Methods for transient assay of gene function in floral tissues.
BACKGROUND
There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.


RESULTS
Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum) chalcone synthase gene (CHS) and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression.


CONCLUSIONS
We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to insert the gene sequence of interest. These techniques should allow analysis of gene function in a much broader range of flower species.
Shang
Yongjin
Y
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.|AgResearch, Private Bag 11008, Palmerston North, New Zealand.|Institute of Molecular BioSciences, Massey University, Private Bag 11222 Palmerston North, New Zealand.
Schwinn
Kathy E
KE
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.
Bennett
Michael J
MJ
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.
Hunter
Donald A
DA
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.
Waugh
Toni L
TL
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.|AgResearch, Private Bag 11008, Palmerston North, New Zealand.
Pathirana
Nilangani N
NN
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.|Institute of Molecular BioSciences, Massey University, Private Bag 11222 Palmerston North, New Zealand.
Brummell
David A
DA
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.
Jameson
Paula E
PE
Institute of Molecular BioSciences, Massey University, Private Bag 11222 Palmerston North, New Zealand.|School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Davies
Kevin M
KM
New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.