Location: Floral and Nursery Plants Research2013 Annual Report
1a. Objectives (from AD-416):
The primary objective of this work is to develop techniques and methods that will lead to sterile or highly infertile cultivars of invasive or potentially invasive nursery crops.
1b. Approach (from AD-416):
The general approach is to use use biotechnology or genetic engineering to create seedless plants or to develop methods that will lead to the creation of seedless plants. One area of study will be to test gene suppression using the AGAMOUS gene and dsRNA-RNAi. The AGAMOUS gene is a floral homeotic gene that, when suppressed or mutated, can result in flowers that have petals in place of pistils and stamens, and are therefore sterile. The second area of study will be to create transgenic plants using several different gene constructs that may induce male and/or female sterility.
3. Progress Report:
The main goal of this work is to develop methods that will lead to sterile or highly infertile cultivars of invasive or potentially invasive woody nursery crops. As a model, transgenic sweetgum trees were produced and planted in the field six years ago. They continue to grow very well, showing similar or greater growth and fall color than the wild-type controls that were also propagated in vitro and transplanted at the same time. Three non-transgenic controls have flowered, and we are expecting that, with their obvious vigorous growth this year, the remaining individuals will flower in 2014. Our previous attempt to design primers to analyse expression of our targeted and housekeeping genes was not successful. The initial primers either gave no amplicon, or multiple amplicons, instead of the desired single amplicon. Therefore, we designed and tested new primer sets for each gene of interest. We have primers that successfully amplify a single amplicon for each AG gene, the AGM3 transgene, and the housekeeping gene GAPC. These primers are currently undergoing testing to optimize their use for quantitative PCR. Our effort to develop successful PCR primers for sweetgum has been hampered by the lack of known gene transcripts. A sweetgum “transcriptome” would provide us with exact sequences of many potential control genes, and would be a valuable resource for the larger scientific community. We plan to use our in-house Illumina HiSeq2000 to obtain transcript sequences from our Worploden control trees, the same cultivar used for transformation. The data will be analysed using both reference-guided assembly (using the poplar genome as a guide) and de-novo assembly tools to generate a set of expressed sweetgum transcripts and their associated sequences. Currently, we are working to optimize RNA extraction methods to obtain the quality and quantity of RNA needed for this procedure. Near the start of this project, we produced an RNAi construct directed against the AGAMOUS homolog in apple. The construct was sent to a cooperator, who produced transgenic Gala apples with it. He has reported the recovery of a number of putatively sterile transgenic flowering trees with multiple whorls of petals, as expected for AG inhibition. The most extreme trees show up to 20 petals, instead of the normal five. These trees were pollinated this past spring to test for correlation between fertility and flower morphology. Additionally, floral tissues were collected from these trees for gene expression analysis. These results suggest that RNAi against AGAMOUS could be an effective means for producing sterile crabapples with showy flowers. We collaborated with a scientist at Oregon State University on a problem analysis of approaches to reducing invasiveness using conventional biotech and transgenic methods. Our resulting manuscript was recently published in the journal HortScience. In addition, we have given several oral presentations about this research.