In vitro growth and content of vincristine and vinblastine of Catharanthus roseus L. hairy roots in response to precursors and elicitors

Catharanthus roseus L. is a medicinal plant that produces numerous indole terpenoid alkaloids, including vincristine and vinblastine, which are used for cancer treatment. The effect of specified precursors (L-phenylalanine, L-tyrosine) and elicitors (chitosan, methyl jasmonate) on C. roseus hairy roots (CHR) growth has been examined in order to increase the content of vincristine and vinblastine. Our results showed that CHR generated by an Agrobacterium rhizogenes strain isolated in Vietnam was capable of producing both vincristine and vinblastine when subjected to precursors, but only vinblastine when exposed to elicitors. However, both precursors and elicitors were evaluated to have an effect on increasing the accumulation of TIAs in CHR. In particular, the use of elicitors required more time to find the appropriate induction conditions, while the use of precursors gave outstanding efficiency in the treatment with 1 µM phenylalanine. The greatest yields of vincristine (51.99 µg g-1 DW) and vinblastine (699.92 µg g-1 DW) were obtained in the 7th week (with 0.306 g DW biomass). This result is the first time we might boost the levels of vincristine and vinblastine in our CHR clone generated by the Vietnam strain of A. rhizogenes.

Detection of Target Sites Edited in Upland Cotton By The CRISPR/Cas9 System Mediated By Agrobacterium Rhizogenes

Background CRIPSR/Cas9 gene editing has the ability to effectively modify plant genomes. Multiple target sites usually were designed and the effective target sites were selected for editing. However, upland cotton is allotetraploid and is commonly considered as difficult and inefficient to transform. Therefore, it’s important to quickly identify feasibility of the target site. In this study, we use Agrobacterium rhizogenes K599 strain to infect cotton shoot meristem and induce them to grow hairy roots to detect the feasibility of a selected target designed in GhMYB25-like gene. Results We designed a sgRNA within the second exons of GhMYB25-likeA and GhMYB25-likeD and constructed the CRISPR vector. Transient hairy root transformation using A. rhizogenes K599 with four OD600s (0.4, 0.6,0.8, 1.0) was performed in Coker 312 (R15). The results show that A. rhizogenes at OD600 = 0.6–0.8 is the best concentration range for inducing cotton hairy roots. The other three cultivars (TM-1, Lumian 21, Zhongmian 49) were injected using A. rhizogenes K599 with OD600 = 0.6-0.8 and all produced hairy roots. We characterized ten R15 plants with hairy roots and detected different degrees of base deletions and insert at the target site in five R15 plants. Conclusion Overall, our data show A. rhizogenes-mediated transient hairy root transformation offers a rapid and efficient method to detect sgRNA feasibility in cotton.

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

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.

Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

Technology and Application of Hairy Root Culture in Monocotyledons

Background: Hairy root culture has been widely used in the production of metabolites in dicotyledons, and a large number of food crops and medicinal plants in monocotyledons need to be developed, but there are many difficulties in the induction of hairy roots in monocotyledons. The purpose of this paper is to introduce the inducing methods, influencing factors and application of hairy roots in monocotyledons, and to promote the development of hairy root system in monocotyledons. Methods: The mechanism of action of Agrobacterium rhizogenes and the current situation of hairy root induction, induction methods and influencing factors of monocotyledons were summarized so as to provide convenience for efficient acquisition of hairy root of monocotyledons. Results: Monocotyledons are not easy to produce phenols, cells are prone to lignification, adverse differentiation and selective response to Agrobacterium rhizogenes strains. It is proposed that before induction, plant varieties and explants should be selected, and different infection strains should be screened. In the process of hairy root induction, exogenous inducers such as acetosyringone can be added. Although these factors can provide some help for the induction of hairy roots in monocotyledons, we still need to pay attention to the disadvantages of monocotyledons from dicotyledons at the cellular level. Conclusion: A large number of food crops and medicinal plants are monocotyledons. Hairy root culture can be used to help the breeding and production of medicinal substances. Therefore, it is necessary to pay attention to the selection of varieties and explants, the selection of Agrobacterium rhizogenes and the addition of acetosyringone in the process of hairy root induction so as to improve the production efficiency and facilitate the development and utilization of monocotyledons.

Characterization of Carnivorous Plants Sarracenia Purpurea L. Transformed with Agrobacterium Rhizogenes

Carnivorous plant of Sarracenia genus are used by human in folk medicine for centuries. The reason for this phenomenon is biochemical composition of Sarracenia plants, which possess many bioactive compounds with anti-inflammatory, antioxidant, antiviral and antibacterial properties.The subject of this research was genetic transformation of Sarracenia purpurea L. with Agrobacterium rhizogenes strain 15834, 9402 and A4 by using two alternatively methods of bacterial injection or co-culture the bacteria with plants explants. These studies confirmed the possibility of hairy roots induction in S. purpurea using the strain of A. rhizogenes 15834 and the injection method. Seven lines of transformed plants, exhibiting the integration of rolB gene, were obtained. The formed hairy roots showed morphological differences in comparison to the roots of unmodified plants. A mathematical model was used to optimize the conditions for the extraction of bioactive compounds. Extracts isolated under optimal conditions from transformed plants showed biochemical changes i.e., the increase in the accumulation of total polyphenols and triterpenes in comparison to untransformed plants, especially when induced roots were analyzed. HPLC analysis showed increase in the level of betulinic acid in some transformed Sarracenia lines. Betulinic acid remains pentacyclic triterpenoid compound with high pharmacological significance. The further work connected with isolation and identification of the other bioactive compounds will be done in the nearest future.