes, and washed with sterile distilled water five (5) times. Finally, they were soaked in sterile distilled water at 4°C for 24 hours. Seeds were germinated on wet tissue papers at 29°C in a dark environment (Fig 1A).
Optimization of in-vitro growth of C. campestris
Two different media were tested; the K medium (modified MS medium containing 1 mg/L Kinetin and 10% coconut water) described by Furuhashi (1991) [36] for C. japonica, and the MMS-1 medium (modified MS medium containing, 1 mg/L BAP and 3 mg/L NAA, indicated as MMS medium) for C. reflexa by Srivastava and Dwivedi (2001) [37]. The MMS medium consisted of BAP (6-Benzylaminopurine) instead of BA (6-Benzyladenine) in the current study. We used three different types of explants of about 1 cm in length, shoot tip without meristem, stem region next to the tip (middle), and the root portion of the seedlings (Fig 1B). The age of the seedlings, 3 days, 5 days, and 7 days was considered as another factor. As such, we designed a three-factor factorial experiment to evaluate the best conditions for C. campestris in-vitro culture.
Each explant was cut sharply and placed horizontally on Petri dishes consisting of appropriate media as 10 cuttings per plate. Each Petri dish 100 mm×15 mm was considered as a technical replicate and each treatment was replicated 5 times. All the cultures were kept in the culture room at 24°C. The number of calli and number of direct regenerated shoots in each replicate were counted after five weeks of initiating cultures and percentage values were calculated accordingly.
Optimization of R. rhizogenes-mediated transformation method for C. campestris
Plasmids and bacteria.
The R. rhizogenes strain MSU440 contains the plasmid pBIN-YFP, which includes the Aequorea victoria enhanced yellow fluorescent protein (EYFP) gene was kindly provided by the Yoder Lab at UC Davis (33). The EYFP gene is driven by the CaMV 35S promoter for expression in plants [38]. The pBIN-YFP plasmid is 11,775 bp and comprises the kanamycin resistance gene for bacterial selection. Within the T-DNA region, the kanamycin resistance gene is included for plant selection.
R. rhizogenes was inoculated onto the MGL plates consisting of Kanamycin from glycerol stock and incubated overnight at 29°C to get a single bacterial colony. Then a single colony from the MGL agar plate was inoculated into a 5 mL MGL medium containing Kanamycin and incubated overnight at 29°C while shaking at 200 rpm. Then 1 mL from the 1-day-old bacterial culture was inoculated into an MGL agar plate and incubated overnight at 29°C until creamy bacterial lawn appeared on the plate.
We selected three types of media K, MMS, and MS, three types of explants, shoot tip and middle segments of a 3-day-old seedling, and root of 1 day-old seedling. The streak infection method previously described by Kanchanamala and Bandaranayake (2019) [39] was used for transformation. After making a sharp cut 1 cm from the tip (excluding the meristem) for the shoot tip, the next 1 cm section was taken as the middle part, and all surfaces were carefully dipped into an approximately 18-hour-old R. rhizogenes culture grown on an MGL plate. Then the cuttings were placed horizontally on the culture plates containing the respective medium.
Controls were performed for all the treatments by dipping the explants into sterilized distilled water. The plates were maintained at 15°C growth chamber for either one week or two weeks for co-incubation. Each Petri dish 100 mm×15 mm consisting of 18–20 explants was considered as a technical replicate and each treatment was replicated at least 5 times.
After the co-incubation period, cuttings were transferred to the same medium supplemented with 300 mg/ L cefotaxime to kill the R. rhizogenes. Cultures were maintained in a culture room at 24°C.
The number of cuttings with YFP expression per plate was counted after 6 weeks from the date of transformation and percentage values were calculated. In addition, we followed the same tissues for 20 weeks with or without subculturing.
Optimization of different plant growth regulators for transformation efficiency and initiation.
We examined the MMS medium with 5 different BAP, 4 different TDZ, and 3 different NAA concentrations (Table 3). Shoot tip explants and a week co-incubation period were used.
We followed the cuttings for about 6 months with subculturing on the same media combinations with 300 mg/L cefotaxime while frequently observing under the above microscope for either direct or indirect regeneration.
