A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes

Area of Science:

• Plant biotechnology
• Microbial genetics

Background:

• Agrobacterium rhizogenes (now Rhizobium rhizogenes) is a key tool for creating transgenic hairy roots.
• Existing methods for generating engineered R. rhizogenes are time-consuming and labor-intensive.

Purpose of the Study:

• To develop a simplified and efficient protocol for generating Rhizobium rhizogenes transconjugants.
• To improve the process of creating genetically modified R. rhizogenes for plant transformation.

Main Methods:

• Utilized bi-parental mating and effective counterselection for R. rhizogenes transconjugant generation.
• Constructed a new Modular Cloning (MoClo) compatible integration vector (pIV101).
• Employed RP4-based mobilization for simplified conjugal transfer.

Main Results:

• The new protocol significantly reduced the time and labor required to create transconjugant R. rhizogenes.
• The pIV101 vector facilitated efficient construction of recombinant vectors and screening of transconjugants.
• Transconjugants were successfully used for transgenic hairy root transformation in Lotus japonicus, supporting rhizobia symbiont infection.

Conclusions:

• The developed protocol offers an expedited method for generating transconjugant R. rhizogenes.
• This approach is efficient for transgenic hairy root transformation and can be applied to other plant species.
• The new vector and mating strategy streamline the genetic engineering of R. rhizogenes.