Development of Efficient Protocols for Stable and Transient Gene Transformation for Wolffia Globosa Using Agrobacterium.

P P M Heenatigala,Sunjeet Kumar,Zuoliang Sun,Gaojie Li,Hongwei Hou,Shiqi Hu,Wei Lin,Lunguang Yao,Jingjing Yang,Anthony Bishopp,Pengfei Duan,Zhigang Wu

doi:10.3389/fchem.2018.00227

Abstract

Members of the Wolffia genus are fascinating plants for many biologists as they are the smallest flowering plants on Earth and exhibit a reduced body plan that is of great interest to developmental biologists. There has also been recent interest in the use of these species for bioenergy or biorefining. Molecular and developmental studies have been limited in Wolffia species due to the high genome complexity and uncertainties regarding the stable genetic transformation. In this manuscript we present new protocols for both stable and transient genetic transformation for Wolffia globosa using Agrobacterium tumefaciens. For the transient transformation, we used Wolffia fronds whereas we used clusters for the stable transformation. As proof of concept we transformed two synthetic promoter constructs driving expression of the GUS marker gene, that have previously been used to monitor auxin and cytokinin output in a variety of species. Using these approaches we obtained a Transformation Efficiency (TE) of 0.14% for the stable transformation and 21.8% for the transient transformation. The efficiency of these two methods of transformation are sufficient to allow future studies to investigate gene function. This is the first report for successful stable transformation of W. globosa.

Highlights

The genus Wolffia is a member of Lemnaceae or duckweed family
We noted that the percentage of GUS positive plants was higher in transient transformation conducted using Agrobacteria strain EHA105 compared with to using LBA4404 strain (Figure 3C) and propose that the Agrobacteria strain EHA105 is more suitable for the transformation of W. globosa
In our stable transformation assays, the Agrobacterium strains EHA 105 worked efficiently, and based upon this observation we propose that the EHA105 is more suitable for W. globosa transformation than the LBA 4404 strain

Summary

Introduction

The genus Wolffia is a member of Lemnaceae or duckweed family. It has been reported that duckweeds absorb nutrients and water through the underside of their fronds (Leng, 1999) most likely making the root functionally redundant (Hillman, 1961; Anderdon et al, 1973). Wolffia increases their biomass mainly through asexual budding by producing daughter fronds within a single side pouch (basal cavity) of the mother frond (Sree et al, 2015; Ziegler et al, 2015). Wolffia plants are able to double their population size within 30 h (Skillicorn et al, 1993)

Methods

Results

Discussion

Conclusion