We defined direct regeneration as growing the YFP expressing shoot without the formation of a callus while indirect regeneration follows a callus stage. The number of cuttings per plate with YFP expressing calli forming new stems and the number of cuttings that directly regenerate new shoots from the cut surface was counted and the percentage values were calculated accordingly.
To investigate the orientation of meristem initiation, we used MMS medium with 0.5 mg/L NAA and three BAP concentrations of 5, 10 and 25 mg/L.
Involvement of host plant on elongation of transgenic plants.
We used 5–7 days-old tomato seedlings of a local cultivar, Thilina reported to be a susceptible host for C. campestris. We conducted an experiment assessing the development of 1 cm long Cuscuta cuttings collected from the wild on tomato host plants grown on wet hand tissues in Petri dishes. We applied the same methodology for cuttings inoculated with R. rhizogenes to test whether those elongate in the presence of the host.
Three days old C. campestris seedlings were used for all these experiments. The shoot tip was cut sharply with a scalpel blade (Fig 11A and 11B). Both surfaces were dipped carefully in about 18 h old R. rhizogenes culture (Fig 11C) and divided into two treatments, (01) placed on host plants facing the tip side towards the shoot tip of the tomato plants grown on sterile hand tissues placed in Petri dishes (Fig 11E) as 3–5 cuttings per plant and (02) in Petri dishes with MMS medium with 5 mg/L of BAP as about 10–20 cuttings per plate (Fig 11D). They were transferred to the host after co-incubation on MMS or post co-incubation on MMS or ½ MS media (Fig 11F and 11G). Both treatments were kept in a controlled growth chamber maintained at 15°C for five to ten days.
(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).
Plates exposed to treatment 01 were taken out from the 15°C growth chamber after seven days and observed under the YFP fluorescence and the number of cuttings with direct regeneration with stable YFP expression, and the number of cuttings with transient expression, were counted. We also examined chimeric expressions. The transient expression occurs when the genetic materials are taken up by the cell but not incorporated into the cell’s genome [40]. The chimeric expression was defined as the introduction and expression of a gene construct that combines genetic elements from different sources [41]. All the plates were kept in the lab at 24°C and 12 h light and 12 h dark and observed daily under the fluorescence microscope.
The treatment 02 plates taken out from the 15°C growth chamber after 10 days were divided into two sets. The cuttings in the first set were transferred onto tomato plants as same as the treatment 01 and transferred to the lab at 24°C observed under the fluorescence microscope after 7 days and the number of YFP expressing shoots that kept growing was counted. The plates were maintained in the lab at 24°C with 12 h light and 12 h dark.
The cuttings in the second set were transferred to plates containing two different combinations of media (1/2 MS and MMS) and four different concentrations of plant growth regulators (0 NAA and 0 BAP, 0 NAA and 10 BAP, 0.5 NAA and 0 BAP, 0.5 NAA and 10 BAP (all the concentration in mg/L) and 300 mg/L cefotaxime. After 5 days of growth in the culture room at 24°C, 16 h light 8 h dark, the cuttings were transferred separately to tomato plants as in treatment 01. Finally, all the explants from different treatments were transferred to the tomato host for elongation of YFP-expressing transgenic shoots (S1 Fig). The plates were maintained in the lab at 24°C 12 h light and 12 h dark for 7 days and observed under the fluorescence microscope.
Both sets of plants from treatment 02 were further assessed two weeks after introducing them to the host for the ability of the tips to branch. The number of branching YFP tips was counted on each plate.
To measure the growth of transgenic shoots, a set of YFP expressing Cuscuta stems was assessed for about 10 days measuring the growth in mm every three days. As a control, young shoot tips harvested from the wild were introduced onto the tomato plants, cutting the tip and without cutting the tip and dipping in water and measuring the growth every three days intervals. Further, the growing transgenic C. campestris shoots and coiled collected shoots were assessed for their ability to form haustoria, counting the number of YFP-expressing stems forming haustoria 10 days after introducing them to the host.
Further, the above experiments with the hosts were also conducted under unsterile conditions by introducing the Cuscuta cuttings onto the host seedlings harvested from the greenhouse and maintained on hand tissues in Petri dishes.
