Volume 5 Supplement 7
Development of Eucalyptus tissue culture conditions for improved in vitro plant health and transformability
© Ma et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform in vitro. Compared to other plant species, few non-commercial laboratories are proficient at Eucalyptus tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.
Five different clones of E. grandis (including the clone of which the genome was recently sequenced – Brasuz1) as well as a single E. grandis x urophylla hybrid were established in the laboratory. Light intensity was studied as a means to reduce browning and promote callus growth and shoot regeneration. Gibberelic acid (GA3) concentrations in the micropropagation medium (Murashige and Skoog’s (MS) basal medium containing 0.05 mg/L benzylaminopurine – BAP) were studied to help produce long internodes to aid transformation in vitro. To mitigate phenolic production we used several different antioxidants including ascorbic acid, PVP and PVPP. Shoot regeneration rates were studied by testing a concentration range of cytokinin (zeatin, benzyl aminopurine – BAP and thidiazuron - TDZ). We tested different Agrobacterium transformation protocol enhancements including the use of acetorsyringone and two different co-cultivation techniques (whole explant immersion versus pipetting of Agrobacterium suspension onto cut leaf edges). We also tested the suitability of kanamycin and hygromycin as selectable markers during transformation.
Results and discussion
There is significant variability in the performance and response of different clones of Eucalyptus during micropropagation, organogenesis and transformation. Testing of multiple Eucalyptus clones confirmed that the genus is relatively unwieldy in tissue culture compared, for example, to Populus. We found that explant type (stem vs leaf for example), and quality (age, general health, absence callus browning) as well as finely-tuned phytohormone concentrations, will play critical roles in enhancing the probability of the successful generation of stable transgenic lines.
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.