Cuscuta cuttings dipped in water were used as the negative control for R. rhizogenes experiments. As the control for the regeneration experiments of YFP-positive tissues on hosts, YFP-expressing cuttings were maintained on respective media with no host. When we use cuttings, each Petri plate with a minimum of 10 cuttings was considered a replicate and each treatment consisted of a minimum of three plates (Fig 11D). For the plant experiments, each plate with 3–4 tomato plants was considered as a replicate and each experiment was replicated 4–5 times. Each host plant was infected with 3–6 C. campestris stems (Fig 11E–11G).
Confirmation of bacterial gene integration in transgenic C. campestris.
Total RNA was extracted from about five weeks old YFP-positive C. campestris using the Trizol method following the manufacturer’s instructions (Invitrogen, Cat No 15596–026). The quality and quantity of RNA were determined with a NanoDrop spectrophotometer and Agarose electrophoresis (1%) followed by DNase treatment (DNA -freeTM kit DNase Treatment and Removal, Invitrogen, Cat No:1906). Then cDNA was prepared from 0.5 μg RNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific™, Cat No:K1622). Reverse-transcribed RNA was amplified using rolB, rolC1, virD2 and rbcL genes (Table 6). These genes and the primers have been successfully used in previous work [42–44]. Primers rolB and rolC1 selectively amplify genes present on the T-DNA and incorporate them into the plant genome, while virD2 is present outside of the T-DNA border and does not integrate into the plant genome and was used as the control to identify the presence of R. rhizogenes cells. The rbcL gene was used as the control for endogenous plant genes.
PCR was carried out in a total of 25 μL containing lX PCR buffer, 1.5 mM MgCl22, 0.2 mM dNTP (Promega, USA), 0.2 μM of each primer (Integrated DNA Technologies, Singapore), 2 μL of 1:1 diluted cDNA and 1 Unit Go Taq Flexi DNA polymerase (Promega, USA). The PCR cycle consisted of initial denaturation at 94°C for 3 minutes followed by 35 cycles at 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 1 minute, and a final extension of 72°C for 5 minutes. A well-grown R. rhizogenes MSU440 colony harboring pBIN-YFP plasmid was suspended in water and used as a positive control. RNA extracted from wild-collected C. campestris was used as the negative control.
PCR products were separated by electrophoresis (5 Vcm-1) on 1.5% agarose gels and stained with ethidium bromide (1 μg/mL). The PCR products of rolB and rolC1 from YFP-positive C. campestris and R. rhizogenes were shipped to Macrogen Inc (Seoul, South Korea– http://dna.macrogen.com) for Sanger sequencing using the primers as used for PCR. Chromatograms of the PCR amplified products were visually inspected using Geneious Prime Software (version 11.0.6) for sequencing errors, and the 5’ and 3’ noisy sequences were removed. Then the multiple sequence alignment was carried out for each gene to check the presence of R. rhizogenes genes in C. campestris. All the sequences were submitted to GenBank (S12 Table).
Data analysis
Transformation efficiency was defined as the percentage of cuttings with YFP expression. The percentage values were calculated based on the numbers counted per plate. Each plate was considered as a replicate and all the experiments were replicated at least three times. The anlysis included the in-vitro growth efficiency, transformation efficiency, efficiency of elongation of YFP expressing shoots, tip growth and shoot growth, and branching. While the average values of all the replicates ± SD were presented, the total number of plates in treatment was presented as (n). The normality of the data was tested for both growth measurement data and the efficiency data and the data were analyzed with the GLM procedure or the t-test using the statistical analysis software SAS OnDemand (Online), SAS Institute Inc. 2024. The mean separation was done using Tukey`s mean separation method.
Supporting information
S1 Fig. The transformation system with different options.
https://doi.org/10.1371/journal.pone.0317347.s001
(TIFF)
S2 Fig. Growth of Cuscuta with fungal infections in in-vivo conditions (Scale bar = 2 mm).
https://doi.org/10.1371/journal.pone.0317347.s002
(TIF)
S4 Fig. Multiple sequence alignment of rolB in YFP positive samples and the R. rhizogenes.
+: R. rhizogenes, T1 and T2: YFP positive C. campestris.
https://doi.org/10.1371/journal.pone.0317347.s004
(TIF)
S5 Fig. Multiple sequence alignment of rolC1 in YFP positive samples and the R. rhizogenes.
+: R. rhizogenes, T1 and T2: YFP positive C. campestris.
https://doi.org/10.1371/journal.pone.0317347.s005
(TIF)
S7 Table. Raw data for Fig 7.
(Treatment 2 –set 1).
https://doi.org/10.1371/journal.pone.0317347.s012
(DOCX)
S8 Table. Raw data for Fig 7.
(Treatment 2 –set 2).
https://doi.org/10.1371/journal.pone.0317347.s013
(DOCX)
S9 Table. Raw data for Fig 9.
(Treatment 2 –set 2).
https://doi.org/10.1371/journal.pone.0317347.s014
(DOCX)
S10 Table. Raw data for Fig 9.
(Treatment 2 –set 1).
https://doi.org/10.1371/journal.pone.0317347.s015
(DOCX)
Acknowledgments
The authors thank Mr. G. D. S. P. Rajapaksha, Research Assistant, Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya for the assistance in setting up experiments and data analysis. The authors appreciate the support of Mrs. I. N. S. Dewapriya, Research Assistant, Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya for the assistance provided in setting up laboratory experiments. The authors would like to thank Ms. Pakkiyarasa Kaviththira undergraduate intern from the Faculty of Technology, Rajarata University of Sri Lanka for the assistance provided during the data collection. Mrs. R. R. M. H. L. Rathnayake, Laboratory attendant, Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya for the assistance provided during laboratory experiments. The authors sincerely thank all the staff members of the Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya for the continuous support given during the project period. The authors acknowledge Grammarly generated responses to improve it.
References
- 1. Dawson JH, Musselman LJ, Wolswinkel PI, Dörr IN. Biology and control of Cuscuta. Reviews of Weed Science, 1994, Vol. 6, 265–317 ref. 303.
- 2.
Mabberley DJ. The plant-book: a portable dictionary of vascular plants. Cambridge university press; 1997 Jun 19.
- 3. Shen H, Xu SJ, Hong L, Wang ZM, Ye WH. Growth but Not Photosynthesis Response of a Host Plant to Infection by a Holoparasitic Plant Depends on Nitrogen Supply. PLoS One. 2013;8(10). pmid:24116055
- 4. Saric-Krsmanovic MM, Bozic DM, Radivojevic LM, Umiljendic JSG, Vrbnicanin SP. Effect of Cuscuta campestris parasitism on the physiological and anatomical changes in untreated and herbicide-treated sugar beet. J Environ Sci Heal—Part B Pestic Food Contam Agric Wastes [Internet]. 2017;52(11):812–6. Available from: pmid:28857671
- 5. Saric-Krsmanovic M, Bozic D, Radivojevic L, Gajic Umiljendic J, Vrbnicanin S. Impact of Field Dodder (Cuscuta campestris Yunk.) on Chlorophyll Fluorescence and Chlorophyll Content of Alfalfa and Sugar Beet Plants. Russ J Plant Physiol. 2018;65(5):726–31.
- 6. Kim G, LeBlanc ML, Wafula EK, DePamphilis CW, Westwood JH. Genomic-scale exchange of mRNA between a parasitic plant and its hosts. Science. 2014 Aug 15;345(6198):808–11.
- 7. Liu N, Shen G, Xu Y, Liu H, Zhang J, Li S, et al. Extensive Inter-plant Protein Transfer between Cuscuta Parasites and Their Host Plants. Mol Plant [Internet]. 2020;13(4):573–85. Available from: https://doi.org/10.1016/j.molp.2019.12.002
- 8. Wu Y, Luo D, Fang L, Zhou Q, Liu W, Liu Z. Bidirectional lncRNA Transfer between Cuscuta Parasites and Their Host Plant. Int J Mol Sci. 2022;23(1). pmid:35008986
- 9. Jhu MY, Sinha NR. Cuscuta species: Model organisms for haustorium development in stem holoparasitic plants. Frontiers in Plant Science. 2022 Dec 12;13:1086384. pmid:36578337
- 10. Sun G, Xu Y, Liu H, Sun T, Zhang J, Hettenhausen C, et al. Large-scale gene losses underlie the genome evolution of parasitic plant Cuscuta australis. Nat Commun [Internet]. 2018;9(1):4–11. Available from: http://dx.doi.org/10.1038/s41467-018-04721-8
- 11. Vogel A, Schwacke R, Denton AK, Usadel B, Hollmann J, Fischer K, et al. Footprints of parasitism in the genome of the parasitic flowering plant Cuscuta campestris. Nat Commun [Internet]. 2018;9(1). Available from: pmid:29955043
- 12. Park I, Song JH, Yang S, Kim WJ, Choi G, Moon BC. Cuscuta species identification based on the morphology of reproductive organs and complete chloroplast genome sequences. Int J Mol Sci. 2019;20(11). pmid:31163646
- 13. Pan H, Zagorchev L, Chen L, Tao Y, Cai C, Jiang M, et al. Complete chloroplast genomes of five Cuscuta species and their evolutionary significance in the Cuscuta genus. BMC Genomics. 2023;24(1):1–13.
- 14. Cordeiro D, Alves A, Ferraz R, Casimiro B, Canhoto J, Correia S. An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus. Plants. 2023;12(5):1–15.
- 15. Rahman SU, Khan MO, Ullah R, Ahmad F, Raza G. Agrobacterium-Mediated Transformation for the Development of Transgenic Crops; Present and Future Prospects. Mol Biotechnol. 2023;(August).
- 16. Bandaranayake PCG, Yoder JI. Factors affecting the efficiency of Rhizobium rhizogenes root transformation of the root parasitic plant Triphysaria versicolor and its host Arabidopsis thaliana. Plant Methods [Internet]. 2018;14(1):1–9. Available from: https://doi.org/10.1186/s13007-018-0327-2
- 17. Ishida JK, Yoshida S, Ito M, Namba S, Shirasu K. Agrobacterium Rhizogenes-Mediated transformation of the parasitic plant Phtheirospermum japonicum. PLoS One. 2011;6(10):1–8. pmid:21991355
- 18. Fernández-Aparicio M, Rubiales D, Bandaranayake PCG, Yoder JI, Westwood JH. Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca. Plant Methods. 2011;7(1):1–10.
- 19. Libiaková D, Ruyter-Spira C, Bouwmeester HJ, Matusova R. Agrobacterium rhizogenes transformed calli of the holoparasitic plant Phelipanche ramosa maintain parasitic competence. Plant Cell Tissue Organ Cult [Internet]. 2018;135(2):321–9. Available from: http://dx.doi.org/10.1007/s11240-018-1466-x
- 20. Borsics T, Mihálka V, Oreifig AS, Bárány I, Lados M, Nagy I, et al. Methods for genetic transformation of the parasitic weed dodder (Cuscuta trifolii Bab. et Gibs) and for PCR-based detection of early transformation events. Plant Sci. 2002;162(2):193–9.
- 21. Švubová R, Blehová A. Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea). Biol. 2013;68(4):633–40.
- 22. Kaštier P, Martinčová M, Fiala R, Blehová A. Transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton. Nov Biotechnol Chim. 2017;16(1):20–5.
- 23. Lachner LAM, Galstyan L, Krause K. A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites. Plant Direct. 2020;4(8):1–11. pmid:32789286
- 24. Adhikari S, Mudalige A, Phillips L, Lee H, Bernal‐Galeano V, Gruszewski H, Westwood JH, Park SY. Agrobacterium‐mediated Cuscuta campestris transformation as a tool for understanding plant–plant interactions. New Phytologist. 2024 Feb 23. pmid:39360397
- 25. Hwang HH, Wang MH, Lee YL, Tsai YL, Li YH, Yang FJ, et al. Agrobacterium-produced and exogenous cytokinin-modulated Agrobacterium-mediated plant transformation. Mol Plant Pathol. 2010;11(5):677–90. pmid:20696005
- 26. Furuhashi K, Iwase K, Furuhashi T. Role of Light and Plant Hormones in Stem Parasitic Plant (Cuscuta and Cassytha) Twining and Haustoria Induction. Photochem Photobiol. 2021;97(5):1054–62. pmid:33934364
- 27. Balios VA, Fischer K, Bawin T, Krause K. One organ to infect them all: the Cuscuta haustorium. Annals of Botany. 2024 Dec 2:mcae208. pmid:39673400
- 28.
Bhatia S, Sharma K, Dahiya R, Bera T. Technical glitches in micropropagation. Cambridge, MA: Academic Press; 2015 Jul 22.
- 29. Hoque ME, Mansfield JW. Effect of genotype and explant age on callus induction and subsequent plant regeneration from root-derived callus of Indica rice genotypes. Plant Cell Tissue Organ Cult. 2004;78(3):217–23.
- 30. Murashige T, Skoog F. Murashige1962 Revised.Pdf. Physiol Plant. 1962;15:474–97.
- 31. Zwanenburg B, Blanco-Ania D. Strigolactones: New plant hormones in the spotlight. J Exp Bot. 2018;69(9):2205–18. pmid:29385517
- 32. Mazumdar P, Basu A, Paul A, Mahanta C, Sahoo L. Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L. South African J Bot [Internet]. 2010;76(2):337–44. Available from: http://dx.doi.org/10.1016/j.sajb.2010.01.001
- 33. Tomilov A, Tomilova N, Yoder JI. Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence. Planta. 2007 Apr;225:1059–71. pmid:17053892
- 34. Bandaranayake PC, Filappova T, Tomilov A, Tomilova NB, Jamison-McClung D, Ngo Q, Inoue K, Yoder JI. A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria. The Plant Cell. 2010 Apr 1;22(4):1404–19. pmid:20424175
- 35. Kaiser B, Vogg G, Fürst UB, Albert M. Parasitic plants of the genus Cuscuta and their interaction with susceptible and resistant host plants. Frontiers in plant science. 2015 Feb 4;6:45. pmid:25699071
- 36. Furuhashi K. Establishment of a successive culture of an obligatory parasitic flowering plant, Cuscuta japonica, in vitro. Plant Sci. 1991;79(2):241–6.
- 37. Srivastava S, Dwivedi UN. Plant regeneration from callus of Cuscuta reflexa–An angiospermic parasite—And modulation of catalase and peroxidase activity by salicylic acid and naphthalene acetic acid. Plant Physiol Biochem. 2001;39(6):529–38.
- 38. Subramanian C, Woo J, Cai X, Xu X, Servick S, Johnson CH, Nebenführ A, Von Arnim AG. A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET). The Plant Journal. 2006 Oct;48(1):138–52. pmid:16925598
- 39. Kanchanamala RWMK Bandaranayake PCG. An efficient and rapid Rhizobium rhizogenes root transformation protocol for Lemna minor. Plant Biotechnol Rep [Internet]. 2019;13(6):625–33. Available from: https://doi.org/10.1007/s11816-019-00558-9
- 40. Tyurin AA, Suhorukova A V., Kabardaeva K V., Goldenkova-Pavlova I V. Transient gene expression is an effective experimental tool for the research into the fine mechanisms of plant gene function: Advantages, limitations, and solutions. Plants. 2020;9(9):1–19.
- 41. Faize M, Faize L, Burgos L. Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation. BMC Biotechnol. 2010;10.
- 42. Medina-Bolivar F, Condori J, Rimando AM, Hubstenberger J, Shelton K, O’Keefe SF, Bennett S, Dolan MC. Production and secretion of resveratrol in hairy root cultures of peanut. Phytochemistry. 2007 Jul 1;68(14):1992–2003. pmid:17574636
- 43. Triplett BA, Moss SC, Bland JM, Dowd MK. Induction of hairy root cultures from Gossypium hirsutum and Gossypium barbadense to produce gossypol and related compounds. In Vitro Cellular & Developmental Biology-Plant. 2008 Dec;44:508–17.
- 44. Wang CT, Liu H, Gao XS, Zhang HX. Overexpression of G10H and ORCA3 in the hairy roots of Catharanthus roseus improves catharanthine production. Plant cell reports. 2010 Aug;29:887–94. pmid:20535474